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COVID-19 Neuropathology: evidence for SARS-CoV-2 invasion of Human Brainstem Nuclei

Neurological manifestations are common in COVID-19, the disease caused by SARS-CoV-2. Despite reports of SARS-CoV-2 detection in the brain and cerebrospinal fluid of COVID-19 patients, it’s still unclear whether the virus can infect the central nervous system, and which neuropathological alterations can be ascribed to viral tropism, rather than immune-mediated mechanisms. Here, we assess neuropathological alterations in 24 COVID-19 patients and 18 matched controls who died due to pneumonia / respiratory failure. Aside from a wide spectrum of neuropathological alterations, SARS-CoV-2-immunoreactive neurons were detected in specific brainstem nuclei of 5 COVID-19 subjects. Viral RNA was also detected by real-time RT-PCR. Quantification of reactive microglia revealed an anatomically segregated pattern of inflammation within affected brainstem regions, and was higher when compared to controls. While the results of this study support the neuroinvasive potential of SARS-CoV-2, the role of SARS-CoV-2 neurotropism in COVID-19 and its long-term sequelae require further investigation.


Introduction
Immunofluorescent staining and confocal microscopy. Fluorescent immunohistochemistry 149 was performed manually. Antigen retrieval was performed on de-paraffinized tissue sections 150 using Dako EnVision PTLink station according to manufacturer recommendations. Following RT-PCR analyses. Viral RNA analysis was performed on 20µm thick paraffin-embedded 174 sections collected in sterile 2ml Eppendorf vials; disposable microtome blades and tongs 175 were changed for each section to reduce contamination risk. Real-time RT-PCR analyses 176 were performed to detect SARS-CoV-2 genome sequences. Briefly, total RNA was purified 177 from selected material using a RecoverAll™ Total Nucleic Acid Isolation kit (Thermo Fisher

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The main cause of death in COVID-19 subjects was diffuse alveolar damage. Twenty-four 225 COVID-19 patients were included in our study. In all patients, SARS-CoV-2 RNA was 226 detected by molecular testing in rhino-pharyngeal swabs. Eleven were females, while 13 227 were males. The mean age of the included subjects was 73±13.7 years. Most included 228 subjects were affected by preexisting chronic medical conditions. Eleven patients (7 female,  Table 1.

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The main cause of death of the control cohort was respiratory failure and pneumonia, aside 238 from other relevant comorbidities. Eighteen age-and sex-matched subjects with comparable 239 ante-mortem medical conditions were included as controls. All patients were negative for 240 SARS-CoV-2 infection or died prior to the COVID-19 pandemic in Italy. Eight were female, 241 while 10 were male. The mean age of included controls was 72±12 years. The mean 242 hospitalization time was 20±15.6 days. Thirteen patients died due to pneumonia, while the 243 remaining subjects died due to respiratory insufficiency, multiorgan failure or ischaemic 244 heart disease. One patient died due to septic shock. Five patients had a clinical diagnosis 245 of cognitive decline. The available clinical data for the control group are reported in Table 2.  Table 1). Furthermore, acute ischaemic injuries  Table 1 and Table 2.  Figure 1D. Other organs were often affected, 283 such as the lungs, liver, intestine, and hypopharynx and even the carotid body 10,27-28 , as 284 summarized in Table 1. In 3 out of 9 cases, microthromboses were identified only within the 285 CNS, while in the remaining 6 subjects, pulmonary thromboses were also detected.

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Interestingly, 3 out of 9 subjects with CNS microthrombi were on antithrombotic medication,     However, as for the medulla, no differences were found between IOT and non-IOT patients, 336 as well as for AD versus non-AD patients ( Figure 5A). Differences in overall microgliosis, as 337 well as differences between anatomical compartments, were not significant between 338 COVID-19 subjects and controls ( Figure 5B). Hence, while there appears to be a higher  At the level of the midbrain, COVID-19 subjects presented marked topographical differences 344 between counting fields comprising the substantia nigra (midbrain tegmentum, FOV1-2 and 345 FOV3, Figure 6C) when compared to counting fields of the midbrain tectum and pes (FOV4-346 6), as seen in Figure 6C-D. This anatomically segregated pattern of inflammation targeting 347 mainly the substantia nigra, but also part of the pre-acqueductal tegmentum, indicates an increasing dorsal-to-ventral gradient of microgliosis which affects the gray matter of the 349 midbrain, sparing counting fields falling within the cerebral peduncle (FOV5-6). Similar to 350 other brainstem levels, no statistically significant differences in overall microgliosis were 351 found when comparing IOT and non-IOT subjects, as well as AD and non-AD subjects 352 ( Figure 6A). When compared to controls, COVID-19 subjects presented significantly higher 353 microglial densities when considering both overall microgliosis, as well as microglial 354 densities within anatomical compartments ( Figure 6E), suggesting for a COVID-19-specific 355 microglial response at the level of the midbrain ( Figure 6F-G). 356 We also found a strong correlation between microglial densities across the different levels       between these studies must be taken into consideration. Our approach to microglial quantification was more similar to Schwabenland et al. 18 , as digitally-assisted manual 528 counting of TMEM119+ cells, a homeostatic microglia-specific marker, was performed to 529 estimate microgliosis; however, we also expanded on these findings by estimating  Unlike previous studies in literature, we have included 18 controls who died due to 590 pneumonia, respiratory insufficiency or multiorgan failure, rather than healthy controls.

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Retrospective selection of control subjects, however, could lead to unwanted selection bias.