key: cord-0775457-t46giw1h
authors: Kantonen, Jonas; Mahzabin, Shamita; Mäyränpää, Mikko I.; Tynninen, Olli; Paetau, Anders; Andersson, Noora; Sajantila, Antti; Vapalahti, Olli; Carpén, Olli; Kekäläinen, Eliisa; Kantele, Anu; Myllykangas, Liisa
title: Neuropathologic features of four autopsied COVID‐19 patients
date: 2020-08-06
journal: Brain Pathol
DOI: 10.1111/bpa.12889
sha: ebe120e89b78a9b23686f48294b5b99dcc08cb60
doc_id: 775457
cord_uid: t46giw1h

Published descriptions of the neuropathological features of COVID‐19 patients have been controversial, ranging from only modest or no pathology to severe hypoxic and hemorrhagic phenotypes, thrombotic complications, acute disseminated encephalomyelitis‐like changes, and encephalitis and meningitis. Here we describe the neuropathological findings of four COVID‐19‐positive patients autopsied at the Helsinki University Hospital during the spring of 2020. While three of the patients (age range 63–90) exhibited merely mild to moderate hypoxia‐associated changes, one 38‐year‐old subject with obesity, diabetes (type 2), Parkinson’s disease, and a very severe clinical course was found to have severe ischemic injury, abundant microhemorrhages and enlarged perivascular spaces most pronounced in the white matter and deep gray matter. The pattern of ischemic changes suggested a defect in microcirculation. In addition, a few small perivascular white matter lesions, with macrophages engulfing myelin, were found. No signs of encephalitis or meningitis were detected in any of the patients. When conducting RT‐PCR and immunohistochemical analyses of brain tissue we could not demonstrate in any of the patients marked injury or presence of SARS‐CoV2 in the olfactory epithelium, olfactory bulbs, or brain areas responsible for respiratory control. In conclusion, our small autopsy series demonstrates various hypoxia‐associated neuropathological features in COVID‐19 patients, but no evidence of neurotropism or meningitis/encephalitis.

Many symptoms of COVID-19, such as stroke, anosmia, and dysregulation of breathing are considered to be directly or partly attributable to neuropathological processes (4) . Furthermore, severe radiologically verified manifestations, such as acute hemorrhagic necrotizing encephalitis (AHLE) and acute disseminated encephalomyelitis (ADEM) have been reported (5) . Here we present neuropathological findings of a series of autopsies performed at the Helsinki University Hospital April 14 through May 18, 2020, comprising four patients who had tested positive for SARS-CoV2 with RT-PCR prior to death. A full neuropathological examination was performed on two patients, while two had extended neuropathological sampling of at least nine CNS areas including olfactory bulbs/tracts during autopsy. Olfactory mucosa was sampled from all four and carotid bodies from three patients.

Clinical autopsies were performed at the Department of Pathology, HUS Diagnostic Center according to Finnish legislation, with consent granted by the next of kin. The modified full autopsies were carried out in an autopsy room appropriate for handling infective decedents, with appropriate personal protective equipment. All autopsies included the complete exploration of the visceral cavity and craniotomy. Modifications included additional virology sampling, extended neuropathological sampling (Cases 1 and 2), samples of the olfactory mucosa with the cribriform plate, and sampling of the carotid bodies where possible (Cases 1-3) (Supplement).

Full neuropathological examination of the brain was performed on Cases 3 and 4. Each brain was dissected after 15 days of formaldehyde-fixation, and at least 20 samples from different brain areas

This article is protected by copyright. All rights reserved were collected. Hematoxylin-eosin, Luxol fast blue, and iron (Prussian blue) stainings were performed using standardized protocols at the Department of Pathology, Helsinki University Hospital.

IHC was performed at the Department of Pathology, University of Helsinki and Helsinki University Hospital using standard methods (selected samples; antibody information and details for IHC for SARS-CoV-2/2019 are shown in Supplement).

RT-PCR was conducted at the Department of Virology, University of Helsinki according to standard protocols. RNA extraction from tissues was carried out by the Trizol method. The primers and probes 

The clinical features of the subjects are summarized in Table 1 . Clinically, only one patient showed severe neurological symptoms (Table 1 , Case 3), and his clinical and neuropathological characteristics will be described in detail below. One patient had ageusia documented in his clinical record without further elaboration on olfaction (Table 1 , Case 1), and two were documented to have only minimal respiratory distress and to lack hyperventilation in response to hypoxia (Table 1, Cases 1 and 3). All patients had several previously diagnosed comorbidities of varying severity ( Table 1 ). The information of the blood group was available for three patients, and, interestingly, they were all A RhD+.

Case 3 was a 38-year-old male with obesity (BMI 38), hypertension, and diabetes (type 2) associated with retinopathy and polyneuropathy. Seven months before his death, a clinical diagnosis of

This article is protected by copyright. All rights reserved Parkinson's disease (PD) was made, with an MRI scan negative for vascular degenerative changes at that time. 23 days prior to death, the patient developed fever, sore throat and cough. He had loose stools for a day. The symptoms persisted, and he developed gradually worsening shortness of breath.

His condition deteriorating, he was taken to the hospital on day 9 after onset of symptoms. On admission, the patient was hypoxemic and in severe respiratory distress. After sedation and intubation he was transferred to the ICU, and later tested positive for SARS-CoV2. Intensive care included antibiotics, dialysis and assisted ventilation, but extracorporeal membrane oxygenation (ECMO) was not used. At first, consciousness was regained during sedation breaks, but after seven days at the ICU the patient no longer responded. Repeated CT scans failed to show explanatory findings and a neurologist was called to assess the patient. On day 14 at the ICU (day 23 after symptom-onset), the neurologic prognosis was considered poor due to suspected hypoxic or encephalitic injury and the patient deceased after withdrawal of support. A clinical autopsy with neuropathological examination was conducted.

At the neuropathological examination the brain weighed 1768 g. Mild brain swelling, depigmentation of the substantia nigra and locus coeruleus, discoloration of the watershed areas, and a few lacunae in inferior putamen (~5 mm in size) were noted. In addition, abundant enlarged perivascular spaces and microhemorrhages were found mostly in the cerebral and cerebellar white matter, in the deep gray matter, and the brain stem. These vascular changes were mostly sized < 1mm, and their quantity was disproportionate to the patient's age (Fig. 1A) .

Microscopically, abundant enlarged perivascular spaces with some hemosiderophages were observed, but we also identified a high density of acute microhemorrhages sized < 1 mm, with a distribution similar to that noted macroscopically (see above). These changes included scattered T lymphocytes with very few B lymphocytes, and some lesions also showed abundant granulocytes, and faint positivity in iron staining (Fig. 1B,D,E) . We only detected minor intravascular deposits of fibrinoid material in some cerebral and subarachnoidal vessels (Fig. 1C) . No thrombotic material was found in cerebral microvessels. Severe hypoxic-ischemic injury was seen, with pyknotic and eosinophilic neurons in hypoxia-sensitive areas. Many axonal spheroids with strong APP-immunopositivity were

This article is protected by copyright. All rights reserved detected, most prominent in the midbrain (peduncle), pons (near the surface of the pontine basis), and medulla (in the pyramid). Supratentorially, there were swollen APP-positive axons particularly in the white matter tracts. In the white matter surrounding the vascular lesions the neuropil was edematous; scattered small, congested vessels and perivascular small nodules of swollen axons with APP positivity (Fig 1F-G) , but very few inflammatory cells, were found. While we observed no vascular changes pathognomonic for hypertension or diabetes, some vessels showed subendothelial hemorrhage (Fig. 1B, D) , and only few vessels exhibited mild inflammation of the vascular wall (not shown). In Luxol fast blue staining, a few perivascular white matter lesions connected with Iba1positive microglia/macrophage-type cells and macrophages engulfing myelin were observed (Fig. 1H -I), with no associated APP-immunopositivity. As expected, we found many alpha-synuclein-positive Lewy bodies and neurites in the substantia nigra, consistent with brainstem-predominant Lewy body disease / PD. It should be noted that no signs of meningitis or encephalitis were seen.

General autopsy findings included partially resolving diffuse alveolar damage in the lungs, with extensive microthrombosis. Outside the lung vasculature microthrombosis was limited to cervical and mediastinal small veins, without other signs of vasculopathy. Hypoxic-ischemic injury was seen in multiple organs.

Other patients in our series had varying, mild to moderate, end-stage hypoxic-ischemic injuries in brain sections (Table 1) . None of our cases showed immunohistochemical or RT-PCR-positivity for SARS-CoV2 in neural tissues or elsewhere outside the respiratory tract (Table 1) .

Thus far, published data on the neuropathological changes associated with COVID-19 have been scarce and controversial, and neuropathological descriptions have ranged from only modest or no pathology to severe hemorrhagic and hypoxic phenotypes, thrombotic complications, ADEM-like changes, and encephalitis and meningitis (6, 7, 8, 10 

This article is protected by copyright. All rights reserved interpretation of the findings. However, precise investigation and description of neuropathological COVID-19-associated changes will significantly contribute to understanding of the neuropathologic processes occurring in the disease progression.

Our small autopsy series covered cases at different stages of disease, with varying comorbidities, and of various ages. Most (3/4) of our patients (age range 63-90) exhibited only modest hypoxia-related neuropathological changes, according with some previous reports (7, 8) . However, one under middleaged patient (38 years old), with obesity, diabetes (type 2) and PD showed abundant perivascular hemorrhages and a few foci of white matter lesions reminiscent of one previous case report (6) . In contrast to it, the hemorrhages observed were smaller (mostly < 1 mm in diameter), there were no neocortical infarcts, and the white matter lesions appeared less pronounced. In addition, another recent report described petechial hemorrhages in severe forms of COVID-19 (10).

All of the CNS samples in our study tested by RT-PCR were found negative for SARS-CoV2. We could not demonstrate marked injury nor presence of SARS-CoV2 by IHC in the brain areas responsible for respiratory control or the carotid bodies. In all patients' specimens, the olfactory neuroepithelial cells appeared intact and none co-localized with SARS-CoV2 immunohistochemistry (Supplement). Thus, we could not provide evidence for neurotropism having affected the CNS, or more specifically being associated with anosmia/ageusia or respiratory dysregulation. Furthermore, contrary to a recent report (10) and according with some other studies (7, 8) , we did not detect any signs of meningitis or encephalitis in any brain areas. Viral antigen could only be detected by IHC in the respiratory epithelium of Case 2 with the shortest illness (6 days) in contrast to the other patients with longer illness (23-26 days). The blood group A RhD+ was overrepresented in our case series, reminiscent of a recent study of SARS-CoV2 pneumonia (3).

The ischemic changes seen in Case 3 were severe, and their pattern suggested a defect in microcirculation. Patients with obesity and associated vasculopathies, such as our Case 3, have been found prone to develop severe forms of the COVID-19 disease. In their pathogenesis some reports have ascribed particular importance to endothelial dysfunction / endotheliitis (2, 9) . Whether the

This article is protected by copyright. All rights reserved changes we detected were caused by possible comorbid small vessel disease, hypoxia, or their combination, and how these changes may be linked to a SARS-CoV2 infection, cannot be determined on the basis of these scanty cases.

In conclusion, our small autopsy series demonstrates various hypoxia-associated neuropathological features, but no evidence for COVID-19 infection involving neurotropism or encephalitis.

Comprehensive neuropathological studies among COVID-19 patients are warranted.

The study was funded by the Academy of Finland (294817, 334812, 308913), the Helsinki University Hospital Fund, the Liv och Hälsa Foundation, the Finnish Medical Foundation, and the Juho Vainio Foundation.

The autopsies were conducted according to Finnish legislation at the Department of Pathology, HUS Diagnostic Center, Helsinki. Consent was given by the next-of-kin. The study is part of the Clin

This article is protected by copyright. All rights reserved COVID project approved by the Ethics committee of the Helsinki University Hospital and HUS Research Center. The authors declare no conflict of interest.

The data supporting the findings of this study are available from the corresponding author on reasonable request. 

HTN= Hypertension, DM2= Diabetes Mellitus type 2, SSS= Sick Sinus Syndrome, CAD= Coronary Artery Disease, MI= Myocardial Infarction, PAD= Peripheral Artery Disease, COPD= Chronic Obstructive Pulmonary Disease

Parkinson's Disease, DLB= Dementia with Lewy Bodies, CAA= cerebral amyloid angiopathy, ICU= Intensive Care Unit

RT-PCR= Reverse Transcriptase -Polymerase Chain Reaction, IHC= Immunohistochemistry, N/A= Not available

Obesity and COVID-19: Immune and metabolic derangement as a possible link to adverse clinical outcomes

Association between ABO blood groups and risk of SARS-CoV-2 pneumonia

Neurological manifestations of COVID-19 and other coronavirus infections: A systematic review

Acute Hemorrhagic Necrotizing Encephalopathy: CT and MRI Features

Neuropathology of COVID-19: a spectrum of vascular and acute disseminated encephalomyelitis (ADEM)-like pathology

Postmortem Examination of Patients with COVID-19

Neuropathological Features of Covid-19

This article is protected by copyright. All rights reserved