key: cord-0915900-2rbmjnua
authors: Cosentino, Giuseppe; Todisco, Massimiliano; Hota, Noy; Della Porta, Giovanni; Morbini, Patrizia; Tassorelli, Cristina; Pisani, Antonio
title: Neuropathological findings from COVID‐19 patients with neurological symptoms argue against a direct brain invasion of SARS‐CoV‐2: A critical systematic review
date: 2021-08-17
journal: Eur J Neurol
DOI: 10.1111/ene.15045
sha: 9421bca4c3e47af5bca2997d2aaa1cf3f02a87fc
doc_id: 915900
cord_uid: 2rbmjnua

BACKGROUND AND PURPOSE: Neuropathological studies can elucidate the mechanisms of nervous system damage associated with SARS‐CoV‐2 infection. Despite literature on this topic is rapidly expanding, correlations between neurological symptoms and brain pathology findings in COVID‐19 patients remain largely unknown. METHODS: We performed a systematic literature review on neuropathological studies in COVID‐19, including 438 patients from 45 articles published by April 22, 2021. We retrieved quantitative data regarding demographic, clinical, and neuropathological findings. We carried out a Wilcoxon rank sum test or χ(2) test to compare patients' subgroups based on different clinical and brain pathology features. RESULTS: Neuropathological findings in COVID‐19 patients were microgliosis (52.5%), astrogliosis (45.6%), inflammatory infiltrates (44.0%), hypoxic‐ischemic lesions (40.8%), edema (25.3%), and hemorrhagic lesions (20.5%). SARS‐CoV‐2 RNA and proteins were identified in brain specimens of 41.9% and 28.3% of subjects, respectively. Detailed clinical information was available from 245 patients (55.9%), and among them, 96 subjects (39.2%) had presented with neurological symptoms in association with typical COVID‐19 manifestations. We found that: (i) the detection rate of SARS‐CoV‐2 RNA and proteins in brain specimens did not differ between patients with versus those without neurological symptoms; (ii) brain edema, hypoxic‐ischemic lesions, and inflammatory infiltrates were more frequent in subjects with neurological impairment; (iii) neurological symptoms were more common among older individuals. CONCLUSIONS: Our systematic revision of clinical correlates in COVID‐19 highlights the pathogenic relevance of brain inflammatory reaction and hypoxic‐ischemic damage rather than neuronal viral load. This analysis indicates that a more focused study design is needed, especially in the perspective of potential therapeutic trials.

The coronavirus disease 2019 (COVID- 19) is an ongoing viral pandemic that emerged from Wuhan province in China and quickly spread to the rest of the world [1] . This infection is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an enveloped, positive-sense, single-stranded RNA betacoronavirus responsible for over 194 million confirmed infections and more than 4 million deaths worldwide as of July 27, 2021 (https://covid 19.who.int). In most cases, COVID-19 presents with fever and upper respiratory symptoms, especially dry cough and often shortness of breath. However, 5% of patients may develop a critical illness with severe pneumonia leading to acute respiratory distress syndrome, disseminated coagulopathy, multiple organ dysfunction syndrome, and eventually death [2, 3] . Morbidity and mortality are more common in the elderly and in patients with comorbidities, including cardiovascular disease, arterial hypertension, obesity, and diabetes mellitus [4] . Nevertheless, young people with no comorbidities can also develop a critical illness [5] .

Although involvement of the respiratory system is the most relevant clinical feature in COVID-19 patients, neurological symptoms have been widely reported [6−8] . Neurological manifestations associated with SARS-CoV-2 infection can affect both the central and peripheral nervous system and range widely from mild taste and smell disturbances to more serious conditions, such as acute cerebrovascular disease, disseminated encephalomyelitis, or Guillain-Barré syndrome [6−10] .

The underlying pathogenic mechanisms of neurological involvement are still unclear. Both direct cytopathic effects mediated by SARS-CoV-2 replication and indirect effects due to respiratory failure with brain hypoxia, cytokine reaction, or parainfectious autoimmune response have been proposed [11] . Although direct and indirect mechanisms could coexist in COVID-19 patients with neurological impairment, in most cases the prevailing pathophysiology remains to be addressed.

Only a few authors have identified SARS-CoV-2 in the cerebrospinal fluid of COVID-19 patients [12−14] . However, in most clinical studies reporting neurological symptoms, the virus was neither searched nor detected in the cerebrospinal fluid.

In this critical review, we focused on neuropathological findings and their clinical correlations in COVID- 19 . Although the number of these postmortem studies is not comparable to the number of reports on neurological manifestations, we believe that an in-depth characterization of SARS-CoV-2-related brain pathology, including assessment of viral proteins and RNA in brain specimens, is crucial for understanding the etiology of neurological symptoms in COVID-19 patients. Moreover, incorporation of neuropathological and molecular findings from brain tissue of subjects with COVID-19

and neurological symptoms could also provide valuable clues for the best management practices and for guiding future research.

This systematic review was conducted according to Preferred

Reporting Items for Systematic Reviews and Meta-Analyses guidelines [15] (Figure 1 All abstracts or full articles without electronic abstracts were reviewed independently by two different authors, who both went through each phase of the review independently (screening, eligibility, inclusion, and quality assessment) to identify potentially relevant studies and evaluate their reliability and risk of bias. The Murad tool for noncomparative cohorts and case reports or case series, and the Newcastle-Ottawa Scale for comparative cohorts and cross-sectional studies were used for quality assessment of the included studies [16, 17] . We considered "poor," "moderate," or "good" quality when three or fewer, four, or five of the criteria were fulfilled in the Murad tool, respectively, and three points or fewer, four to five, or six and above in the Newcastle-Ottawa Scale, respectively. Disagreement was resolved by discussion with the other investigator.

The following main features from individual patients were considered when reported: (i) demographic characteristics (i.e., age and gender), (ii) presence or absence of cardiovascular risk factors from medical history (at least one among arterial hypertension, diabetes mellitus, dyslipidemia, coronary artery disease, or previous stroke), ( iii) presence or absence of neurological symptoms concurrent to or following the onset of typical COVID-19 manifestations, (iv) presence or absence of SARS-CoV-2 RNA or proteins in brain specimens, and (v) presence or absence of main alterations at gross and microscopic examination of brain samples (i.e., edema, hypoxic-ischemic lesions, hemorrhagic lesions, microgliosis, astrogliosis, and inflammatory infiltrates). Patients' subgroups featured by different brain pathology findings were compared by age, gender, and presence or absence of cardiovascular risk factors or neurological symptoms.

Comparisons of brain pathology findings based on detection rate of SARS-CoV-2 RNA or proteins in brain specimens were also carried out. Differences between subgroups were analyzed by means of Wilcoxon rank sum test or Pearson χ 2 test, as appropriate. Level of statistical significance was set at a 0.05. Because of the exploratory nature of these analyses, we did not use correction for multiple comparisons to avoid type II error. Statistical analyses were performed using JMP Pro 14.0 (SAS Institute, Cary, NC).

We selected a final number of 45 articles, which provided a cumulative sample of 438 COVID-19 patients with available brain pathology (see Appendix S1 for the list of articles, and Appendix S2 for a summary of the main characteristics of these studies). The quality was rated "good" in 18 articles (40.0%), "moderate" in 20 articles (44.4%), and "poor" in seven articles (15.6%) (see Appendix S2

for details). In one article, the histopathological examination was Table 4 for details). Patients with neurological symptoms were older than those without neurological impairment (median age: 69 vs. 66 years, respectively; p = 0.038), whereas no significant differences were observed in regard to gender (56 males/26 females vs. 106 males/43 females, respectively; p = 0.596), and presence (Y) or absence (N) of cardiovascular risk factors (54 Y/18 N vs. 106 Y/42 N, respectively; p = 0.598).

TA B L E 1 Cumulative brain pathology findings and comparisons based on neurological symptoms in COVID-19 patients (Tables 1 and 5) . Instead, a significant difference emerged when considering age, because patients with SARS-CoV-2 RNA-positive brain specimens were older than subjects with negative brain samples (median age: 71.5 vs. 66 years) ( Identification of hemorrhagic lesions did not differ with regard to the presence of neurological symptoms, age, gender, or history of cardiovascular risk factors (Tables 1 and 5 ).

Evidence of microgliosis or astrogliosis was not related to the occurrence of neurological symptoms, gender, or cardiovascular risk factors (Tables 1 and 5) . Instead, these brain pathology findings were associated with patients' age, because subjects with microgliosis or astrogliosis were older than patients without these neuropathological alterations (median age: 71 vs. 64 years for microgliosis, 70 vs. 63 years for astrogliosis; Table 5 ).

Identification of inflammatory infiltrates was not related to gender or history of cardiovascular risk factors, but was associated with the presence of neurological symptoms and patients' age (Tables 1 and 5 ). Neurological symptoms were more often observed in individuals with inflammatory infiltrates. Moreover, subjects with inflammatory infiltrates were older than those without this neuropathological feature (median age: 71 vs. 64 years).

Comparisons of brain pathology findings based on presence or absence of SARS-CoV-2 RNA or proteins in COVID-19 patients are shown in Tables 2 and 3 , respectively. The only significant association was found between edema and presence of SARS-CoV-2 proteins (Table 3 ). In particular, edema was more common among brain specimens positive for SARS-CoV-2 proteins.

Several studies analyzed samples from different brain areas, whereas in other cases the sampling site was not specified. A quantitative summary of these available neuropathological data is depicted in Tables 6 and 7 . tection in brain specimens [19, 20] . Of relevance, the relatively low identification rates (about 42%) and the low levels of viral RNA cast doubt on the real presence of SARS-CoV-2 in the brain. It has been argued that detection of SARS-CoV-2 RNA in brain samples could derive from hematogenous viral RNA or viral contamination during different stages of the autopsy [20] . This suggestion is further corroborated by the even lower detection rates of SARS-CoV-2 proteins (about 28%) as well as of viral RNA when using in situ hybridization, which is a more reliable detection technique with respect to the most widely used RT-PCR [20] .

On the other hand, edema, hypoxic-ischemic lesions, and inflammatory infiltrates were more frequently observed in brain specimens from COVID-19 patients with neurological impairment as compared to subjects without neurological symptoms. This evidence would support a role of brain inflammatory reaction and hypoxic-ischemic TA B L E 6 Detection rate of SARS-CoV-2 RNA and proteins in COVID-19 patients in different brain areas damage rather than neuronal viral invasion in determining the neurological involvement associated with SARS-CoV-2 infection.

More than 25% of COVID-19 patients have shown varying degrees of brain edema, leading to flattened brain surface, widened gyri, narrowed sulci, and meningeal congestion with diffuse discoloration of the gray-white matter junction [6, 21] . Although the pathogenesis of brain edema remains to be elucidated, the presence of inflammatory cell infiltration surrounding the edematous tissues suggests that edema might result from a host-specific inflammatory response [6, 21] . SARS-CoV-2 can induce an exaggerated immune-mediated response to viral infection capable of damaging damage blood vessel walls and increasing vascular permeability in the brain [22] .

Hypoxic-ischemic lesions were found in brain specimens of about 41% of cases. This finding is not unexpected, considering that COVID-19 patients may develop severe hypoxia due to respiratory failure or as a complication of protracted hypotension during cardiac arrest [23] . Hypoxia per se might cause a hypercoagulable condition leading to microthrombotic brain vessel occlusion and ischemic damage [24] . Direct activation of the coagulation cascade by a cytokine storm and endothelial dysfunction may also contribute to a procoagulant state in COVID-19 [25] . Of interest, the evidence of megakaryocytes in cortical capillaries from COVID-19 patients could play a major role in causing brain ischemic alterations by obstructing microvascular blood flow [26] . It is noteworthy that hypoxic-ischemic lesions were more commonly reported in patients with cardiovascular risk factors. On the one hand, hypoxic ischemic lesions could be at least partly preexisting in some cases. On the other hand, microvascular changes due to cardiovascular risk factors, such as arterial hypertension or diabetes mellitus, could foster the occurrence of hypoxic-ischemic lesions in COVID-19 patients. This latter hypothesis is in keeping with the evidence of an increased risk of vascular events in subjects with COVID-19 [27] , and with findings of worse outcome of COVID-19 when premorbid vascular risk factors and diseases are present [28] .

T-cell lymphocytic infiltrates were found in 44% of the brain samples from COVID-19 patients. Again, both direct viral infection and host-specific inflammatory response could be involved [19, 29, 30] .

The second hypothesis may be corroborated by the finding of no association between detection rate of SARS-CoV-2 RNA or proteins and inflammatory infiltration in our analysis. This suggestion is further supported by the evidence that inflammatory cell infiltration similar to that of COVID-19 can be encountered in patients with sepsis or systemic inflammation [29] .

The cumulative examination of brain specimens revealed hemorrhagic lesions with different extension, from perivascular microhemorrhages to larger intracerebral and subarachnoid hemorrhages, in more than 20% of subjects with COVID-19, in the absence of any association with neurological manifestations. The mechanisms responsible for COVID-19 patients' susceptibility to develop intracranial hemorrhages remain unclear [31] . The tropism of SARS-CoV-2 toward the endothelial cells via their angiotensin-converting enzyme II receptors could play a role [32, 33] , also according to the evidence of viral particles within endothelial cells and accumulation of inflammatory cells leading to the death of endothelial cells [25] . Additional pathogenic mechanisms underlying cerebral hemorrhagic lesions associated with SARS-CoV-2 infection could include disseminated intravascular coagulation and concomitant anticoagulation therapy [34] .

A variable degree of microgliosis and astrogliosis was seen in about half of the cases, often in association with T-cell lymphocytic infiltrates, but without any association with the presence of neurological symptoms. It has been supposed that in COVID-19 patients, microglial activation could be induced by increased levels of systemic cytokines, including interleukin-6 and interferonγ [35, 36] .

Microglial activation would allow the phagocytosis of dying neurons as described in hypoxic and systemic inflammatory conditions [37, 38] . As for microgliosis, activation of astrocytes represents an essential part of the response of the central nervous system to injury, being involved in mechanisms of neural protection and repair after damage of different etiologies [39] . As both microgliosis and astrogliosis occur in a variety of medical conditions, and critical illness can contribute to their induction, the causal connection to SARS-CoV-2 infection remains uncertain [19, 37] . Our findings showed no association between detection rate of SARS-CoV-2 RNA or proteins and microgliosis or astrogliosis, thus supporting the hypothesis that these neuropathological alterations may represent nonspecific responses of the brain. In line with this view, Deigendesch et al. [29] failed to detect differences when comparing the extent of microglial activation between subjects with COVID-19 and patients who died from septic conditions.

Our analysis for different brain areas has highlighted that microgliosis, and to a lesser extent astrogliosis and inflammatory cell infiltration, were more frequent in the brainstem. Whether this is a consequence of an increased vulnerability to inflammatory stress of brainstem structures, or of a greater susceptibility to a direct cytopathic viral damage, remains to be ascertained [19] . Furthermore, we observed a greater prevalence of brain specimens featured by microgliosis, astrogliosis, and inflammatory infiltrates among older patients, whereas no association between these neuropathological findings and gender or cardiovascular risk factors was found. This finding may be not surprising considering that age is a well-recognized negative prognostic factor for morbidity and mortality in COVID-19 [40] . Also, the detection rate of SARS-CoV-2 RNA, but not of SARS-CoV-2 proteins, was higher in the older population, though RNA levels were always low and more likely compatible with contamination from blood viremia [37, 41] . It may be supposed that an increased presence of viral RNA in the blood within brain samples could be detected in older patients, who more frequently present a severe disease and higher viral load [42] . Moreover, age might correlate with the evidence of neurological manifestations, as supported by our finding that patients with COVID-19-related neurological symptoms were older than subjects who did not shown any evidence of neurological impairment.

Thus, age could represent a risk factor not only for COVID-19 severity [40] , but also for the onset of neurological symptoms associated with SARS-CoV-2 infection.

Gender differences in hospitalization and mortality rate of COVID-19 patients have emerged since the beginning of the pandemic, with evidence that SARS-CoV-2 infection causes more severe symptoms and higher mortality among men [43, 44] . Differences in immune phenotypes between men and women are supposed to be involved [45] . An unexpected finding of our analysis was that detection rate of SARS-CoV2 proteins was greater in brain specimens from women. In agreement, Mahallawi et al. [46] have shown that females have a higher SARS-CoV-2 viral load in the blood. Taking into account the observations in animal models that SARS-CoV-2 proteins can cross the blood-brain barrier [47] , it may be speculated that a greater number of viral proteins could cross the blood-brain barrier in women given their higher SARS-CoV-2 viral load.

A strength of this review is that we followed a systematic ap- Unfortunately, these pieces of information were not provided in the selected articles, thus preventing further elaboration on these issues.

Hopefully, the cumulative findings from this review will stimulate the accurate collection of more detailed data on these specific aspects in future studies. Moreover, some of the articles included in this review failed to report patients' neurological symptoms, whereas the retrospective nature of the studies precluded the systematic use of validated and standardized tools to collect reliable clinical data, also including information on neurological manifestations. A separate analysis of neuropathological findings based on type of neurological involvement was hampered by the lack of detailed clinical information in a large number of the selected articles. In addition, almost all neurological symptoms or disorders in patients with available brain pathology refer to central nervous system impairment (Table 4) In conclusion, although prevalence and meaning of neuropathological findings in patients who died from COVID-9 should be interpreted with caution given the abovementioned limitations, the results of this systematic review shed light on the pathogenic underpinning of COVID-19-related neurological complications.

The evidence from brain pathology that edema, hypoxic-ischemic lesions, inflammatory infiltrates, but not viral RNA or proteins, were associated with the presence of neurological symptoms in COVID-19 patients corroborates the hypothesis that neurological impairment is likely due to brain inflammatory and hypoxicischemic damage rather than to the controversial neurotropism 

All authors declare no conflicts of interest regarding the content of this article. 

No ethical approval or informed consent was required given the nature of this study.

All data supporting the findings of this study are available in this article.

https://orcid.org/0000-0002-6527-3558

Cristina Tassorelli https://orcid.org/0000-0003-1513-2113

Antonio Pisani https://orcid.org/0000-0002-8432-594X

A pneumonia outbreak associated with a new coronavirus of probable bat origin

Clinical characteristics of Covid-19 in New York City

Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in Wuhan

Risk factors for Covid-19 severity and fatality: a structured literature review

Baseline characteristics and outcomes of 1591 patients infected With SARS-CoV-2 admitted to ICUs of the Lombardy region

Neurologic features in severe SARS-CoV-2 infection

Neurological and neuropsychiatric complications of COVID-19 in 153 patients: a UK-wide surveillance study

Neuro-COVID frequency and short-term outcome in the Northern Portuguese population

Guillain-Barré syndrome and COVID-19: an observational multicentre study from two Italian hotspot regions

Clinical and electrophysiological outcome measures of patients with post-infectious neurological syndromes related to COVID-19 treated with intensive neurorehabilitation

Does SARS-Cov-2 invade the brain? Translational lessons from animal models

First case of SARS-COV-2 sequencing in cerebrospinal fluid of a patient with suspected demyelinating disease

SARS-CoV-2 detected in cerebrospinal fluid by PCR in a case of COVID-19 encephalitis

A first case of meningitis/encephalitis associated with SARS-Coronavirus-2

Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015 statement

Methodological quality and synthesis of case series and case reports

The Newcastle-Ottawa scale (NOS) for assessing the quality of nonrandomised studies in metaanalyses

Isolated bulbar palsy after SARS-CoV-2 infection

Neuropathology of patients with COVID-19 in Germany: a post-mortem case series

COVID-19 neuropathology at Columbia University Irving Medical Center/New York Presbyterian Hospital. Brain. 2021:awab148. Epub ahead of print. 21. van den Enden AJM, van Gils L, Labout JAM, et al. Fulminant cerebral edema as a lethal manifestation of COVID-19

SARS-CoV-2 and viral sepsis: observations and hypotheses

German recommendations for critically ill patients with COVID-19

The stimulation of thrombosis by hypoxia

Endothelial cell infection and endotheliitis in COVID-19

Assessing brain capillaries in coronavirus disease 2019

Vascular events, vascular disease and vascular risk factors-strongly intertwined with COVID-19

The effect of vascular risk factor burden on the severity of COVID-19 illness, a retrospective cohort study

Correlates of critical illnessrelated encephalopathy predominate postmortem COVID-19 neuropathology

Viral presence and immunopathology in patients with lethal COVID-19: a prospective autopsy cohort study

Intracranial hemorrhage in critically ill patients hospitalized for COVID-19

COVID-19 and the cardiovascular system: implications for risk assessment, diagnosis, and treatment options

COVID-19 and intracerebral haemorrhage: causative or coincidental?

COVID-19-associated coagulopathy and disseminated intravascular coagulation

Detectable serum severe acute respiratory syndrome coronavirus 2 viral load (RNAemia) is closely correlated with drastically elevated interleukin 6 level in critically ill patients with Coronavirus Disease

Longitudinal characteristics of lymphocyte responses and cytokine profiles in the peripheral blood of SARS-CoV-2 infected patients

Neuronophagia and microglial nodules in a SARS-CoV-2 patient with cerebellar hemorrhage

Microglial phagocytosis of live neurons

Astrocyte barriers to neurotoxic inflammation

Prognostic factors for severity and mortality in patients infected with COVID-19: a systematic review

Neuropathological features of Covid-19

SARS-CoV-2 viral load is associated with increased disease severity and mortality

Impact of sex and gender on COVID-19 outcomes in Europe

Sex-specific clinical characteristics and prognosis of coronavirus disease-19 infection in Wuhan, China: a retrospective study of 168 severe patients

Sex differences in immune responses that underlie COVID-19 disease outcomes

Association of viral load in SARS-CoV-2 patients with age and gender

The S1 protein of SARS-CoV-2 crosses the blood-brain barrier in mice