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.

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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1000 Peer Reviewed Studies Questioning Covid-19 Vaccine Safety

Peer Reviewed Medical Papers Submitted To Various Medical Journals, Evidencing A Multitude Of Adverse Events In Covid-19 Vaccine Recipients.

The list includes studies published as of January 20, 2022 concerning the potential adverse reaction from COVID-19 vaccines, such as myocarditis, thrombosis, thrombocytopenia, vasculitis, cardiac, Bell’s Palsy, immune-mediated disease, and many more.

  1. Myocarditis after mRNA vaccination against SARS-CoV-2, a case series: https://www.sciencedirect.com/science/article/pii/S2666602221000409
  2. Myocarditis after immunization with COVID-19 mRNA vaccines in members of the US military. This article reports that in “23 male patients, including 22 previously healthy military members, myocarditis was identified within 4 days after receipt of the vaccine”: https://jamanetwork.com/journals/jamacardiology/fullarticle/2781601
  3. Association of myocarditis with the BNT162b2 messenger RNA COVID-19 vaccine in a case series of children: https://pubmed.ncbi.nlm.nih.gov/34374740/
  4. Acute symptomatic myocarditis in seven adolescents after Pfizer-BioNTech COVID-19 vaccination: https://pediatrics.aappublications.org/content/early/2021/06/04/peds.2021-052478
  5. Myocarditis and pericarditis after vaccination with COVID-19 mRNA: practical considerations for care providers: https://www.sciencedirect.com/science/article/pii/S0828282X21006243
  6. Myocarditis, pericarditis and cardiomyopathy after COVID-19 vaccination: https://www.sciencedirect.com/science/article/pii/S1443950621011562
  7. Myocarditis with COVID-19 mRNA vaccines: https://www.ahajournals.org/doi/pdf/10.1161/CIRCULATIONAHA.121.056135
  8. Myocarditis and pericarditis after COVID-19 vaccination: https://jamanetwork.com/journals/jama/fullarticle/2782900
  9. Myocarditis temporally associated with COVID-19 vaccination: https://www.ahajournals.org/doi/pdf/10.1161/CIRCULATIONAHA.121.055891.
  10. COVID-19 Vaccination Associated with Myocarditis in Adolescents: https://pediatrics.aappublications.org/content/pediatrics/early/2021/08/12/peds.2021-053427.full.pdf
  11. Acute myocarditis after administration of BNT162b2 vaccine against COVID-19: https://pubmed.ncbi.nlm.nih.gov/33994339/
  12. Temporal association between COVID-19 vaccine Ad26.COV2.S and acute myocarditis: case report and review of the literature: https://www.sciencedirect.com/science/article/pii/S1553838921005789
  13. COVID-19 vaccine-induced myocarditis: a case report with review of the literature: https://www.sciencedirect.com/science/article/pii/S1871402121002253
  14. Potential association between COVID-19 vaccine and myocarditis: clinical and CMR findings: https://www.sciencedirect.com/science/article/pii/S1936878X2100485X
  15. Recurrence of acute myocarditis temporally associated with receipt of coronavirus mRNA disease vaccine 2019 (COVID-19) in a male adolescent: https://www.sciencedirect.com/science/article/pii/S002234762100617X
  16. Fulminant myocarditis and systemic hyper inflammation temporally associated with BNT162b2 COVID-19 mRNA vaccination in two patients: https://www.sciencedirect.com/science/article/pii/S0167527321012286.
  17. Acute myocarditis after administration of BNT162b2 vaccine: https://www.sciencedirect.com/science/article/pii/S2214250921001530
  18. Lymphohistocytic myocarditis after vaccination with COVID-19 Ad26.COV2.S viral vector: https://www.sciencedirect.com/science/article/pii/S2352906721001573
  19. Myocarditis following vaccination with BNT162b2 in a healthy male: https://www.sciencedirect.com/science/article/pii/S0735675721005362
  20. Acute myocarditis after Comirnaty (Pfizer) vaccination in a healthy male with previous SARS-CoV-2 infection: https://www.sciencedirect.com/science/article/pii/S1930043321005549
  21. Acute myocarditis after vaccination with SARS-CoV-2 mRNA-1273 mRNA: https://www.sciencedirect.com/science/article/pii/S2589790X21001931
  22. Acute myocarditis after SARS-CoV-2 vaccination in a 24-year-old man: https://www.sciencedirect.com/science/article/pii/S0870255121003243
  23. A series of patients with myocarditis after vaccination against SARS-CoV-2 with mRNA-1279 and BNT162b2: https://www.sciencedirect.com/science/article/pii/S1936878X21004861
  24. COVID-19 mRNA vaccination and myocarditis: https://pubmed.ncbi.nlm.nih.gov/34268277/
  25. COVID-19 vaccine and myocarditis: https://pubmed.ncbi.nlm.nih.gov/34399967/
  26. Epidemiology and clinical features of myocarditis/pericarditis before the introduction of COVID-19 mRNA vaccine in Korean children: a multicenter study https://search.bvsalud.org/global-literature-on-novel-coronavirus-2019-ncov/resourc e/en/covidwho-1360706.
  27. COVID-19 vaccines and myocarditis: https://pubmed.ncbi.nlm.nih.gov/34246566/
  28. Myocarditis and other cardiovascular complications of COVID-19 mRNA-based COVID-19 vaccines https://www.cureus.com/articles/61030-myocarditis-and-other-cardiovascular-complications-of-the-mrna-based-covid-19-vaccines
  29. Myocarditis and other cardiovascular complications of COVID-19 mRNA-based COVID-19 vaccines https://www.cureus.com/articles/61030-myocarditis-and-other-cardiovascular-complications-of-the-mrna-based-covid-19-vaccines
  30. Myocarditis, pericarditis, and cardiomyopathy after COVID-19 vaccination: https://pubmed.ncbi.nlm.nih.gov/34340927/
  31. Myocarditis with covid-19 mRNA vaccines: https://www.ahajournals.org/doi/10.1161/CIRCULATIONAHA.121.056135
  32. Association of myocarditis with COVID-19 mRNA vaccine in children: https://media.jamanetwork.com/news-item/association-of-myocarditis-with-mrna-co vid-19-vaccine-in-children/
  33. Association of myocarditis with COVID-19 messenger RNA vaccine BNT162b2 in a case series of children: https://jamanetwork.com/journals/jamacardiology/fullarticle/2783052
  34. Myocarditis after immunization with COVID-19 mRNA vaccines in members of the U.S. military: https://jamanetwork.com/journals/jamacardiology/fullarticle/2781601%5C
  35. Myocarditis occurring after immunization with COVID-19 mRNA-based COVID-19 vaccines: https://jamanetwork.com/journals/jamacardiology/fullarticle/2781600
  36. Myocarditis following immunization with Covid-19 mRNA: https://www.nejm.org/doi/full/10.1056/NEJMc2109975
  37. Patients with acute myocarditis after vaccination withCOVID-19 mRNA: https://jamanetwork.com/journals/jamacardiology/fullarticle/2781602
  38. Myocarditis associated with vaccination with COVID-19 mRNA: https://pubs.rsna.org/doi/10.1148/radiol.2021211430
  39. Symptomatic Acute Myocarditis in 7 Adolescents after Pfizer-BioNTech COVID-19 Vaccination: https://pediatrics.aappublications.org/content/148/3/e2021052478
  40. Cardiovascular magnetic resonance imaging findings in young adult patients with acute myocarditis after COVID-19 mRNA vaccination: a case series: https://jcmr-online.biomedcentral.com/articles/10.1186/s12968-021-00795-4
  41. Clinical Guidance for Young People with Myocarditis and Pericarditis after Vaccination with COVID-19 mRNA: https://www.cps.ca/en/documents/position/clinical-guidance-for-youth-with-myocarditis-and-pericarditis
  42. Cardiac imaging of acute myocarditis after vaccination with COVID-19 mRNA: https://pubmed.ncbi.nlm.nih.gov/34402228/
  43. Case report: acute myocarditis after second dose of mRNA-1273 SARS-CoV-2 mRNA vaccine: https://academic.oup.com/ehjcr/article/5/8/ytab319/6339567
  44. Myocarditis / pericarditis associated with COVID-19 vaccine: https://science.gc.ca/eic/site/063.nsf/eng/h_98291.html
  45. The new COVID-19 mRNA vaccine platform and myocarditis: clues to the possible underlying mechanism: https://pubmed.ncbi.nlm.nih.gov/34312010/
  46. Myocarditis associated with COVID-19 vaccination: echocardiographic, cardiac tomography, and magnetic resonance imaging findings: https://www.ahajournals.org/doi/10.1161/CIRCIMAGING.121.013236
  47. In-depth evaluation of a case of presumed myocarditis after the second dose of COVID-19 mRNA vaccine: https://www.ahajournals.org/doi/10.1161/CIRCULATIONAHA.121.056038
  48. Occurrence of acute infarct-like myocarditis after COVID-19 vaccination: just an accidental coincidence or rather a vaccination-associated autoimmune myocarditis?: https://pubmed.ncbi.nlm.nih.gov/34333695/

This list is not meant to be all inclusive of all peer-reviewed potential harms from mRNA vaccines. To access any of the 1,000 Vaccine Harms published in Medical journals Click The Link Below:

https://www.informedchoiceaustralia.com/post/1000-peer-reviewed-studies-questioning-covid-19-vaccine-safety

OR VIEW AND DOWNLOAD FULL LIST PDF

Updated_Peer_Reviewed_medical_papers_submitted_to_various_medical.pdfDownload PDF • 1.01MB

Post-COVID ‘brain fog’ could be result of virus changing patients’ spinal fluid

Authors: by John Andere JANUARY 19, 2022

Cases of “brain fog” among COVID patients are becoming more and more common, even among people recovering from mild infections. Now, new research is finally providing some potential answers to why people have difficulty concentrating, thinking clearly, and completing easy daily tasks after battling COVID. A team from the University of California-San Francisco say brain fog may result from how the virus alters a person’s spinal fluid — just like other diseases which attack the brain.

Their study finds certain patients who develop cognitive symptoms following a mild case of COVID-19 display abnormalities in their cerebrospinal fluid, similar to the kinds which appear in patients with diseases like Alzheimer’s. While this is only a start, study authors are optimistic this work is an important first step toward understanding what exactly SARS-CoV-2 can do to the human brain.

“They manifest as problems remembering recent events, coming up with names or words, staying focused, and issues with holding onto and manipulating information, as well as slowed processing speed,” explains senior study author Joanna Hellmuth, MD, MHS, of the UCSF Memory and Aging Center, in a university release.

Post-COVID brain fog is likely much more common than most people realize. One recently released study focusing on a post-COVID clinic in New York found that a staggering 67 percent of 156 recovered COVID-19 patients experienced some form of brain fog.

Brain fog patients experience more brain inflammation

This latest research featured 32 adults. All participants had recovered from a COVID-19 infection but did not require hospitalization. Twenty-two exhibited genuine cognitive symptoms, while the rest served as a healthy control group.

Among the entire group, 17 (including 13 with brain fog symptoms) agreed to have their cerebrospinal fluid analyzed. Scientists extracted the fluids from the lower back, on average, about 10 months after each patient’s first COVID symptoms.

Those tests showed 10 of the 13 participants with cognitive symptoms had anomalies within their cerebrospinal fluid. Importantly, the other four cerebrospinal fluid samples collected from people without brain fog showed no anomalies whatsoever. Participants experiencing cognitive issues tended to be older, with an average age of 48, while the control group’s average age was younger: 39 years-old.

All of the patients come from the Long-term Impact of Infection with Novel Coronavirus (LIINC) study, which tracks and assesses adults recovering from SARS-CoV-2.

Further analyses performed on the cerebrospinal fluid samples showed higher-than-normal protein levels and the presence of some unexpected antibodies usually found in an activated immune system. Researchers say these observations suggest a high level of inflammation. Some of these antibodies were seen in the blood and cerebrospinal fluid, implying a systemic inflammatory response. Some antibodies, however, were unique to the cerebrospinal fluid, which hints at brain inflammation specifically.

Study authors don’t know the intended target of these antibodies yet, but theorize they may attack the body itself, like an autoimmune disease.

“It’s possible that the immune system, stimulated by the virus, may be functioning in an unintended pathological way,” explains Dr. Hellmuth, who is the principal investigator of the UCSF Coronavirus Neurocognitive Study. “This would be the case even though the individuals did not have the virus in their bodies.”

Pre-existing conditions raise the risk of COVID brain fog

Notably, patients dealing with brain fog symptoms had an average of 2.5 cognitive risk factors, such as diabetes, high blood pressure, or a history of ADHD, in comparison to an average of less than one average risk factor for participants without brain fog symptoms.

These cognitive risk factors are relevant because they potentially raise an individual’s risk of stroke, mild cognitive impairment, vascular dementia, and generally make the mind more susceptible to executive functioning issues. Additional risk factors include drug use, learning disabilities, anxiety, and depression.

Additionally, all participants underwent a series of cognitive tests with a neuropsychologist modeled after the criteria used for HIV-associated neurocognitive disorder (HAND). To the research team’s surprise, 59 percent of patients dealing with brain fog met HAND criteria, while 70 percent of the control subjects did the same.

“Comparing cognitive performance to normative references may not identify true changes, particularly in those with a high pre-COVID baseline, who may have experienced a notable drop but still fall within normal limits,” Dr. Hellmuth concludes. “If people tell us they have new thinking and memory issues, I think we should believe them rather than require that they meet certain severity criteria.”

The study is published in the journal Annals of Clinical and Translational Neurology.

Neurologic Manifestations Associations of COVID-19

High-quality epidemiologic data is still urgently needed to better understand neurologic effects of COVID-19.

Authors: Shraddha Mainali, MD; and Marin Darsie, MD VIEW/PRINT PDF

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection continues to prevail as a deadly pandemic and unparalleled global crisis. More than 74 million people have been infected globally, and over 1.6 million have died as of mid-December 2020. The virus transmits mainly through close contacts and respiratory droplets.1 Although the mean incubation period is 3 to 9 days (range, 0-24 days), transmission may occur prior to symptom onset, and about 18% of cases remain asymptomatic.2 The highest rates of coronavirus disease 2019 (COVID-19) in the US have been reported in adults age 18 to 29 and 50 to 64 years, representing 23.8% and 20.5% of cases, respectively.3 Although adults age 65 and older make up only 14.6% of total cases in the US, they account for the vast majority of deaths (79.9%).3 Similarly, men appear to be more vulnerable to the disease, accounting for 69% of intensive care unit (ICU) admissions and 58% of deaths despite nearly equal disease prevalence between men and women.4 In terms of ethnicity, Black Americans account for 15.6% of COVID-19 infections and 19.7% of related deaths, whereas Hispanic/Latinx Americans account for 26.3% of COVID-19 infections and 15.7% of COVID-19 deaths, despite these groups comprising 13.4% and 16.7% of the US population, respectively.3,5

The most commonly reported symptoms are fever, dry cough, fatigue, dyspnea, and anorexia.2 Numerous studies have also reported a spectrum of neurologic dysfunctions, including mild symptoms (eg, headache, anosmia, and dysgeusia) to severe complications (eg, stroke and encephalitis). Despite the prolific reports of neurologic associations and complications of COVID-19 in the face of a raging pandemic with limited resources, there is a significant lack of control for important confounders including the severity of systemic disease, exacerbation or recrudescence of preexisting neurologic disease, iatrogenic complications, and hospital-acquired conditions. Moreover, given the ubiquity of the virus, it is challenging to parse COVID-19–related complications from coexisting conditions. There is an urgent need for high-quality epidemiologic data reflecting COVID-19 prevalence by age, sex, race, and ethnicity on a local, state, national, and international level.

Neurologic and Neuropsychiatric Manifestations of COVID-19

Prevalence estimates of acute neurologic dysfunctions caused by COVID-19 are widely variable, with reports ranging from 3.5% to 36.4%.6 A recent study from Chicago showed that in those with COVID-19 who develop neurologic complications, 42% had neurologic complaints at disease onset, 63% had them during hospitalization, and 82% experienced them during the course of illness.7 Considering the widespread nature of the pandemic, with millions infected globally, neurologic complications of COVID-19 could lead to a significant increase in morbidity, mortality, and economic burden.

People over age 50 with comorbidities (eg, hypertension, diabetes, and cardiovascular disease) are prone to neurologic complications.2,8 Common nonspecific symptoms include headache, fatigue, malaise, myalgia, nausea, vomiting, confusion, anorexia, and dizziness. COVID-19 is known characteristically to affect taste (dysgeusia) and smell (anosmia) in the absence of coryza with variable prevalence estimates ranging from 5% to 85%.9 Since the first report on hospitalized individuals in Wuhan, China, numerous other reports have indicated a spectrum of mild-to-severe neurologic complications, including cerebrovascular events, seizures, demyelinating disease, and encephalitis.8,10-13 As a result of fragmented data from across the world with diverse neurologic manifestations and multiple potential mechanisms of injury, the classification of neurologic dysfunctions in COVID-19 is complex and varies across the literature. Here we present 2 pragmatic classification approaches based on 1) type and site of neurologic manifestations disease categories.

For More Information: https://practicalneurology.com/articles/2021-jan/neurologic-manifestations-associations-of-covid-19

Long COVID or Post-acute Sequelae of COVID-19 (PASC): An Overview of Biological Factors That May Contribute to Persistent Symptoms

Authors: Amy D. Proal1 and Michael B. VanElzakker1,2*

The novel virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic of coronavirus disease 2019 (COVID-19). Across the globe, a subset of patients who sustain an acute SARS-CoV-2 infection are developing a wide range of persistent symptoms that do not resolve over the course of many months. These patients are being given the diagnosis Long COVID or Post-acute sequelae of COVID-19 (PASC). It is likely that individual patients with a PASC diagnosis have different underlying biological factors driving their symptoms, none of which are mutually exclusive. This paper details mechanisms by which RNA viruses beyond just SARS-CoV-2 have be connected to long-term health consequences. It also reviews literature on acute COVID-19 and other virus-initiated chronic syndromes such as post-Ebola syndrome or myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) to discuss different scenarios for PASC symptom development. Potential contributors to PASC symptoms include consequences from acute SARS-CoV-2 injury to one or multiple organs, persistent reservoirs of SARS-CoV-2 in certain tissues, re-activation of neurotrophic pathogens such as herpesviruses under conditions of COVID-19 immune dysregulation, SARS-CoV-2 interactions with host microbiome/virome communities, clotting/coagulation issues, dysfunctional brainstem/vagus nerve signaling, ongoing activity of primed immune cells, and autoimmunity due to molecular mimicry between pathogen and host proteins. The individualized nature of PASC symptoms suggests that different therapeutic approaches may be required to best manage care for specific patients with the diagnosis.

Introduction

The novel virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in a global pandemic of coronavirus disease 2019 (COVID-19) (Hiscott et al., 2020). Classic cases of acute COVID-19 are characterized by respiratory symptoms, fever, and gastrointestinal problems (Larsen et al., 2020). However, patients can present with a wide range of other symptoms, including neurological issues suggesting central nervous system (CNS) involvement (Harapan and Yoo, 2021). Acute COVID-19 cases range in length and severity. Many patients are asymptomatic, while others require hospitalization and ventilation (Cunningham et al., 2021). Overall, an average case of COVID-19 lasts between 1 and 4 weeks. However, across the globe, a subset of patients who sustain an acute SARS CoV-2 infection are developing a wide range of persistent symptoms that do not resolve over the course of many months (Carfì et al., 2020Davis et al., 2020Huang C. et al., 2021) (Figure 1). One study of COVID-19 patients who were followed for up to 9 months after illness found that approximately 30% reported persistent symptoms (Logue et al., 2021). These patients are being given the diagnosis Long COVID, post-acute COVID-19 syndrome (PACS), or post-acute sequelae of COVID-19.

For More Information: https://www.frontiersin.org/articles/10.3389/fmicb.2021.698169/full

Potential mechanisms of cerebrovascular diseases in COVID-19 patients

Authors: Manxue Lou 1Dezhi Yuan 2 3Shengtao Liao 4Linyan Tong 1Jinfang Li 5Affiliations expand

Abstract

Since the outbreak of coronavirus disease 2019 (COVID-19) in 2019, it is gaining worldwide attention at the moment. Apart from respiratory manifestations, neurological dysfunction in COVID-19 patients, especially the occurrence of cerebrovascular diseases (CVD), has been intensively investigated. In this review, the effects of COVID-19 infection on CVD were summarized as follows: (I) angiotensin-converting enzyme 2 (ACE2) may be involved in the attack on vascular endothelial cells by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), leading to endothelial damage and increased subintimal inflammation, which are followed by hemorrhage or thrombosis; (II) SARS-CoV-2 could alter the expression/activity of ACE2, consequently resulting in the disruption of renin-angiotensin system which is associated with the occurrence and progression of atherosclerosis; (III) upregulation of neutrophil extracellular traps has been detected in COVID-19 patients, which is closely associated with immunothrombosis; (IV) the inflammatory cascade induced by SARS-CoV-2 often leads to hypercoagulability and promotes the formation and progress of atherosclerosis; (V) antiphospholipid antibodies are also detected in plasma of some severe cases, which aggravate the thrombosis through the formation of immune complexes; (VI) hyperglycemia in COVID-19 patients may trigger CVD by increasing oxidative stress and blood viscosity; (VII) the COVID-19 outbreak is a global emergency and causes psychological stress, which could be a potential risk factor of CVD as coagulation, and fibrinolysis may be affected. In this review, we aimed to further our understanding of CVD-associated COVID-19 infection, which could improve the therapeutic outcomes of patients. Personalized treatments should be offered to COVID-19 patients at greater risk for stroke in future clinical practice.

For More Information: https://pubmed.ncbi.nlm.nih.gov/33534131/

The OC43 human coronavirus envelope protein is critical for infectious virus production and propagation in neuronal cells and is a determinant of neurovirulence and CNS pathology

Authors:Jenny K.Stodola1GuillaumeDubois1AlainLe CoupanecMarcDesforgesPierre J.Talbot

Highlights

Coronavirus structural envelope (E) protein specific motifs involved in protein-protein interaction or in homo-oligomeric ion channel formation are needed for optimal production of recombinant infectious virus.•

Fully functional E protein of HCoV-OC43 is crucial for viral propagation in the CNS and neurovirulence.•

Fully functional E protein of HCoV-OC43 is crucial for efficient viral propagation in the central nervous system and thereby for neurovirulence.

Abstract

The OC43 strain of human coronavirus (HCoV-OC43) is an ubiquitous respiratory tract pathogen possessing neurotropic capacities. Coronavirus structural envelope (E) protein possesses specific motifs involved in protein-protein interaction or in homo-oligomeric ion channel formation, which are known to play various roles including in virion morphology/assembly and in cell response to infection and/or virulence. Making use of recombinant viruses either devoid of the E protein or harboring mutations either in putative transmembrane domain or PDZ-binding motif, we demonstrated that a fully functional HCoV-OC43 E protein is first needed for optimal production of recombinant infectious viruses. Furthermore, HCoV-OC43 infection of human epithelial and neuronal cell lines, of mixed murine primary cultures from the central nervous system and of mouse central nervous system showed that the E protein is critical for efficient and optimal virus replication and propagation, and thereby for neurovirulence.

For More Information: https://www.sciencedirect.com/science/article/pii/S0042682217304361

Long-Haul COVID

 Authors: Avindra Nath

Modern medicine has faced its biggest challenge from the smallest of organisms. It is becoming increasingly apparent that many patients who recovered from the acute phase of the SARS-CoV-2 infection have persistent symptoms. This includes clouding of mentation, sleep disturbances, exercise intolerance and autonomic symptoms (table 1). Some also complain of persistent low grade fever and lymphadenopathy. Although there are no peer reviewed papers at the moment on these patients, many news articles have been written about this phenomenon1,,4 and there are Facebook groups with several thousand patients describing these symptoms. They call the illness, “Long-Haul COVID” or “Long-tail COVID.” Many of these patients are health care workers who had massive exposure to the virus early in the pandemic and describe having symptoms for “100+ days.”5

Most of these patients were in excellent health prior to getting infected with SARS-CoV-2. They all had a myriad of symptoms during the acute phase. However as the fever and respiratory symptoms improved, they are left with persistent systemic symptoms some of which are gradually improving but not all are following that course. Still others feel they had nearly recovered from the acute illness and then a few days later, developed a plethora of symptoms that are now persisting. Some describe a cyclical nature to their symptoms where they improve and then worsen every few days. While some were admitted to the hospital due to pulmonary symptoms, the majority were isolated at home. Access to testing and medical care has been limited and most appointments with physicians are being done via telemedicine which has its limitations. Some patients have had extensive testing by internists, infectious disease specialists, cardiologists and pulmonary medicine experts but nothing has been found to explain the symptoms.5 These patients, some of whom are physicians themselves are concerned that they could be stigmatized as being “functional.” Many of these symptoms overlap with those of patients with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS).6,7 However one needs to be careful not jump to the conclusion that they have ME/CFS unless other possible causes of their symptoms have been investigated (table 2).

Table 2

The cause of ME/CFS remains unknown despite decades of research on the syndrome. Many patients with ME/CFS similarly report a viral infection as a trigger but since they come to our attention often years after symptom onset, it is impossible to know what may have triggered the symptoms.8 Long-Haul COVID thus represents an excellent opportunity to study the pathophysiology of ME/CFS and in doing so may have broader implications.

For More Information: https://n.neurology.org/content/95/13/559