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


Brain. 2021;144(9):2696-2708. 

In This Article

Materials and Methods

This study was approved by the Columbia University Irving Medical Center (CUIMC) Institutional Review Board and is in line with the Declaration of Helsinki. The requirement for written informed consent for chart review was waived as the study design was deemed to cause no more than minimal risk. The Institutional Review Board approved this study (AAAS9987) on 4 May 2020. Consent for autopsy was obtained from patient surrogates through standardized consenting procedures via telephone, given that no visitors were allowed in hospital during the study time. We included all consecutive autopsies with brain removal starting during the fourth week of March 2020 and ending in the third week of June 2020. All cases met the Centre for Disease Control and Prevention case definition for definitive COVID-19 infection ( Clinical data including demographics, clinical, laboratory, radiographic and treatment data were obtained retrospectively through an electronic medical record review. All laboratory tests, neuroradiology assessments and interventions were performed at the discretion of the treating physicians.

Autopsy Procedures

All autopsies were conducted in a negative pressure room. The brain with the attached uppermost spinal cord was removed after the general autopsy using a bone saw with a vacuum attachment to minimize personnel exposure to bone dust. Tissue samples (up to ~3 cm in the greatest dimension) from the olfactory bulb/gyrus rectus, superior frontal gyrus, mesial temporal lobe with the anterior hippocampus and amygdala, cerebellum and medulla oblongata were placed in clean plastic bags, sealed, marked, frozen on dry ice and stored at −80°C in the Columbia University BioBank laboratory dedicated to storing and processing COVID-19 autopsy tissues. After removing tissue for freezing, each brain was fixed in 10% buffered formalin for 10 days. After brain removal, parallel longitudinal cuts were made in the medial anterior cranial fossa to circumferentially excise the cribriform plate of the ethmoid bones along with underlying olfactory and nasal epithelium. A small piece of nasal epithelial tissue was sampled for qRT-PCR, and the remainder divided for freezing and fixation in 10% formalin.

Tissue Processing

After ~10 days in formalin fixation, brains were externally examined, the cerebral hemispheres were sliced in the coronal plane, the brainstem in the transverse plane and the cerebellum in the sagittal plane. An extensive set of tissue blocks were processed for paraffin embedding in cassettes, which included olfactory bulb/tracts with adjacent gyrus rectus, superior frontal cortex, striatum at the level of the globus pallidus, anterior thalamus, anterior hippocampus, hippocampal formation at the level of the lateral geniculate body, amygdala, striatum at the level of the nucleus accumbens, calcarine cortex, cerebellum with dentate nucleus, corpus callosum (genu), corpus callosum (body with cingulate cortex), corpus callosum (splenium), rostral midbrain, caudal midbrain, rostral pons, caudal pons, four sequential slices of the medulla, cervical spinal cord, pituitary gland, pineal gland, choroid plexus from the lateral ventricle, nasal epithelium and vessels of the circle of Willis. Additional blocks were obtained depending on gross pathology, such as infarcts or haemorrhages.

Tissue Staining

Sections of paraffin blocks were cut at a thickness of 7 μm and stained with haematoxylin and eosin. As routine measures, immunostaining for CD3, CD68 and glial fibrillary acidic protein (GFAP) were performed on sections of olfactory bulb with adjacent gyrus rectus, temporal lobe with hippocampus, pons, medulla and cerebellum. Immunostaining for SARS-CoV-2 nucleocapsid (N) protein (Sino Biological©; Cat #40143-R001) was performed on sections of nasal epithelium, olfactory bulb and medulla. Other sections were stained with these antibodies when appropriate. Immunostaining for the majority of cases was conducted in the Department of Pathology Immunohistochemistry Core Laboratory using routine protocols with the Leica™ Bond autostainer. All antibodies used are available for clinical diagnostics, including herpes simplex virus type 1 (HSV-1, Cell Marque #361 A-ASR).

In a subset of cases, additional immunoperoxidase staining was performed on paraffin sections of the pons and choroid plexus from the lateral ventricle, as described.[30] Sequential sections were immunostained for claudin 5 (CLDN5) (ThermoScientific 1:200), CD3 (Novus Biological, 1:100), CD31 (Dako, 1:50), CD68 (Novus Biologicals, 1:200), IBA1 (Wako, 1:250), collagen IV (Abcam, 1:300), laminin (Sigma; 1:60), vascular cell adhesion molecule-1 (VCAM1) (Abcam; 1:250) and zona occludens-1 (ZO-1) (ThermoScientific 1:200). Images were acquired with a Zeiss Axioimager using a colour camera and 10× or 20× objective.

Tissue Analysis

Each brain was examined by at least two board-certified neuropathologists, and the key pathological findings were reviewed and discussed at a divisional conference (which includes eight board-certified neuropathologists). The findings reflect the consensus of our group.

Detection of SARS-CoV-2 by qRT-PCR in Brain Autopsy Tissues

Quantitative RT-PCR was performed on 125 brain tissue samples from 25 autopsy cases. Samples were taken from different anatomic locations including the nasal epithelium (n = 21), olfactory bulb (n = 25), superior frontal gyrus (n = 7), temporal lobe (n = 25), cerebellum (n = 23) and medulla oblongata (n = 24). The samples were obtained from either fresh or frozen sections and immediately placed in DNA/RNA shield (ZYMO Research; Cat# R1200-25). RNA was extracted using an RNeasy® Mini Kit (Qiagen) and qRT-PCR was performed using TaqMan™ 4× master mix using SARS-CoV-2 primer/probe sets (IDT) against the N region to detect the presence of virus per CDC recommendations [2019 Novel Coronavirus Real-Time Reverse Transcriptase (RT)-PCR Diagnostic Panel]. Each assay included a standard curve to determine the viral load (log10 copies/sample).

Detection of SARS-CoV-2 by RNAscope® in Brain Autopsy Tissues

RNAscope® was performed on 21 fresh frozen brain samples. These were from the medulla (n = 16), four olfactory bulbs and one cerebellum. For positive controls we used three lung specimens from patients that had high SARS-CoV-2 viral loads [cycle threshold (Ct) values from 18 to 19 for the N region]. Five ready-to-use probes were acquired commercially: an antisense probe for the N gene of SARS-CoV2 (#846081; cross-reacts with SARS and MERS), an antisense probe for the spike (S) gene of SARS-CoV-2 (#848561; unique to SARS-CoV2), a negative control antisense probe for the LAT gene of HSV-1 (#315651), a negative control antisense probe for the dapB gene of Bacillus subtilis strain SMY (#310043) and a positive control antisense probe for the human CLDN5 gene (#517141; a vascular marker) [ACD Biotechne (ACD]. Probes were composed of 20–40 ZZ oligo-probes, except the dapB probe, which was composed of 10 ZZ oligo probes; the sequences of probes are proprietary. RNAscope® was performed using a 2.5 HD Red Detection Kit according to the manufacturer's recommendations (ACD). Briefly, fresh frozen sections were fixed in 4% paraformaldehyde for 10–15 min, washed 3× with phosphate-buffered saline (PBS) for 5 min each wash and dehydrated in graded ethanol series (50%, 70% and 100% ethanol) for 5 min. Slides were processed immediately or stored in 100% ethanol at −20°C for up to 1 week. Slides were dried for 5–10 min at room temperature, pretreated with 3% hydrogen peroxide for 10 min and protease IV for 15 min (ACD) and incubated with probe solution for 2 h at 40°C in the HybEZ™ oven (ACD). In situ hybridization (ISH) signal was amplified following the manufacturer's recommendation, except that AMP5 incubation was increased to 45 min. Slides were developed in WarpRed chromogen (Biocare Medical) for 10–15 min at room temperature, counterstained with haematoxylin (Gill II formulation, Ricca Chemical Company) and mounted with Permount™ (Sigma Aldrich). Images were acquired with a Zeiss Axioimager using a colour camera and 20× objective. RNA ISH was also performed in the Department of Pathology clinical laboratory as previously described using two probes to the SARS-CoV-2 RNA encoding the spike protein.[31]

Data Availability

All data are available upon reasonable request.