COVID-19 Neuropathology at Columbia University Irving Medical Center/New York Presbyterian Hospital

Kiran T. Thakur; Emily Happy Miller; Michael D. Glendinning; Osama Al-Dalahmah; Matei A. Banu; Amelia K. Boehme; Alexandra L. Boubour; Samuel S. Bruce; Alexander M. Chong; Jan Claassen; Phyllis L. Faust; Gunnar Hargus; Richard A. Hickman; Sachin Jambawalikar; Alexander G. Khandji; Carla Y. Kim; Robyn S. Klein; Angela Lignelli-Dipple; Chun-Chieh Lin; Yang Liu; Michael L. Miller; Gul Moonis; Anna S. Nordvig; Jonathan B. Overdevest; Morgan L. Prust; Serge Przedborski; William H. Roth; Allison Soung; Kurenai Tanji; Andrew F. Teich; Dritan Agalliu; Anne-Catrin Uhlemann; James E. Goldman; Peter Canoll

Disclosures

Brain. 2021;144(9):2696-2708. 

In This Article

Discussion

Gaps in our understanding of SARS-CoV-2 infection remain. There is a paucity of detailed neuropathological data, critical to understanding the neuro-invasive capacity of SARS-CoV-2 and mechanisms of neurological injury. In this retrospective study of 41 patients, we provide detailed investigations of the clinicopathological and molecular characteristics of COVID-19 in post-mortem brain samples. Strengths of our study include the multi-ethnic group of patients, detailed clinicopathologicalstudies, the wide spectrum of hospital courses from less than 1 day to many weeks and a multi-pronged effort to detect viral RNA and protein in the brains in conjunction with histopathology. Many of the pathological changes can be attributed to the effects of hypoxia, coagulopathy and multi-organ damage in severe infection, accompanied by virus-mediated inflammatory processes such as systemic cytokine release, while other changes reflect the age range and comorbidities of our patients.

RNAscope® and Immunocytochemistry did not Detect Viral RNA and Protein in the Brains

Quantitative RT-PCR analyses of nasal epithelium and brain tissues provide evidence for the presence of viral RNA, albeit at very low levels in the brain. The high positive levels in the nasal epithelium were unexpected in some patients, given that nine patients were sampled, at the time of autopsy, more than 1 month after their initial diagnosis by nasal pharyngeal swabs. While several RT-PCR studies have suggested prolonged shedding in some patients,[34,35] our findings indicate that either individuals who died have prolonged viral or that viral persistence in the nasal epithelium can last even longer than previously anticipated.

We detected low viral loads of SARS-CoV-2 in at least one CNS section from a substantial proportion of patients. While there is some variation among sites, the relatively low levels of viral RNA suggest that there is poor CNS tropism of SARS-CoV-2, compared with SARS or MERS. The low viral load is in concordance with recent studies,[28,33,36] which report low viral loads in some, but not all of the brain sections tested by RT-PCR. Convincing microscopic evidence of virus in the brain is lacking in our study, since RNAscope® and immunohistochemistry failed to identify viral RNA or protein for the N or S regions in the brain or choroid plexus. Quantitative RT-PCR may be more sensitive, since our RNAscope® detected viral RNA in lungs that had Ct values of 18–19 for the N gene (data not shown). However, a possible source of SARS-CoV-2 in brain tissues is haematogenous viral RNA within CNS blood vessels or haemorrhages, as we demonstrated in a recent case report on one of our patients (Patient 10).[37] It is also possible the viral RNA in some leptomeningeal vessels, as seen by RNAscope® (Figure 6K), contributes to the low levels of virus seen by RT-PCR in some tissue samples. Furthermore, viral contamination during the different stages of the autopsy cannot be excluded. However, the nasal epithelium and the CNS sections were obtained during different autopsy procedures, making it unlikely that contamination from the nasal epithelium contributed to positive qRT-PCR findings. Moreover, appropriate negative controls were included in qRT-PCR assays, and repeat qRT-PCR of select sections confirmed their positive and negative status.

We interpret these very low levels of SARS-CoV-2 seen in some samples with caution. While we cannot completely exclude the possibility that viral protein or RNA is present in these brains, our observations indicate that if there, the levels must be extremely low, supporting our conclusion that the neuropathological findings are unlikely to be caused by viral infection of brain tissue. Furthermore, we found no correlations between qRT-PCR results in medullary sections and the presence of microglial activation and microglial nodules, since medullas that tested negative by qRT-PCR still contained activated microglia. Several studies have also failed to detect viral proteins by immunostaining.[28,38,39] Rare positive cells of undefined nature have been reported.[40] Further studies examining CSF or brain from newly infected individuals or the use of double-stranded RNA probes to identify remnants of replicating virus may help determine any neurotropism for SARS-CoV-2.

Microglial Activation is Widespread, but Most Common in the Brainstem

A large majority of brains had prominent microglial activation in multiple areas. As expected, activation often corresponded to areas containing hypoxic damage or infarcts. However, we observed many areas of focal microglial activation characterized by microglial nodules, many of which contained neurons. These were most prevalent in the lower brainstem, although more rarely in other areas. In the medulla, both the inferior olivary nucleus and a number of tegmental nuclei were involved. Microglial nodules are characteristically present in viral and autoimmune encephalitis, in which neurons contain viral antigens or autoimmune antigens,[41] and in animal models of recovery from neurotropic viruses such as Zika virus, neuronophagia is observed in brain regions with demonstrated viral targeting.[42] Despite the presence of microglial nodules and neuronophagia, we were unable to detect viral RNA or protein in these areas. Thus, it seems likely that interactions between microglia and neurons were not a direct response to viral infection of neurons but rather were secondary to hypoxic/ischaemic injury in the setting of a systemic inflammatory process.

All patients suffered some degree of hypoxia, a condition known to produce signals in neurons that attract and activate microglia,[43] leading to phagocytosis. Microglial nodules are not commonly associated with diffuse or focal hypoxic damage, although our findings suggest that hypoxia may contribute to this type of microglial activation, particularly in the setting of a systemic inflammatory process. Furthermore, microglial activation accompanies sepsis.[44] Although a minority of our patients were known to be bacteraemic, others may have met criteria for septic shock and were receiving antibiotics. A careful review of the brains of patients with acute respiratory distress syndrome, prolonged coma, associated bacteraemia and hypoxia in pre-COVID years would be important future efforts.

An immunological cause of activated microglia and neuronophagia is a further consideration, perhaps made worse by hypoxia. The smaller microglial nodules had little or no T cell component, while larger ones included some T cells. Such pathology could represent a spectrum of inflammatory development as postulated in Rasmussen's encephalitis,[45] a model in which microglia first attacked neurons and secondarily attracted T cells, or T cell-mediated microglial activation and phagocytosis, as shown in flavivirus infections.[42] Otherwise, we found little T cell presence in the parenchyma, perivascular areas, meninges or choroid plexus. Notably, with the exception of the one patient who had HSV-1 encephalitis, none of the COVID autopsy brains showed histopathological features of limbic encephalitis. Furthermore, considering the sparse lymphocytic infiltrate, the histopathological findings were not suggestive of autoimmune encephalitis.

Neuronal damage and loss in the lower brainstem could cause a number of clinical features, including altered cardiac and respiratory regulation, cranial nerve motor findings, somnolence, insomnia and other signs and symptoms. It was unlikely that these were discovered in such ill patients, many of whom were heavily sedated on ventilatory support. The reason for a predominant brainstem localization of microglial nodules is not clear but has been described in other reports (Supplementary Table 1). The involvement of multiple brainstem areas and nuclei are characteristic of several infectious and autoimmune disorders.[46]

Infarcts are Common

A number of our patients suffered acute or subacute infarcts, which were likely to have occurred during the course of the disease, and many of these were diagnosed post-mortem. The locations of infarcts did not conform to a stereotypic pattern, since many different areas were involved in different individuals. The small infarcts associated with haemorrhages were most consistent with thrombotic or thromboembolic events and reperfusion. However, we only observed thrombi in a small number of brains (3/41; 7%). Furthermore, we did not find vasculitis, defined either as fibrinoid destruction of vessel walls or inflammatory cells within vessel walls. Indeed, immunostaining for structural components of the vascular basement membrane revealed intact blood vessels.

Many Patients Have Pathological Comorbidities

Given the patients' mean age of 74, we were not surprised to see atherosclerosis and arteriolar sclerosis. These could have limited blood flow and oxygenation diffusely or focally in patients with respiratory or cardiac failure. Several brains contained cerebral amyloid angiopathy, but none was associated with haemorrhage. Some of the older patients had amyloid plaques and tau tangles and three brains contained Lewy bodies. These neurodegenerative pathologies must have preceded the SARS-CoV-2 infection, but whether or not they contributed to the acute and subacute pathologies is not clear.

Tissue Sampling in Space and Time

We submitted 20–30 sections per brain, including many regions. We only sampled the upper cervical spinal cord, so any lower cord pathology remains uncharacterized. Thus, even sampling many areas, we could have failed to identify relevant, local pathologies in unsampled areas.

Our patients suffered a broad time range of illness, from expiration on arrival at the hospital up to several months in the hospital (Figure 1). However, we found similar neuropathology marked by prominent microglial activation with microglial nodules and neuronophagia in patients with both short and lengthy courses. Furthermore, the fact that we did not see detectable levels of virus by RNAscope® or immunohistochemistry in any of these cases, and detected only very low levels of virus by qRT-PCR, argues against the possibility that we have systematically missed some time point or location at which there is abundant virus after which the virus might be cleared from the brain.

Our Findings may Have Implications for COVID-19 Survivors

It is important to consider the potential impact of the neuropathological changes we, and others, have found in autopsies if such changes are present in the brains of patients who survive COVID-19. In light of the brainstem and hippocampal distribution of microglial activation, the latter of which has been linked to virus-induced cognitive deficits,[47] it is notable that some COVID-19 survivors develop neuropsychiatric symptoms, including memory disturbances, somnolence, fatigue and insomnia, and that similar symptoms are reported in both the acute and recovery phases. Critical to future work is understanding the short- and long-term consequences in survivors. This study included only patients who were severely ill and died. These changes may not be seen in patients with mild illness, and understanding the roles of contributing factors such as inflammation, multi-organ damage and hypoxia will require further studies.

Since the initial submission of this study, there have been other papers published describing the neuropathological findings in COVID-19 brain autopsies.[48–50] By and large, the findings of these other recent publications are consistent with our findings, and support the conclusion that the neuropathological findings of COVID-19 are most likely related to the systemic infection and hypoxic/ischaemic CNS damage, rather than direct viral invasion.

Limitations

This study has several limitations. Our patients, a multi-ethnic group, drawn from a single centre in New York City, may not represent the general COVID-19 population. Autopsy studies over-represent severe cases, and our findings may not be generalizable to less severe cases. There are currently limited data on the sensitivity and specificity of SARS-CoV-2 qRT-PCR in various tissues including brain, and positive qRT-PCR in the brain may be due to virions in the blood rather than brain tissue. Additionally, clinical data for this study were obtained through retrospective chart review and thus fully reliant on electronic medical records. Most patients had significant pre-existing comorbidities, which confound our ability to attribute our findings to COVID-19 infection. Future studies with appropriate controls are needed to delineate which neuropathological findings result from COVID-19 infection and treatment and are not caused by other processes.

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