Abstract

1. Manuscript

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an enveloped, single-stranded positive-sense RNA betacoronavirus, is the causative agent of coronavirus disease 2019 (COVID-19), for which there have been over 50 million confirmed cases and 1.2 millions deaths worldwide as of November 8, 2020 [1,2]. Morbidity and mortality are more common in older individuals and those with comorbidities, including cardiovascular disease, hypertension, obesity, and diabetes, although young people with no comorbidities are also at risk for critical illness [[3], [4], [5]]. While many SARS-CoV-2 infected individuals are asymptomatic or experience predominantly respiratory symptoms, extrapulmonary manifestations, including neurological symptoms and conditions, are increasingly recognized [[6], [7], [8]]. The majority of current studies on neurological manifestations are case reports or retrospective series focused on hospitalized patients through the extraction of medical record data, which have described disorders of consciousness, delirium, and neuromuscular and cerebrovascular complications [[7], [8], [9], [10]]. Smell and taste disturbances in the absence of nasal obstruction are particularly characteristic of COVID-19, leading to speculation regarding the olfactory nerve as a possible route of central nervous system entry [11,12]. Other neurological findings include headache, myalgia, rhabdomyolysis, Guillain-Barre syndrome, encephalopathy, and myelopathy with rare cases of encephalitis based on imaging or cerebrospinal fluid [8,[13], [14], [15], [16], [17], [18]]. SARS-CoV-2 has not been detected in cerebrospinal fluid in the majority of patients tested [8,19], highlighting the need for studies of autopsy brain tissue to understand COVID-19 neuropathogenesis and develop neurocognitive preserving treatment strategies.

Autopsies provide a wealth of information about the decedents, regardless of whether a likely cause of death was identified pre-mortem [20,21]. Due to initial uncertainties regarding the infectious properties of SARS-CoV-2 and limitations in personnel and personal protective equipment availability, autopsies for COVID-19 patients have been limited, although an increasing number of studies are now being published (reviewed in [[22], [23], [24]]). Reports of detailed neuropathological examinations have lagged behind general autopsy series, in part due to the initial focus on lung pathology combined with the longer (2–3 weeks) formalin fixation time preferred by most neuropathologists before cutting brains. Additional factors include the reluctance of some institutions to perform brain removal in COVID-19 cases due to concerns over electric bone saw generated aerosols, which can be effectively contained through the use of vacuum filters or hand saws [25,26]. Included in this review are peer-reviewed studies of autopsy findings published in English between January 1, 2020, and November 5, 2020. Two different databases (PubMed, Google Scholar) were searched for key terms, including COVID-19, nCoV-2019, and SARS-CoV-2, crossed with autopsy, histology, histopathology, neuropathology, and post-mortem. This search was complemented with three review articles [[22], [23], [24]], text word searching and examining references in identified articles. A total of 24 studies were identified that included 149 individuals (range 1–43 subjects per series). Reported gross and microscopic findings and results of SARS-CoV-2 targeted studies are summarized in Table 1 . Representative gross, microscopic, and ultrastructural findings are illustrated in Fig. 1 .

Gross brain autopsy findings were reported individually or in aggregate for 142 subjects. In keeping with the high prevalence of comorbidities in this patient population, evidence of prior brain disease was frequently identified, including neurodegeneration, prior strokes, tumor resection, demyelinating disease, and atherosclerosis. Acute gross abnormalities were much more limited, and a direct causal relationship with SARS-CoV-2 infection was not always straightforward to identify. A total of 92 (65 %) of the gross brain examinations reported either no significant findings or no acute abnormalities. Of the remaining 50 cases, multiple findings were often described in individual brains. Hemorrhage was the most common abnormality reported, ranging from petechial bleedings and punctate subarachnoid hemorrhages (n = 9) [14,[27], [28], [29], [30], [31]], to large cerebral/cerebellar hemorrhages (n = 4) [27,32,33], hemorrhagic conversion of middle cerebral artery stroke (n = 1) [34], and a recently drained subdural hematoma (n = 1) [32]. Large acute and/or subacute infarcts (n = 11) [29,31,33,35] as well as lacunar infarcts/microinfarcts and watershed infarcts (n = 2) [29,30] were identified in several cases. Severe edema resulting in herniation (n = 5) [27,31,33] as well as mild to moderate edema without herniation (n = 34) [14,30,31,35,36] were also present.

Microscopic findings were reported for 146 of the cases in these studies. Similar to the gross examinations, histopathology identified correlates of pre-existing disease, including neurodegeneration, chronic/subacute strokes, hepatic encephalopathy, and arteriolosclerosis. No specific findings were reported for 25 (17 %) of the cases. Mild to moderate acute hypoxic injury was the most common abnormality (n = 58) [14,27,30,31,33,34,[36], [37], [38]], while severe hypoxic-ischemic injury (n = 1) [30] and infarcts/focal ischemic necrosis (n = 22) [14,29,31,32,35] were identified in several cases. Focal microhemorrhage or hemorrhagic suffusion was also frequently reported (n = 23) [14,[28], [29], [30], [31], [32], [33]], although intravascular microthrombi (n = 12) [31,39] or neutrophilic plugs (n = 3) [38] were less common. Mild focal perivascular, parenchymal, and leptomeningeal T-cell predominant lymphocytic infiltrates were identified in a large number of cases without clear evidence of vasculitis or meningoencephalitis (n = 81) [27,[29], [30], [31],[33], [34], [35], [36], [37], [38], [39]]. Moderate to intense microglial activation was noted, particularly in the brainstem (n = 73), although similar results were also reported in COVID-19-negative individuals with systemic inflammatory/septic clinical courses [31,[33], [34], [35], [36],38]. Axonal damage was identified in a few cases (n = 5) [14,27,30]. Acute disseminated encephalomyelitis (ADEM)-like lesions were reported in a single case [14]. The olfactory system was examined to varying degrees, identifying prominent acute and chronic inflammation in the olfactory epithelium (n = 14) [33,38,39], microglial activation (n = 18) [36] and red neurons (n = 1) [33] in the olfactory bulb, and only unremarkable age-related corpora amylacea in olfactory tracts.

Researchers across the globe have employed multiple strategies to directly assess for the presence of SARS-CoV-2 in brain tissue, including immunohistochemistry, in situ hybridization (ISH), targeted quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), and transmission electron microscopy. At this time, immunohistochemistry, using antibodies that recognize the viral nucleocapsid (N) or spike (S) proteins, have been negative in most attempted human cases (n = 58) [30,33,35,37,38], with the exception of a recent case series that reported positive staining in vagus and glossopharyngeal nerves and scattered cells in the medulla in a total of 16 cases [35]; in situ hybridization for viral RNA has been negative (n = 1) [33]. Viral spike protein has been reported to be present in the olfactory epithelium in 5/6 patients; however, brain findings from these cases were not discussed [40]. A number of qRT-PCR assays have been employed targeting the N, S, envelope (E), open reading frame (ORF) 1/a, ORF1ab, or RNA-dependent reverse transcriptase (RdRp) genes, identifying low levels of virus in frozen or formalin-fixed paraffin-embedded brain tissue (34/84; 41 %) [12,[30], [31], [32], [33], [34], [35], [36], [37],41] and olfactory bulb/tract (n = 9/36; 25 %) [31,33,36,37]. Viral subgenomic RNA, a marker of actively replicating virus, was positive in a single case (n = 1/5; 20 %) [34]. Transmission electron microscopy (TEM) without immunolabeling reported virus-like particles in the frontal lobe (n = 1) [12].

While additional COVID-19 autopsy series continue to be published, the overall picture of acute hypoxic-injury, hemorrhage, and mild to moderate non-specific inflammation is unlikely to change significantly. Evidence of direct viral involvement in the brain or olfactory nerve is limited to the detection of low levels of viral RNA and rare viral antigen in cranial nerves and scattered brainstem cells. Diagnosis of coronavirus particles by electron microscopy is challenging due to similar appearing normal cellular structures, which has created significant controversy in the literature [42,43]. Due to the inherent bias of autopsy studies for severe, fatal disease, and additional institutional restrictions for which cases include brain evaluation, the frequency and extent of neuropathological findings are likely to be overestimated relative to the average COVID-19 patient. At the time of this review, pediatric autopsies, including individuals with multisystem inflammatory syndrome in children (MIS-C), remain extremely limited. While the number of pediatric COVID-19 cases accounts for <2 % of all cases [44], data obtained from brain tissue in this age-group can help address the unique pathophysiology of SARS-CoV-2 infection, including age-dependent immune-responses, hypercoagulability, and degree of hypoxic-ischemic injury.

Additional remaining areas of interest include characterizing the effects of remdesivir and other potential antiviral therapeutics, immunomodulatory medications including dexamethasone, anti-IL-6 or other monoclonal antibodies, and anticoagulants on brain tissue. Given that the therapeutic response to COVID-19 vastly differs between institutions, it remains a challenge to understand how therapeutic choices during acute hospitalization are responsible for the variability in observed neurological manifestations and neuropathological findings. Also, while not surprisingly this early in the pandemic, long-term neuropathological sequelae in COVID-19 survivors remain unstudied. There is evidence that neurological symptoms, including fatigue and headaches, linger for weeks to months in a subset of affected patients [45,46] and studies determining mechanisms for persistent neurological symptoms are needed.

There have been several efforts for sharing COVID-19 brain tissue, including the International Society of Neuropathology (ISN) Collaborative Efforts [47] and the COVID-19 Virtual Biobank at the University of Nebraska Medical Center [48]. To address many of the remaining unanswered questions regarding the neuropathological effects of COVID-19, large scale integrated studies from multiple institutions with relevant clinical metadata will be crucial. The ongoing collection of neurological tissue will be critical to inform best practice management guidance and to direct research priorities as it relates to neurological morbidity from COVID-19.

Funding sources

S.S.M. was supported by the National Institute of Mental Health at the National Institutes of Health [grant number K23MH115812], James S. McDonnell Foundation and the Harvard University Eleanor and Miles Shore Fellowship Program. I.H.S. was supported by the National Institute of Neurological Disorders and Stroke at the National Institutes of Health [grant number R21NS119660].

Acknowledgements

References