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

Results

Clinical Data

The patients' mean age was 74 years (range 38–97); 27 patients (66%) were male and 24 (83%) were of Hispanic/Latinx ethnicity. The most frequent presenting symptoms included dyspnoea (27/41; 66%), cough (18/41; 44%) and confusion (13/41; 34%) (Supplementary Tables 2 and 3). The average time from symptom onset to hospital presentation was 6 days (range 0–12 days), and the average length of hospitalization was 19 days (range 0–69 days) (Figure 1). Three (7%) patients presented in acute cardiopulmonary distress with rapid progression to death. Dementia or mild cognitive impairment was the most common neurological comorbidity (8/41; 20%), identified by review of electronic medical records. Chest X-rays on admission showed multifocal pneumonia in 33 (81%) of patients. Twenty-four patients (59%) were admitted to the intensive care unit. Hospital-associated complications were common, including deep-vein thrombosis/pulmonary embolism in eight (20%) patients, acute renal failure requiring dialysis in seven (17%) and bacteraemia in 10 (24%). Seven (17%) patients had a neurology consult during their hospital admission, and one was admitted to the stroke service. Eight (20%) patients died less than 24 h after hospital admission, seven (17%) in less than 1 week, 15 (37%) in 1–4 weeks and 11 (27%) over 4 weeks after hospital admission (Figure 1). The average time between death and autopsy was 26 h (range 2–177 h); 31/41 (76%) of the autopsies were performed within 24 h. The majority of patients for whom laboratory testing was performed showed elevated inflammatory markers. In the 30 patients with C-reactive protein measurements; 12 (40%) patients had peak values over 300 mg/l and 18 (60%) had an average of 186.2 (range 12.9–285.2). Peak interleukin (IL)-6 levels were markedly elevated in 26 (96%) of the 27 patients tested (Supplementary Table 4).

Figure 1.

Clinical course of COVID-19 patients by days. Bar graph showing the length of the clinical course of our patients, including symptoms prior to presentation (blue) and days in the hospital before death, either in the intensive care unit (grey) or not in the intensive care unit (orange). Dots represent the last positive qRT-PCR result prior to death. Patients are numbered 1–41 and displayed from shortest to longest clinical course.

Ante-mortem and Post-mortem Neuroimaging

Head CTs were performed on 11 (27%) patients during their hospital stays and two (5%) also underwent a brain MRI (Supplementary Table 2). Parenchymal haemorrhages/haemorrhagic infarcts were identified in three patients and multiple cortical and deep grey nuclei early subacute infarcts in one patient. Five patients had diffuse cerebral oedema and hypoxaemic injury, three of whom exhibited concurrent or prior haemorrhages, and one of these also showed bilateral basal ganglionic petechial haemorrhages. Nine brains (22%) were imaged post-mortem: one brain with multiple cortical haemorrhagic infarcts and a right basal ganglionic infarct, one with mild cortical haemorrhage, one with minimal bilateral basal ganglionic haemorrhages, one with a right occipital parenchymal haemorrhage and two with intraventricular haemorrhages. An example of premortem, post-mortem and corresponding neuropathology of acute haemorrhagic infarcts is shown in Figure 2.

Figure 2.

Acute, focal, haemorrhagic infarcts in COVID-19 patients. Acute, focal, haemorrhagic infarcts of (AD) right inferior frontal and (EH) left lateral parietal lobes. (A and E) Premortem CAT scans; (B and F) post-mortem MRI; (C and D) gross photographs of coronal brain slices through the corresponding infarcts; and (D and H) microscopic images of the corresponding infarcts. The insets in E and F show the parietal infarct in the axial plane, red boxes outline the lesions on the scans and brain slices. (D and H) Areas of fresh haemorrhage (number symbol) and acute necrosis (asterisk) shown with haemoxylin and eosin stain. Scale bar in H = 500 μm for D and H; B, C, F and G = 1 cm.

Neuropathological Findings

Hypoxic/Ischaemic Injury was the Most Common Pathology. All brains contained hypoxic damage, varying from acute to subacute. Acute changes included neuronal shrinkage and eosinophilia with or without neuronal loss, reactive astrocytosis, highlighted with GFAP immunostaining, and subacute hypoxic changes manifested as subacute infarcts with variable macrophage infiltrates, reactive astrocytosis and neovascularization. These findings were widespread in most brains, but in a few patients (9/41; 22%) were more focal, predominantly involving the isocortex, hippocampus, cerebellum and/or brainstem (Table 1).

Vascular Pathology was Also Common. Eighteen (44%) brains contained infarcts, acute, subacute, or chronic in isocortex, striatum, thalamus, hippocampus, corpus callosum and brainstem, with 10 (24%) containing one or multiple small infarcts (Table 1). None of the larger infarcts appeared to represent watershed infarcts. Eight (19%) brains contained haemorrhages involving isocortex, white matter, cerebellum, brainstem and/or the subarachnoid space. These ranged from multifocal, perivenular haemorrhages, to large haemorrhages, the largest in the cerebellum. The majority of haemorrhages appeared to represent ischaemic infarcts that became haemorrhagic, evidenced by small haemorrhages adjacent to infarcted tissue (Figure 2A–H).

The majority of brains (36/41; 88%) showed atherosclerotic changes in the circle of Willis and arteriolosclerosis in smaller intraparenchymal arteries and arterioles. We did not detect vasculitis, defined as fibrinoid necrosis of vessel walls or the destruction of vessels walls with intramural inflammatory cells. Immunostaining for the vascular basement membrane proteins such as Collagen IV and Laminin or the tight junction proteinZO-1 showed intact capillary and venular walls (Supplementary Figure 1A–F). One patient, who had disseminated HSV-1 infection with CNS involvement (see below), showed a clear loss of vascular basement membrane proteins and ZO-1 immunostaining (Supplementary Figure 1G–I) and an increase in VCAM1 (data not shown).

Microglial Activation was Present in the Majority of COVID-19 Brains. We defined microglial activation by enlargement of cell soma and thickening of processes detected by either IBA1 or CD68 immunostaining. Diffuse microglial activation was present in the majority of the brains (34/41; 81%), variably involving many brain areas (Table 1). Microglia also appeared in clusters (microglial nodules) in over half of the brains (26/41; 63%). Small clusters of CD3+/CD8+ T cells were associated with prominent microglial nodules in a few cases (Figure 3C, D and Supplementary Figure 2A, B, E and F). Neurons were present in some of these microglial clusters (Figure 3A, B, E–I and Supplementary Figure 2A, B, E and F), representing neuronophagia. The microglial nodules were most prevalent in the brainstem, where they appeared particularly common in the inferior olivary nucleus and the tegmental nuclei of the medulla and pons, including the locus coeruleus, hypoglossal nucleus, dorsal vagal motor nucleus, solitary nucleus and midline raphe (Figure 3A–G and Supplementary Figure 2A–F). They were also present in the cerebellar deep nuclei (Figure 3H and I) and white matter, although not in the cerebellar cortex, except one patient who had concomitant HSV-1 encephalitis and diffuse microglial activation (Supplementary Figure 2I and J). The microglial nodules were less frequent in the hippocampus (8/41; 20%), where they preferentially localized to the pyramidal cell layer (Figure 3J and K) and in the isocortex (2/41; 5%) and olfactory bulb (2/41; 5%).

Figure 3.

Inflammatory pathology in COVID-19 brains. (A) Section of the hypoglossal nucleus shows several motor neurons and a microglial module (arrow). (B) An adjacent section stained for CD68, showing clustered microglia in the nodule. Inset: Microglia in close apposition to a hypoglossal neuron (CD68). (C) An adjacent section stained for CD3, showing scattered T cells in the tissue and associated with the microglial nodule. (D) An adjacent section stained for CD8 showing that many of the T cells are CD8+. (E) The locus coeruleus contains a microglial nodule with a degenerating neuron in the centre, identified by its residual neuromelanin (arrow). (F and G) Neurons of the dorsal motor nucleus of the vagus surrounded by CD68+ microglia. (H and I) Microglial nodules in the dentate nucleus (arrows in H), neuron in the middle of a nodule (arrow in I), CD68. Scale bar in D = 200 μm for AD; in E = 10 μm; F and G = 50 μm; H = 100 μm; I = 50 μm; J = 1 mm; and K = 250 μm.

Perivascular Lymphocytic Inflammation and Infiltration Into the Brain Parenchyma was Sparse. In 38 (93%) brains, we found scant lymphocytic infiltration, predominantly around blood vessels, with very few CD3+ T lymphocytes penetrating into the brain parenchyma and meninges (Figures 3C, D and 4A and C). Immunostaining for CD20 revealed no B lymphocyte infiltration (data not shown). One brain showed very large perivascular and intraparenchymal T cell and macrophage infiltrates concomitant with an HSV-1 infection (Figure 4E and Supplementary Figure 2I and J), providing a stark contrast to the modest level of lymphocytic infiltration seen in COVID-19 brains. We also observed few lymphocytes in the choroid plexuses from lateral ventricles of 16 brains, except one with HSV-1 encephalitis that contained prominent T cells and activated microglia (Figure 4B, D, F and Supplementary Figs 2K, L and 5D–F). Consistent with sparse immune cell infiltrations in the CNS of COVID-19 cases, we also found that expression of tight junction proteins in the choroid plexus epithelial cells was intact with the exception of the HSV-1 encephalitis case, where tight junction staining was completely lost (Supplementary Figure 3).

Figure 4.

Immunocytochemical staining for CD3 + T cells in COVID-19 brains. (A and C) Sparse perivascular CD3+ T cells in the pons of COVID-19 Patients 27 and 30. (B and D) Sparse CD3+ T cells in the choroid plexus from the lateral ventricle of Patients 27 and 30. (E and F) CD3+ T cell infiltrates around a pontine vessel (E) and in the choroid plexus (F) of Patient 25 with HSV-1 encephalitis; arrows indicate CD3+ T cell infiltrates. All sections are counterstained with haematoxylin. Scale bars = 200 μm.

Many Patients Showed the Pathology of Neurodegenerative Diseases. Given the ages of our patients and the ante-mortem histories of dementia/mild cognitive impairment and Parkinson's disease in some patients (Table 1), it was not unexpected that 19 brains contained neurofibrillary tangles, with and without amyloid plaques (17/41, 41.4%) and Lewy bodies of Parkinson's disease (3/41, 7%). One of these 18 patients had both Alzheimer's and Parkinson's pathology. Of note, only eight had an ante-mortem diagnosis of dementia/mild cognitive impairment and three a history of Parkinson's disease.

Demyelination was not Evident. Because of a report of acute disseminated encephalomyelitis-like pathology in a COVID-19 patient,[24] we examined all brains for demyelination but did not find evidence of it. While we cannot rule out the possibility that some of the areas of brains that were not sampled contained foci of demyelination, we note that brains were extensively sampled in 20–30 regions for histopathology.

Multifocal Necrotizing Leukoencephalopathy in one Patient. We found lesions consistent with multifocal necrotizing leukoencephalopathy in the pons in one patient, showing small foci of necrosis, myelin loss, oedema and axonal swellings (Supplementary Figure 4). This 74-year-old female with hypertension and hypothyroidism was admitted after 1 week of fever, cough and chills in hypoxaemic respiratory failure. She developed acute kidney injury requiring renal replacement therapy, severe hypoxaemia requiring paralysis and pronation and multiple other infections (Escherichia coli urinary tract infection, methicillin-susceptible Staphylococcus aureus pneumonia and recurrent candidiasis in respiratory and urine cultures). The patient's status acutely worsened with septic shock in the context of Pseudomonas aeruginosa ventilator-associated pneumonia and bacteraemia.

Pathology in the Olfactory Bulbs was Mild. Because of widespread speculation that SARS-CoV-2 may enter the brain via the olfactory route,[33] we examined olfactory bulbs in all patients. We found variable, albeit generally sparse numbers of T cells in the parenchyma, mild-to-moderate patchy microglial activation and very few microglial nodules, indicating no major histopathology in this region (data not shown).

One Brain Contained HSV-1 Encephalitis. One of our patients also had a disseminated HSV-1 infection with CNS involvement, diagnosed only post-mortem (Patient 22). This 70-year-old female with hypertension, living independently, developed acute respiratory distress and vasodilatory shock in the context of COVID-19. She developed acute renal failure, Staphylococcus epidermidis bacteraemia, P. aeruginosa ventilator-associated pneumonia and cytomegalovirus viraemia during her prolonged hospitalization. Because of her refractory hypoxaemia after 44 days of hospitalization, she was palliatively extubated and died within minutes. We found disseminated HSV-1 infection with CNS involvement, proven by immunocytochemistry (Supplementary Figure 5C). In contrast to COVID-19 neuropathology, the brain had prominent lymphocytic infiltrates in the parenchyma, meninges, and choroid plexus, vasculitis, severe microglial activation and microglial nodules (Figure 4E, F and Supplementary Figs 1G–I, 2I–J and 5). In addition, there was a reduction or loss of collagen IV and laminin in the basement membrane of blood vessels, loss of ZO-1 expression in endothelial cells in proximity to massive T cell infiltrates (Supplementary Figure 1G–I) and loss of CLDN5 expression in the epithelial barrier of the choroid plexus (Supplementary Figure 3D and D').

Molecular Findings

Quantitative RT-PCR Detected Very low Levels of Viral RNA in Some Brains. To determine whether SARS-CoV-2 was present in the brain, we conducted qRT-PCR for the N gene in four different brain areas in the first 25 patients of this series. We also examined the nasal epithelium for most of these patients as an internal control. Nearly all available nasal epithelium samples (19/21; 91%) were highly positive for SARS-CoV-2 according to the qRT-PCR analysis. The median viral copy/sample for nasal epithelium was 43 840 (Supplementary Table 5). For CNS samples, the proportion of positive samples as well as the number of viral copies was significantly lower (Figure 5 and Supplementary Table 5). In 7/25 (28%) patients, all CNS sites were negative for SARS-CoV-2, whereas 18/25 (72%) patients had at least one very low but positive CNS site and 9/25 (36%) patients had multiple very low but positive CNS sites (Figure 5). The cerebellum was most commonly positive (10/23; 44%), followed by samples from the olfactory bulb (10/25; 40%), the temporal lobe (9/25; 36%) and the medulla (8/24; 33%). We did not find any correlation between detection of viral RNA by qRT-PCR and the histopathological findings discussed above. For example, no microglial nodules were seen in any of the olfactory bulb samples that were analysed by qRT-PCR (0/25), whereas the medulla samples showed prominent microglial activation, with the majority of samples analysed by qRT-PCR containing microglia nodules (14/24), yet there was no significant difference in the viral levels detected in olfactory bulb samples compared to medulla samples (paired sample t-test: P = 0.309). Furthermore, we compared the viral levels in medulla samples with microglial nodules to those without microglial nodules, and again found no significant difference (Mann Whitney U-test: P = 0.710, independent sample t-test: P = 0.770).

Figure 5.

SARS-CoV-2 qRT-PCR results from the nasal epithelia and brains of COVID-19 patients. Heat map of cycle threshold (Ct) values of brain autopsy sample qRT-PCR for detection of SARS-CoV-2. Nasopharyngeal swab at the time of admission and nasal epithelium are included as samples outside of the CNS. Ct values are presented in quintiles based on the distribution in samples tested. 'X' denotes sample not included for that patient.

SARS-CoV-2 mRNA was not Detected in Brain Tissue by RNAscope®. The presence of low levels of viral RNA in at least one brain region in 18/25 (72%) COVID-19 patients raised the question as to whether the virus is present in the brain or associated with the vasculature or blood components, including infiltrating macrophages. We performed RNAscope® on fresh frozen COVID-19 brain sections for 16 cases, including sections from the medulla (n = 16), olfactory bulb (n = 3) and cerebellum (n = 1) (Supplementary Table 6). The brains were selected based on qRT-PCR data to include areas that were either high, low positive, or negative for the viral RNA by RT-PCR. Lung sections from three COVID-19-positive cases in a different series that had high CT values (18–19 for the N gene) according to RT-PCR were selected as positive controls for the RNA scope. In contrast to the RT-PCR data, we could not detect viral RNA on fresh frozen brain sections using either an antisense probe for the S or N region, or a combination of both probes in cases that were either positive or low positive by RT-PCR (Figure 6A, B and D–G). However, we could detect abundant RNA for SARS-CoV-2 S or the N region in the COVID-19 lung sections (Figure 6C, F and I). As a positive control for brain sections, we detected CLDN5 mRNA (an endothelial cell marker) in medullas (Figure 6J). Notably, in one case (Patient 18), we detected viral RNA for the S region in perivascular cells in the adventitia of a large blood vessel outside the medulla, suggesting sporadic infection of blood vessel cells (Figure 6K). Overall, our findings suggest that if SARS-CoV-2 is present in brain tissue, either its levels are very low and below the limits of detection by RNAscope®, or the virus had already been cleared in some brains.

Figure 6.

RNAscope® results from the brains and lungs of COVID-19 patients. RNAscope® with SARS-CoV-2 S region probe on (A) medulla Patient 6, (B) medulla Patient 5, and (C) lung, positive control. SARS-CoV-2 N region probe on (D) medulla Patient 6, (E) medulla Patient 5, and (F) lung, positive control. SARS-CoV-2 N + S region probes on (G) medulla Patient 6, (H) medulla Patient 5, and (I) lung, positive control. (J) CLDN5 probe on medulla Patient 6 showing a positive signal in endothelial cells. (K) SARS-CoV-2 S region probe gives a positive signal in the adventitia of a meningeal vessel outside of medulla Patient 18. All sections were counterstained with haematoxylin. Arrows indicate positive RNAscope® signal. Scale bars = 200 μm.

Immunohistochemistry did not Detect Viral Proteins in COVID-19 Brains. We performed immunocytochemistry for SARS-CoV-2 N protein on sections of olfactory bulb and medulla on all brains. Other sections of brains were stained when appropriate, such as those that showed high numbers of microglial nodules or infarcts. All brain sections showed no staining; however, the nasal epithelium was positive (Supplementary Figure 6). N protein antibody staining of lung tissues of some of our patients also stained positively (data not shown).

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