key: cord-0955907-wwml8b3g authors: Kirschenbaum, Daniel; Imbach, Lukas L.; Rushing, Elisabeth J.; Frauenknecht, Katrin B. M.; Gascho, Dominic; Ineichen, Benjamin V.; Keller, Emanuela; Kohler, Sibylle; Lichtblau, Mona; Reimann, Regina R.; Schreib, Katharina; Ulrich, Silvia; Steiger, Peter; Aguzzi, Adriano; Frontzek, Karl title: Intracerebral endotheliitis and microbleeds are neuropathological features of COVID‐19 date: 2020-12-14 journal: Neuropathol Appl Neurobiol DOI: 10.1111/nan.12677 sha: ba1da991dd350f89b61117d9d41b8a58884562c0 doc_id: 955907 cord_uid: wwml8b3g Coronavirus disease 19 (COVID‐19) is a rapidly evolving pandemic caused by the coronavirus Sars‐CoV‐2. Clinically manifest central nervous system symptoms have been described in COVID‐19 patients and could be the consequence of commonly associated vascular pathology, but the detailed neuropathological sequelae remain largely unknown. A total of six cases, all positive for Sars‐CoV‐2, showed evidence of cerebral petechial hemorrhages and microthrombi at autopsy. Two out of six patients showed an elevated risk for disseminated intravascular coagulopathy according to current criteria and were excluded from further analysis. In the remaining four patients, the hemorrhages were most prominent at the grey and white matter junction of the neocortex, but were also found in the brainstem, deep grey matter structures and cerebellum. Two patients showed vascular intramural inflammatory infiltrates, consistent with Sars‐CoV‐2‐associated endotheliitis, which was associated by elevated levels of the Sars‐CoV‐2 receptor ACE2 in the brain vasculature. Distribution and morphology of patchy brain microbleeds was clearly distinct from hypertension‐related hemorrhage, critical illness‐associated microbleeds and cerebral amyloid angiopathy, which was ruled out by immunohistochemistry. Cerebral microhemorrhages in COVID‐19 patients could be a consequence of Sars‐ CoV‐2‐induced endotheliitis and more general vasculopathic changes and may correlate with an increased risk of vascular encephalopathy. This patient harboured fresh juxtacortical haemorrhages and multiple subacute haemorrhages in the corpus callosum. Sars-CoV-infected patients. 8 Similarly, it is a recurrent feature in the lungs and other peripheral organs of Sars-CoV-2-infected patients 9 but has not yet been reported in the central nervous system. We speculated that cerebrovascular pathology in COVID-19 patients could be a direct consequence of hitherto unidentified cerebral endotheliitis caused by Sars-CoV-2. We retrospectively analysed all brain autopsies from Sars-CoV-2-infected patients referred to our department, for the presence of vasculopathic changes and cerebral haemorrhage (Table 1) . We excluded two patients with overt disseminated intravascular coagulopathy 10 (DIC, one patient was described previously 11 ). Accordingly, we present a detailed neuropathological work-up of four Sars-CoV- Detailed clinical information is provided in Table 1 and Data S1. Four patients (three males, one female, age range 70-81 years) presented with progressive respiratory symptoms, three were diagnosed with bilateral COVID-19 pneumonia during hospitalisation. Patient 2 also presented with bilateral pneumonia, consistent with COVID-19, and the lung specimen taken at autopsy tested positive for Sars-CoV-2, although two previous nasopharyngeal swabs were negative. None of the patients showed laboratory signs of overt DIC (Table 1) . 10 Ante-mortem cranial tomography studies performed in patient 1 were unremarkable. All patients died 5-15 days after admission. Endotheliitis in the lungs of patient 1 9 and inflammatory olfactory neuropathy in patients 1 and 3 have been described earlier. 12 Gross examination of the brains was unremarkable except in two cases (patients 1 and 4), which revealed diffuse petechial haemorrhage, most prominent at the grey-white matter junction of the neocortex ( Figure 1A , Table 1 ). Corresponding post-mortem magnetic resonance imaging (MRI) of one brain showed multiple cerebral petechial haemorrhages on susceptibility weighted imaging (SWI) (patient 1, Figure 1B ). On histology, the vast majority of the haemorrhages were fresh ( Figure 1C ), without evidence of haemoglobin breakdown products on Prussian blue stains. Juxtacortical microbleeds were observed in all patients, most conspicuous in the frontal lobe. Additionally, petechial haemorrhages were observed in the thalamus, mesencephalon and pons (Table 1 ). In one case, multiple intraparenchymal subacute haemorrhages were found in the corpus callosum (patient 4, Figure 1D ). Fresh haemorrhages were both perivascular, as well as intraparenchymal without relation to vasculature. In all cases, there was evidence of diffuse intravascular thrombosis. In small veins of the basal ganglia of two patients (1 and 3), intra-endothelial lymphocytic and monocytic inflammation with occasional apoptotic figures were observed, consistent with the previously reported "endotheliitis" in Sars-CoV-2-infected patients 9 (patients 1 and 3, Figure 1E ). Immunohistochemistry for ACE2 consistently revealed detectable expression in all but one patient diagnosed with COVID-19 ( Figure S1 ). Additional staining of three pre-pandemic autopsy controls revealed very faint or absent ACE2 expression ( Figure S1 and Data S1). To test whether the younger age of the controls confounded ACE2 expression, we compared ACE2 transcripts from basal ganglia from the Genotype-Tissue Expression project (GTEx, total of n = 205 patients). We did not see increased gene expression during ageing (Data S1). Additional lymphocytic "cuffing" was observable in two cases (patients 1 and 3). Congo red stains and immunohistochemistry for beta-amyloid were negative in all cases. has not yet been reported in the brain. 6, 7 We report here for the first time the presence of intracerebral endotheliitis in two patients diagnosed with Sars-CoV-2 infection. The observed endotheliitis could be an autoimmune, late-onset phenomenon or a direct effect of endothelial infection as angiotensinconverting enzyme 2 (ACE2), the Sars-CoV-2 receptor, is expressed in the brain vasculature ( Figure S1 and Ref. 13 ). Here, we found higher ACE2 expression in the brain vasculature of patients with endotheliitis than in COVID-19 patients without endotheliitis or than in control patients ( Figure S1 ). Although control patients were younger on average, comparison of publicly available gene expression data did not show increased ACE2 expression in the basal ganglia during ageing (Data S1). The small number of patients analysed, however, precludes a causal inference. Recently, the concept of critical illness-associated microbleeds (CRAM) was introduced. 14 The topology of the microbleeds described in this condition is somewhat similar, but not identical, to the patients in our cohort as well as in hypoxaemic patients after high-altitude exposure. 14 All patients suffered from arterial hypertension and hypertensive microangiopathy, however, hypertensive microbleeds favour the deep grey matter and the infratentorial region. 15 Elevated risk for thrombosis and pulmonary embolism is well-documented in COVID-19 patients, and all the patients received prophylactic anticoagulants and/or antiplatelet therapy, which may have predisposed them to haemorrhagic events. Hydroxychloroquine, which was administered to three patients, has also been reported to cause bleeding. 16 On autopsy, no haemorrhage was seen at predilection sites such as the gastrointestinal tract, thus making solely drug-induced haemorrhage unlikely. 16 In both patients suffering from prolonged coma and negative wake-up attempts, intraparenchymal haemorrhages had already been observed grossly suggesting a positive correlation between the severity of the vasculopathy and acute sleep-wake dysregulation. Accordingly, the distribution of microbleeds showed a neuroanatomical preference for central modulators of wakefulness (pons, mesencephalon and paramedian thalamus, Figure 1E) . A similar distribution of brainstem lesions leading to disturbed sleep-wake regulation can be observed in neurodegenerative diseases, traumatic brain injury and acute vascular events. 17 A major drawback of our study is the plethora of co-morbidities, such as acute respiratory distress syndrome, hypertension and prophylactic anticoagulation, all of which increase the risk of intracerebral haemorrhage. It seems likely that we investigated an at-risk cohort, which may explain the unusually high occurrence of intracerebral bleeding in our case series compared to another recently published study. 18 Although it is tempting to deduce a causal connection between intracerebral haemorrhage, Sars-CoV-2-induced endothelial inflammation and hypoxaemic damage, the retrospective nature of this study and the small number of patients allows for limited conclusions and necessitates further studies. Neurological symptoms associated with COVID-19 have been described with manifold aetiologies, such as ischaemic stroke, haemorrhagic encephalopathy and others. 19 In contrast to a recently published case report, we did not observe signs of perivascular demyelination. 20 Cerebral microbleeds have been associated with increased risk for cardiovascular mortality 21 and cognitive deterioration. 22 Additionally, emerging evidence suggests rapidly waning humoral anti-Sars-CoV-2 immunity might be associated with a risk of recurrent infection and subsequent cognitive dysfunction. 23 The temporal evolution of COVID-19-associated cerebrovascular pathology remains unclear. Future studies could clarify whether endothelial inflammation is self-limiting or if similar pathological changes can be observed in COVID-19 patients without neurological symptoms. The authors wish to express their grateful thanks to the patients' families without whose contribution this case series would not have been possible. The authors would like to thank Daniela Meir and Fabian Baron for excellent technical assistance. Informed consent for autopsy and publication was given by next-ofkin in all cases. Case series do not need institutional review board approval according to Swiss legislation. The peer review history for this article is available at https://publo ns.com/publo n/10.1111/nan.12677. ACE2 stained slides and the detailed analysis pipeline for ACE2 COVID-19: too little, too late? 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