Multivariate profile and acute-phase correlates of cognitive deficits in a COVID-19 hospitalized cohort

Authors: AdamHampshireaDoris A.ChatfieldbAnne ManktelowMPhilbAmyJollyaWilliamTrenderaPeter J.HellyeraMartina DelGiovaneaVirginia F.J.NewcombebJoanne G. Outtrimb BenWarneb JunaidBhattid LindaPointond AnneElmere NyarieSitholebf JohnBradleybgh NathalieKingston lStephen J.Sawceri Edward T.Bullmorecdj…David K.Menonbck1

eClinicalMedicine

Volume 47, May 2022, 101417

Summary

Background

Preliminary evidence has highlighted a possible association between severe COVID-19 and persistent cognitive deficits. Further research is required to confirm this association, determine whether cognitive deficits relate to clinical features from the acute phase or to mental health status at the point of assessment, and quantify rate of recovery.

Methods

46 individuals who received critical care for COVID-19 at Addenbrooke’s hospital between 10th March 2020 and 31st July 2020 (16 mechanically ventilated) underwent detailed computerised cognitive assessment alongside scales measuring anxiety, depression and post-traumatic stress disorder under supervised conditions at a mean follow up of 6.0 (± 2.1) months following acute illness. Patient and matched control (N = 460) performances were transformed into standard deviation from expected scores, accounting for age and demographic factors using N = 66,008 normative datasets. Global accuracy and response time composites were calculated (G_SScore & G_RT). Linear modelling predicted composite score deficits from acute severity, mental-health status at assessment, and time from hospital admission. The pattern of deficits across tasks was qualitatively compared with normal age-related decline, and early-stage dementia.

Findings

COVID-19 survivors were less accurate (G_SScore=-0.53SDs) and slower (G_RT=+0.89SDs) in their responses than expected compared to their matched controls. Acute illness, but not chronic mental health, significantly predicted cognitive deviation from expected scores (G_SScore (p=​​0.0037) and G_RT (p = 0.0366)). The most prominent task associations with COVID-19 were for higher cognition and processing speed, which was qualitatively distinct from the profiles of normal ageing and dementia and similar in magnitude to the effects of ageing between 50 and 70 years of age. A trend towards reduced deficits with time from illness (r∼=0.15) did not reach statistical significance.

Interpretation

Cognitive deficits after severe COVID-19 relate most strongly to acute illness severity, persist long into the chronic phase, and recover slowly if at all, with a characteristic profile highlighting higher cognitive functions and processing speed.

Funding

This work was funded by the National Institute for Health Research (NIHR) Cambridge Biomedical Research Centre (BRC), NIHR Cambridge Clinical Research Facility (BRC-1215-20014), the Addenbrooke’s Charities Trust and NIHR COVID-19 BioResource RG9402. AH is funded by the UK Dementia Research Institute Care Research and Technology Centre and Imperial College London Biomedical Research Centre. ETB and DKM are supported by NIHR Senior Investigator awards. JBR is supported by the Wellcome Trust (220258) and Medical Research Council (SUAG/051 G101400). VFJN is funded by an Academy of Medical Sciences/ The Health Foundation Clinician Scientist Fellowship. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health and Social Care.

Evidence before this study

A PubMed search for articles using the terms ‘COVID-19′, ‘chronic’ and ‘cognitive impairment’ returns 85 results between 2020 and 2022, reflecting growing concern that people may suffer persistent cognitive problems after SARS-CoV-2 infection. However, most of these studies have built on either subjective report of cognitive problems or brief pen-and paper assessment scales that lack sensitivity to mild deficits and precision regarding affected cognitive domains.

Added value of this study

Using precision computerised cognitive assessment tools, we observed that 46 COVID-19 patients matched for age, gender, education and first language, 6–10 months after admission for care at Addenbrookes hospital perform less well than controls in terms of cognition. Critically, the scale of their cognitive deficits correlated with acute illness severity as recorded during the hospital stay, but not fatigue or mental health status at the time of cognitive assessment.

Implications of all the available evidence

These results suggest that the patients who have recovered from severe COVID-19 may need longer term support for cognitive deficits that persist into the chronic phase. More research is required to understand the basis of these deficits. Future work will be focused on mapping these cognitive deficits to underlying neural pathologies and inflammatory biomarkers, and to longitudinally track recovery into the chronic phase.

Introduction

There is growing evidence that COVID-19 can cause lasting cognitive and mental health problems. Recovered patients reporting psychological symptoms including fatigue, cognitive difficulties (“brain fog” and “problems finding the words”), sleep disturbances breathlessness and psychiatric disorders months after infection.1 In the UK alone, 13.7% of 20,000 individuals reported having symptoms inclusive of cognitive difficulties 12 weeks after a positive COVID-19 test (UK Office for National Statistics, April 2021). Mild cases can report persistent cognitive symptoms; however, prevalence is higher in severe cases,2 with ∼33–76% of patients suffering cognitive symptoms 3–6 months post hospitalisation.3,4

The neurobiological and psychological bases of these deficits remain unclear. Imaging biomarker studies indicate multiple likely candidates. Indeed, drawing parallels with serious acute respiratory syndrome (SARS), middle eastern respiratory syndrome (MERS) and post-critical illness/intensive care syndrome, a range of neurological/ central nervous system (CNS) complications can arise from infection.5,6 Most notably, encephalitis, ischaemia, haemorrhage, microstructural and functional changes and cerebrovascular disease (CVD) have been observed in COVID-19 patients, and more recently, evidence of brainstem inflammation using 7 Tesla magnetic resonance imaging (MRI) has been reported.7 There has been concern regarding whether cognitive deficits will remain for years as a chronic syndrome, and whether patients who develop CVD as a result of infection will experience neurodegeneration and dementia in the long-term,789 despite recovery of other acute and sub-acute symptoms.10

Key limitations for much of this early work include a reliance on self-report as opposed to objective assessment of cognitive deficits, the application of neuropsychological scales that lack sensitivity to detect subtle deficits in the formerly unimpaired or precision to differentiate deficits across cognitive domains, and uncertainty regarding longevity of deficits. Furthermore, depression, anxiety, fatigue and post-traumatic stress are elevated post COVID-19 illness,11 which might mediate the association with cognitive sequelae.

Recently, we provided preliminary results addressing some of these limitations. Specifically, we used computerised cognitive assessment technology,12,13 which has superior sensitivity and precision to gold-standard neuropsychological scales,14 to investigate objectively measurable deficits across multiple cognitive domains in a large online cohort15,16 that incidentally included people who reported infection with COVID-19 of varying severity.17 Higher cognitive functions such as spatial planning and analogical reasoning appeared to be disproportionately impaired, especially in hospitalised patients. However, our earlier analyses lacked clinical-record corroboration of self-reported illness severity or hospital treatment. Furthermore, participants primarily were in the early chronic phase ∼2–3 months post illness, which limited insight into the longevity of deficits.

Here, we use the same technology to assess patients at timepoints ranging from between ∼1 and 10 months post admission to hospital for severe COVID-19. We sought to determine whether (i) the finding of higher cognitive deficits after COVID-19 infection can be replicated in a hospital confirmed cohort, (ii) the cognitive deficits relate to features of acute illness vs. mood, anxiety, tiredness or post-traumatic stress disorder (PTSD) at the point of assessment, (iii) the deficits negatively correlate with time since illness and (iv) the scale and profile of deficits is qualitatively comparable to that observed in normal age-related decline or dementia.

Methods

Data collection

All patients admitted to Addenbrookes Hospital with COVID-19 between 10th March 2020 and 31st July 2020, who survived and consented to take part were eligible for this cohort study. This comprised 489 patients, of whom 49 were consented to the NIHR COVID-19 BioResource to participate in the study and were administered the follow up battery. The study was approved by the Cambridge Central Research Ethics Committee (17/EE/0025 0025 & IRAS ID: 220277). Of these, 46 patients (27 females, 19 males, age mean=51 years standard deviation (SD)=14 years, range 28–83 years) completed the study protocol adequately to allow analysis (Tables S1–3). Based on the effect size observed in our previous citizen science dataset,17 where people were assessed using the same technology, expected effect size for critically ill hospitalised patients would be >0.5 standard deviations. At n-46, power was sufficient to detect with one-tailed alpha at p < 0.05 a 0.5SD effect size difference as gauged by DfE scores from the linear model at 96% relative to zero and at 94% relative to the matched control group. There was statistical power of 95% to detect medium strength correlations of r = 0.50 at two tailed alpha p < 0.05. Participants completed a custom computerised cognitive assessment battery under supervised conditions via the Cognitron platform,17,18 comprising 8 tasks deployed on an iPad (Supplemental Methods), as well as standard mood, anxiety and post-traumatic stress scales, specifically, the Generalized Anxiety Disorder 7 (GAD-7),19 the Patient Health Questionnaire 9 (PHQ-9)20,21 and the PTSD Checklist for Diagnostic and Statistical Manual of Mental Disorders 5 (PCL-5)22 in a return visit to the hospital on average 179 days after illness onset (SD=62 inter-quartile range=81).

Statistical methods

All analyses were conducted in MATLAB R2020a. To enable correlation of deficit magnitude with clinical and mental health measures whilst accounting for population variables, accuracy and median reaction times were extracted for each task, comprising 16 measures (Table S4), were transformed to deviation from expected (DfE) scores (see below definition) relative to N = 66,008 normative datasets (Table S5), comprising individuals who had performed the same set of tasks. Specifically, to calculate DfE scores, linear models were trained to predict performance for each task within the normative dataset from age decade, sex (male, female, other), education level, handedness (left, right ambidextrous) and first language (English, other). The trained models were then applied to the patient demographics, to which they were naive, providing expected scores for each individual. DfE score was quantified as the difference between observed minus predicted score divided by the control standard deviation. Non-compliant individuals from the normative dataset already had been identified and removed based on responding unfeasibly fast given the response time distribution; applying the same threshold identified no non-compliant participants within the patient dataset. Four patients could not complete Verbal Analogies and one could not complete Spatial Planning as they found them too challenging. Control and patient datasets were concatenated, and composites were then calculated by taking the first unrotated principal component (Table S6) across the eight summary score measures (G_SScore), focused on accuracy, and across the eight response time scores (G_RT), focussing on speed of response. Component scores were calculated for each subject via regression of the component loadings matrix across the above measures, excluding any unavailable datapoints, and transformed to DfE score as described above. For further comparison, a set of matched controls was identified from within the normative database and processed in the same manner as the patients. Specifically, for each patient, ten unique control datasets were randomly selected who exactly matched them in terms of age decade, sex, handedness, first language and education level.

All statistical analyses applied a prior significance cut-off set to p < 0.05. T-tests, performed one-tailed with family wise error (FWE) correction for multiple comparisons, evaluated whether patient composite and individual task DfE scores were consistently poorer than expected relative to the matched normative group. Multiple regression determined whether G_SScore and G_RT DfE scores could be predicted from clinical features during the acute hospital stay or mental health measures at the time of assessment. Clinical features were World Health Organisation (WHO) COVID-19 severity score,23 highest C-reactive protein (CRP), mechanical ventilation, extrapulmonary support, days ventilated, tracheostomy, and highest D-dimer; as well as age, sex and time since illness. Mental health scores were the GAD7, PHQ9 and PCL5. Due to high correlations between some of these clinical features, the feature matrix was reduced by applying Principal Component Analysis with varimax rotation, where the number of components was defined according to the Kaiser convention of retaining components with eigenvalues >1. The relationship between G_SScore and G_RT to time since illness was further examined in isolation using bivariate correlations with one-tailed significance.

To qualitatively gauge whether the profile of COVID-19 related cognitive deficits was similar in pattern or scale to age-related decline, standard deviation differences were extracted from the normative models (that is, accounting for the other population variables listed above) for each task between people at ages aged 70–79 minus those 20–29 or 50–59 within the control dataset. For further qualitative comparison, performance data from a previously collected group of 28 early-mid stage dementia patients were submitted to the same DfE pipeline as described above and effect sizes plotted (clinical and demographic details provided in Table S7).

Role of the funding source

The funder of the study had no role in the design of the study, data collection, data analysis, interpretation or writing of the report. All authors had full access to all data within the study. The corresponding authors had final responsibility for the decision to submit for publication.

Results

T-tests of global summary score and response time composites (Figure 1a) confirmed that participants who had been hospitalised due to COVID-19 scored significantly lower and were slower in their responses than would be expected given the control population as gauged by DfE scores (G_SScore estimate=-0.538 SDs, t=-4.214 p < 0.0001; G_RT estimate=0.726SDs, t = 4.507, p < 0.0001). Repeating the analysis for the 43 chronic-phase patients >90 days post symptom onset showed a similar result (G_SScore estimate=-0.524 SDs, t=-3.875 p = 0.0004; G_RT estimate=0.715SDs, t = 4.194, p < 0.0001). Contrasting the DfE scores directly against 460 precisely matched individuals (Figure 2), 10 per patient, from the control database reinforced this observation (mean difference in G_SScore estimate=-0.525SDs, t=-4.327, p < 0.0001; mean difference in G_RT estimate=0.887SDs, t = 5.803, p < 0.0001).

Fig 1
Fig 2

Application of Principal Component Analysis to the matrix of clinical and mental health features identified three components with eigenvalues >1 capturing 74% of the variance (Figure 1b). After varimax rotation, Component 1 captured variance pertaining to general severity of acute illness, including heavy positive loadings from WHO COVID-19 severity score, highest CRP, and requirement for mechanical ventilation, extrapulmonary organ support and days ventilated, moderate positive loading with age and requirement of tracheostomy and moderate negative loading for days since illness. Component 2 had heavy positive loading of requirement for tracheostomy and days ventilated, moderate positive loading for highest D-dimer and mechanical ventilation and extrapulmonary support, and moderate negative loading for females vs. males and time from illness onset. Component 3 had heavy positive loadings for the three mental health scales.

Multiple regression of the component scores onto DfE performance composites (Figure 1c) showed a significant negative correlation between G_SScore and Component 1 (Estimate=-0.346, F(1,42)= 9.392 p = 0.00380), but not Component 2 (Estimate=0.140, F(1,42)=1.841 p = 0.18208) or Component 3 (Estimate=-0.153, F(1,42)=1.855 p = 0.18041). There was also a threshold level negative correlation between G_RT and Component 1 (Estimate=0.305, F(1,42)=4.008 p=​ 0.05178), but not Component 2 (Estimate=-0.177, F(1,42)=1.791 p = 0.21044) or Component 3 (Estimate=0.111, F(1,42)=0.592 p = 0.46861).

Bivariate correlations (Table S8) showed significant associations between G_SScore and Severity WHO COVID-19 ordinal scale, mechanical ventilation, extrapulmonary organ dysfunction support and highest CRP during admission at the one tailed uncorrected threshold. However, the hypothesised trends towards reduced underperformance over time were of small effect size and were statistically non-significant (G_SScore r = 0.15 p = 0.1542, G_RT r=-0.16 p = 0.1486 one tailed and uncorrected). Reanalysing the data focusing exclusively on either those who were or were not ventilated relative to their respective controls showed significant cognitive deficits in both sub-groups (Fig. S1 & Table S9).

Finally, DfE scores were examined at the individual task level. There was a broad pattern of reduced accuracy and slowed response compared to the 460 matched controls (Table 1Figure 2a), with multiple tasks surviving the p < 0.05 one-tailed and family wise error (FWE) corrected for multiple comparisons threshold. As predicted,17 underperformance was more substantial for tasks challenging higher cognitive functions such as Analogical Reasoning (score -0.85SDs RT +1.34SDs) and Spatial Planning (score +0.28SDs RT +0.89SDs), as well as 2D Manipulations (score -0.58SDs RT +0.57SD) and word recall (immediate score -0.43SDs RT +0.43SDs delayed score -0.051SDs RT +0.46SDs).

Table 1. T-tests contrasting patients vs. 460 matched controls (one-tailed and FWE corrected for multiple comparisons).

Empty CellEmpty CellEffect size (DfE)tp (corrected)
AccuracyVerbal analogies-0.854-6.205<0.00001
2D manipulation-0.575-4.2210.00026
Words immediate-0.432-2.8690.03863
Spatial span-0.405-3.6050.00309
Target detection-0.176-1.4501.32876
3D rotation-0.076-0.9962.87946
Words delayed-0.051-0.4585.82405
Spatial planning0.2831.5101.18614
LatencySpatial span0.2311.5431.11135
Words immediate0.4313.0350.02276
Target detection0.4442.5680.09468
Words delayed0.4632.9420.03070
2D manipulation0.5703.8790.00107
3D rotation0.6203.5220.00421
Spatial planning0.8884.7790.00002
Verbal analogies1.3377.018<0.00001
GlobalG_SScore-0.525-4.3270.00016
G_RT0.8875.803<0.00001

For comparison (Figure 2), the pattern of mean age-related differences in performance of people in their 70s minus 20s or 70s minus 50s was quite distinct, with age related differences being most pronounced for 2D Manipulations, Spatial Span and Target Detection as opposed to Spatial Planning or Verbal Analogies. Furthermore, the 28 dementia patients who undertook 6 of the tasks showed the greatest DfE score on the Word Memory task with notably higher effect size.

Discussion

Individuals who survive severe COVID-19 illness have objectively measurable cognitive deficits, lasting many months, with respect to age- and demographic-adjusted norms.17,24252627282930 Taking Cohen’s notion of effect sizes as a gauge, the scale of those deficits was large; on average the 0.52SD and 0.89SD levels of underperformance on global accuracy and response time composite measures span the medium to large effect size range. For individuals who required mechanical ventilation, both composites were in the large range at 0.90SDs and 1.0 SDs, respectively, which is somewhat larger than our previous online study using the same assessment tools.17 The deficits within specific cognitive domains were even greater, e.g., Verbal Analogies response times were 1.3SDs longer on average for all patients and 1.7SDs for those who had required mechanical ventilation. Notably, when analysing only those individuals for whom English was the native language, the same pattern of deficits was still evident. Furthermore, our analyses accounted for both first language and education level. These results accord with self-reported problems ‘finding words’31 and neuropsychological case studies indicating verbal fluency deficits in severe COVID-19 patients post recovery.32

By using a large pre-existing normative dataset to correct for normal population variability in cognitive performance, we were able to begin the process of disentangling potential contributors to cognitive deficits post COVID-19. In particular, measures of mood, post-traumatic stress and mental health at the point of assessment were sufficiently dissociable from acute illness severity to be evaluated within the predictor matrix. This distinction is critical, because it is now well established that people who have recovered from severe COVID-19 illness can have a broad spectrum of symptoms of poor mental health11 as do those suffering from Long Covid,1 which could conceivably contribute to both self-perceived and objectively measured cognitive deficits. These include problems with depression, anxiety post-traumatic stress, low motivation, fatigue, low mood, and disturbed sleep. Here, it was clearly the case that acute illness severity was the better predictor of objectively measurable global cognitive deficits during the chronic phase. At the level of individual clinical features, WHO COVID-19 severity score, highest CRP and the requirement for mechanical ventilation and multiple organ support were predictive of poorer cognitive performance.

All patients were recruited from the same hospital and following illness within a narrow timeframe, which given differences in patient treatment and virus variants across time limits our confidence when generalising these results. We believe that this limitation is somewhat mitigated by the concordance between the results presented here and our previous citizen science dataset, published in this journal.17 Nonetheless, future research should seek to determine the relationship between variants, treatment strategies and cognitive outcomes at larger scale.

Regarding how representative the cohort was, the recruited population were younger, and more frequently female, and with a higher proportion of critical care admissions (WHO Ordinal Scale >6) than those who came through the centre (Tables S10–S14). A significant proportion, though not all, of these differences is attributable to the mortality of 24% in the overall admitted population, since non-survivors were older (median age=80 inter quartile range =73–87), more often male (64%), and may have included patients in whom treatment limitation decisions may have been in place.

Our analysis of fatigue post COVID-19 illness was not in the original analysis plan. However, scores capturing self-report of fatigue in the months post illness were available for 38 patients (Tables S1–3) from the Post-Intensive Care Unit Presentation Screen, a brief functional screening tool to inform the rehabilitation needs after treatment in intensive care settings. 28 of them endorsed some level of fatigue. Fatigue score correlated robustly with the mental health composite score (r=-0.45 p = 0.005) but not with the acute illness composite score (r = 0.03 p = 0.852) or either of the cognitive composite scores (G_ACC r = 0.19 p = 0.240; G_RT r=-0.16 p = 0.343). These results indicate that although both fatigue and mental health are prominent chronic sequalae of COVID-19, their severity is likely to be somewhat independent from the observed cognitive deficits.

A further limitation was that the acute clinical features were too highly correlated with each other to dissociate. All but two of the participants requiring mechanical ventilation also required multiple organ support, and the requirement for mechanical ventilation correlated with highest CRP, a measure of inflammation, at r∼=0.8. The observed correlation with a marker of acute inflammation may reflect a causal relationship beyond the severity of respiratory problems; however, given the high correlation to other clinical features of the acute phase, work seeking to disentangle underlying clinical causes of the observed cognitive deficits will require either substantial sized cohorts with sufficient power to delineate highly correlated predictors or additional data types, such as brain imaging in order to detect associations with markers in specific types of neuropathology.

Some previous studies have observed significant recovery across time in terms of cognitive symptoms18 and imaging measures of brain function.33 In accordance with these studies, we did observe slow and non-significant trends towards reduced deficits in both accuracy and response latency as a function of time from illness. We conclude that any recovery in cognitive faculties is at best likely to be slow. It also is important to consider that trajectories of cognitive recovery may vary across individuals depending on illness severity and the neurological or psychological underpinnings, which are likely complex. Plotting recovery trajectories and untangling their multivariate relationships to clinical features will require multi-timepoint studies in larger cohorts.

At a finer multivariate grain, the profile of deficits replicates our previous report in an online cohort of disproportionate underperformance within certain cognitive domains. In concordance with a previous large scale online study this pattern includes tasks designed to assess performance accuracy of attention, memory, difficult word-based reasoning and planning.17 However, we also observed slowed processing speed. On a neurological level, this pattern of impairment aligns with the observation of sub-acute phase hypometabolism within frontoparietal systems after COVID-19 illness26 that are known to be recruited in different combinations and configurations during the performance of these tasks.12,13,34

In this latter respect, the application of an assessment battery that provides a dimensional profile spanning multiple cognitive domains is of value when offering interoperability across studies. Indeed, it was informative to note that this profile of cognitive dysfunction was quite distinct to the normal pattern of age-related decline and to the pattern of deficits observed in early-stage dementia patients. On average, the scale of deficits was most similar to that observed in normal cognitive decline between the ages of 50–70; however, when examined in more detail the pattern of cognitive deficits was most pronounced for different tasks than either age-related decline or the dementia group. These more detailed results highlight the potential value of cross comparing multivariate profile of COVID-19 cognitive deficits to a wider variety of populations in order to identify potential similarities to other neurological conditions. Future work should also expand the repertoire of disorders, especially populations who have recovered from other critical illnesses, and cross relate these detailed cognitive profiles to imaging and blood biomarker measures of neuropathology and tracking recovery and decline trajectories over a longer temporal scale.

In summary, severe COVID-19 illness is associated with significant objectively measurable cognitive deficits that persist into the chronic phase. The scale of the deficits correlates with clinical severity during the acute phase as opposed to mental health status at the time of assessment, shows at best a slow recovery trajectory and the multivariate profile of deficits is consistent with higher cognitive dysfunction as opposed to accelerated ageing or dementia.

Funding

This work was funded by the National Institute for Health Research (NIHR) Cambridge Biomedical Research Centre (BRC), NIHR Cambridge Clinical Research Facility (BRC-1215-20014), the Addenbrooke’s Charities Trust and NIHR COVID-19 BioResource RG9402. AH is funded by the UK Dementia Research Institute Care Research and Technology Centre and Imperial College London Biomedical Research Centre. ETB and DKM are supported by NIHR Senior Investigator awards. JBR is supported by the Wellcome Trust (220258) and Medical Research Council (SUAG/051 G101400). VFJN is funded by an Academy of Medical Sciences/ The Health Foundation Clinician Scientist Fellowship. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health and Social Care.

Contributors

AH and DKM had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Concept and design: AH, JB, ETB, JBR, DKM

Acquisition, analysis, or interpretation of data: AH, DAC, AM, AJ, WT, PH, MDG, VFJN, JGG, JB, LP, AE, NS, JB, NK, SJS, DKM.

Drafting of the manuscript: AH.

Critical revision of the manuscript for important intellectual content: All authors.

Statistical analysis: AH.

Obtained funding: JB, JBR, ETB, DKM.

Supervision: AH, ETB, JBR, DKM.

Data sharing statement

Requests for data should directed to the corresponding authors. Data will be available upon reasonable request.

Declaration of Interests

Dr. Hampshire reports grants from UK Dementia Research Institute, grants from NIHR Imperial Biomedical Research Centre, and grants from NIHR, outside the submitted work; and is Co-director and owner of H2 Cognitive Designs Ltd and director and owner of Future Cognition Ltd, which support online cognitive studies and develop custom cognitive assessment software, respectively. Ms. Chatfield has nothing to disclose. Ms. Manktelow has nothing to disclose. Dr. Jolly has nothing to disclose. Mr. Trender has nothing to disclose. Dr. Hellyer reports being Chief Executive of H2 Cognitive Designs LTD, which provides a platform for online cognitive tests for remote assessment and receives remuneration for role. Ms. Del Giovane has nothing to disclose. Dr. Newcombe reports grants from Academy of Medical Sciences / The Health Foundation Clinician Scientist Fellowship during the conduct of the study. Ms. Outrim has nothing to disclose. Mr. Warne has nothing to disclose. Mr. Bhatti has nothing to disclose. Ms. Pointon has nothing to declare. Ms. Elmer has nothing to disclose. Dr. Sithole has nothing to disclose. Dr. Bradley reports grants from Funding for NIHR BioResource (IS-BRC-1215-20014) during the conduct of the study. Dr. Kingston has nothing to disclose. Dr. Sawcer has nothing to disclose. Dr. Bullmore reports personal fees from GlaxoSmithKline, personal fees from Sosei Heptares, outside the submitted work; and is Honorary Treasurer and member of Council for the Academy of Medical Sciences. Dr. Rowe reports grants from Wellcome Trust, grants from NIHR, grants from Medical Research Council, during the conduct of the study. Dr. Menon reports grants from Lantmannen AB, grants from GlaxoSmithKline Ltd, personal fees from Calico LLC, personal fees from GlaxoSmithKline Ltd, personal fees from Lantmannen AB, other from Integra Neurosciences, outside the submitted work; and reports leadership and fiduciary roles for Queens’ College, Cambridge, Intensive Care National Audit and Research Centre, London, and European Brain Injury Consortium.

Acknowledgments

We thank NIHR BioResource volunteers for their participation, and gratefully acknowledge NIHR BioResource centres, NHS Trusts and staff for their contribution. We thank the National Institute for Health Research, NHS Blood and Transplant, and Health Data Research UK as part of the Digital Innovation Hub Programme. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health and Social Care.

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References

1A. Nalbandian, K. Sehgal, A. Gupta, et al.Post-acute COVID-19 syndromeNat Med, 27 (4) (2021), pp. 601-615 View PDFCrossRefView Record in ScopusGoogle Scholar2D. Huang, X. Lian, F. Song, et al.Clinical features of severe patients infected with 2019 novel coronavirus: a systematic review and meta-analysisAnn Transl Med, 8 (9) (2020), p. 576 View PDFCrossRefView Record in ScopusGoogle Scholar3C. Huang, Y. Wang, X. Li, et al.Clinical features of patients infected with 2019 novel coronavirus in Wuhan, ChinaLancet, 395 (10223) (2020), pp. 497-506ArticleDownload PDFGoogle Scholar4V. Chopra, S.A. Flanders, M. O’Malley, A.N. Malani, H.C. PrescottSixty-day outcomes among patients hospitalized with COVID-19Ann Intern Med, 174 (4) (2021), pp. 576-578 View PDFCrossRefView Record in ScopusGoogle Scholar5S. Inoue, J. Hatakeyama, Y. Kondo, et al.Post-intensive care syndrome: its pathophysiology, prevention, and future directionsAcute Med Surg, 6 (3) (2019), pp. 233-246 View PDFCrossRefView Record in ScopusGoogle Scholar6M.H. Lam, Y.K. Wing, M.W. Yu, et al.Mental morbidities and chronic fatigue in severe acute respiratory syndrome survivors: long-term follow-upArch Intern Med, 169 (22) (2009), pp. 2142-2147 View PDFCrossRefView Record in ScopusGoogle Scholar7S. Miners, P.G. Kehoe, S. LoveCognitive impact of COVID-19: looking beyond the short termAlzheimers Res Ther, 12 (1) (2020), p. 170View Record in ScopusGoogle Scholar8D. Bougakov, K. Podell, E. GoldbergMultiple neuroinvasive pathways in COVID-19Mol Neurobiol, 58 (2) (2021), pp. 564-575 View PDFCrossRefView Record in ScopusGoogle Scholar9E. Goldberg, K. Podell, D.K. Sodickson, E. FieremansThe brain after COVID-19: compensatory neurogenesis or persistent neuroinflammation?EClinicalMedicine, 31 (2021), Article 100684ArticleDownload PDFView Record in ScopusGoogle Scholar10L.A. Jason, M.F. Islam, K. Conroy, et al.COVID-19 symptoms over time: comparing long-haulers to ME/CFSFatigue Biomed Health Behav (2021)Google Scholar11S.R. Chamberlain, J.E. Grant, W. Trender, P. Hellyer, A. HampshirePost-traumatic stress disorder symptoms in COVID-19 survivors: online population surveyBJPsych Open, 7 (2) (2021), p. e47View Record in ScopusGoogle Scholar12E. Soreq, I.R. Violante, R.E. Daws, A. HampshireNeuroimaging evidence for a network sampling theory of individual differences in human intelligence test performanceNat Commun, 12 (1) (2021), p. 2072View Record in ScopusGoogle Scholar13A. Hampshire, R.R. Highfield, B.L. Parkin, A.M. OwenFractionating human intelligenceNeuron, 76 (6) (2012), pp. 1225-1237ArticleDownload PDFView Record in ScopusGoogle Scholar14H. Brooker, G. Williams, A. Hampshire, et al.FLAME: a computerized neuropsychological composite for trials in early dementiaAlzheimers Dement, 12 (1) (2020), p. e12098(Amst)View Record in ScopusGoogle Scholar15Hampshire A. Great British intelligence test protocol. Research Square. 2020.Google Scholar16A. Hampshire, P.J. Hellyer, E. Soreq, et al.Associations between dimensions of behaviour, personality traits, and mental-health during the COVID-19 pandemic in the United KingdomNat Commun, 12 (1) (2021), p. 4111View Record in ScopusGoogle Scholar17A. Hampshire, W. Trender, S.R. Chamberlain, et al.Cognitive deficits in people who have recovered from COVID-19EClinicalMedicine (2021), Article 101044ArticleDownload PDFView Record in ScopusGoogle Scholar18Zhao S, Shibata K, Hellyer PJ. et al. Rapid vigilance and episodic memory decrements in COVID-19 survivors. medrXiv 2021.Google Scholar19R.L. Spitzer, K. Kroenke, J.B. Williams, B. LoweA brief measure for assessing generalized anxiety disorder: the GAD-7Arch Intern Med, 166 (10) (2006), pp. 1092-1097 View PDFCrossRefView Record in ScopusGoogle Scholar20K. Kroenke, R.L. Spitzer, J.B. Williams, B. LoweThe patient health questionnaire somatic, anxiety, and depressive symptom scales: a systematic reviewGen Hosp Psychiatry, 32 (4) (2010), pp. 345-359ArticleDownload PDFView Record in ScopusGoogle Scholar21R.L. Spitzer, K. Kroenke, J.B. WilliamsValidation and utility of a self-report version of PRIME-MD: the PHQ primary care study. Primary care evaluation of mental disorders. Patient health questionnaireJAMA, 282 (18) (1999), pp. 1737-1744Google Scholar22C.A. Blevins, F.W. Weathers, M.T. Davis, T.K. Witte, J.L. DominoThe posttraumatic stress disorder checklist for DSM-5 (PCL-5): development and initial psychometric evaluationJ Trauma Stress, 28 (6) (2015), pp. 489-498 View PDFCrossRefGoogle Scholar23WHO Working Group on the Clinical Characterisation and Management of COVID-19 infectionA minimal common outcome measure set for COVID-19 clinical researchLancet Infect Dis, 20 (8) (2020), p. e192-e7Google Scholar24V. Beaud, S. Crottaz-Herbette, V. Dunet, et al.Pattern of cognitive deficits in severe COVID-19J Neurol Neurosurg Psychiatry, 92 (5) (2021), pp. 567-568 View PDFCrossRefView Record in ScopusGoogle Scholar25J. Hellmuth, T.A. Barnett, B.M. Asken, et al.Persistent COVID-19-associated neurocognitive symptoms in non-hospitalized patientsJ Neurovirol, 27 (1) (2021), pp. 191-195 View PDFCrossRefView Record in ScopusGoogle Scholar26J.A. Hosp, A. Dressing, G. Blazhenets, et al.Cognitive impairment and altered cerebral glucose metabolism in the subacute stage of COVID-19Brain, 144 (4) (2021), pp. 1263-1276 View PDFCrossRefView Record in ScopusGoogle Scholar27A. Jaywant, W.M. Vanderlind, G.S. Alexopoulos, C.B. Fridman, R.H. Perlis, F.M. GunningFrequency and profile of objective cognitive deficits in hospitalized patients recovering from COVID-19Neuropsychopharmacology (2021)Google Scholar28K.W. Miskowiak, S. Johnsen, S.M. Sattler, et al.Cognitive impairments four months after COVID-19 hospital discharge: pattern, severity and association with illness variablesEur Neuropsychopharmacol, 46 (2021), pp. 39-48ArticleDownload PDFView Record in ScopusGoogle Scholar29B. Raman, M.P. Cassar, E.M. Tunnicliffe, et al.Medium-term effects of SARS-CoV-2 infection on multiple vital organs, exercise capacity, cognition, quality of life and mental health, post-hospital dischargeEClinicalMedicine, 31 (2021), Article 100683ArticleDownload PDFView Record in ScopusGoogle Scholar30M.S. Woo, J. Malsy, J. Pottgen, et al.Frequent neurocognitive deficits after recovery from mild COVID-19Brain Commun, 2 (2) (2020), p. fcaa205View Record in ScopusGoogle Scholar31J. Seessle, T. Waterboer, T. Hippchen, et al.Persistent symptoms in adult patients one year after COVID-19: a prospective cohort studyClin Infect Dis (2021)Google Scholar32D.M. Whiteside, V. Oleynick, E. Holker, E.J. Waldron, J. Porter, M. KasprzakNeurocognitive deficits in severe COVID-19 infection: case series and proposed modelClin Neuropsychol, 35 (4) (2021), pp. 799-818 View PDFCrossRefView Record in ScopusGoogle Scholar33G. Blazhenets, N. Schroeter, T. Bormann, et al.Slow but evident recovery from neocortical dysfunction and cognitive impairment in a series of chronic COVID-19 patientsJ Nucl Med, 62 (7) (2021), pp. 910-915 View PDFCrossRefView Record in ScopusGoogle Scholar34A.E. Jolly, G.T. Scott, D.J. Sharp, A.H. HampshireDistinct patterns of structural damage underlie working memory and reasoning deficits after traumatic brain injuryBrain, 143 (4) (2020), pp. 1158-1176 View PDFCrossRefView Record in ScopusGoogle Scholar

COVID-19 infection could age brain by 20 years, lower IQ significantly

Authors: Chris Melore Study Finds May 3, 2022

A severe coronavirus infection could leave patients with the brain of a 70-year-old, lowering someone’s IQ by 10 points, according to a new study. Researchers from the University of Cambridge and Imperial College London found that COVID patients are dealing with levels of cognitive impairment which are similar to the decline a healthy person sees between the ages of 50 and 70. Disturbingly, the team warns that this damage may never fully heal.

Long-term cognitive and mental health problems have been a growing issue during the pandemic. Even after the infection passes, a large number of patients continue to experience “brain fog,” problems recalling words, sleep issues, PTSD, and dozens of other symptoms for months — a condition doctors call long COVID. A recent study found that up to six in 10 recovering patients develop long COVID.

Even a mild case of the virus can lead to lingering cognitive issues. Study authors say as many as three-quarters of hospitalized COVID patients could suffer from some level of brain damage and cognitive decline.

Brain aging 20 years — in 6 months

Their new study examined 46 coronavirus patients entering the hospital or an intensive care unit between March and July 2020. Sixteen of these individuals ended up needing mechanical ventilation due to severe infection.

Six months later, the team conducted a series of computerized cognitive tests using the Cognitron platform. The system measures different aspects of brain health, including memory, attention, and reasoning skills. Researchers also examined each person’s levels of anxiety, depression, and PTSD following their infection.

In comparison to over 66,000 healthy people from the general public, results show severe COVID patients were less accurate and had slower response times on cognitive tests. These deficits were even worse among patients needing ventilation while in the hospital.

Estimates show that the damage from a COVID-19 infection led to the same amount of cognitive decline that the average person sees after 20 years of aging — between ages 50 and 70. That’s also the equivalent of losing 10 IQ points during their illness and the months following.

Specifically, COVID survivors did poorly on tasks involving verbal analogical reasoning — which translates to problems with finding the right words in conversation. The patients also displayed slower processing speeds, which the team says connects to the decreases in brain glucose consumption within the frontoparietal network of the brain doctors are seeing during the pandemic. This area of the brain controls a person’s attention span and complex problem-solving skills. It’s also important to their working memory.

“Cognitive impairment is common to a wide range of neurological disorders, including dementia, and even routine ageing, but the patterns we saw – the cognitive ‘fingerprint’ of COVID-19 – was distinct from all of these,” Professor David Menon says in a university release.

Some COVID-19 patients will ‘never fully recover’

Although studies continue to find ties between long COVID and lingering mental health issues, the study authors say they’ve discovered a clearer link between the severity of someone’s infection and cognitive decline. Concerningly, the team found that patients are only gaining some of these skills back over time.

“We followed some patients up as late as ten months after their acute infection, so were able to see a very slow improvement. While this was not statistically significant, it is at least heading in the right direction, but it is very possible that some of these individuals will never fully recover,” Prof. Menon adds.

As for what’s causing COVID to take such a toll on the human brain, researchers say there are a number of possibilities. COVID could be directly infecting the brain, but the team notes this is not a major cause. It’s more likely that a combination of inadequate oxygen or blood supply to the brain, blood clots, and microscopic bleeds are all contributing to the brain damage in coronavirus patients.

There is also growing evidence that COVID-19 produces inflammation which is similar to what people experience while developing Alzheimer’s disease.

“Around 40,000 people have been through intensive care with COVID-19 in England alone and many more will have been very sick, but not admitted to hospital. This means there is a large number of people out there still experiencing problems with cognition many months later. We urgently need to look at what can be done to help these people,” concludes Professor Adam Hampshire from the Department of Brain Sciences at Imperial College London.

The study is published in the journal eClinicalMedicine.The contents of this website do not constitute advice and are provided for informational purposes only.

Alzheimer’s-like signaling in brains of COVID-19 patients

Authors: Steve Reiken,Leah Sittenfeld,Haikel Dridi,Yang Liu,Xiaoping Liu,Andrew R. Marks First published: 03 February 2022  https://doi.org/10.1002/alz.12558

Abstract

Introduction

The mechanisms that lead to cognitive impairment associated with COVID-19 are not well understood.

Methods

Brain lysates from control and COVID-19 patients were analyzed for oxidative stress and inflammatory signaling pathway markers, and measurements of Alzheimer’s disease (AD)-linked signaling biochemistry. Post-translational modifications of the ryanodine receptor/calcium (Ca2+) release channels (RyR) on the endoplasmic reticuli (ER), known to be linked to AD, were also measured by co-immunoprecipitation/immunoblotting of the brain lysates.

Results

We provide evidence linking SARS-CoV-2 infection to activation of TGF-β signaling and oxidative overload. The neuropathological pathways causing tau hyperphosphorylation typically associated with AD were also shown to be activated in COVID-19 patients. RyR2 in COVID-19 brains demonstrated a “leaky” phenotype, which can promote cognitive and behavioral defects.

Discussion

COVID-19 neuropathology includes AD-like features and leaky RyR2 channels could be a therapeutic target for amelioration of some cognitive defects associated with SARS-CoV-2 infection and long COVID.

1 NARRATIVE

1.1 Contextual background

Patients suffering from COVID-19 exhibit multi-system organ failure involving not only pulmonary1 but also cardiovascular,2 neural,3 and other systems. The pleiotropy and complexity of the organ system failures both complicate the care of COVID-19 patients and contribute, to a great extent, to the morbidity and mortality of the pandemic.4 Severe COVID-19 most commonly manifests as viral pneumonia-induced acute respiratory distress syndrome (ARDS).5 Respiratory failure results from severe inflammation in the lungs, which arises when SARS-CoV-2 infects lung cells. Cardiac manifestations are multifactorial and include hypoxia, hypotension, enhanced inflammatory status, angiotensin-converting enzyme 2 (ACE2) receptor downregulation, endogenous catecholamine adrenergic activation, and direct viral-induced myocardial damage.67 Moreover, patients with underlying cardiovascular disease or comorbidities, including congestive heart failure, hypertension, diabetes, and pulmonary diseases, are more susceptible to infection by SARS-CoV-2, with higher mortality.67

In addition to respiratory and cardiac manifestations, it has been reported that approximately one-third of patients with COVID-19 develop neurological symptoms, including headache, disturbed consciousness, and paresthesias.8 Brain tissue edema, stroke, neuronal degeneration, and neuronal encephalitis have also been reported.2810 In a recent study, diffuse neural inflammatory markers were found in >80% of COVID-19 patient brains, processes which could contribute to the observed neurological symptoms.11 Furthermore, another pair of frequent symptoms of infection by SARS-CoV-2 are hyposmia and hypogeusia, the loss of the ability to smell and taste, respectively.3 Interestingly, hyposmia has been reported in early-stage Alzheimer’s disease (AD),3 and AD type II astrocytosis has been observed in neuropathology studies of COVID-19 patients.10

Systemic failure in COVID-19 patients is likely due to SARS-CoV-2 invasion via the ACE2 receptor,9 which is highly expressed in pericytes of human heart8 and epithelial cells of the respiratory tract,12 kidney, intestine, and blood vessels. ACE2 is also expressed in the brain, especially in the respiratory center and hypothalamus in the brain stem, the thermal center, and cortex,13 which renders these tissues more vulnerable to viral invasion, although it remains uncertain whether SARS-CoV-2 virus directly infects neurons in the brain.14 The primary consequences of SARS-CoV-2 infection are inflammatory responses and oxidative stress in multiple organs and tissues.1517 Recently it has been shown that the high neutrophil-to-lymphocyte ratio observed in critically ill patients with COVID-19 is associated with excessive levels of reactive oxygen species (ROS) and ROS-induced tissue damage, contributing to COVID-19 disease severity.15

Recent studies have reported an inverse relationship between ACE2 and transforming growth factor-β (TGF-β). In cancer models, decreased levels of ACE2 correlated with increased levels of TGF-β.18 In the context of SARS-CoV-2 infection, downregulation of ACE2 has been observed, leading to increased fibrosis formation, as well as upregulation of TGF-β and other inflammatory pathways.19 Moreover, patients with severe COVID-19 symptoms had higher blood serum TGF-β concentrations than those with mild symptoms,20 thus further implicating the role of TGF-β and warranting further investigation.

Interestingly, reduced angiotensin/ACE2 activity has been associated with tau hyperphosphorylation and increased amyloid beta (Aβ) pathology in animal models of AD.2122 The link between reduced ACE2 activity and increased TGF-β and tau signaling in the context of SARS-CoV-2 infection needs further exploration.

Our laboratory has shown that stress-induced ryanodine receptor (RyR)/intracellular calcium release channel post-translational modifications, including oxidation and protein kinase A (PKA) hyperphosphorylation related to activation of the sympathetic nervous system and the resulting hyper-adrenergic state, deplete the channel stabilizing protein (calstabin) from the channel complex, destabilizing the closed state of the channel and causing RyR channels to leak Ca2+ out of the endoplasmic/sarcoplasmic reticulum (ER/SR) in multiple diseases.2329 Increased TGF-β activity can lead to RyR modification and leaky channels,30 and SR Ca2+ leak can cause mitochondrial Ca2+ overload and dysfunction.29 Increased TGF-β activity31 and mitochondrial dysfunction32 are also associated with SARS-CoV-2 infection.

Here we show that SARS-CoV-2 infection is associated with adrenergic and oxidative stress and activation of the TGF-β signaling pathway in the brains of patients who have succumbed to COVID-19. One consequence of this hyper-adrenergic and oxidative state is the development of tau pathology normally associated with AD. In this article, we investigate potential biochemical pathways linked to tau hyperphosphorylation. Based on recent evidence that has linked tau pathology to Ca2+ dysregulation associated with leaky RyR channels in the brain,333 we investigated RyR2 biochemistry and function in COVID-19 patient brains.

RESEARCH-IN-CONTEXT

  1. Systematic review: The authors reviewed the literature using PubMed. While the mechanisms that lead to cognitive impairment associated with COVID-19 are not well understood, there have been recent reports studying SARS-CoV-2 infection and brain biochemistry and neuropathology. These relevant citations are appropriately cited.
  2. Interpretation: Our findings link the inflammatory response to SARS-CoV-2 infection with the neuropathological pathways causing tau hyperphosphorylation typically associated with Alzheimer’s disease (AD). Furthermore, our data indicate a role for leaky ryanodine receptor 2 (RyR2) in the pathophysiology of SARS-CoV-2 infection.
  3. Future directions: The article proposes that the alteration of cellular calcium dynamics due to leaky RyR2 in COVID-19 brains is associated with the activation of neuropathological pathways that are also found in the brains of AD patients. Both the cortex and cerebellum of SARS-CoV-2–infected patients exhibited a reduced expression of the Ca2+ buffering protein calbindin. Decreased calbindin could render these tissues more vulnerable to cytosolic Ca2+ overload. Ex vivo treatment of the COVID-19 brain using a Rycal drug (ARM210) that targets RyR2 channels prevented intracellular Ca2+ leak in patient samples. Future experiments will explore calcium channels as a potential therapeutic target for the neurological complications associated with COVID-19.

1.2 Study conclusions and disease implications

Our results indicate that SARS-CoV-2 infection activates inflammatory signaling and oxidative stress pathways resulting in hyperphosphorylation of tau, but normal amyloid precursor protein (APP) processing in COVID-19 patient cortex and cerebellum. There was reduced calbindin expression in both cortex and cerebellum rendering both tissues vulnerable to Ca2+-mediated pathology. Moreover, COVID-19 cortex and cerebellum exhibited RyR Ca2+ release channels with the biochemical signature of ‘‘leaky’’ channels and increased activity consistent with pathological intracellular Ca2+ leak. RyR2 were oxidized, associated with increased NADPH oxidase 2 (NOX2), and were PKA hyperphosphorylated on serine 2808, both of which cause loss of the stabilizing subunit calstabin2 from the channel complex promoting leaky RyR2 channels in COVID-19 patient brains. Furthermore, ex vivo treatment of COVID-19 patient brain samples with the Rycal drug ARM210, which is currently undergoing clinical testing at the National Institutes of Health for RyR1-myopathy (ClinicalTrials.gov Identifier: NCT04141670), fixed the channel leak. Thus, our experiments demonstrate that SARS-CoV-2 infection activates biochemical pathways linked to the tau pathology associated with AD and that leaky RyR Ca2+ channels may be a potential therapeutic target for the neurological complications associated with COVID-19.

The molecular basis of how SARS-CoV-2 infection results in ‘‘long COVID’’ is not well understood, and questions regarding the role of defective Ca2+ signaling in the brain in COVID-19 remain unanswered. A recent comprehensive molecular investigation revealed extensive inflammation and degeneration in the brains of patients that died from COVID-19,34 including in patients with no reported neurological symptoms. These authors also reported overlap between marker genes of AD and genes that are upregulated in COVID-19 infection, consistent with the findings of increased tau pathophysiology reported in the present study. We propose a potential mechanism that may contribute to the neurological complications caused by SARS-CoV-2: defective intracellular Ca2+ regulation and activation of AD-like neuropathology.

TGF-β belongs to a family of cytokines involved in the formation of cellular fibrosis by promoting epithelial-to-mesenchymal transition, fibroblast proliferation, and differentiation.35 TGF-β activation has been shown to induce fibrosis in the lungs and other organs by activation of the SMAD-dependent pathway. We have previously reported that TGF-β/SMAD3 activation leads to NOX2/4 translocation to the cytosol and its association with RyR channels, promoting oxidization of the channels and depletion of the stabilizing subunit calstabin in skeletal muscle and in heart.2830 Alteration of Ca2+ signaling may be particularly crucial in COVID-19-infected patients with cardiovascular/neurological diseases due, in part, to the multifactorial RyR2 remodeling after the cytokine storm, increased TGF-β activation, and increased oxidative stress. Moreover, SARS-CoV-2–infected patients exhibited a hyperadrenergic state. The elevated expression of glutamate carboxypeptidase 2 (GCPII) in COVID-19 brains reported in the present study could also contribute directly to increased PKA signaling of RyR2 by reducing PKA inhibition via metabotropic glutamate receptor 3 (mGluR3).36 Hyperphosphorylation of RyR2 channels can promote pathological remodeling of the channel and exacerbate defective Ca2+ regulation in these tissues. The increased Ca2+/cAMP/PKA signaling could also open nearby K+ channels which could potentially weaken synaptic connectivity, reduce neuronal firing,36 and could activate Ca2+ dependent enzymes.

Interestingly, both the cortex and cerebellum of SARS-CoV–2-infected patients exhibited a reduced expression of the Ca2+ buffering protein calbindin. Decreased calbindin could render these tissues more vulnerable to the cytosolic Ca2+ overload. This finding is in accordance with previous studies showing reduced calbindin expression levels in Purkinje cells and the CA2 hippocampal region of AD patients3739 and in cortical pyramidal cells of aged individuals with tau pathology.3340 In contrast to the findings in the brains of COVID-19 patients in the present study, calbindin was not reduced in the cerebellum of AD patients, possibly protecting these cells from AD pathology.3941

Leaky RyR channels, leading to increased mitochondrial Ca2+ overload and ROS production and oxidative stress, have been shown to contribute to the development of tau pathology associated with AD.3232933 Recent studies of the effects of COVID-19 on the central nervous system have found memory deficits and biological markers similar to those seen in AD patients.4243 Our data demonstrate increased activity of enzymes responsible for phosphorylating tau (pAMPK, pGSK3β), as well as increased phosphorylation at multiple sites on tau in COVID-19 patient brains. The tau phosphorylation observed in these samples exhibited some differences from what is typically observed in AD, occurring in younger patients and in areas of the brain, specifically the cerebellum, that usually do not demonstrate tau pathology in AD patients. Taken together, these data suggest a potential contributing mechanism to the development of tau pathology in COVID-19 patients involving oxidative overload-driven RyR2 channel dysfunction. Furthermore, we propose that these pathological changes could be a significant contributing factor to the neurological manifestations of COVID-19 and in particular the “brain fog” associated with long COVID, and represent a potential therapeutic target for ameliorating these symptoms. For example, tau pathology in the cerebellum could explain the recent finding that 74% of hospitalized COVID-19 patients experienced coordination deficits.44 The data presented also raise the possibility that prior COVID-19 infection could be a potential risk factor for developing AD in the future.

The present study was limited to the use of existing autopsy brain tissues at the Columbia University Biobank from SARS-CoV-2–infected patients. The number of subjects is small and information on their cognitive function as well as their brain histopathology and levels of Aβ in cerebrospinal fluid and plasma are lacking. Furthermore, we did not have access to a suitable animal model of SARS-CoV-2 infection in which to test whether the observed biochemical changes in COVID-19 brains and potential cognitive and behavioral deficits associated with the brain fog of long COVID could be reversed or attenuated by therapeutic interventions. The design of future studies should include larger numbers of subjects that are age- and sex-matched. The cognitive function of SARS-CoV-2–infected patients who presented cognitive symptoms should be assessed and regularly monitored. Moreover, it is important to know whether the observed neuropathological signaling is unique to SARS-CoV-2 infection or are common to all other viral infections. Previous studies have reported cognitive impairment in Middle East respiratory syndrome45 as well as Ebola4647 patients. Retrospective studies comparing the incidence and the magnitude of cognitive impairments caused by these different viral infections would improve our understanding of these neurological complications of viral infections.

2 CONSOLIDATED RESULTS AND STUDY DESIGN

There were increased markers of oxidative stress (glutathione disulfide [GSSG]/ glutathione [GSH]) in the cortex (mesial temporal lobe) and cerebellum (cerebellar cortex, lateral hemisphere) of COVID-19 tissue. Kynurenic acid, a marker of inflammation, was increased in COVID-19 cortex and cerebellum brain lysates compared to controls, is in accordance with recent studies showing a positive correlation between kynurenic acid and cytokines and chemokine levels in COVID-19 patients.4850

To determine whether SARS-CoV-2 infection also increases tissue TGF-β activity, we measured SMAD3 phosphorylation, a downstream signal of TGF-β, in control and COVID-19 tissue lysates. Phosphorylated SMAD3 (pSMAD3) levels were increased in COVID-19 cortex and cerebellum brain lysates compared to controls, indicating that SARS-CoV-2 infection increased TGF-β signaling in these tissues. Interestingly, brain tissues from COVID-19 patients exhibited activation of the TGF-β pathway, despite the absence of the detectable (by immunohistochemistry and polymerase chain reaction, data not shown) virus in these tissues. These results suggest that the TGF-β pathway is activated systemically by SARS-CoV-2, resulting in its upregulation in the brain, as well as other organs. In addition to oxidative stress, COVID-19 brain tissues also demonstrated increased PKA and calmodulin-dependent protein kinase II association domain (CaMKII) activity, most likely associated with increased adrenergic stimulation. Both PKA and CaMKII phosphorylation of tau have been reported in tauopathies.5152

The hallmarks of AD brain neuropathology are the formation of Aβ plaques from abnormal APP processing by BACE1, as well as tau ‘‘tangles’’ caused by tau hyperphosphorylation.53 Brain lysates from COVID-19 patients’ autopsies demonstrated normal BACE1 and APP levels compared to controls. The patients analyzed in the present study were grouped by age (young ≤ 58 years old, old ≥ 66 years old) to account for normal, age-dependent changes in APP and tau pathology. Abnormal APP processing was only observed in brain lysates from patients diagnosed with AD. However, AMPK and GSK3β phosphorylation were increased in both the cortex and cerebellum in COVID-19 brains. Activation of these kinases in SARS-CoV-2–infected brains leads to a hyperphosphorylation of tau consistent with AD tau pathology in the cortex. COVID-19 brain lysates from older patients showed increased tau phosphorylation at S199, S202, S214, S262, and S356. Lysates from younger COVID-19 patients showed increased tau phosphorylation at S214, S262, and S356, but not at S199 and S202, demonstrating increased tau phosphorylation in both young and old individuals and suggesting a tau pathology similar to AD in COVID-19–affected patients. Interestingly, both young and old patient brains demonstrated increased tau phosphorylation in the cerebellum, which is not typical of AD.

RyR channels may be oxidized due to the activation of the TGF-β signaling pathway.30 NOX2 binding to RyR2 causes oxidation of the channel, which activates the channel, manifested as an increased open probability that can be assayed using 3[H]ryanodine binding.54 When the oxidization of the channel is at pathological levels, there is destabilization of the closed state of the channel, resulting in spontaneous Ca2+ release or leak.2730 To determine the effect of the increased TGF-β signaling associated with SARS-CoV-2 infection on NOX2/RyR2 interaction, RyR2 and NOX2 were co-immunoprecipitated from brain lysates of COVID-19 patients and controls. NOX2 associated with RyR2 in brain tissues from SARS-CoV-2–infected individuals were increased compared to controls.

Given the increased oxidative stress and increased NOX2 binding to RyR2 seen in COVID-19 brains, RyR2 post-translational modifications were investigated. Immunoprecipitated RyR2 from brain lysates demonstrated increased oxidation, PKA phosphorylation on serine 2808, and depletion of the stabilizing protein subunit calstabin2 in SARS-CoV-2–infected tissues compared to controls. This biochemical remodeling of the channel is known as the ‘‘biochemical signature’’ of leaky RyR2235556 that is associated with destabilization of the closed state of the channel. This leads to SR/ER Ca2+ leak, which contributes to the pathophysiology of a number of diseases including AD.232426305557 RyR channel activity was determined by binding of 3[H]ryanodine, which binds only to the open state of the channel. RyR2 was immunoprecipitated from tissue lysates and ryanodine binding was measured at both 150 nM and 20 μM free Ca2+. RyR2 channels from SARS-CoV-2–infected brain tissue demonstrated abnormally high activity (increased ryanodine binding) compared to channels from control tissues at physiologically resting conditions (150 nM free Ca2+), when channels should be closed. Interestingly, cortex and cerebellum of SARS-CoV-2–infected patients also exhibited a reduced expression of the Ca2+ binding protein calbindin. Calbindin is typically not reduced in the cerebellum of AD patients, possibly providing some protection against AD pathology. The low calbindin levels in the cerebellum of COVID-19 brains could contribute to the observed tau pathology in this brain region. An additional atypical finding in the COVID-19 brains studied in this investigation is an increased level of GCPII. This could contribute to the observed RyR PKA phosphorylation by increasing cAMP and inhibiting the metabotropic glutamate receptor type 3.36

3 DETAILED METHODS AND RESULTS

3.1 Methods

3.1.1 Human samples

De-identified human heart, lung, and brain tissue were obtained from the COVID BioBank at Columbia University. The cortex samples were from the mesial temporal lobe and the cerebellum samples were from the cerebellar cortex, lateral hemisphere. The Columbia University BioBank functions under standard operating procedures, quality assurance, and quality control for sample collection and maintenance. Age- and sex-matched controls exhibited absence of neurological disorders and cardiovascular or pulmonary diseases. Sex, age, and pathology of patients are listed in Table 1.TABLE 1. Sex, age, and pathology of COVID-19 patients

Patient NumberSexAgePathology
1Male57Acute hypoxic-ischemic injury in the hippocampus, pons, and cerebellum.
2Female38Hypoxic ischemic encephalopathy, severe, global.
3Male58Hypoxic/ischemic injury, global, widespread astrogliosis/microgliosis.
4Male84Dementia. Beta-amyloid plaques are noted in cortex and cerebellum.
5Female80Severe hypoxic ischemic encephalopathy, severe. Global astrogliosis and microgliosis. Mild Alzheimer-type pathology.
6Female74Acute hypoxic-ischemic encephalopathy, global, moderate to severe. Arteriolosclerosis, mild. Metabolic gliosis, moderate
7Male66Left frontal subacute hemorrhagic infarct. Multifocal subacute infarcts in pons and left cerebral peduncle. Global astrogliosis and microgliosis (see microscopic description). Alzheimer’s pathology.
8Female76Hypoxic ischemic encephalopathy, moderate. Alzheimer’s pathology. Atherosclerosis, moderate. Arteriolosclerosis, moderate
9Male72Hypoxic/ischemic injury, acute to subacute, involving hippocampus, medulla and cerebellum. Mild atherosclerosis. Mild arteriolosclerosis
10Male71Hypoxic-ischemic encephalopathy, acute, global, mild to moderate. Diffuse Lewy body disease, neocortical type, consistent with Parkinson disease dementia. Atherosclerosis, severe. Arteriolosclerosis, mild.

Lysate preparation and Western blots

Tissues (50 mg) were isotonically lysed using a Dounce homogenizer in 0.25 ml of 10 mM Tris maleate (pH 7.0) buffer with protease inhibitors (Complete inhibitors from Roche). Samples were centrifuged at 8000 × g for 20 minutes and the protein concentrations of the supernatants were determined by Bradford assay. To determine protein levels in tissue lysates, tissue proteins (20 μg) were separated by 4% to 20% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblots were developed using the following antibodies: pSMAD3 (Abcam, 1:1000), SMAD3 (Abcam, 1:1000), AMPK (Abcam, 1:1000), tau (Thermo Fisher, 1:1000), pTauS199 (Thermo Fisher, 1:1000), pTauS202/T205 (Abcam, 1:1000), pTauS262 (Abcam, 1:1000), GSK3β (Abcam, 1:1000), pGSK3βS9 (Abcam, 1:1000), pGSK3βT216 (Abcam, 1:1000), APP (Abcam, 1:1000), BACE1 (Abcam, 1:1000), GAPDH (Santa Cruz Biotech, 1:1000), CTF-β (Santa Cruz Biotechnology, Inc., 1:1000), Calbindin (Abcam, 1:1000), and GCPII (Thermo Fisher, 1:4000).

Analyses of ryanodine receptor complex

Tissue lysates (0.1 mg) were treated with buffer or 10 μM Rycal (ARM210) at 4°C. RyR2 was immunoprecipitated from 0.1 mg lung, heart, and brain using an anti-RyR2 specific antibody (2 μg) in 0.5 ml of a modified radioimmune precipitation assay buffer (50 mm Tris-HCl, pH 7.2, 0.9% NaCl, 5.0 mm NaF, 1.0 mm Na3VO4, 1% Triton X-100, and protease inhibitors; RIPA) overnight at 4°C. RyR2-specific antibody was an affinity-purified polyclonal rabbit antibody using the peptide CKPEFNNHKDYAQEK corresponding to amino acids 1367–1380 of mouse RyR2 with a cysteine residue added to the amino terminus. The immune complexes were incubated with protein A-Sepharose beads (Sigma) at 4°C for 1 hour, and the beads were washed three times with RIPA. The immunoprecipitates were size-fractionated on SDS-PAGE gels (4%–20% for RyR2, calstabin2, and NOX2) and transferred onto nitrocellulose membranes for 1 hour at 200 mA. Immunoblots were developed using the following primary antibodies: anti-RyR2 (Affinity BioReagents, 1:2500), anti-phospho-RyR-Ser(pS)-2808 (Affinity BioReagents 1:1000), anti- calstabin2 (FKBP12 C-19, Santa Cruz Biotechnology, Inc., 1:2500), and anti-NOX2 (Abcam, 1:1000). To determine channel oxidation, the carbonyl groups in the protein side chains were derivatized to DNP by reaction with 2,4-dinitrophenylhydrazine. The DNP signal associated with RyR2 was determined using a specific anti-DNP antibody according to the manufacturer using an Odyssey system (LI-COR Biosciences) with infrared-labeled anti-mouse and anti-rabbit immunoglobulin G (IgG; 1:5000) secondary antibodies.

Ryanodine binding

RyR2 was immunoprecipitated from 1.5 mg of tissue lysate using an anti-RyR2 specific antibody (25 μg) in 1.0 ml of a modified RIPA buffer overnight at 4°C. The immune complexes were incubated with protein A-Sepharose beads (Sigma) at 4°C for 1 hour, and the beads were washed three times with RIPA buffer, followed by two washes with ryanodine binding buffer (10 mM Tris-HCl, pH 6.8, 1 M NaCl, 1% CHAPS, 5 mg/ml phosphatidylcholine, and protease inhibitors). Immunoprecipitates were incubated in 0.2 ml of binding buffer containing 20 nM [3H] ryanodine and either of 150 nM and 20 μm free Ca2+ for 1 hour at 37°C. Samples were diluted with 1 ml of ice-cold washing buffer (25 mm Hepes, pH 7.1, 0.25 m KCl) and filtered through Whatman GF/B membrane filters pre-soaked with 1% polyethyleneimine in washing buffer. Filters were washed three times with 5 ml of washing buffer. The radioactivity remaining on the filters is determined by liquid scintillation counting to obtain bound [3H] ryanodine. Nonspecific binding was determined in the presence of 1000-fold excess of non-labeled ryanodine.

GSSG/GSH ratio measurement and SMAD3 phosphorylation

Approximately 20 mg of tissue suspended in 200 μL of ice-cold phosphate-buffered saline/0.5% NP-40, pH6.0 was used for lysis. Tissue was homogenized with a Dounce homogenizer with 10 to 15 passes. Samples were centrifuged at 8000 × g for 15 minutes at 4°C to remove any insoluble material. Supernatant was transferred to a clean tube. Deproteinizing of the samples was accomplished by adding 1 volume ice-cold 100% (w/v) trichloroacetic acid (TCA) into five volumes of sample and vortexing briefly to mix well. After incubating for 5 minutes on ice, samples were centrifuged at 12,000 × g for 5 minutes at 4°C and the supernatant was transferred to a fresh tube. The samples were neutralized by adding NaHCO3 to the supernatant and vortexing briefly. Samples were centrifuged at 13,000 × g for 15 minutes at 4°C and supernatant was collected. Samples were then deproteinized, neutralized, TCA was removed, and they were ready to use in the assay. The GSSG/GSH was determined using a ratio detection assay kit (Abcam, ab138881). Briefly, in two separate assay reactions, GSH (reduced) was measured directly with a GSH standard and Total GSH (GSH + GSSG) was measured by using a GSSG standard. A 96-well plate was set up with 50 μL duplicate samples and standards with known concentrations of GSH and GSSG. A Thiol green indicator was added, and the plate was incubated for 60 minutes at room temperature (RT). Fluorescence at Ex/Em = 490/520 nm was measured with a fluorescence microplate reader and the GSSG/GSH for samples were determined comparing fluorescence signal of samples with known standards.

Kynurenic acid assay

Kynurenic acid (KYNA) concentration in brain lysates was determined using an enzyme-linked immunosorbent assay (ELISA) kit for KYNA (ImmuSmol). Briefly, samples (50 μl) were added to a microtiter plate designed to extract the KCNA from the samples. An acylation reagent was added for 90 minutes at 37°C to derivatize the samples. After derivatization, 50 μl of the prepared standards and 100 μl samples were pipetted into the appropriate wells of the KYNA microtiter plate. KYNA Antiserum was added to all wells and the plate was incubated overnight at 4°C. After washing the plate four times, the enzyme conjugate was added to each well. The plate was incubated for 30 minutes at RT on a shaker at 500 rpm. The enzyme substrate was added to all wells and the plate was incubated for 20 minutes at RT. Stop solution was added to each well. A plate reader was used to determine the absorbance at 450 nm. The sample signals were compared to a standard curve.

PKA activity assay

PKA activity in brain lysates was determined using a PKA activity kit (Thermo Fisher, EIAPKA). Briefly, samples were added to a microtiter plate containing an immobilized PKA substrate that is phosphorylated by PKA in the presence of ATP. After incubating the samples with ATP at RT for 2 hours, the plate was incubated with the phospho-PKA substrate antibody for 60 minutes. After washing the plate with wash buffer, goat anti-rabbit IgG horseradish peroxidase (HRP) conjugate was added to each well. The plate was aspirated, washed, and TMB substrate was added to each well, which was then incubated for 30 minutes at RT. A plate reader was used to determine the absorbance at 450 nm. The sample signals were compared to a standard curve.

CaMKII activity assay

CaMKII activity in brain lysates was determined using the CycLex CaM kinase II Assay Kit (MBL International). Briefly, samples were added to a microtiter plate containing an immobilized CaMKII substrate that is phosphorylated by CaMKII in the presence of Mg2+ and ATP. After incubating the samples in kinase buffer containing Mg2+ and ATP at RT for 1 hour, the plate was washed and incubated with the HRP conjugated anti-phospho-CaMKII substrate antibody for 60 minutes. The plate was aspirated, washed, and TMB substrate was added to each well, which was then incubated for 30 minutes at RT. A plate reader was used to determine the absorbance at 450 nm. The sample signals were compared to a standard curve.

Statistics

Group data are presented as mean ± standard deviation. Statistical comparisons between the two groups were determined using an unpaired t-test. Values of P < .05 were considered statistically significant. All statistical analyses were performed with GraphPad Prism 8.0.

3.2 Results

3.2.1 Oxidative stress and TGF-β, PKA, and CaMKII activation

Oxidative stress levels were determined in brain tissues (cortex, cerebellum) from COVID-19 patient autopsy tissues and controls by measuring the ratio of GSSG to GSH by an ELISA kit. COVID-19 patients exhibited significant oxidative stress with a 3.8- and 3.2-fold increase in GSSG/GSH ratios in cortex (Ctx) and cerebellum (CB) compared to controls, respectively (Figure 1A). High circulating levels of kynurenine have been reported in COVID-19.4850 However, the expression of KYNA in COVID-19 brain tissue has not been examined. Levels in the Ctx and CB were measured using an ELISA kit. COVID-19 brains had a significant increase in the Ctx and CB compared to controls (Figure 1A). An additional marker of tissue inflammation is increased cytokine expression. SMAD3 phosphorylation, a downstream signal of TGF-β, was increased in COVID-19 Ctx and CB tissue lysates compared to controls (Figure 1B and 1C). Increased adrenergic activation in the brain of patients infected with SARS-CoV-2 was also demonstrated by measuring PKA activity in the Ctx and CB and CaMKII activity was increased as well (Figure 1D).

Details are in the caption following the image
FIGURE 1Open in figure viewerIncreased oxidative stress, inflammatory and adrenergic signaling in brains of COVID-19 patients. A, Bar graph depicting the glutathione disulfide (GSSG)/ glutathione (GSH) ratio and kynurenic acid (KYNA) enzyme-linked immunsorbent assay signal from control (n = 6) and COVID-19 (n = 6) tissue lysates. CB, cerebellum; Ctx, cortex. Data are mean ± standard deviation (SD). *P < .05 control versus COVID-19. B, Western blots showing phospho-SMAD3 and total SMAD3 from control (n = 4) and COVID-19 (n = 7) brain lysates. C, Bar graphs depicting quantification of pSMAD3/SMAD3 from Western blot signals in B. D, Calmodulin-dependent protein kinase II association domain (CaMKII) and protein kinase A (PKA) activity of brain tissue lysates. Data are mean ± SD. *P < .05 control versus COVID-19

Activation of AD-linked signaling

Both PKA and CaMKII have been directly implicated in the increased phosphorylation of tau associated with AD.5152 Because COVID-19 brain lysates had increased PKA and CaMKII activity, AD-linked biochemistry was evaluated in the COVID-19 brain lysates. Normal APP processing was observed in COVID-19 brain lysates as demonstrated by normal BACE1 and APP levels compared to controls (Figure 2A and B). Abnormal APP processing was only observed in brain lysates from patients diagnosed with AD (see Table 1 for patient details). However, phosphorylation/activation of AMPK and GSK3β was observed in SARS-CoV-2–infected patient brain lysates. Activation of these kinases along with the activation of PKA and CaMKII (Figure 1) leads to a hyperphosphorylation of tau at multiple residues (Figure 2C and D). Tau hyperphosphorylation in the cerebellum is not typical of AD pathology. The CB tau pathology demonstrated in COVID-19 warrants further investigation.

Details are in the caption following the image
FIGURE 2Open in figure viewerHyperphosphorylation of tau but normal amyloid precursor protein (APP) processing in COVID-19 brains. A, Brain (CB, cerebellum; Ctx, cortex) lysates were separated by 4% to 20% polyacrylamide gel electrophoresis. Immunoblots were developed for pAMPK, AMPK, GSK3β, pGSK3β (T216), APP, BACE1, and GAPDH loading control. The numbers (1–10) above immunoblots refer to patient numbers listed in Table 1. B, Bar graphs showing quantification of pAMPK, pGSK3β, APP/GAPDH, and BACE1/GAPDH from Western blots in (A). Data are mean ± standard deviation (SD). *P < .05 control versus COVID-19; **P < .05 CB versus Ctx; #P < .05 COVID (Young) versus COVID (Old). C, Immunoblots of brain lysates showing total tau and tau phosphorylation on residues S199, S202/T205, S214, S262, and S356. D, Bar graphs showing quantification phosphorylated tau at the residues shown on Western blots in (C). Data are mean ± SD. *P < .05 control versus COVID-19; **P < .05 CB versus Ctx; #P < .05 COVID (Young) versus COVID (Old)

RyR2 channel oxidation and leak

RyR2 biochemistry was investigated to determine whether RyR2 in COVID-19 brain tissues demonstrated a “leaky” phenotype. Increased NOX2/RyR2 binding was shown in Ctx and CB lysates from SARS-CoV-2–infected individuals compared to controls using co-immunoprecipitation (Figure 3A and B). In addition, RyR2 from SARS-CoV-2–infected brains had increased oxidation, increased serine 2808 PKA phosphorylation, and depletion of the stabilizing protein subunit calstabin2 compared to controls (Figure 3A and B). RyR channels exhibiting these characteristics can be inappropriately activated at low cytosolic Ca2+ concentrations resulting in a pathological ER/SR Ca2+ leak. 3[H]Ryanodine binding to immunoprecipitated RyR2 was measured at both 150 nM and 20 μM free Ca2+. Because ryanodine binds only to the open state of the channel under these conditions, 3[H]Ryanodine binding may be used as a surrogate measure of channel open probability. The total amount of RyR immunoprecipitated was the same for control and COVID-19 samples (data not shown). Increased RyR2 channel activity at resting conditions (150 nM free Ca2+) was observed in COVID-19 channels compared to controls (Figure 3C). Under these conditions, RyR channels should be closed. Rebinding of calstabin2 to RyR2, using a Rycal, has been shown to reduce SR/ER Ca2+ leak, despite the persistence of the channel remodeling. Indeed, calstabin2 binding to RyR2 was increased when COVID-19 patient brain tissue lysates were treated ex vivo with the Rycal drug ARM210 (Figure 3A and B). Abnormal RyR2 activity observed at resting Ca2+ concentration was also decreased by Rycal treatment (Figure 3C).

Details are in the caption following the image
FIGURE 3Open in figure viewerDysregulation of calcium-handling proteins in COVID-19 brains. A, Western blots depicting ryanodine receptor 2 (RyR2) oxidation, protein kinase A (PKA) phosphorylation, and calstabin2 or NADPH oxidase 2 (NOX2) bound to the channel from brain (CB, cerebellum; Ctx, cortex) lysates. B, Bar graphs quantifying DNP/RyR2, pS2808/RyR2, and calstabin2 and NOX2 bound to the channel from the Western blots. Data are mean ± standard deviation (SD). *P < .05 control versus COVID-19; # P < .05 COVID-19 versus COVID-19+ARM210. C, 3[H]ryanodine binding from immunoprecipitated RyR2. Bar graphs show ryanodine binding at 150 nM Ca2+ as a percent of maximum binding (Ca2+ = 20 μM). Data are mean ± SD. *P < .05 control versus COVID-19; #P < .05 COVID-19 versus COVID-19+ARM210. D, Western blots showing the levels of glutamate carboxypeptidase 2 (GCPII), calbindin, and GAPDH loading control in brain (Ctx, CB). E, Bar graphs quantifying GCPII/GAPDH and calbindin/GAPDH from the western blots. Data are mean ± SD. *P < .05 control versus COVID-19

An interesting finding concerning the tau phosphorylation in brain lysates from SARS-CoV-2 patients was the increase of phosphorylation at multiple sites in the cerebellum. This is atypical of AD. One potential mechanism to explain this finding is the significantly decreased levels of calbindin expressed in COVID-19 cerebellum (Figure 3D3E). The decreased cerebellar calbindin levels could make this area of the brain more susceptible to Ca2+-induced activation of enzymes upstream of tau phosphorylation. Moreover, increased GCPII expression was observed in COVID-19 cortex and cerebellar lysates (Figure 3D3E), which would reduce mGluR3 inhibition of PKA signaling and could contribute to the PKA hyperphosphorylation of RyR2.

Model for the role for leaky RyR2 in the pathophysiology of SARS-CoV-2 infection

Our data indicate a role for leaky RyR2 in the pathophysiology of SARS-CoV-2 infection (Figure 4). In addition to the brain of COVID-19 patients, we observed increased systemic oxidative stress and activation of the TGF-β signaling pathway in lung, and heart, which correlates with oxidation-driven biochemical remodeling of RyR2 (Figure 3 and S1 in supporting inormation). This RyR2 remodeling results in intracellular Ca2+ leak, which can play a role in heart failure progression, pulmonary insufficiency, as well as cognitive dysfunction.232628 The alteration of cellular Ca2+ dynamics has also been implicated in COVID-19 pathology.5859 Taken together, the present data suggest that leaky RyR2 may play a role in the long-term sequelae of COVID-19, including the “brain fog” associated with SARS-CoV-2 infection which could be a forme fruste of AD,60 and could predispose long COVID patients to developing AD later in life. Leaky RyR2 channels may be a therapeutic target for amelioration of some of the persistent cognitive deficits associated with long COVID.

Details are in the caption following the image
FIGURE 4Open in figure viewerSARS-CoV-2 infection results in leaky ryanodine receptor 2 (RyR2) that may contribute to cardiac, pulmonary, and cognitive dysfunction. SARS-CoV-2 infection targets cells via the angiotensin-converting enzyme 2 (ACE2) receptor, inducing inflammasome stress response/activation of stress signaling pathways. This results in increased transforming growth factor-β (TGF-β) signaling, which activates SMAD3 (pSMAD) and increases NADPH oxidase 2 (NOX2) expression and the amount of NOX2 associated with RyR2. Increased NOX2 activity at RyR2 oxidizes the channel, causing calstabin2 depletion from the channel macromolecular complex, destabilization of the closed state, and ER/SR calcium leak that is known to contribute to cardiac dysfunction,55 arrhythmias,61 pulmonary insufficiency,2325 and cognitive and behavioral abnormalities associated with neurodegenreation.2426 Decreased calbindin in COVID-19 may render brain more susceptible to tau pathology. Rycal drugs fix the RyR2 channel leak by restoring calstabin2 binding and stabilizing the channel closed state. Fixing leaky RyR2 may improve cardiac, pulmonary, and cognitive function in COVID-19.

Warning to anyone who’s had Covid over ‘irreversible’ damage to the brain

Authors: Vanessa Chalmers, Digital Health Reporter  Apr 11 2022

COVID survivors have been warned that the brain could be irreversibly harmed by the virus.

The major organ has been shown in dozens of studies to be damaged in even the mildest forms of Covid illness.

‘Brain fog’, difficulty concentrating and memory problems have all been reported, with some encouraging studies suggesting most people see improvements in six to nine months.

The new study, by researchers at the University of Oxford, looked at people in the UK over the age of 50 who had a mild case of Covid.

All 785 participants were in the UK Biobank, a large database for medical research, and had two brain scans 38 months aparts.

A total of 401 participants had tested positive for Covid in between the two scans.

The study found a number of effects on the brain, on average 4.5 months following infection.

Covid survivors had a greater reduction in grey matter thickness and tissue damage in regions of the brain associated with smell.

They had a reduction in whole brain size and, after performing a number of tests, showed a drop in cognitive function.

The effects ranged from 0.2 to 2 per cent additional change compared with the participants who had not been infected.

Professor Gwenaëlle Douaud, lead author on the study, said: “Despite the infection being mild for 96 per cent of our participants, we saw a greater loss of grey matter volume, and greater tissue damage in the infected participants.

“They also showed greater decline in their mental abilities to perform complex tasks, and this mental worsening was partly related to these brain abnormalities. 

“All these negative effects were more marked at older ages. 

“A key question for future brain imaging studies is to see if this brain tissue damage resolves over the longer term.”

It is not clear at this stage if the effects on the brain are reversible.

Professor Stephen Smith, senior author on the study, said: “The fact that we have the pre-infection scan helps us distinguish brain changes related to the infection from differences that may have pre-existed in their brains.”

The evidence is stacking up

The study, published in the journal Nature in March, echoes the findings of a number of others.

Researchers at Tulane University reported findings last week based on studying primates, which are used in studies for the likeness to humans. 

They found severe brain swelling and injury linked to reduced blood flow or oxygen to the brain.

They also found evidence of small bleeds, neuron damage and death – even in primates that didn’t have a severe illness.

Lead investigator Dr Tracy Fischer said: “Because the subjects didn’t experience significant respiratory symptoms, no one expected them to have the severity of disease that we found in the brain.

“But the findings were distinct and profound, and undeniably a result of the infection.”

Meanwhile, researchers – including from the universities of Imperial College London and Cambridge – found that Covid can cause a “substantial drop” in intelligence.

The findings came from a series of tests on memory, reasoning, planning and problem solving on more than 81,300 people.

People who had been on a ventilator during their Covid sickness were most likely to see a decline in scores.

In a classic intelligence test, they would have lost the equivalent of seven points in IQ, the team claimed.

The study said: “These results accord with reports of long-Covid, where ‘brain fog’, trouble concentrating and difficulty finding the correct words are common.

“The deficits were of substantial effect size for people who had been hospitalised.”

Another study reassured that “brain fog” shouldn’t persist for more than a year.

Covid patients scored significantly worse in episodic memory and in their ability to sustain attention on a task over time.

However, Professor Masud Husain, of Oxford University, said it was “encouraging” that most people’s attention and memory return “largely to normal in six to nine months”.

He said: “We still do not understand the mechanisms that cause these cognitive deficit.”

Explain this…

A team in the US suggested brain fog symptoms were the result of the organ being starved of oxygen.

After autopsying Covid victims, scientists at Johns Hopkins University School of Medicine found that large cells called megakaryocytes were taking up space and leaving less room for blood to pass through the brain freely.

According to Professor James Goodwin, the Director of Science and Research Impact at the Brain Health Network, it is thought that Covid gets into the brain through tightly sealed blood vessels which surround the organ.

But there is another explanation, he wrote in The Telegraph, and our own immune systems are to blame.

Sometimes the immune system goes into overdrive in response to a virus, releasing too many inflammatory molecules called cytokines.

This phenomenon, known as a cytokine storm, can injure healthy organs, including the brain, as well as the lungs and heart. 

It has led to the death of many Covid victims, and those who survive may have long-term damage.

The cytokine storm is typically more common in people who are unhealthy, have a long-term illness, are older or who have a high viral load, Prof Goodwin said.

Neuropathology and virus in brain of SARS-CoV-2 infected non-human primates

Authors: Ibolya RutkaiMeredith G. MayerLinh M. HellmersBo NingZhen HuangChristopher J. MonjureCarol CoyneRachel SilvestriNadia GoldenKrystle HensleyKristin ChandlerGabrielle LehmickeGregory J. BixNicholas J. ManessKasi Russell-Lodrigue, Tony Y. HuChad J. RoyRobert V. BlairRudolf BohmLara A. Doyle-MeyersJay Rappaport & Tracy Fischer 

Nature Communications volume 13, Article number: 1745 (2022)  Published: 

Abstract

Neurological manifestations are a significant complication of coronavirus disease (COVID-19), but underlying mechanisms aren’t well understood. The development of animal models that recapitulate the neuropathological findings of autopsied brain tissue from patients who died from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are critical for elucidating the neuropathogenesis of infection and disease. Here, we show neuroinflammation, microhemorrhages, brain hypoxia, and neuropathology that is consistent with hypoxic-ischemic injury in SARS-CoV-2 infected non-human primates (NHPs), including evidence of neuron degeneration and apoptosis. Importantly, this is seen among infected animals that do not develop severe respiratory disease, which may provide insight into neurological symptoms associated with “long COVID”. Sparse virus is detected in brain endothelial cells but does not associate with the severity of central nervous system (CNS) injury. We anticipate our findings will advance our current understanding of the neuropathogenesis of SARS-CoV-2 infection and demonstrate SARS-CoV-2 infected NHPs are a highly relevant animal model for investigating COVID-19 neuropathogenesis among human subjects.

Introduction

Multiple and continuing reports demonstrate a substantial number of patients with coronavirus disease 2019 (COVID-19) develop new-onset neurological symptoms. Several case reports have, in fact, identified neurological complications as the initial presentation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, particularly among those who develop stroke1,2,3. Among the more urgent COVID-19-associated neurological presentations, stroke, meningoencephalitis, and hemorrhagic necrotizing encephalopathies have been associated with more severe disease2,4,5,6; however, even comparatively mild neurological symptoms, such as dizziness or unresolving headache4,7, may be indicative of neuropathological processes in the context of infection and disease. Notably, individuals across the lifespan, with and without significant comorbidities, and with all disease severities, including asymptomatic patients, have suffered the variety of reported neurological manifestations8.

While damage to the central nervous system (CNS) of patients with COVID-19 is increasingly evident, the neuropathogenesis remains unclear. Here, we provide a comprehensive assessment of brain pathology associated with SARS-CoV-2 infection in two non-human primate (NHP) models of infection with varied disease severity. This work reveals neuroinflammation, brain hypoxia, microhemorrhages, and pathology consistent with hypoxic-ischemic injury with rare infection of brain vasculature in SARS-CoV-2 infected NHPs and provides key insights into SARS-CoV-2-associated neuropathogenesis. Our findings are consistent with those reported on autopsied brain of human subjects who died with SARS-CoV-2 infection. Additional molecular analyses on brain from our animal models suggest reduced oxygen to the CNS may contribute significantly to injury in the context of infection. Importantly, animals that did not develop acute respiratory distress syndrome (ARDS) demonstrated neuropathology that may lead to long-term neurological symptoms of post-acute sequelae of COVID-19 (PASC), or “long COVID”.

Results

Significant inflammation in brain

Eight adult NHPs, including four Rhesus macaques (RM), 13–15 years of age, and four wild-caught African green monkeys (AGMs), approximately 16 years of age, were inoculated with the 2019-nCoV/USA-WA1/2020 strain of SARS-CoV-29 via a multi-route mucosal or aerosol challenge (Table 1). Two animals of each species were inoculated via aerosol and two by multi-route exposure. Multi-route mucosal exposure included conjunctival, nasal, pharyngeal, and intratracheal routes. Control animals included two RMs, approximately 18–22 years of age, and two AGMs, approximately 17 years of age. Control animals were mock-infected through multi-route mucosal exposure of the same growth media used for virus propagation. All study animals underwent the same clinical tests and procedures.Table 1 Study animals.Full size table

All animals exposed to SARS-CoV-2 developed infection within the first week of exposure, as demonstrated by the detection of the viral nucleocapsid (N) mRNA in nasal swabs taken within 3–7 days after challenge (Table 1). No differences in infection were noted between the two inoculation strategies. Further verification of infection is seen through detection of the virus by immunohistochemistry (IHC) in lung (Supplementary Data Fig. 1). Additional detailed findings in lung and clinical measures have been previously reported10.

All animals survived to study endpoint, except for AGM1 and AGM2. At 8 days post infection, AGM1 was found recumbent and marginally responsive to stimuli. This animal also presented with dyspnea/tachypnea (respiratory rate of 72 breaths per minute), hypothermia (<32.2 °C), and hypoxemia [blood oxygen saturation (SpO2) = 77%] and was euthanized. At 22 days post infection, shortly before its scheduled study endpoint, AGM2 developed severe tachypnea, hypothermia, and hypoxemia, with a respiratory rate of 96 breaths per minute and SpO2 = 77% and was subsequently euthanized.

Seven regions of the CNS, including frontal, parietal, occipital, and temporal lobes, basal ganglia, cerebellum, and brainstem were collected at necropsy from all animals and investigated for neuroinflammation through histopathological and immunohistochemical methods. A summary of the neuropathological findings is included in Table 2.Table 2 CNS Pathology and Summary of Findings.Full size table

Neuroinflammation was seen in all study animals but was greater in those with SARS-CoV-2, as compared to age-matched mock-infected controls (Fig. 1). The pan-microglial protein, ionized calcium-binding adapter molecule 1 (Iba-1), was upregulated in the context of infection and revealed morphological alterations indicative of microglial activation, with retracted, thickened processes and a large cell body (Fig. 1b, d). Occasional, small perivascular cuffs were observed in infected (Fig. 1f, h) but not control animals (Fig. 1e, g). In contrast, nodular lesions were seen more frequently than cuffs and were present in both infected (Fig. 1j, l) and mock-infected (Fig. 1i, k) animals, however, these appeared larger in the context of infection.

figure 1
Fig. 1: Prominent neuroinflammation in brain of SARS-CoV-2 infected NHPs.

To further characterize microglial activation, tissues were investigated for the MHC class II cell surface receptor, HLA-DR (Fig. 1m–p). Similar to findings in brain of aged human subjects11, microglial expression of HLA-DR was observed in animals without SARS-CoV-2 infection (Fig. 1m, o). Expression was also seen in brain of infected animals (Fig. 1n, p); however, this did not appear greater than those that were mock-infected. HLA-DR did highlight nodular lesions in all animals, which were larger in infection, as seen with Iba-1.

Additional evidence of increased neuroinflammation in infection was seen through glial fibrillary acidic protein (GFAP) IHC, which was upregulated in infected animals (Fig. 1r, t), as compared to age-matched controls (Fig. 1q, s). GFAP immunopositivity revealed astrocytic hypertrophy in the context of aging, suggestive of astrocyte activation, however, this was more pronounced in infection, which also displayed significant loss of individual astrocytic domains.

Neuronal injury and apoptosis

Hematoxylin and eosin (H&E; Fig. 2) staining revealed marked changes in neuronal morphology, which was most often observed in cerebellum and brainstem (Fig. 2b–d). Neuronal degeneration was characterized by pyknotic and karyorrhectic nuclei with shrunken cytoplasm and vacuolation in the surrounding neuropil (Fig. 2b–d). The cerebellum contained several regions of degenerate Purkinje neurons that exhibited cellular blebs and debris and cytoplasmic vacuoles (Fig. 2b, c). Contiguous with areas of degenerate Purkinje cells, neurons and glia within the molecular and granular layers appeared pyknotic with condensed, basophilic nuclei (Fig. 2b). Similar morphologic changes were noted in glial cells adjacent to apoptotic neurons in the brainstem (Fig. 2d). In both brainstem and cerebellum, neurons are seen at various stages of nuclear dissolution (Fig. 2b–d). Degeneration of Purkinje cells was further confirmed with FluoroJade C (Fig. 2e, f).

figure 2
Fig. 2: Neuronal pathology and cell death in SARS-CoV-2 infection.

Given the prominent morphologic changes noted within Purkinje cells, we sought to identify the mechanisms underlying these degenerative changes by investigating all brain regions for the presence of cleaved caspase 3, the activated form of this key executioner of apoptosis. Cleaved caspase 3 was seen in at least one CNS region from all infected animals except AGM1, which did not have any positive cells (Fig. 2i, Supplementary Data Fig. 2). Three animals, RM1, AGM3, and AGM4 showed positivity in more than one brain region, while RM2 had cleaved caspase 3 positive cells in all regions examined (Fig. 2i; Supplementary Data Fig. 2). In cerebellum, cytoplasmic and nuclear-cleaved caspase 3 was predominantly restricted to cells within and proximal to the Purkinje cell layer (Fig. 2g). Other CNS regions, including brainstem, had foci of cleaved caspase 3 positivity (Fig. 2h). In comparison to infected animals, mock-infected controls showed little-to-no positivity (Fig. 2i; Supplementary Data Fig. 2). Unbiased quantitation revealed a statistically significant difference in cleaved caspase 3 positivity between infected and mock-infected animals in all brain regions investigated (Fig. 2i). When stratified by species, statistical significance was not achieved by Mann–Whitney U Test, which is likely due to the low number of each species (Supplementary Data Fig. 2). Interestingly, cleaved caspase 3 was not detected in any CNS region examined from AGM1, who was euthanized at 8 days post infection due to advanced illness. This may suggest programmed cell death in the CNS occurs later in the disease process.

While vacuolation was at times observed in the cerebellar gray and white matter (Supplementary Data Fig. 3a, b), significant demyelination was not a major finding in this study. Luxol Fast Blue (LFB) did reveal localized myelin pallor, suggestive of oligodendrocyte injury and/or loss, in the cerebellum of RM3 and occipital lobe of AGM3 (Supplementary Data Fig. 3c, d).

Brain microhemorrhages

Microhemorrhages, as suggested by the presence of erythrocyte extravasation into the brain parenchyma, were identified in all study animals and seen with and without ischemic injury of adjacent tissues, characterized by localized/regional tissue pallor (Fig. 3a–f). Although the number of bleeds varied, all animals were observed to have at least one. Infected animals appeared to have larger bleeds than mock-infected controls, with more dense accumulation of red blood cells on the parenchymal side of the blood vessel (Fig. 3, compare a–d with e, f). Quantitation of microhemorrhages was determined on Axio Scan.Z1 (Zeiss) scanned slides and HALO software (Indica Labs, v2.3.2089.70 and v3.1.1076.405) and normalized by tissue area (Fig. 3g). The whole brain showed a higher increase in the number of microbleeds in infection which reached statistical significance in the basal ganglia (Fig. 3h and Supplementary Data Fig. 4).

figure 3
Fig. 3: Multiple microhemorrhages in CNS of SARS-CoV-2 infected NHPs.

Accumulation of cerebral microhemorrhages occurs with aging and are seen most frequently in deep brain structures, including brainstem, basal ganglia, and cerebellum12. This may be due to age-associated decrease in arterial elasticity and increased blood pressure on brain microvasculature, as well as other risk factors for vascular injury, such as diabetes and dyslipidemia. Vascular injury can promote thrombosis, or blood clot formation within a blood vessel, which may aid in stopping the brain microbleed or, conversely may underlie microhemorrhages and result in more serious brain injury by impeding the flow of blood in the brain, leading to stroke. To assess the potential contribution of thrombosis to microhemorrhage development in SARS-CoV-2 infection, we examined all brain regions for luminal accumulation of the platelet glycoprotein, CD61 (aka, integrin b-3). This revealed multiple blood vessels with aggregated platelets in both infected and mock-infected animals, which were seen with and without associated microbleeds (Fig. 4a–d). Microhemorrhages without CD61 accumulation were also observed (Fig. 4e, f). Quantitation of total brain microhemorrhages with and without associated CD61 positivity revealed a greater frequency without thrombi (CD61 positivity) in the context of infection, apart from AGM5 who had many bleeds without visible thrombi (Fig. 4g, h). These findings suggest that in the context of infection, leakage of blood vessels without vascular damage/injury occurs more frequently.

figure 4
Fig. 4: Reduced CD61 positive associated-microhemorrhages in SARS-CoV-2 infected NHPs.

Chronic hypoxemia/brain hypoxia

Microhemorrhages and ischemia appear to play a central role in neuronal injury observed in this study. The brain is a highly metabolic organ with a limited capacity for energy storage. Due to the significant energy demands of the brain and neurons, a prolonged reduction in blood flow and concomitant reduction in oxygen and glucose can be detrimental to neuronal vitality, in addition to the resulting neurotoxicity of erythrocyte breakdown products and inflammation. Of particular interest is the finding that AGM1, who was found recumbent and minimally responsive to stimuli at 8 days post infection, had a substantial number of microbleeds in the cerebellum, basal ganglia, and brainstem (Table 2). These findings suggest AGM1 suffered multiple acute microhemorrhages that may have contributed to her rapid decline. Alternatively, AGM1’s rapid pulmonary decline may have promoted end stage microhemorrhages. The timing of acute microhemorrhages in the disease process is unclear and warrants further investigation.

In addition to localized ischemic injury, all infected animals experienced variations in SpO2 that fluctuated between 89 and 99% but stayed below 95% for most over the study course (Fig. 5a). Correspondingly, blood carbon dioxide (CO2) ranged from 24 to 33 mEq/L, remaining above the physiological range for most of the study animals (Fig. 5b). While these levels are not immediately alarming, they may suggest mild hypoxemia and impaired gas exchange in the lungs. As such, chronic hypoxemia may contribute to impairment of the endothelium and/or neurovascular unit leading to increased vascular permeability. The brain requires aerobic metabolism of glucose for ATP production and any prolonged or intermittent reductions of blood O2 may contribute to localized CNS hypoxia and energy failure. Even minor reductions in oxygen may promote injury, particularly among neurons, which appear to have suffered the greatest insult in this study. In support of this notion, large regions of Purkinje cells, which are especially vulnerable to hypoxic insult13,14, as well as cells in their immediate proximity, appear degenerate or committed to undergoing apoptosis.

figure 5
Fig. 5: Reduced blood oxygen may contribute to brain hypoxia in SARS-CoV-2 infection.

To assess brain tissue for evidence of hypoxia, we performed IHC against the oxygen-regulated alpha subunit of hypoxia inducible factor-1 (HIF-1a), which is upregulated and stabilized under hypoxic conditions. For this analysis, only basal ganglia, brainstem, and cerebellum were investigated because our earlier studies demonstrated these brain regions had the greatest injury/pathology. This study demonstrated marked upregulation of HIF-1a in brain of infected animals, as compared to mock-infected controls (Fig. 5f–m). Areas of intense positivity, suggestive of HIF-1a accumulation, were predominantly seen in and around blood vessels, which extended into the brain parenchyma in infection (Fig. 5g, i, k, m). Areas of HIF-1a positivity were noted in mock-infected animals but were less intense than that seen in brain of infected animals and/or did not extend appreciably into the parenchyma (Fig. 5f, h, j, l). Non-biased quantitation of HIF-1a intensity [optical density (OD)] around blood vessels, which excluded the blood vessel lumen, revealed a statistically significant increase in HIF-1a by cells comprising the vasculature and neighboring parenchymal cells of infected animals, as compared to controls, in brainstem (Fig. 5c, *p = 0.0154) and basal ganglia (Fig. 5d, **p = 0.0016) but not cerebellum (Fig. 5ep = 0.0940). Our approach for quantifying HIF-1a expression around the vasculature, while excluding the blood vessel lumen, is shown in Supplementary Data Figs. 5 and 6. Statistical significance was only retained in the basal ganglia when stratified by species (RMs *p = 0.049, AGMs *p = 0.034; Supplementary Data Fig. 7).

Rare virus in brain-associated endothelium

The potential for direct virus involvement in CNS pathology was explored through IHC and RNAscope analyses of all brain regions. Using an antibody against SARS-CoV-2 nucleocapsid protein (SARS-N), IHC studies revealed rare virus infection in brain that, when seen, appeared to be restricted to the vasculature (Fig. 6a). Sparse virus was detected most frequently within the basal ganglia, cerebellum, and/or brainstem and seen less often within the temporal, parietal, and occipital lobes (Table 2). This was verified further through in situ hybridization (ISH) analyses, employing RNAscope Technology with enhanced signal amplification. Using an anti-sense probe to the viral spike protein RNA (SARS-S), cytoplasmic positivity was seen in brain of infected animals but not in mock-infected controls (Fig. 6c–h; Supplementary Data Fig. 8). The specificity of the probe used in these studies is demonstrated in lung, which only showed positivity in the context of infection (Supplementary Data Fig. 8).

figure 6
Fig. 6: SARS-CoV-2 detection in the brain.

The single-label studies suggested SARS-CoV-2 infection in brain is limited to the brain vasculature and appeared to be restricted to endothelial cells. Suspected endothelial cell infection is supported by colocalization of SARS-N with von Willebrand factor (vWF; Fig. 6i–k). A blood vessel in close proximity to that shown in Fig. 6i–k but without detectable virus is included to demonstrate the specificity of the SARS-N antibody (Fig. 6l–n).

Using a highly sensitive CRISPR-based fluorescent detection system (CRISPR-FDS)15, virus was not identified in the cerebrospinal fluid (CSF) (Fig. 6o), consistent with most findings among human subjects, except in rare cases of encephalitis16,17,18. In contrast, this method detected limited viral RNA in whole brain, frozen at the time of necropsy, that was largely representative of our IHC/IF findings (Fig. 6o). Similar to our findings in fixed tissues, virus was more frequently observed in basal ganglia, cerebellum, and brainstem. CRISPR-FDS analysis also revealed viral RNA in the frontal lobe of one animal, AGM1, which was not convincingly seen by IHC/IF for this region in any study animal. This may reflect differences in sampling error that is inherently present in the two methods, where the amount of tissue used for the CRISPR-FDS studies is greater than that used in IHC, and/or extracerebral virus that may have been present in the blood vessel lumen.

Together, our findings demonstrate scarce SARS-CoV-2 infection in brain-associated endothelial cells in deep brain structures of NHPs, even in the absence of severe disease or overt neurological symptoms.

Discussion

Neurological manifestations are commonly seen in the context of SARS-CoV-2 infection but are highly varied and range in severity from impaired smell and/or taste to stroke2,19. As such, the mechanisms underlying SARS-CoV-2-associated neurological complications are likely complex. Relevant animal models of infection and CNS involvement that reflect human disease are critical for elucidating these mechanisms, as well as identifying and/or developing effective therapeutic strategies. In our two models of aged NHPs infected with SARS-CoV-2, we found evidence of prominent neuroinflammation, microhemorrhages with and without associated microthrombi, and neuronal injury and death consistent with hypoxic-ischemic injury but without substantial virus detection in brain. Our findings are largely in line with those reported in autopsy studies of individuals who died from infection20,21,22,23,24,25,26. Like human disease, reactive astrocytes and microglia were a common feature, seen throughout the entirety of the brain in infected animals. This appeared greater in basal ganglia, brainstem, and cerebellum, which contained the majority of cuffs and nodular lesions observed. Lymphocyte infiltrate, which has been reported in human brain22,24, was not observed in any brain region investigated from our study animals. This may reflect a shorter time with severe disease in our animal model. Additional life-saving efforts were not made for animals that developed serious disease (e.g., ARDS), as would be done with humans, and were quickly euthanized to minimize pain and suffering of the animal. It is worth noting that autopsy reports of significant lymphocyte infiltration into the CNS or COVID-associated encephalitis are relatively few and may be a less common complication of disease8.

Our findings of hypoxic-ischemic injury in brain of NHPs are also in agreement with autopsy studies of brain from human subjects21,27. This may arise from chronic, peripheral hypoxemia, as well as reduced cerebral blood flow due to acute microhemorrhages. The brain is a highly metabolic organ and requires aerobic metabolism of glucose for adenosine triphosphate (ATP) production. Any prolonged or chronic intermittent reductions of blood SpO2 may contribute to localized CNS hypoxia and energy failure. Even minor, but sustained, reductions in oxygen may promote injury, particularly among neurons, which appear to have suffered the greatest insult in this study. In support of this notion, large stretches of Purkinje cells, which are especially vulnerable to hypoxic insult13,14, as well as cells within their immediate proximity, appear degenerate and/or committed to undergoing apoptosis. Areas of injured neurons at various stages of nuclear dissolution were noted in other brain regions, including brainstem. Moreover, neuronal injury did not appear to be a direct consequence of virus infection, as only limited virus was convincingly detected in brain vasculature and did not appear to involve parenchymal cells. Instead, neuronal injury and death most likely occur as a result of energy failure, which is an early consequence of hypoxic-ischemic events. Multiple microhemorrhages, microinfarcts, and hypoxemia appear to play a role in neuronal injury and death observed in these animals.

Consistent with a hypoxic environment, we detected upregulation/stabilization of HIF-1a in infected animals that localized to the brain vasculature and was significantly greater than mock-infected controls in the deep brain regions assessed. This was observed in all infected animals, regardless of disease severity, suggesting reduced brain oxygen may be a common complication of infection. While the mechanism is not yet elucidated, chronic hypoxemia, as well as an exaggerated and prolonged immune response likely play an important role. Indeed, several inflammatory mediators and growth factors have been reported to stabilize and/or promote expression of HIF-1a, including nitric oxide, interleukin 1b, and tumor necrosis factor-a28,29,30.

Interestingly, we did not observe HIF-1a upregulation in cerebellar Purkinje cells in any animal. This may be due to the kinetics of HIF-1a expression, which has been shown in a mouse model of chronic hypoxia to peak in Purkinje cells at 4–5 h and return to normoxic levels after 9–12 h in a continual hypoxic environment31. These findings suggest that any potential upregulation and/or stabilization of HIF-1a in Purkinje cells had returned to normal levels by the time the animals were euthanized. It is also likely that degenerate Purkinje cells no longer produce HIF-1a. Our conflicting findings in the brain vasculature may be due to continued exposure of these cells to peripheral factors that promote HIF-1a stabilization and/or expression.

A direct role for the virus in HIF-1a upregulation cannot be ruled out, however, the negligible frequency of SARS-CoV-2 infected cells seen in the CNS compartment argues against the virus being a significant factor. A recent RNAseq analysis, however, found increased HIF-1a mRNA in peripheral blood mononuclear cells (PBMC) acquired from SARS-CoV-2 infected human subjects, as compared to healthy, non-infected controls32. Additional in vitro analyses suggested SARS-CoV-2 ORF3a protein induces HIF-1a production in transfected cells, as well as several cytokines upregulated in the context of infection32. How this translates to HIF-1a expression in vivo, however, remains unclear.

In agreement with most reports of living subjects and those who died from COVID-198, we did not detect virus in CSF and found only minimal virus in the brain that appeared to be limited to the vasculature, suggestive of hematological dissemination of virus to the brain. Infection of pericytes, perivascular macrophages, and/or cells within the brain parenchyma cannot be ruled out but was not convincingly demonstrated in these studies. Instead, virus appeared to be restricted to the endothelium, which is consistent with a previous study of human biopsy tissues that demonstrated the principal receptor for SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2), is expressed by endothelial cells throughout the body, including brain33,34. More recently, a large autopsy series out of Mount Sinai demonstrated robust ACE2 expression by brain vasculature in patients who died from SARS-CoV-2 infection20. This may suggest a greater vulnerability of the brain to infection in the context of severe disease but was not observed in NHPs that developed ARDS in this study. One autopsy report identified virus in a subset of cranial nerves22, however, these were not available for investigation. Additionally, the olfactory bulb, which was not recovered from our study animals, may also be an important site for virus entry into the CNS and requires additional investigation.

Notably, the animals in this study were of advanced age, which is associated with a higher risk for the development of cerebrovascular disease among infected patients8. Indeed, aging, itself, is the greatest risk factor for cerebrovascular disease, due, at least in part to age-related changes of cerebral vascular structure and/or function that contribute to reduced cerebral blood flow, which may be further compounded by underlying vascular pathology35,36. This may predispose the aging vasculature to cerebrovascular events, particularly in the context of prolonged systemic inflammation and hypoxemia, which have been shown to contribute to increased vascular permeability through microglia and astrocyte responses37,38.

Here, we show substantial pathological changes in brain of SARS-CoV-2 infected NHPs that are compatible with autopsy and imaging reports of infected human subjects. Additionally, our pathological investigation suggests a significant role for brain hypoxia in the neuropathogenesis of COVID-19, including animals without severe disease. It is reasonable to anticipate that similar findings may occur among human subjects, particularly those with continuing neurological symptoms after recovery from infection39,40,41. For example, an increasing number of retrospective neuroimaging reports have reported cerebral microhemorrhages in critically ill patients with COVID-1942,43,44. Many patients, however, including those who do not require hospitalization, report comparatively milder neurological symptoms that are not evaluated through neuroimaging. As such, neuropathology among these individuals remains unclear but likely contributes to lingering neurocognitive difficulties reported by a number of convalesced/convalescing patients45 and warrants further investigation. This further increases the significance of NHPs as a viable model for elucidating the mechanisms that underlie SARS-CoV-2-associated neuropathology that are translatable to human disease, as neuropathogenesis can be more closely examined in animals that do not experience mortal disease. Additionally, neuropathological complications may contribute to worsening disease among infected patients. For example, damage to the brainstem, which modulates the respiratory cycle by regulating inspiratory and expiratory muscle activity, may contribute to worsening respiratory distress and failure in patients with COVID-19. Additional studies, employing relevant animal models, are warranted and likely to reveal important insight into human disease.

While SARS-CoV-2 neuropathogenic processes are poorly understood, this work reveals infected NHPs are a viable animal model for understanding the neuropathogenesis and potential long-term consequences of infection. We also provide important insight into the mechanisms underlying CNS disease, which was seen even in the absence of severe respiratory disease and may suggest that vascular leakage and hypoxic brain injury is a common complication of SARS-CoV-2 infection and COVID-19. Neuronal degeneration and activation of caspase 3 observed in this study supports this notion and indicates non-reversible neuronal injury may be significant to individuals suffering from PASC. Finally, our findings and conclusions presented herein suggest the need for long-term neurological follow-up of persistently symptomatic convalescent patients.

Methods

Ethics and biosafety statement

All animal studies were approved by the Tulane University Institutional Animal Care and Use Committee and carried out in the Regional Biocontainment Laboratory at the Tulane National Primate Research Center (TNPRC) within an animal biosafety level 3 facility. The TNPRC is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). All animals were cared for in accordance with the Institute for Laboratory Animal Research Guide for the Care and Use of Laboratory Animals, 8th edition. The Tulane University Institutional Biosafety Committee (IBC) approved all procedures for sample handling, inactivation, and removal from BSL3 containment.

Animal study design

A total of twelve NHPs, including six Indian-origin RMs (ages 13–21 years) and six AGMs of Caribbean origin (all approximately 16–17 years of age), were included in this study. Four of each species was inoculated with SARS-CoV-2 strain 2019-nCoV/USA-WA1/2020 (MN985325.1) and two of each species were mock-infected with culture media used for virus propagation (Table 1). The viral strain used was isolated from the first confirmed SARS-CoV-2 case in the United States and deposited by the Centers for Disease Control9. All animals underwent the same procedures and biological sampling.

All RMs were acquired from the TNPRC specific pathogen-free breeding colony and confirmed negative for simian type D retrovirus (SRV), simian immunodeficiency virus (SIV), simian T cell lymphotropic virus type 1 (STLV1), measles virus (MV), Macacine herpesvirus 1 (MHV1/B virus), and tuberculosis (TB). The AGMs were wild-caught and also confirmed negative for SRV, SIV, STLV, MV, and TB. The AGMs were housed at the Center for over a year before assignment to this study. All animals were tested and found negative for SARS-CoV-2 (antibody and virus) prior to experimental infection.

Two routes of virus exposure, multi-route mucosal and aerosol, were employed to mimic major routes of infection among humans. Two animals from each species were randomly subjected to the different routes of exposure for a total of four animals in each species challenge group. Multi-route exposure included conjunctival, nasal, pharyngeal, and intratracheal routes for a cumulative dose of 3.61 × 106 PFU (plaque-forming unit). Animals exposed to virus by aerosol received an approximate inhaled dose of 2 × 103 TCID50 (50% tissue culture infectious dose). Study animals were euthanized for necropsy at 24–28 days post infection unless humane endpoints required euthanasia at an earlier time (Table 1). Postmortem examination was performed by a board-certified veterinary pathologist (R.V.B.).

Quantification of Nasal Swab SARS-CoV-2 subgenomic nucleocapsid mRNA (sg-N mRNA)

Nasal swab specimens were collected in 200 µL DNA/RNA Shield (Zymo Research) and extracted for viral RNA (vRNA) using the Quick-RNA Viral kit (Zymo Research). Viral RNA Buffer (Zymo) was dispensed directly to the swab in the DNA/RNA Shield (Zymo). A modification to the manufacturers’ protocol was to insert the swab directly into the spin column to centrifugate, allowing all the solution to cross the spin column membrane. The vRNA was eluted (45 µL), from which 5 µL was added to a 0.1 mL fast 96-well optical microtiter plate format (Thermo Fisher) for a 20 µL RT-qPCR reaction. The RT-qPCR reaction used TaqPath 1-Step Multiplex Master Mix (Thermo Fisher) along with the following primers and probe: Forward primer: (sgm-N FOR) 5′-CGATCTCTTGTAGATCTGTTCTC-3′; Probe: (sgm-N PRB) 5′-FAM TAACCAGAATGGAGAACGCAGTGGG-BHQ1-3′; Reverse primer: (sgm-N REV) 5′-GGTGAACCAAGACGCAGTAT-3′. The reaction master mix was added using an X-Stream repeating pipette (Eppendorf) to the microtiter plates. Loaded plates were covered with optical film (Thermo Fisher), vortexed, and pulse centrifuged. The RT-qPCR reaction employed the following program: UNG incubation at 25 °C for 2 min, RT incubation at 50 °C for 15 min, and an enzyme activation at 95 °C for 2 min, followed by 40 cycles of denaturation at 95 °C for 3 s and annealing at 60 °C for 30 s. Fluorescence signals were detected with an Applied Biosystems QuantStudio 6 Sequence Detector. Data were captured and analyzed with Sequence Detector Software v1.3 (Applied Biosystems). Equivalent viral copy numbers were calculated by plotting Cq values obtained from unknown (i.e., test) samples against a standard curve representing known viral copy numbers. The limit of detection of the assay was ten copies per reaction volume. A 2019-nCoV positive control (IDTDNA) was analyzed in parallel with every set of test samples to verify the RT-qPCR master mix and reagents were prepared correctly. A non-template control was included in the qPCR to ensure there was no cross-contamination between reactions.

Immunohistochemistry

IHC was performed on 5 µm zinc formalin-fixed paraffin-embedded (FFPE) brain sections46. Sections were deparaffinized in xylenes and rehydrated through an ethanol series ending in distilled water. Heat-mediated antigen retrieval was carried out in a vacuum oven with Tris-EDTA buffer (10 mM Trizma base, 1 mM EDTA, 0.05% Tween 20, pH 9.0) or sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0). All washes were performed using tris buffered saline containing Tween 20 (TTBS; 0.1 M Trizma base, 0.15 M NaCl, 0.1% Tween 20, pH 7.4). Following antigen retrieval, tissues were blocked with 20% normal horse or goat serum. Endogenous biotin was blocked with Avidin-Biotin Solution (Vector Labs). Titrated primary antibodies included anti-cleaved caspase 3 (rabbit polyclonal, 1:250, Abcam, ab2302), anti-von Willebrand factor (vWF, rabbit EPR12010, 1:62.5, Abcam, ab179451), anti-HIF-1a (mouse mgc3, 1:1600, Abcam, ab16066), anti-CD61 (rabbit RM382, 1:125, Invitrogen, MA5-33041), anti-ionized calcium-binding adapter molecule 1 (Iba-1, goat polyclonal, 1:200, Abcam, ab5076), anti-GFAP (rabbit EPR1034Y, 1:500, Abcam, ab68428), anti-HLA-DR (mouse TAL.1B5, 1:400, Novus, NB600989), and anti-SARS-CoV-2 nucleocapsid (rabbit polyclonal, 1:125, Novus, NB100-56576). Tissues were incubated with primary antibody overnight at room temperature and detected using the appropriate biotinylated secondary antibody (1:200, Vector Labs, BA-1100, BA-2000, BA-9500) and alkaline phosphatase-Vector Red according to manufacturer instructions (Vector Labs). Tissues were counterstained with Mayer’s hematoxylin and coverslipped.

Double labeling of 5 µm FFPE brain tissue was performed by sequential application of primary antibodies with their corresponding secondary47. SARS-CoV-2 nucleocapsid was detected with Alexa Fluor 555 goat anti-rabbit IgG (1:500, Invitrogen, A21428). Von Willebrand factor was detected with Alexa Fluor 488 goat anti-rabbit IgG (1:500, Invitrogen, A11008). Controls consisted of brain tissue incubated in blocking buffer only, tissue incubated with one primary and the corresponding secondary antibody, and tissue incubated with fluorophore-conjugated secondaries only. Tissues were coverslipped with Vectashield® HardSet™ Antifade mount with DAPI (Vector Labs).

In situ hybridization (RNAscope)

ISH was carried out on 5 µm FFPE tissues using RNAScope® Multiplex Fluorescent V2 Assay Kit (Advanced Cell Diagnostics), according to the manufacturer’s directions. Briefly, sections were deparaffinized in xylenes and dried, followed by incubation with hydrogen peroxide. Heat-mediated antigen retrieval was carried out in a steamer with the provided kit buffer. A hydrophobic barrier was drawn around the tissues before treatment with the kit-provided protease reagent and hybridized with the V-nCoV2019-S probe (Advanced Cell Diagnostics) in a HybEZ oven (Advanced Cell Diagnostics). All washes were performed with the kit wash buffer. Signal amplification was accomplished with three successive AMP solutions and HRP channel (Advanced Cell Diagnostics) and visualized with Opal 570 (1:1000, Akoya Biosciences). Autofluorescence was quenched with TrueVIEW® Autofluorescence Quenching Kit (Vector Labs). Positive and negative control tissues and tissues without probe exposure were included in every run to ensure the specificity of staining and assess background.

Hematoxylin and eosin

Deparaffinized and rehydrated slides were taken through Hemalast and hematoxylin, followed by differentiator and bluing solutions. After which, slides were dehydrated in 95% EtOH and stained with eosin. Stained slides were dehydrated, cleared, and coverslipped.

Luxol fast blue

Slides were deparaffinized and rehydrated through 95% EtOH, then incubated in warmed 0.1% LFB solution. Afterward, slides were washed, dipped in 0.05% lithium carbonate, differentiated in 70% EtOH, and rinsed. Following a check under microscope, the slides were oxidized in 0.5% periodic acid solution, then immersed in Schiff’s reagent before rinsing, dehydration, clearing, and coverslipping.

FluoroJade C

Five micrometers FFPE tissues were immersed in 0.06% KMNO4 for 10 min and washed. Tissues were then immersed in 0.0002% FluoroJade C (Histo-Chem) containing 0.1% acetic acid in the dark for 20 min, counterstained with 4′,6-diamidino-2-phenylindole (DAPI), washed, and dried at 60 °C. Cleared tissues were coverslipped with DPX mount (Sigma).

Imaging and quantitation

Slides were scanned with the Axio Scan.Z1 digital slide scanner (Zeiss). Brightfield images were acquired using HALO (Indica Labs, v2.3.2089.70 and v3.1.1076.405). Fluorescent images were acquired on a Leica DMi8 automated confocal microscope, model SP8, equipped with a Leica imaging software application suite X model LAS X, software v3.5.7.23225 and an Olympus IX73 inverted microscope with cellSens Dimension 3 software v3.1. Colocalization images were created in Photoshop (Adobe, v21.2.0) by overlaying the same image acquired through the appropriate fluorophore filter. Presented images were subjected to brightness, contrast, and/or darken midtones enhancement in Photoshop, applied to the entire image to reduce background.

Threshold and multiplex analyses were performed with HALO algorithms for non-biased quantitation of proteins of interest, without processing. For active caspase 3 hematoxylin-stained nuclei were used to quantify the number of cells and Vector Red intensity above a rigorous threshold accounted for the cells positive. Quantitation of HIF-1a was performed using an area quantification algorithm for Vector Red intensity. Annotations were drawn to outline blood vessel-associated parenchymal stain based on the algorithm results. The annotated area was analyzed for OD of Vector Red staining. The average OD within the annotated area was calculated in HALO per tissue section.

Microhemorrhages were independently counted and annotated within HALO on seven distinct 5 µm CD61-immunostained regions of the CNS from all infected and control animals by two individuals. Counts were normalized by area of each tissue section. Microhemorrhages were defined by the presence of blood vessels with red blood cell extravasation (>10 red blood cells on the parenchymal side of an unbroken blood vessel). Normalized microhemorrhage counts were plotted for each specific brain region and total regions investigated. CD61+ aggregates within blood vessels were counted and annotated on HALO on seven distinct 5 µm sectioned regions of the CNS from all infected and control animals by two individuals. Blood vessel-associated CD61+ thrombi were defined by aggregated CD61 stained platelets within a vessel.

RNA isolation from whole tissues

Dissected frontal lobe, basal ganglia, cerebellum, and brainstem were collected fresh and immediately frozen at necropsy. One milliliter of Trizol LS (Thermo Fisher) was added to 100 mg of thawed tissue and homogenized in gentleMACS M tubes using a gentleMAC Dissociator (Miltenyi Biotec). The resulting lysate was then centrifuged at 3000 × g for 5 min and supernatant transferred into a 2 mL microcentrifuge tube. An equal volume of ethanol (95–100%) was added to the sample in Trizol LS (1:1) and mixed well. The resulting mixture was transferred to a Zymo-Spin III CG Column in a 2 mL collection tube (Zymo) and centrifuged for 30 s. The column was washed with RNA Wash Buffer (Zymo), followed by treatment with DNase I for 30 min to remove residual genomic DNA. The column was washed with RNA Wash Buffer (Zymo) and RNA eluted with 45 μL of DNase/RNase-free water (Thermo Fisher).

CRISPR-based fluorescent detection system (CRISPR-FDS)

CRISPR-FDS reaction was carried out with the following steps15. Isolated RNA samples were mixed with one-step RT-PCR mix containing 2× PlatinumTM SuperFiTM RT-PCR Master Mix (Thermo Fisher), forward primer (10 μM), reverse primer (10 μM), SuperScriptTM IV RT Mix (Thermo Fisher), and nuclease-free water. Samples were then incubated in a T100 thermocycler (Bio-Rad) using a cDNA synthesis protocol, immediately followed by a DNA amplification protocol. CRISPR-FDS reactions were performed as follows: a sample RT-PCR reaction was transferred to a 96-well half-area plate and mixed with CRISPR reaction mixture containing 10X NEBuffer™ 2.1, gRNA (300 nM), EnGen® Lba Cas12a (1 μM), fluorescent probe (10 μM), and nuclease-free water. After incubation at 37 °C for 20 min in the dark, fluorescence signal was detected using SpectraMax i3x Multi-Mode Microplate Reader (Molecular Devices). A positive sample was defined as any specimen with a CRISPR-FDS signal that was greater than the cut-off threshold of 3.6 × 106 photoluminescence (PL) intensity (arb. units).

Statistics

Kolmogorov-Smirnov normality test, Mann–Whitney U test, and Student’s unpaired two-tailed t-tests were performed with GraphPad Prism software, v9.0.2. When separated by species the number of controls was below the detectable limit for the Kolmogorov–Smirnov normality test. Data were defined as gaussian or non-gaussian based on the overall distribution. P values ≤ 0.05 were considered significant.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

https://figshare.com/articles/dataset/COVID_NHP_CNS_Source_Data/19241727https://doi.org/10.6084/m9.figshare.19241727Source data are provided with this paper.

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COVID can cause lingering brain damage — even in mild cases

Authors: Chris Melore Study Finds APRIL 1, 2022

COVID-19 started as a serious threat to the lungs, but it’s become just as synonymous with brain issues throughout the pandemic. Now, a new study is revealing exactly how the virus damages the central nervous system. Researchers at Tulane University say even a mild COVID infection can leave lingering damage in a patient’s brain.

Previous studies have documented several cognitive issues like headaches, confusion, and “brain fog” during a COVID patient’s infection and in the months following — as a symptom of long COVID. Until now, however, scientists haven’t fully understood how the illness targets the brain.

The new study found severe brain inflammation and injury resulting from reduced blood flow or oxygen traveling to the brain. This included neuron damage and cell death. The team also discovered microhemorrhages (small bleeds) in the brains of nonhuman primates who died after a coronavirus infection.

To the research team’s surprise, even primates who did not suffer from severe respiratory disease showed the same damage in the brain.

“Because the subjects didn’t experience significant respiratory symptoms, no one expected them to have the severity of disease that we found in the brain,” says lead investigator Tracy Fischer in a university release. “But the findings were distinct and profound, and undeniably a result of the infection.”

COVID brain damage may lead to a more severe infection

Fischer has been studying brains for decades. After Tulane’s National Primate Research Center launched a COVID pilot program in early 2020, the study author started examining the brain tissue of several animals infected with coronavirus.

Fischer’s initial findings were so shocking that she spent the next year refining the results to make sure COVID-19 was truly responsible for this severe brain damage. Concerningly, these new findings are in line with those coming from human autopsies during the pandemic.

Study authors say this suggests that nonhuman primates can serve as a reliable model as scientists continue to explore how COVID-19 damages the human body.

The team also notes that neurological problems are some of the first symptoms people experience during a COVID infection. These symptoms also impact patients of all ages, whether they have pre-existing conditions or not.

As for how brain damage from COVID may impact a patient’s chances of survival, researchers say the brain plays a major role in controlling a person’s respiratory system — a main target of COVID.

“Neuropathological complications may contribute to worsening disease among infected patients. For example, damage to the brainstem, which modulates the respiratory cycle by regulating inspiratory and expiratory muscle activity, may contribute to worsening respiratory distress and failure in patients with COVID-19,” the researchers write in the journal Nature Communications.

Uptake of SARS-CoV-2 spike protein in human cerebrovascular cells

Authors:  Bhavana Kunkalikar Mar 24 2022 Reviewed by Danielle Ellis, B.Sc. BioRxiv

A recent study posted to the bioRxiv* preprint server assessed the uptake of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein in human cerebrovascular cells via the lipid raft ganglioside.  

Study: SARS-CoV-2 spike proteins uptake mediated by lipid raft ganglioside GM1 in human cerebrovascular cells. Image Credit: Dotted Yeti
Study: SARS-CoV-2 spike proteins uptake mediated by lipid raft ganglioside GM1 in human cerebrovascular cells. Image Credit: Dotted Yeti

Various studies have reported the effects of coronavirus disease 2019 (COVID-19) on the respiratory organs and non-respiratory ones, including the brain. However, there is still a lack of knowledge about the SARS-CoV-2 uptake mechanism involved in the viral entry into the human cerebrovasculature cells. 


About the study

The present study investigated the mechanism of SARS-CoV-2 S protein uptake by three types of cerebrovascular cells: endothelial cells, smooth muscle cells, and pericytes.

The team obtained human cerebral microvascular endothelial cells (hCMEC/D3), human
brain vascular smooth muscle cells (HBVSMCs), and human brain vascular pericytes (HBVPs). The cells were expanded separately and then pre-incubated with the inhibitor before being exposed to SARS-CoV-2 S protein in an incubator. S protein uptake without the inhibitor was then estimated as a percentage of control wells. 

The in vitro cell viability of the unlabeled wild type (WT) S protein was evaluated by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The team also used immobilized recombinant human angiotensin-converting enzyme 2 (ACE2) to assess its binding ability to the S protein.  

Images of the whole well were taken, followed by all fluorescence quantification. The custom pixel classifiers measured the fluorescence intensity while the cell count was calculated using Qupath’s cell counter. Furthermore, primary antibodies were used in the immunocytochemistry (ICC) and imaged.


Results 

The study results showed that the uptake mechanism associated with the SARS-CoV-2 WT S proteins (SP-555) was mostly observed on the hCMEC/D3 cell surface while the HBVP and HBVSMC cells showed more internalization. Also, each cell type reached equilibrium after six hours since the 100 nanometers (nm) SP-555 signal started. Furthermore, the endothelial cells showed the lowest ability to uptake S protein compared to the smooth muscle cells and the pericytes, possibly because of the different cell sizes.

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Similar binding affinities were observed for the HBVMC and the hCMEC/D3 cells, while ACE2 and SP-555 binding showed lower values than the other two. Notably, SARS-CoV-2 S was not toxic towards the type of cells tested in this study. Also, in the MTT assay, increased levels of formazan were found at higher S concentrations in the hCMEC/D3 cells, indicating elevated metabolic activity in these cells because of the higher mitochondria content present in the cerebral endothelium. 

The team also noted receptor binding in the S uptake patterns in the cell types, highlighting ACE2 as the major binding site for the viral protein. Notably, interactions between ACE2 and SP-555 were also confirmed, while these interactions were localized twice more in the hCMEC/D3 cells than in the other two cell types studied. 

A 40% reduction of SP-555 uptake in the presence of excess unlabeled αACE2 indicated the co-localization of SP-555 with αACE2. On the other hand, excess unlabeled S protein reduced the bound labeled S protein uptake by 50% to 60% for the three cell types. The assessment of S uptake at different temperatures showed that the uptake was 2.2 to 5.5 times higher at 37oC than at 4oC, suggesting greater protein interaction with the cell types at 37oC.

The study showed that a sialic acid-binding lectin, called wheat germ agglutinin (WGA), increased the uptake of SP-555 by 2.4 times in the hCMEC/D3, 3.2 times in the HBVP, and 1.4 times in the HBVMC cell types. In contrast, a polysaccharide glycosaminoglycans (GAGs) called heparin reduced the S uptake by 30% to 60% in the HBVMC and the HBVP cells, while no such change was observed in the hCMEC/D3 cells. Anti-ganglioside 1 (GM1) antibody (αGMI) also reduced the S protein uptake by 60% to 80% in the three cell types. Moreover, the decrease in uptake observed in the presence of both αACE2 and αGM1 antibodies was similar to that observed with αGM1 alone.

Furthermore, in the hCMEC/D3 cells, an increased S uptake of 1.5, 1.9, and 2.8 times was noted for mutations found in SARS-CoV-2 variants of concern, including D614G, N501Y, and E484K, respectively, as compared to the WT S protein. In the HBVP cells, while D614G showed no difference in uptake, an increase of 1.7 times was found for both N501Y and E484K mutants. Lastly, in the HBVSMC cells, an increase of 3.2, 5.0, and 3.8 times was observed in the uptake of the D614G, N501Y, and E484K mutants.  

Conclusion

To summarize, the study findings showed that the mechanism of SARS-CoV-2 S protein uptake via the GM1/lipid raft is crucial as the inhibition of this entry point can serve as a potential target against SARS-CoV-2 infections.

A Case Of Shrunken Brains: How Covid-19 May Damage Brain Cells

Authors: William A. Haseltine Forbes March 22, 2022

Thanks to a new study from the UK we are now beginning to uncover the effects of SARS-CoV-2 infections in the brain. Comparing brain volume before and after individuals were exposed to SARS-CoV-2, this study documents significant cortical gray matter loss, equivalent to nearly 10 years of aging. Gweanaelle Douaud, the study’s first author and Professor at the University of Oxford, says that infected individuals display structural “differences over time above and beyond any potential baseline differences.” Most strikingly, individuals that experienced no or only mild symptoms with Covid-19 displayed specifically significant changes, but cortical damage seems to occur regardless of disease severity, age, sex, or vaccination status. It may be years before the long-term consequences of these structural differences are fully understood.

Douand et al had unique access to an extensive biomedical database, called the UK BioBank. The UK BioBank provided pre-pandemic brain scans from 785 individuals that were used as a baseline for normal size and structure relative to each participant. About three years later, in May 2021, the same population of participants returned for new brain scans. From the total cohort, 401 individuals were infected with Covid-19 between scans and the remaining served recruited as controls.

Between the first and second brain scans, individuals previously infected with Covid-19 experienced a 0.7% reduction in overall cortical gray matter on average, compared to the control group. To put that into perspective, people middle aged and beyond only lose 0.2% to 0.3% of volume per year.

Although it is clear that SARS-CoV-2 can damage the brain, how this damage occurs without directly infecting nerve cells remains a mystery. Current theories suggest there may be multiple factors contributing to these deficits. Structural abnormalities in the brain may in fact be secondary to infection occurring in other sites around the body, including the olfactory epithelium. Considering how close the nose is to the brain, this Oxford University Study considered whether losing the sense of smell may be linked to other neurological damage.

Loss of smell is a consistent clinical feature of Covid-19, with recent studies suggesting that 86% of individuals exposed to the virus may experience partial or complete loss of smell. A smaller percentage of people also develop additional neurological complications, including brain fog, fatigue, impaired cognitive function, and encephalography, characterized by impairments in brain structure and function. For some, these symptoms may continue to linger anywhere from a few months to more than a year after the initial infection. These effects, however, do not discriminate between mild and severe disease.

First, Douand et al. found reduced gray matter in several regions associated with olfaction, consistent with symptoms of prolonged loss of smell. Individuals exposed to Covid-19 experienced the most significant neural damage to the primary olfactory cortex, or piriform cortex. This structure receives input directly from olfactory receptors that pass through the olfactory bulb, making it the first neural target for processing and perceiving odors. Reduced cortical thickness was also observed in a connected region called the orbitofrontal cortex. Illustrated in Figure 1, the orbitofrontal cortex (OFC) receives inputs from the primary olfactory cortex and is commonly referred to as the secondary olfactory cortex.

Schematic-view-of-the-human-olfactory-system-The-primary-and-secondary-olfactory
Figure 1: Schematic view of the human olfactory system. The primary and secondary olfactory cortices … [+] FROM: “ A REVIEW ON THE NEURAL BASES OF EPISODIC ODOR MEMORY: FROM LABORATORY-BASED TO AUTOBIOGRAPHICAL APPROACHES” SAIVE ET AL. 2014.

Damage to these areas may be linked to loss of smell, but there is no evidence of causation. Instead, researchers speculate that the loss of volume to brain regions associated with olfaction may be partially attributed to widespread damage to the olfactory epithelium that disrupts neural pathways and impairs function. As with many other neural systems, pathways that are not being used over a long period of time cease to exist, a process neuroscientists often call “use it or lose it” that causes brain tissues to shrink.

Surprisingly, Douand et al. also found additional abnormalities in regions not normally associated with the sense of smell. In particular, they observed reduced gray matter volume in some regions of the limbic system, involving several structures important for producing behavioral and emotional responses. The largest differences, ranging from 0.2% to 2% reductions, were seen in the left parahippocampal gyrus and the entorhinal cortex. These regions play an important role in the hippocampal memory system, so gray matter loss could signify future memory impairments. Structures are shown below in Figure 2 for reference.

Blausen_0614_LimbicSystem
Figure 2: Illustration of the limbic system. The parahippocampal gyrus and hippocampus, which … [+] BRUCEBLAUS, WIKIPEDIA COMMONS

Are the differences seen in these limbic structures also linked to deficits in the olfactory system during Covid-19? Douand et al. argues that the parahippocampal gyrus, the orbitofrontal cortex and other parts of the limbic system are in some way connected to the olfactory cortex. Since sensory inputs, including those for olfaction, are transmitted and integrated all over the brain to guide a range of behavioral responses, damage to the olfactory epithelium may also have disastrous consequences on regions of the brain not exclusively involved in olfaction. More likely, however, these and other changes in brain structure may be a consequence of a robust immune response occurring all around the brain, albeit the mechanisms underlying inflammation-induced brain damage remain unclear.

Additional exploratory analyses found gray matter loss in the amygdala, the insula which borders the temporal cortex, and the front-most portion of the cingulate gyrus, known as the anterior cingulate gyrus. Interestingly, all these regions play a role in emotion processing and regulation. More research is needed to determine whether deficits in these brain regions may be linked to mood disorders associated with long-haul Covid-19, including depression and anxiety.

ApolloMed_2017_14_4_198_224728_f1 (1)
Figure 3:Reference locations for amygdala, anterior cingulate cortex, and insula. APOLLO MEDICINE 2020

It is important to note that not every individual infected with Covid-19 will experience a reduction in brain volume, while others will experience much greater losses. Those hospitalized with Covid-19, for example, had more widespread tissue damage and atrophy, compared to those not hospitalized for infection. Beyond hospitalized vs. non-hospitalized, there was limited data from this study showing how severity of infection may contribute to these effects.

Finally, Douand et al. asked whether these structural changes in the cerebral cortex are linked to new neurological symptoms following Covid-19 infection. Interestingly, they did not find any significant correlations. No correlation between structural changes and the prevalence of new neurological symptoms, however, does not mean that these changes will not impact brain function.

For a vast majority of people, the regenerative properties of the olfactory bulb restores the sense of smell within a few weeks or months. What about the rest of the brain? Damage to brain cells cannot be reversed. When tissues die, cerebrospinal fluid and other biomolecules fill the excess space to maintain the integrity of the brain. Perhaps, this explains why neurological complications associated with long-haul Covid-19 show little improvement over time. Years of additional research are needed before the consequences of losing so much gray matter are fully understood. Identifying these changes now will help us to better support and treat what will be a growing class of people with cognitive impairments.

Although the loss of smell is often one of the first symptoms of Covid-19 preceding any respiratory complications, the hypothesis that the SARS-CoV-2 damages the brain when it infects cells in the olfactory epithelium remains heavily-debated. Researchers do seem confident that the virus does not directly infect brain cells. If the nose is a window to the brain, it may be time to develop new vaccines that aim to close it off from the SARS-CoV-2 virus.

7 in 10 long COVID patients are dealing with memory and concentration problems

Authors: Study Finds MARCH 17, 2022

The vast majority of people dealing with “long COVID” are experiencing memory and concentration problems — months after their actual coronavirus infection, a new study warns. Researchers at the University of Cambridge say seven in 10 people experiencing the lingering effects of COVID are now struggling mentally.

The study finds long COVID patients are also performing worse on cognitive exams. Moreover, three in four people with a severe case of long COVID say they have been unable to work because of it.

The team also found a link between the severity of symptoms and how much fatigue, dizziness, and headache pain patients experienced during their initial bout with the virus. Worryingly, half of long COVID sufferers claim they’ve struggled to get doctors to take their condition seriously.

Long COVID has received very little attention politically or medically. It urgently needs to be taken more seriously, and cognitive issues are an important part of this. When politicians talk about ‘Living with COVID’ – that is, unmitigated infection, this is something they ignore. The impact on the working population could be huge,” says study senior author Dr. Lucy Cheke in a university release.

“People think that long COVID is ‘just’ fatigue or a cough, but cognitive issues are the second most common symptom – and our data suggest this is because there is a significant impact on the ability to remember.”

Long COVID patients dealing with brain fog, forgetfulness

Researchers say there is growing evidence that COVID-19 impacts the brain, with multiple studies likening its impact to Alzheimer’s disease.

“Infection with the virus that causes COVID-19 can lead to inflammation in the body, and this inflammation can affect behavior and cognitive performance in ways we still don’t fully understand, but we think are related to an early excessive immune response,” says Dr. Muzaffer Kaser.

“It’s important that people seek help if they’re concerned about any persistent symptoms after COVID infection. COVID can affect multiple systems and further assessment is available in long COVID clinics across the UK, following a GP referral.”

Of the 181 people who took part in the study, 78 percent reported difficulty concentrating, 69 percent said they experienced “brain fog,” 68 percent had moments of forgetfulness, and three in five had problems finding the right words while speaking. These self-reported symptoms were confirmed by the significantly lower ability among long COVID sufferers to remember words and pictures in cognitive tests.

Severe cases of COVID leading to more cognitive issues

During the study, participants took part in several tasks to assess their decision-making abilities and memory. These included remembering words in a list and remembering which two images appeared together. Results revealed a consistent pattern of ongoing memory problems in those who previously suffered a coronavirus infection.

Study authors say these problems were more pronounced in people whose overall ongoing symptoms were more severe. The researchers investigated other symptoms that could have a link to long COVID to help them pinpoint their causes.

They found people who experienced fatigue and neurological symptoms, such as dizziness and headache, during their initial illness were more likely to have cognitive symptoms later on. They also found that those who were still experiencing neurological symptoms particularly struggled on cognitive tests.

Results show that, even among people who did not need to go to the hospital, those with worse initial symptoms of COVID-19 were more likely to have a variety of ongoing long COVID symptoms including nausea, abdominal pain, chest tightness, and breathing issues weeks and months later. Those symptoms were likely to be more severe than in people whose initial illness was mild.

‘A huge impact on my life’

Study authors also found that people over 30 were more likely to have severe ongoing symptoms than younger COVID patients. The findings are of particular concern given the prevalence of long COVID, which health experts estimate could affect between 10 and 25 percent of people who test positive for COVID.

“Having been fit and active all my life, after catching COVID-19 during the first wave, my son (then 13) and I didn’t seem to recover. We were left with debilitating fatigue and a confusing mix of strange and life changing symptoms. I was also left with significant neurological symptoms, including speech and language issues, which had a huge impact on my life,” explains long COVID patient Lyn Curtis.

“My other children also experienced significant ongoing symptoms every time we were re-infected, such as changes to periods, fatigue, insomnia, changes in mood, nausea, vomiting, diarrhea, and nose bleeds,” Curtis continues. “The acknowledgement of long COVID and a greater understanding of the associated symptoms is essential both for identifying treatments and the management of existing symptoms. The work into the effects on cognition are especially important to me, as this is the ongoing symptom that impacts the most on my quality of life and ability to work.”

The researchers add long COVID is causing and will continue to cause high rates of workplace absences and disruptions to society. They say it is important not just for sufferers themselves but for society as a whole to understand what causes the condition and how to treat it.

The findings are published in the journal Frontiers in Aging Neuroscience.

COVID-19 and cerebrovascular diseases: a comprehensive overview

Authors: Georgios TsivgoulisLina PalaiodimouRamin Zand

First Published December 8, 2020 Review Article Find in PubMed https://doi.org /10.1177/1756286420978004

Abstract

Neurological manifestations are not uncommon during infection with the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A clear association has been reported between cerebrovascular disease and coronavirus disease 2019 (COVID-19). However, whether this association is causal or incidental is still unknown. In this narrative review, we sought to present the possible pathophysiological mechanisms linking COVID-19 and cerebrovascular disease, describe the stroke syndromes and their prognosis and discuss several clinical, radiological, and laboratory characteristics that may aid in the prompt recognition of cerebrovascular disease during COVID-19. A systematic literature search was conducted, and relevant information was abstracted. Angiotensin-converting enzyme-2 receptor dysregulation, uncontrollable immune reaction and inflammation, coagulopathy, COVID-19-associated cardiac injury with subsequent cardio-embolism, complications due to critical illness and prolonged hospitalization can all contribute as potential etiopathogenic mechanisms leading to diverse cerebrovascular clinical manifestations. Acute ischemic stroke, intracerebral hemorrhage, and cerebral venous sinus thrombosis have been described in case reports and cohorts of COVID-19 patients with a prevalence ranging between 0.5% and 5%. SARS-CoV-2-positive stroke patients have higher mortality rates, worse functional outcomes at discharge and longer duration of hospitalization as compared with SARS-CoV-2-negative stroke patients in different cohort studies. Specific demographic, clinical, laboratory and radiological characteristics may be used as ‘red flags’ to alarm clinicians in recognizing COVID-19-related stroke.

Introduction

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and has been declared as a Public Health Emergency of International Concern by the World Health Organization.1 Since its outbreak in December 2019, SARS-CoV-2 has spread in more than 235 countries, and as of 25 September 2020, it has infected 32,029,704 patients.2 Characteristically, COVID-19 affects the respiratory system, producing symptoms ranging from mild upper-airway manifestations to pneumonia and severe acute respiratory distress syndrome.3 COVID-19 is increasingly recognized as a multi-system disease with apparent involvement of other organ systems and clinical gastrointestinal, cardiological, dermatological and other extrapulmonary manifestations.48

COVID-19 affection of the nervous system has also been reported, including both the central nervous system (CNS) and peripheral nervous system (PNS).9 A growing number of case series and cohort studies have been published identifying neurological symptoms associated with COVID-19. CNS manifestations may include headache, dizziness, seizures, confusion, delirium, and coma. PNS involvement may be presented as hypogeusia, hyposmia, other cranial neuropathies or generalized weakness due to Guillain–Barré and intensive-care-unit-acquired polyneuropathy or myopathy.

In the first case series reporting on the neurological manifestations of COVID-19, cerebrovascular disease was reported with a higher prevalence in COVID-19 patients who were more seriously infected.10 Since then, multiple studies have been published, arguing whether there is a causal or coincidental relationship between COVID-19 and cerebrovascular disease.

In this narrative review, we present the possible pathophysiological mechanisms linking cerebrovascular disease and COVID-19, describe the clinical syndromes and their prognosis and provide a ‘red flag’ system to alert clinicians for prompt recognition of cerebrovascular disease during COVID-19.

Methods

We systematically searched the literature through MEDLINE and EMBASE, using the following keywords and their combination: SARS-CoV-2, COVID-19, cerebrovascular disease, stroke, ischemic stroke, intracranial hemorrhage, subarachnoid hemorrhage, cerebral venous thrombosis, cerebral sinus, and vein thrombosis. We evaluated only peer-reviewed articles that had been published or had been officially accepted for publication. References of retrieved articles were also screened. Case reports, case series, editorials, reviews, case-control, and cohort studies were evaluated, and relevant information was abstracted. The literature search protocol was conducted by three independent authors (GT, LP, and AHK). The last literature search was conducted on 1 September 2020. Characteristic images of cerebrovascular disease manifestations in COVID-19 patients have been provided by co-authors using previously unpublished data from COVID-19 tertiary care referral centers from Europe, North America, and Asia.

Results

Possible pathophysiological mechanisms linking cerebrovascular disease and COVID-19

Several common cerebrovascular risk factors have been associated with severe COVID-19 as well, including cardiovascular disease, diabetes mellitus, hypertension, smoking, advanced age, and previous history of stroke.11 This raises the question whether their relationship is causal or if they just coincide. Several possible pathophysiological mechanisms have been recently described (Figure 1).

Figure 1. Potential pathophysiological mechanisms underlying cerebrovascular involvement in COVID-19.

SARS-CoV-2 binds to angiotensin-converting enzyme 2 (ACE2) receptors, leading to the receptors’ inactivation. ACE2 dysregulation contributes to the post-ischemic inflammation cascade, resulting in decreased perfusion in the ischemic zone and the development of larger infarct volume in the case of ischemic stroke (IS). In addition, ACE2 dysfunction may subsequently cause hypertensive peaks and impairment of cerebrovascular endothelium, contributing to the pathogenesis of intracerebral hemorrhage (ICH). Virus-related cardiac injury, including myocardial ischemia and cardiac arrhythmias such as atrial fibrillation, may cause cardio-embolism and subsequently, IS. COVID-19-related hypercoagulability may determine in situ arterial thrombosis and IS. Another potential IS mechanism related to hypercoagulability is paradoxical emboli of generated venous thrombi through right-to-left shunts. Cerebral venous thrombosis may also be caused by hypercoagulability and in situ thrombosis. Furthermore, COVID-19-related coagulopathy may present as dysfunctional hemostasis and predispose to ICH, especially when therapeutic anticoagulation is administered. Cytokine storm-mediated endotheliitis and vasculitis of the CNS due to SARS-CoV-2 infection causes vessel remodeling, leading to vessel occlusion or injury and IS or ICH, respectively. Finally, a primarily immune-mediated critical illness during COVID-19, hypoxemia, and systemic hypotension may induce hypoxic/ischemic encephalopathy or cerebral microbleeds with or without leukoencephalopathy.

CNS, central nervous system; COVID-19, coronavirus disease 2019; DVT, deep venous thrombosis; PE, pulmonary embolism; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Targeting angiotensin-converting enzyme 2 (ACE2) receptor

SARS-CoV-2 is known to bind to the ACE2 receptor, causing the inactivation of the receptor and leading to dysfunction in blood pressure regulation.12,13 In turn, this could lead to hypertensive peaks that could be particularly important in the pathogenesis of intracerebral hemorrhage (ICH). However, data to date have suggested that patients with ICH and COVID-19 present with lower systolic blood pressure, relative to spontaneous ICH.14 Furthermore, ACE2 dysregulation may also contribute to the post-ischemic inflammation cascade through the accumulation of angiotensin 2, resulting in decreased perfusion in the ischemic zone and the development of larger infarct volumes.15,16 The impaired endothelial function in cerebral arteries caused by ACE2 inactivation has also been implicated in the pathogenesis of cerebrovascular events, including both ischemic and hemorrhagic stroke.17,18

Cardiovascular complications associated with COVID-19

Virus-related cardiac injury in patients with COVID-19 has been associated with ACE2 dysregulation, imbalanced immune response leading to cytokine storm, hypoxia related to respiratory dysfunction and treatment-adverse events.6 Specifically, SARS-CoV-2 infection can be complicated with decompensated heart failure, myocarditis, acute myocardial infarction and cardiac arrhythmias, including incident atrial fibrillation.6,19,20 The aforementioned cardiac manifestations of COVID-19 may lead to subsequent cardio-embolism and cerebral infarction.

Coagulopathy associated with COVID-19

Various reports have been published regarding hypercoagulability associated with severe COVID-19 illness, due to immobilization, dehydration, inflammation, elevated fibrinogen, endothelial cell injury, and platelet activation.2124 The hypercoagulability may further add to the risk of developing cerebral venous thrombosis (CVT) or ischemic stroke (IS).2325 The COVID-19-associated prothrombotic state is accompanied by high D-dimer levels, elevated ferritin, and in some cases, detectable lupus anticoagulant, anticardiolipin immunoglobulin A (IgA), and antiphospholipid IgA and immunoglobulin M (IgM) autoantibodies directed against β2-glycoprotein-1.22,26 On the other hand, it is well established that such autoantibodies appear in many conditions characterized by profound immune activation with no apparent pathogenic role.

Hypercoagulability due to SARS-CoV-2 infection has also been associated with a higher incidence of deep vein thrombosis.27,28 Right-to-left shunt and paradoxical embolism of generated venous thrombi through a patent foramen ovale might act as an etiopathogenic mechanism in younger patients with cryptogenic cerebral ischemia without any vascular risk factors for stroke. In addition to intracardiac shunts, the right-to-left shunt may be associated with intrapulmonary causes, such as pulmonary vascular dilatations or pulmonary arteriovenous malformations. In a recently published study, contrast-enhanced transcranial Doppler was performed in mechanically ventilated patients with COVID-19 pneumonia and detected microbubbles in 83% of the patients.29 The detection of microbubbles was attributable to pulmonary vasodilatations which may also explain the disproportionate hypoxemia seen in COVID-19 patients.29

On the other hand, coagulopathy associated with COVID-19 may present as dysfunctional hemostasis, prolonged prothrombin time and bleeding disorder, especially in severely infected patients. The characteristics of COVID-19 coagulopathy are similar but not perfectly matched to those of disseminated intravascular coagulopathy.30 Characteristically, COVID-19 coagulopathy manifests with significantly elevated D-dimers, but only mild thrombocytopenia and slightly prolonged prothrombin time, and rarely meets the diagnostic criteria of disseminated intravascular coagulopathy according to the International Society on Thrombosis and Haemostasis.31,32 Nevertheless, the disruption of hemostasis in COVID-19 may contribute to an increased risk of secondary intracranial hemorrhage, especially when therapeutic anticoagulation is also administered.

Triggering CNS vasculitis and endotheliitis

SARS-CoV-2 viral-like particles may be detected in brain capillary endothelium, as was recently noted in an autopsy study.33 This finding supports the viral neurotropism and potentially implicates SARS-CoV-2 in a direct effect on cerebral vessels with subsequent endothelium dysfunction and degeneration.34 SARS-CoV-2 has also been implicated in triggering CNS vasculitis, possibly through an inflammatory response mediated by the cytokine storm and specifically interleukin-6.3538 This is not new for viral infections, since other viruses (e.g. varicella zoster, human immunodeficiency virus, hepatitis C virus, hepatitis B, cytomegalovirus, parvovirus b19) may trigger such a response.39 Inflammation of cerebral vasculature may lead to arterial remodeling with either stenosed or dilated, fragile vessels with subsequent ischemic or hemorrhagic stroke, respectively. Reversible cerebral vasoconstriction syndrome and posterior reversible encephalopathy syndrome may mimic primary angiitis of the CNS and have been recently described in COVID-19 patients.4042 IS, ICH, and convexal subarachnoid hemorrhage are common manifestations of reversible cerebral vasoconstriction syndrome.43

Critical illness due to COVID-19

A significant percentage of patients with SARS-CoV-2 infection present with serious manifestations and may need intubation, mechanical ventilation, and prolonged hospitalization in intensive care units, mostly due to pulmonary complications. Hypoxemia and systemic hypotension due to primarily immune-mediated critical illness may further induce hypoxic/ischemic encephalopathy and contributes to IS, mostly in watershed territories or presenting as cortical laminar necrosis.44 Additionally, prolonged hypoxemia and respiratory failure have been associated with cerebral microbleeds and/or leukoencephalopathy.45

Cerebrovascular manifestations associated with COVID-19

Reports of cerebrovascular complications associated with COVID-19 are continuously increasing.

Ischemic stroke

A recently published, prospective, multinational study reported 123 patients who presented with acute IS out of a total of 17,799 SARS-CoV-2-infected patients.46 This result corresponds to a non-weighted risk of 0.7% for IS among patients hospitalized for COVID-19. Other cohort studies reporting IS risk among hospital admissions for COVID-19 also presented similar results (Table 1). These data underscore that COVID-19 may be associated with a small but non-negligible risk for IS. Another important fact is that IS is reported as the initial manifestation and reason for hospitalization in 26% of COVID-19-confirmed patients.47

Table 1. Cohort studies reporting cerebrovascular events in COVID-19 patients during general hospital admission.

Table 1. Cohort studies reporting cerebrovascular events in COVID-19 patients during general hospital admission.View larger version

Regarding the IS subtype according to Trial of ORG 10172 in Acute Stroke Treatment classification, COVID-19 was reported to be associated with a higher incidence of cryptogenic stroke.47,48,56,60 According to a retrospective cohort study performed in a major health system in New York, cryptogenic stroke was twice more prevalent in COVID-19 positive patients compared with both a contemporary control group consisting of COVID-19 negative patients and a historical control group derived from patients treated in the same period in 2019.48 The presented high rates of cryptogenic stroke could further alarm clinicians regarding hypercoagulability state, in situ arterial thrombosis from endothelitis and occult cardioembolism or paradoxical embolism, both of which require deeper investigation and consideration of therapeutic anticoagulation.6163 Other studies, which also included hospitalized COVID-19 patients, reported an incidence of up to 35% for cryptogenic stroke among IS patients.51,52,64 Finally, it should be noted that different case series of stroke complicating COVID-19 patients have reported a decreased prevalence of lacunar infarction (⩽10%) among IS patients.46,48 This observation indicates the potential lack of association between COVID-19 and intrinsic small-vessel disease. However, since lacunar strokes are generally associated with less severe symptoms compared with large-vessel occlusion (LVO) strokes, the patients may have not undergone neuroimaging evaluation with brain MRI and ascertainment of acute cerebral ischemia mechanism leading to the under-representation of lacunar strokes in patient cohorts.

Another important observation regarding IS incidence in patients with COVID-19 is the report of younger patients without known risk factors presenting with stroke due to LVO.65 Also, COVID-19 patients with LVO were younger compared with both contemporary controls of COVID-19-negative patients and historical controls, as investigated in different studies.66,67 In another case series, it was shown that among patients hospitalized for stroke due to LVO, more than half tested positive for SARS-CoV-2.67 Almost a quarter of COVID-19 patients admitted for acute IS is reported to be due to LVO.52,68 An illustrative case of a patient with LVO stroke and concurrent COVID-19 is presented in Figure 2. Multifocal LVOs is another matter of concern in those patients, since multivessel obstruction is presented significantly more frequently in SARS-CoV-2 positive patients.66,68

Figure 2. Imaging evaluation of a patient with acute proximal occlusion of the right middle cerebral artery during hospitalization for COVID-19.

A 65-year-old man with a history of hypertension and diabetes mellitus presented with acute left-sided hemiplegia, dysarthria, neglect, and right-gaze deviation. He had minimal respiratory symptoms. Emergency brain CT and CTA scanning were performed. Acute proximal right middle cerebral artery (MCA) occlusion was demonstrated on CTA (panel A, arrow) with evolving right MCA infarction. Right MCA occlusion was also confirmed on CTA 3D reconstruction (panel B, dotted circle). At that time, D-dimer levels were 2.8 ng/ml (normal values <500 ng/ml). The patient was not eligible for either intravenous thrombolysis due to delayed presentation or mechanical thrombectomy due to an unfavorable perfusion profile. Brain MRI was subsequently performed, showing restricted diffusion in right MCA territory, confirmative of large right MCA infarct (panel C). SARS-CoV-2 infection was confirmed at day 9 of hospitalization. On the 3-month follow up, the patient had modified Rankin Scale score of 4 with persistent severe left hemiparesis.

COVID-19, coronavirus disease 2019; CT, computed tomography; CTA, computed tomography angiography; MRI, magnetic resonance imaging; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

In patients admitted with acute IS during the period of COVID-19 restrictions, significant concern has been raised regarding the delivery of acute reperfusion treatments. This gains even more importance, especially in the aforementioned patients with LVO, who may need mechanical thrombectomy. Initially, patients were thought to be reluctant to seek medical help for stroke symptoms due to their fear of contracting COVID-19, and subsequently presented with substantial delay to the emergency department, outside the time window for available acute reperfusion therapies.69,70 Different cohort studies evaluated the management in the acute phase of stroke patients during COVID-19 restrictions compared with historical controls treated in the same periods before the pandemic. Several of them have underscored the negative effect of lockdown on the management of IS, depicting reductions of stroke admissions, the total number of thrombolysis and/or thrombectomy and significant increases in treatment times.7175

However, the American Heart Association/American Stroke Association Stroke Council Leadership responded promptly with emergency guidance to the need of addressing those issues and securing the delivery of acute stroke treatments.70 A ‘protected code stroke’ was recommended and a focused framework was provided with the aim of COVID-19-specific screening, personal protective equipment, and crisis resource management during acute stroke treatment.76 In addition, the European Society of Neurosonology and Cerebral Hemodynamics issued practice recommendations for neurovascular investigations of acute stroke patients during the COVID-19 pandemic aiming to highlight the utility of ultrasound as a non-invasive, easily repeatable bedside real-time examination of cerebral vessels.77 Finally, the European Stroke Organization and other organizations addressed to the public and underscored that patients with stroke symptoms should seek medical help as soon as possible, despite COVID-19 restrictions.78 Hopefully, such measures will contribute to the stabilization of stroke treatment delivery despite those unprecedented times.

Cerebral hemorrhage

Several case reports and cohort studies have recently been published presenting COVID-19 patients with parenchymal hemorrhage,58,59,7985 subarachnoid hemorrhage,14,58,59,86 and subdural hematoma.59 A retrospective case series of five patients showed that COVID-19 patients with ICH were younger than expected and mostly suffered from lobar ICH.85 One of the patients described in this report had multifocal ICH without any underlying vascular abnormality.85 Similar results were presented in a retrospective cohort study, that showed 0.5% of hospitalized COVID-19 patients to be diagnosed with hemorrhagic stroke, with coagulopathy being the most common etiology.58 A large, deep intracerebral hematoma with an irregular, multi-lobular shape identified in a COVID-19 patient is presented in Figure 3.

Figure 3. Imaging evaluation of a COVID-19 patient with large, multi-lobular intracerebral hemorrhage.

A 55-year-old woman with a history of diabetes mellitus and hypertension was quarantined at home due to SARS-CoV-2 infection with mild respiratory symptoms. At 12 days later, she deteriorated, presenting dyspnea and respiratory failure. She was admitted to the ICU for mechanical ventilation. On day 11 of hospitalization, she became apneic on the ventilator, with fixed, dilated pupils. An emergent brain CT scan was performed showing a large, left-sided intracerebral hemorrhage causing compression of the ipsilateral lateral ventricle and midline shift to the right (panel A). The hematoma had an irregular, multi-lobular shape (panel A and B). No hypertensive spike was confirmed. The patient expired 1 day after neurological worsening.

COVID-19, coronavirus disease 2019; CT, computed tomography; ICU, intensive care unit; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

However, among general hospital admissions of COVID-19 patients, only a minor proportion exhibited ICH (Table 1). Similarly, COVID-19 patients who were hospitalized in neurological wards did not have significantly more cerebral hemorrhagic events compared with non-COVID-19 patients.87 Based on these observations, it remains unknown whether COVID-19 has a causal association with ICH through ACE2 inactivation, endothelial dysfunction/degeneration, coagulopathy or hypocoagulability, or rather, whether secondary effects of COVID-19 such as renal failure/cirrhosis with concomitant therapeutic anticoagulation in a critically ill older population is the culprit. The atypical multifocal nature of many of the reported ICH cases to date would suggest some form of underlying vasculopathy which likely acts synergistically with the aforementioned factors in causing ICH. One pathological report to date has confirmed underlying endothelial reactivity, as well as endothelial and neuropil degeneration in a COVID-19 patient with ICH.34

Cerebral microbleeds have also been demonstrated in critically ill COVID-19 patients and can present with or without leukoencephalopathy.45,88 The atypical location of cerebral microbleeds in the corpus callosum and juxtacortical region may raise the suspicion of SARS-CoV-2 infection in critically ill patients.42 However, a very similar pattern has previously been presented in critically ill non-COVID-19 patients with respiratory failure.89,90 These neuroimaging findings are associated with worse neurological status and longer hospitalization in COVID-19, and likely reflect a more advanced stage of critical illness.45

Cerebral venous thrombosis

Venous thromboembolic events, such as pulmonary embolism and deep venous thrombosis, are detected with high frequency in COVID-19 patients hospitalized in intensive care units, even despite anticoagulation treatment.91 The risk of thrombosis associated with COVID-19 may also be responsible for CVT. Numerous case reports have been published about COVID-19 patients presenting with CVT.9299 In addition, cases with more atypical presentations, such as cortical or deep cerebral venous thrombosis, have been described.100,101 Six patients were diagnosed with CVT among 17,799 hospitalized SARS-CoV-2 patients.46 Headache and impaired consciousness may complicate or even be the presenting symptoms of both COVID-19 and CVT (Figure 4). For that reason, clinicians should be quite vigilant in order to timely diagnose CVT-complicating COVID-19.102 Clinicians should also be able to differentiate COVID-19 patients with primary ICH and hemorrhagic infarction due to CVT, since the latter requires anticoagulant treatment in therapeutic dosage.103 Finally, CVT involving the internal cerebral veins may be challenging to differentiate from acute hemorrhagic necrotizing encephalitis (Weston–Hurst syndrome), which is another COVID-19 CNS complication that may symmetrically affect basal ganglia and thalami with hemorrhagic lesions.104

Figure 4. Imaging evaluation of a COVID-19 patient with cerebral venous thrombosis.

A 59-year-old woman presented with a thunderclap headache followed by a severe progressive headache. Her neurological examination revealed bilateral papilledema. In the brain CT scan, she had an ischemic occipital lesion (panel A). Brain MRV demonstrated lack of flow in the left transverse and sigmoid sinuses, confirming the diagnosis of cerebral venous sinus thrombosis (panel B). In addition, the patient reported she had a fever, cough, and sore throat 10 days before her neurological symptoms. Chest CT showed diffuse ground-glass opacification (panel C). A positive SARS-CoV2 PCR confirmed the diagnosis of COVID-19.

COVID-19, coronavirus disease 2019; CT, computed tomography; MRV, magnetic resonance venography; PCR, polymerase chain reaction; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

Red flags for COVID-19-associated stroke diagnosis

Prompt diagnosis and treatment of acute cerebrovascular disease complicating COVID-19 are essential since co-existing stroke appears to negatively influence the outcome in patients with SARS-CoV-2 infection.50,105 Several clinical and neuroimaging characteristics are present and can raise suspicion in COVID-19 associated stroke diagnosis. The adjunctive use of artificial intelligence may expedite an accurate diagnosis and is already developing in the field of COVID-19-related brain injury.106

Ischemic strokes in COVID-19 patients often present with multi-territorial arterial distribution, embolic pattern, and hemorrhagic transformation.64,107114 A COVID-19 patient diagnosed with multi-territorial arterial infarctions of an embolic pattern in the absence of vascular risk factors is shown in Figure 5. Large cerebral vessel occlusions with significant thrombus burden and a propensity for clot fragmentation is well documented in the literature.46,65,67,68,108,110,115 Characteristically, LVO may affect younger patients without known risk factors for stroke, it is associated with higher in-hospital mortality and may represent the initial manifestation leading to hospitalization during SARS-CoV-2 infection.65,67,115,116 Hypercoagulable state, cardioembolism, or paradoxical embolism due to COVID-19 may be potential reasons for such a stroke presentation. Furthermore, unexpected IS locations, such as in the corpus callosum or a frequent involvement of posterior circulation, have been associated with COVID-19.117119 Increased incidence of hemorrhagic transformation of ischemic infarcts has also been observed in COVID-19 stroke patients.56,111 Whether the dysfunctional hemostasis or the increased use of anticoagulants could be associated with the likelihood of hemorrhagic transformation remains currently unknown. Finally, level of consciousness was recently reported as an important component in a risk stratification score related to severe COVID-19.120 Confusion may not only be an important parameter relating to the severity of a multisystem disease like COVID-19, but in addition, it may underlie the presence of cerebrovascular disease. Unsuccessful recovery after ventilation weaning should alarm clinicians about the possibility of cerebrovascular disease development, and mandates appropriate neuroimaging studies.111

Figure 5. Imaging evaluation of a COVID-19 patient with multi-territorial ischemic infarcts.

An 83-year-old man with a history of hypertension, diabetes, hyperlipidemia, and prior ischemic stroke with no residual symptoms, presented to the emergency department with seizure-like activity and acute respiratory failure, likely due to aspiration. Brain MRI was performed showing restricted diffusion in the territories of both the right middle cerebral artery (MCA; panel A) and the right posterior cerebral artery (PCA; panel B), indicating right MCA and PCA acute ischemic stroke. He was intubated in the emergency department for decreased level of consciousness, hypoxia, and airway protection and initially admitted to the medical ICU. On presentation and the day after presentation, nasopharyngeal swab tests were performed and were both negative for SARS-CoV-2. At 10 days after initial presentation, a third nasopharyngeal swab was performed and found positive, confirming SARS-CoV-2 infection. Despite initial hypoxia and multiorgan failure, the patient improved systematically and was eventually weaned off the ventilator after 1 month of ICU hospitalization. However, the neurological examination did not improve accordingly, and the patient did not fully regain his level of consciousness. For that reason, a brain MRI was repeated and disclosed acute left MCA (panel C and D) and additional right MCA (panel D) territory recurrent ischemic infarcts. The patient was finally discharged to a long-term nursing facility and expired approximately 2.5 months after his initial presentation.

COVID-19, coronavirus disease 2019; ICU, intensive care unit; MRI, magnetic resonance imaging; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

ICH may complicate in COVID-19 patients that are younger than expected for conventional ICH.85 Coagulopathy was reported as the most common etiology of ICH, whereas COVID-19-negative patients most often suffered from hypertension-related ICH.58 Coagulopathy is reflected by the significantly higher levels of D-dimers, which may be considered as a ‘red flag’ for COVID-19-associated ICH.58 Furthermore, although spontaneous ICH most typically presents as deep hemorrhage in patients with negative COVID-19 history, lobar and multi-focal hematomas without underlying macrovascular abnormalities are commonly reported in COVID-19 patients.58,85 Cerebral microbleeds represent a recently described neuroimaging finding in COVID-19 patients, which is associated with critical illness.45,88 In addition, anticoagulation treatment prior to the manifestation of hemorrhagic stroke in COVID-19 patients has been consistently reported in a recent case series from New York City.75

Development of CVT in patients without other risk factors of hypercoagulability should alert clinicians to the possibility of SARS-CoV-2 infection, especially in the presence of excessively high (>2.0 ng/ml) D-dimer levels. Persisting headache and altered consciousness with confusion and/or agitation, coupled with high levels of D-dimers, may be a hint for CVT complication that should be promptly diagnosed and anticoagulated.103

Prognosis of cerebrovascular events associated with COVID-19

Data regarding the long-term prognosis of COVID-19 patients with cerebrovascular events are scarce. Most studies report clinical outcomes at discharge. In a single-center study, it was shown that positive patients had higher National Institutes of Health Stroke Scale score and were less likely to achieve functional independency at discharge.87 Moreover, the mortality rate in COVID-19 stroke patients was higher compared with contemporary SARS-CoV-2-negative stroke patients.87 Similar results are confirmed in other cohort studies investigating cerebrovascular disease in COVID-19 patients compared with COVID-19-negative patients (Table 2). A high mortality rate was also reported in a multinational cohort study, for both ischemic and hemorrhagic COVID-19 stroke patients.56 Additionally, in the same study it was shown that 17.4% of COVID-19 IS patients suffered from a hemorrhagic transformation of the infarction.56

Table 2. Cohort studies reporting clinical outcomes at discharge of COVID-19 patients with cerebrovascular disease compared with COVID-19-negative patients.

Table 2. Cohort studies reporting clinical outcomes at discharge of COVID-19 patients with cerebrovascular disease compared with COVID-19-negative patients.View larger version

Irrespective of SARS-CoV-2 positivity, the COVID-19 pandemic and the subsequently imposed restrictions had a negative influence on mortality and functional outcomes in stroke patients in general. Higher incidence of mortality and worse functional outcomes at discharge were reported in different cohort studies evaluating stroke patients who were admitted during the COVID-19 pandemic, compared with historical controls of the pre-COVID-19 period.60,64,71,72,122125 These alarming results may be attributable to delays in the presentation of stroke patients and subsequent lower rates of recanalization therapies. Furthermore, longer hospitalization stay was needed for the acute management of stroke patients during COVID-19 pandemic.60,64,71,72,122 Severe neurological impairment of the stroke patients, limited medical and laboratory resources, assignment, and transfer of medical personnel from stroke wards to COVID-19 designated wards might explain the need for the extension of hospitalization.

Apart from the observation that SARS-CoV-2 is a risk factor of mortality in stroke patients, it has also been shown that COVID-19 patients who experienced an acute IS during the infection had significantly lower rates of survival compared with those without a stroke.50,105 In addition, a previous history of stroke was an independent risk factor for severe pneumonia leading to critical illness, the need for ventilation, and mortality in COVID-19 patients.126,127

In ICH patients, COVID-19 was negatively associated with prognosis. A higher mortality rate was observed in COVID-19 patients compared to both contemporary and historical negative controls with ICH.58 In another cohort, all COVID-19 patients with hemorrhagic lesions on brain MRI suffered from acute respiratory distress syndrome and were hospitalized in intensive care units.88 A total of 20% patients with hemorrhagic lesions died during hospitalization, compared with 6% of COVID-19 patients with other non-hemorrhagic lesions.88

Impact of COVID-19 pandemic on stroke management

The COVID-19 pandemic and the imposed restrictions severely impacted stroke management.9,69 Since the first weeks of the outbreak, declining numbers of stroke admissions have been reported in the literature.128,129 Patients with transient or minor stroke symptoms are more likely to avoid seeking medical help due to their fear of contracting the virus.130 Moreover, the availability of acute reperfusion therapies, including both intravenous thrombolysis and mechanical thrombectomy has been limited, according to recent reports from North America and Europe.7175 When administered, treatment time metrics are prolonged, negatively impacting the effectiveness of the treatments.131 The performance of the more time-consuming, multiparametric stroke neuroimaging techniques is reported to have also declined, which, in turn, impedes the recognition of eligible patients for acute reperfusion therapies.132

In order to safely implement acute stroke management during the COVID-19-imposed restrictions, a ‘protected code stroke’ has been proposed.76 When a stroke patient presents to the emergency department, the clinicians should specifically ask for signs and symptoms compatible with COVID-19 infection, such as a history of fever, cough, dyspnea, diarrhea, hyposmia, or hypogeusia.133 History taking regarding COVID-19 should not be limited to the stroke patient, but should also include patients’ close contacts. Temperature checks can be used as an initial, feasible, and inexpensive screening tool upon patient presentation. In cases of positive COVID-19 history or confirmed fever, a nasopharyngeal swab should be tested for SARS-CoV-2 and repeated if negative and clinical suspicion is high, according to local COVID-19 protocols. Rapid antigen tests with high sensitivity and specificity can be used while in triage and may be a game changer in this regard. However, in cases of acute stroke, the management should not be delayed, and the patient should be treated as suspected COVID-19 by the minimum number of medical personnel and appropriate infection control measures, in order to minimize exposure.134

When indicated, intravenous thrombolysis with bolus tenecteplase might be considered as an alternative to alteplase bolus, and 1 h infusion to reduce the acute treatment duration and staff exposure.135 In addition, a minimum number of medical personnel, who should be experienced in donning and doffing personal protective equipment with safety, should perform mechanical thrombectomy procedures. If possible, mechanical thrombectomy under conscious sedation should be considered as the first line if the patient is stable.136 If general anesthesia and endotracheal intubation are needed, extreme caution is mandatory, since the latter is an aerosol and airborne-generating procedure. Ideally, intubation, mechanical ventilation, and extubation of the patient should be performed in negative-pressure rooms.136 Finally, acute stroke care should be provided by experienced stroke teams and the involved specialized personnel should not be redeployed to other hospital departments due to COVID-19-specific demands.137

During further hospitalization, transcranial ultrasound may be used to monitor intracranial vessel patency in IS patients who have received recanalization treatments, since it is a feasible, bedside test and can limit patient transportation.77 If further neuroimaging is needed, brain MRI may be performed for the differential diagnosis of COVID-19 patients with neurological manifestations.138 Moreover, regarding secondary prevention treatment in the subacute phase, prophylactic anticoagulant treatment is indicated in COVID-19 stroke patients.139,140 Finally, possible underlying mechanisms and deteriorating factors, such as hypoxia, coagulation disorder, and electrolyte imbalance should be managed, and cardiac function should be supported if needed during stroke hospitalization.

As for the long-term management and functional improvement, the COVID-19 stroke patients will not only require rehabilitation for their stroke, but also for the long-term consequences associated with a severe COVID-19 infection. The multidisciplinary stroke rehabilitation team needs to adapt to cater for both needs. It is of importance to initiate and maintain an inpatient rehabilitation program for the most severely affected patients.141 Tele-rehabilitation through electronic communication technologies may be a viable procedure for milder cases and their caregivers, limiting unnecessary transportation and close contacts, and providing protection against SARS-CoV-2 transmission.142,143 Finally, mental health should also be preserved. Stroke patients may often experience depression and anxiety, while anxiety itself may act as an additional trigger factor for acute stroke.144,145 Psychiatric support becomes even more important during the COVID-19 pandemic, since self-isolation, physical distancing, imposed restrictions and the fear of contracting the virus may pose additional emotional threats.146

Discussion

The causative versus incidental relationship between SARS-CoV-2 and stroke may still be debatable. Our narrative review presents the potential underlying mechanisms of the association between COVID-19 and cerebrovascular disease. ACE2 receptor dysregulation, excessive immune response, coagulation disorders, cardiac complications, and critical illness can lead to the development of different cerebrovascular disease manifestations during SARS-CoV-2 infection. However, stroke incidence in COVID-19 patients appears to be lower (0.5–1.5%) than what was originally reported from the Wuhan outbreak (5–6%).10,4648 This discrepancy might partially be explained by the fact that during the first weeks of the pandemic, prophylactic anticoagulation treatment was not administered as a standard of care in every COVID-19 patient.

Several key aspects should be summarized based on the presented review. Cryptogenic ischemia due to LVO appears to be the most common manifestation of acute cerebral ischemia, while lacunar stroke is rarely reported to complicate SARS-CoV-2 infection. Coagulation disorders, in situ arterial thrombosis, paradoxical embolization through right-to-left shunts, and occult paroxysmal atrial fibrillation should be scrutinized as indicated. Lobar or subcortical ICH that may be temporally associated with the initiation of anticoagulation therapy appears to be the most prevalent hemorrhagic CNS manifestation of COVID-19. CVT may also present as a hemorrhagic transformation of the cerebral venous infarction and should be included in the differential diagnosis of hemorrhagic lesions in COVID-19 patients. Importantly, CVT may seldom (≈0.5%) represent the initial manifestation of COVID-19 in patients with underlying hypercoagulability.

The adverse outcomes of COVID-19 patients with co-existing cerebrovascular disease mandate physician alertness for timely diagnosis and swift delivery of available acute stroke therapies. Several clinical and neuroimaging characteristics may be utilized as red flags to promote diagnosis. Younger age of patients, the absence of known stroke risk factors, difficulties in weaning from mechanical ventilation, high D-dimer levels, spontaneously prolonged international normalized ratio (INR)/partial thromboplastin time (PTT), multi-territorial acute infarctions, unexpected stroke locations (e.g. splenium of the corpus callosum), and LVOs should alarm clinicians about the co-existence of stroke and SARS-CoV-2 infection. Despite COVID-19-imposed restrictions, acute treatment should be offered as indicated through a safe pathway for both the patients and the medical personnel. Tele-neurology and remote imaging access may minimize exposure of vascular neurologists during the management of SARS-CoV-2 infected patients and should be used when feasible.147,148

The limitations of the present review should be acknowledged. First, this is a narrative review of the literature with the aim of discussing the association between COVID-19 and stroke. Cohort studies and case series included in this review are highly heterogenous regarding stroke diagnostic work-up, acute management, study populations, and use of contemporary versus historical controls. Furthermore, we did not review studies that have been submitted but not accepted to international medical journals. Ideally, an up-to-date systematic review and meta-analysis of included cohort studies may be conducted, investigating the incidence, treatment, and outcomes of cerebrovascular disease in COVID-19 patients and providing future directions in stroke diagnosis and management in the COVID-19 era.

Conclusion

Neurological manifestations are not uncommon during SARS-CoV-2 infection. Among those, cerebrovascular disease was initially reported with an alarming incidence of 6% in a small Chinese cohort.10 However, recent studies in the international larger dataset report a smaller but non-negligible risk of stroke (0.5–1.5%) in infected patients.4648 Whether this smaller risk could be attributed to the standard prophylactic anticoagulation given in every hospitalized patient is not known. ACE2 receptor dysregulation, inflammation, coagulopathy, COVID-19-associated cardiac involvement with subsequent cardio-embolism, and critical illness may all act as pathogenetic mechanisms for developing IS, hemorrhagic stroke, and CVT. Longer duration of hospitalization, worse functional outcomes at discharge, and higher in-hospital mortality are reported in COVID-19 associated stroke. For that reason, prompt stroke diagnosis and preservation of high-quality acute stroke treatment should be emphasized. Several clinical characteristics, such as younger age of patients and lack of known stroke risk factors, as well as neuroimaging (multi-territorial cerebral ischemia of embolic pattern due to underlying LVO) and laboratory (excessive elevation of D-dimer levels and spontaneously prolonged INR/PTT at hospital admission) caveats could further assist clinicians in diagnosing cerebrovascular disease in patients with SARS-CoV-2 infection.

Funding
The authors received no financial support for the research, authorship, and/or publication of this article.

Conflict of interest statement
The authors declare that there is no conflict of interest.

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