SARS-CoV-2 Causes Neuroinflammation, Brain Hypoxia, And Microhemorrhages

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A new study finds that SARS-CoV-2 Causes neuroinflammation, brain hypoxia, and micro-hemorrhages.

Source: https://www.nature.com/articles/s41467-022-29440-z

 

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Table 1 Study animals​

Animal ID	Age (years)	Sex	Species	Route of challenge	Virus exposure	SARS-CoV-2 N mRNA (Eq. VC/mL)	Necropsy (days PI)
RM1	14.01	Male	M. mulatta	Multi-route	3.61 × 106 PFU	1.46 × 104	27
RM2	12.97	Female	M. mulatta	Multi-route	3.61 × 106 PFU	2.86 × 106	27
RM3	13.06	Male	M. mulatta	Aerosol	2 × 103 TCID50a	2.97 × 107	28
RM4	15.03	Male	M. mulatta	Aerosol	2 × 103 TCID50a	1.29 × 109	28
RM5	17.97	Female	M. mulatta	Multi-route	TC media	n/a	29
RM6	21.62	Male	M. mulatta	Multi-route	TC media	n/a	29
AGM1	16.28	Female	C.a. sabaeus	Aerosol	2 × 103 TCID50a	9.33 × 104	8
AGM2	16.29	Female	C.a. sabaeus	Multi-route	3.61 × 106 PFU	6.94 × 104	22
AGM3	16.3	Male	C.a. sabaeus	Multi-route	3.61 × 106 PFU	7.62 × 103	26
AGM4	16.33	Male	C.a. sabaeus	Aerosol	2 × 103 TCID50a	2.58 × 104	24
AGM5	17.34	Female	C.a. sabaeus	Multi-route	TC media	n/a	28
AGM6	17.34	Male	C.a. sabaeus	Multi-route	TC media	n/a	28

 

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Table 2 CNS Pathology and Summary of Findings.​

ID	Route of infection	Age (years)	Sex	CNS pathology and summary of findings
RM1	Multi-route mucosal	14.01	Male	Multiple acute microhemorrhages (++++) were observed in cerebellum, BG, and brainstem. Marked neuronal and surrounding cell injury was seen within cerebellum and brainstem. Cleaved caspase 3 positivity was mostly limited to Purkinje cells and immediate neighbors (+++) in cerebellum. BG had a single region of cell with sporadic caspase 3 positivity within the area. Limited vascular caspase 3 positivity was also observed in the BG (+). No caspase 3 positivity was observed in brainstem despite large regions with abnormal neuronal morphology. Limited SARS-N positivity was found in endothelium of cerebellum, brainstem, and BG (+).
RM2	Multi-route mucosal	12.97	Female	Acute microhemorrhages were seen in cerebellum and BG (+). Marked neuronal injury was present in cerebellum and brainstem. Cleaved caspase 3 positivity was observed in Purkinje cells and immediate neighbors in cerebellum and parenchymal cells in brainstem (+++). Rare caspase 3 positivity was observed in cerebellar endothelium, with much greater EC positivity seen in brainstem (+++). Caspase 3 was also observed in BG endothelium, but to a lesser degree (+). BG also showed limited caspase 3 positivity of parenchymal cells with apparent nuclear dissolution and surface blebs (+). Parietal lobe showed rare cleaved caspase 3 positivity in ECs and parenchymal cells. This was localized to blood vessels within associated areas of tissue damage that contained cells at different stages of nuclear dissolution with apparent blebbing. The temporal lobe had several foci with high cleaved caspase 3 positivity (+++). Cleaved caspase 3 was also seen with moderate frequency in temporal lobe ECs (++). Limited SARS-N positivity in endothelium of cerebellum and brainstem (+), with infrequent positivity observed in BG.
RM3	Aerosol	13.06	Male	An acute microhemorrhage was seen in the BG but not in cerebellum or brainstem, in contrast to the majority of our study animals. Moderate neuronal injury with vacuoles in WM were seen in cerebellum. Rare cleaved caspase 3 positivity was seen in Purkinje cells and immediate neighbors. Limited pyknotic neurons were observed in brainstem, however, infrequent caspase 3 positivity was restricted to the endothelium. Rare cleaved caspase 3 positivity was also observed in parietal lobe, despite apparent areas of cell injury/death. Rare SARS-N positivity was detected in endothelium of brainstem, BG, and parietal and temporal lobes.
RM4	Aerosol	15.03	Male	A moderate number of acute microhemorrhages (++) were seen in cerebellum, BG, and brainstem. Marked neuronal injury was observed in cerebellum with WM vacuolation. Active caspase 3 positivity was not detected. In brainstem, foci of cell injury/apoptosis were seen with active caspase 3 positivity in parenchymal cells and endothelium (++). Rare SARS-N positivity was found in endothelium of cerebellum and temporal lobe, with infrequent positivity in parietal and occipital lobes.
RM5	(mock) Multi-route mucosal	17.97	Female	Acute areas of neuronal injury in the Purkinje cell layer of the cerebellum (+). No microhemorrhages were seen in any regions. All brain regions were negative for cleaved caspase 3 and SARS nucleocapsid.
RM6	(mock) Multi-route mucosal	21.62	Male	Healthy brain morphology was generally observed. A microhemorrhage was noted in the brainstem, basal ganglia, and cerebellum. All brain regions were negative for cleaved caspase 3 and SARS nucleocapsid.
AGM1	Aerosol	16.28	Female	Extensive acute microhemorrhages (++++) were seen in cerebellum, BG, and brainstem. Marked neuronal and neighboring cell injury were also seen in cerebellum, BG, and brainstem but without cleaved caspase 3 positivity. Likewise, cleaved caspase 3 was not seen in parietal lobe, despite obvious cell/tissue injury and/or death. Rare SARS-N positivity was detected in endothelium of cerebellum, brainstem, and BG, which was infrequent and dim in the temporal lobe.
AGM2	Multi-route mucosal	16.29	Female	A considerable number of acute microhemorrhages (++++) were observed in cerebellum, BG, and brainstem. While marked neuronal injury was observed in cerebellum, cleaved caspase 3 positivity in Purkinje cells and immediate neighbors was moderate (++). Brainstem had foci of caspase 3 positivity (++), whereas the parietal lobe contained regions of apparent cell injury/death without cleaved caspase 3 positivity. Rare SARS-N positivity was noted in endothelium of cerebellum, brainstem, BG, and parietal lobe.
AGM3	Multi-route mucosal	16.3	Male	Several acute microhemorrhages (+++) were seen in cerebellum, BG, and brainstem. Marked neuronal caspase 3 positivity was seen in Purkinje cells and immediate neighboring cells (+++) within the cerebellum, whereas EC-associated positivity was rare. Considerable caspase 3 positivity was present in parenchymal and ECs of brainstem (++++) and parietal lobe [parenchymal (+++); ECs (+)]. Rare SARS-N positivity was observed in the endothelium of cerebellum, brainstem, BG, and temporal lobe.
AGM4	Aerosol	16.33	Male	Several acute microhemorrhages (+++) were seen in cerebellum and brainstem. Cerebellum showed marked neuronal injury with moderate cleaved caspase 3 positivity in Purkinje cells and immediate neighbors (++). Rare caspase 3 positivity was seen in cerebellar and BG endothelial cells. Focal regions of parenchymal cell injury were present in brainstem with active caspase 3 positivity in parenchymal cells and ECs (+). A single region within the parietal lobe had considerable cleaved caspase 3 positivity in the parenchyma (++++ for this area only). Limited SARS-N positivity was seen in endothelium of cerebellum, brainstem, and BG (+), which was infrequent in temporal, parietal, and occipital lobe endothelium.
AGM5	(mock) Multi-route mucosal	17.34	Female	Several microhemorrhages seen in the cerebellar white matter and granular layer (+). Rare cleaved caspase 3 positivity in the frontal lobe. All brain regions were negative for SARS nucleocapsid.
AGM6	(mock) Multi-route mucosal	17.34	Male	Microhemorrhages were noted in the basal ganglia, brainstem, and cerebellum with larger bleeds in the white matter (+). Rare vacuoles in cerebellum (+). Sparse cleaved caspase 3 positivity in the cerebellum, basal ganglia, and brainstem but not near the areas of microhemorrhages. All investigated brain regions were negative for SARS nucleocapsid.

 

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Neuroinflammation was seen in all study animals but was greater in those with SARS-CoV-2, as compared to age-matched mock-infected controls (Fig. 1). The pan-microglial protein, ionized calcium-binding adapter molecule 1 (Iba-1), was upregulated in the context of infection and revealed morphological alterations indicative of microglial activation, with retracted, thickened processes and a large cell body (Fig. 1b, d). Occasional, small perivascular cuffs were observed in infected (Fig. 1f, h) but not control animals (Fig. 1e, g). In contrast, nodular lesions were seen more frequently than cuffs and were present in both infected (Fig. 1j, l) and mock-infected (Fig. 1i, k) animals, however, these appeared larger in the context of infection.

Fig. 1: Prominent neuroinflammation in brain of SARS-CoV-2 infected NHPs.​

Representative images identify microglia through Iba-1 immunopositivity in basal ganglia of mock-infected animals RM6 and AGM5 (a, c) that was upregulated in SARS-CoV-2 infected parenchyma, as shown in RM2 and AGM4 (b, d). Mild-moderate accumulation of microglia was often observed around blood vessels (RM1 f, AGM1 h). Nodular lesions were also frequently observed in brain of infected animals, represented here in RM4 and AGM4 (j, l). Microglial accumulation around blood vessels was not seen in age-matched mock-infected controls (RM6 e, AGM5 g), however, nodules (RM5 i, AGM5 k) were seen. These were less frequent and smaller than those observed in infection. Iba-1 immunopositivity also revealed morphological changes in microglia indicative of increased activation in infected animals, as compared to mock-infected controls, including large cell bodies with short, thickened processes (b, d, f, h, j, l). Microglial expression of HLA-DR was upregulated in the context of infection (n, p) seen in RM2 and AGM2, however, expression was also seen in control animals (m, o) represented by RM6 and AGM5. GFAP expression by astrocytes is upregulated and reveals morphological changes in the context of infection (cerebellum from RM4 r, AGM2 t), indicative of astrogliosis. Cerebellum from non-infected controls RM6 and AGM5 (q, s). Each immunohistochemical stain was performed twice on all brain regions. Abbreviations: AGM African green monkey, RM Rhesus macaque. Scale bars = 100 µm (a–d, m–t) and 50 µm (e–l).

 

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To further characterize microglial activation, tissues were investigated for the MHC class II cell surface receptor, HLA-DR (Fig. 1m–p). Similar to findings in brain of aged human subjects11, microglial expression of HLA-DR was observed in animals without SARS-CoV-2 infection (Fig. 1m, o). Expression was also seen in brain of infected animals (Fig. 1n, p); however, this did not appear greater than those that were mock-infected. HLA-DR did highlight nodular lesions in all animals, which were larger in infection, as seen with Iba-1.

Additional evidence of increased neuroinflammation in infection was seen through glial fibrillary acidic protein (GFAP) IHC, which was upregulated in infected animals (Fig. 1r, t), as compared to age-matched controls (Fig. 1q, s). GFAP immunopositivity revealed astrocytic hypertrophy in the context of aging, suggestive of astrocyte activation, however, this was more pronounced in infection, which also displayed significant loss of individual astrocytic domains.

Neuronal injury and apoptosis​

Hematoxylin and eosin (H&E; Fig. 2) staining revealed marked changes in neuronal morphology, which was most often observed in cerebellum and brainstem (Fig. 2b–d). Neuronal degeneration was characterized by pyknotic and karyorrhectic nuclei with shrunken cytoplasm and vacuolation in the surrounding neuropil (Fig. 2b–d). The cerebellum contained several regions of degenerate Purkinje neurons that exhibited cellular blebs and debris and cytoplasmic vacuoles (Fig. 2b, c). Contiguous with areas of degenerate Purkinje cells, neurons and glia within the molecular and granular layers appeared pyknotic with condensed, basophilic nuclei (Fig. 2b). Similar morphologic changes were noted in glial cells adjacent to apoptotic neurons in the brainstem (Fig. 2d). In both brainstem and cerebellum, neurons are seen at various stages of nuclear dissolution (Fig. 2b–d). Degeneration of Purkinje cells was further confirmed with FluoroJade C (Fig. 2e, f).

 

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Fig. 2: Neuronal pathology and cell death in SARS-CoV-2 infection.​

Representative H&E images show a healthy Purkinje cell layer in the cerebellum of a non-infected control RM6 (a) and reveal cell death-associated neuronal changes in cerebellum of infected animals, (AGM4 b, AGM3 c), and brainstem from AGM3 (d). Arrows indicate pyknotic and karyolitic Purkinje cells and cellular blebs. Asterisks denote areas of tissue necrosis/vacuolation on H&E sections (b) and (d). H&E was performed and assessed twice on all brain regions. Neuronal degeneration in cerebellum was only seen in the context of infection, visualized by positive, green FluoroJade C-stained neurons (AGM2 e, AGM1 f). FluoroJade C staining was performed twice on the brain regions investigated. Abnormal neuronal morphology and cleaved caspase 3 positivity is demonstrated in cerebellum (AGM3 g) and brainstem (RM2 h). Summary data of cleaved caspase 3 positive cells stratified by brain region (i) where n = 4 biologically independent samples/brain region in the control group and n = 8 biologically independent samples/brain region in the infected group, except OL where n = 7 infected animals. Immunohistochemical staining for cleaved caspase 3 was performed twice on all brain regions. Statistics were performed with a two-tailed Mann–Whitney U test. *p ≤ 0.05 and **p ≤ 0.005 comparing mock-infected to infected animals. Data are expressed as mean ± SEM. p values: CER = 0.0081, BS = 0.0182, FL = 0.0323, OL = 0.0061, TL = 0.0485, PL = 0.0485, BG = 0.0040 (control vs. infected). Source data are provided as a Source Data file. Abbreviations: CER cerebellum, BS brainstem, FL frontal lobe, OL occipital lobe, TL temporal lobe, PL parietal lobe, BG basal ganglia, C control, I infected, AGM African green monkey, RM Rhesus macaque. Scale bars = 100 µm (a, c–f, h) and 50 µm (b, g).

 

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Given the prominent morphologic changes noted within Purkinje cells, we sought to identify the mechanisms underlying these degenerative changes by investigating all brain regions for the presence of cleaved caspase 3, the activated form of this key executioner of apoptosis. Cleaved caspase 3 was seen in at least one CNS region from all infected animals except AGM1, which did not have any positive cells (Fig. 2i, Supplementary Data Fig. 2). Three animals, RM1, AGM3, and AGM4 showed positivity in more than one brain region, while RM2 had cleaved caspase 3 positive cells in all regions examined (Fig. 2i; Supplementary Data Fig. 2). In cerebellum, cytoplasmic and nuclear-cleaved caspase 3 was predominantly restricted to cells within and proximal to the Purkinje cell layer (Fig. 2g). Other CNS regions, including brainstem, had foci of cleaved caspase 3 positivity (Fig. 2h). In comparison to infected animals, mock-infected controls showed little-to-no positivity (Fig. 2i; Supplementary Data Fig. 2). Unbiased quantitation revealed a statistically significant difference in cleaved caspase 3 positivity between infected and mock-infected animals in all brain regions investigated (Fig. 2i). When stratified by species, statistical significance was not achieved by Mann–Whitney U Test, which is likely due to the low number of each species (Supplementary Data Fig. 2). Interestingly, cleaved caspase 3 was not detected in any CNS region examined from AGM1, who was euthanized at 8 days post infection due to advanced illness. This may suggest programmed cell death in the CNS occurs later in the disease process.

While vacuolation was at times observed in the cerebellar gray and white matter (Supplementary Data Fig. 3a, b), significant demyelination was not a major finding in this study. Luxol Fast Blue (LFB) did reveal localized myelin pallor, suggestive of oligodendrocyte injury and/or loss, in the cerebellum of RM3 and occipital lobe of AGM3 (Supplementary Data Fig. 3c, d).

Brain microhemorrhages​

Microhemorrhages, as suggested by the presence of erythrocyte extravasation into the brain parenchyma, were identified in all study animals and seen with and without ischemic injury of adjacent tissues, characterized by localized/regional tissue pallor (Fig. 3a–f). Although the number of bleeds varied, all animals were observed to have at least one. Infected animals appeared to have larger bleeds than mock-infected controls, with more dense accumulation of red blood cells on the parenchymal side of the blood vessel (Fig. 3, compare a–d with e, f). Quantitation of microhemorrhages was determined on Axio Scan.Z1 (Zeiss) scanned slides and HALO software (Indica Labs, v2.3.2089.70 and v3.1.1076.405) and normalized by tissue area (Fig. 3g). The whole brain showed a higher increase in the number of microbleeds in infection which reached statistical significance in the basal ganglia (Fig. 3h and Supplementary Data Fig. 4).

 

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Fig. 3: Multiple microhemorrhages in CNS of SARS-CoV-2 infected NHPs.​

H&E examination of infected animals revealed microhemorrhages, as demonstrated in cerebellum (AGM1 a), brainstem (AGM2 b, AGM4 c), and basal ganglia (RM4 d), which tended to be larger and packed with red blood cells, as compared to non-infected controls, (RM6 brainstem e, AGM5 cerebellum f). Erythrocyte extravasation into the brain parenchyma is indicated by black arrows. Asterisks denote tissue injury around damaged blood vessels. A dotted line outlines the vessel lumen in each panel. H&E was performed and assessed twice on all brain regions. The number of microbleeds/mm2 was assessed in all brain regions (g) and found to be significantly greater in the basal ganglia (h, *p = 0.0263), where n = 4 biologically independent samples in the control group and n = 8 biologically independent samples in the infected group. Statistics were performed with a two-tailed Mann–Whitney U test. *p ≤ 0.05. Data are expressed as mean ± SEM. Source data are provided as a Source Data file. Abbreviations: C control, I infected, AGM African green monkey, RM Rhesus macaque. Scale bars = 100 µm except outset of b where scale bar = 500 µm.

 

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Accumulation of cerebral microhemorrhages occurs with aging and are seen most frequently in deep brain structures, including brainstem, basal ganglia, and cerebellum12. This may be due to age-associated decrease in arterial elasticity and increased blood pressure on brain microvasculature, as well as other risk factors for vascular injury, such as diabetes and dyslipidemia. Vascular injury can promote thrombosis, or blood clot formation within a blood vessel, which may aid in stopping the brain microbleed or, conversely may underlie microhemorrhages and result in more serious brain injury by impeding the flow of blood in the brain, leading to stroke. To assess the potential contribution of thrombosis to microhemorrhage development in SARS-CoV-2 infection, we examined all brain regions for luminal accumulation of the platelet glycoprotein, CD61 (aka, integrin b-3). This revealed multiple blood vessels with aggregated platelets in both infected and mock-infected animals, which were seen with and without associated microbleeds (Fig. 4a–d). Microhemorrhages without CD61 accumulation were also observed (Fig. 4e, f). Quantitation of total brain microhemorrhages with and without associated CD61 positivity revealed a greater frequency without thrombi (CD61 positivity) in the context of infection, apart from AGM5 who had many bleeds without visible thrombi (Fig. 4g, h). These findings suggest that in the context of infection, leakage of blood vessels without vascular damage/injury occurs more frequently.

 

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Fig. 4: Reduced CD61 positive associated-microhemorrhages in SARS-CoV-2 infected NHPs.​

Representative images show CD61 positivity in intact vessels in parietal lobe from control animal AGM5 (a) and temporal lobe (b) from infected AGM4. CD61 positivity was also seen in association with microhemorrhages, as shown in control cerebellum of AGM5 (c) and brainstem of infected RM2 (d). Microhemorrhages without CD61 positivity were also observed in both non-infected (RM5 brainstem e) and infected (RM1 temporal lobe f) but was more frequent in infection. The percent frequency of microhemorrhages with and without CD61-associated platelet aggregates is shown for each species (g and h). Immunohistochemical staining for CD61 was performed twice on all brain regions. Source data are provided as a Source Data file. Abbreviations: AGM African green monkey, RM Rhesus macaque. Scale bars = 50 µm.

 

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Chronic hypoxemia/brain hypoxia​

Microhemorrhages and ischemia appear to play a central role in neuronal injury observed in this study. The brain is a highly metabolic organ with a limited capacity for energy storage. Due to the significant energy demands of the brain and neurons, a prolonged reduction in blood flow and concomitant reduction in oxygen and glucose can be detrimental to neuronal vitality, in addition to the resulting neurotoxicity of erythrocyte breakdown products and inflammation. Of particular interest is the finding that AGM1, who was found recumbent and minimally responsive to stimuli at 8 days post infection, had a substantial number of microbleeds in the cerebellum, basal ganglia, and brainstem (Table 2). These findings suggest AGM1 suffered multiple acute microhemorrhages that may have contributed to her rapid decline. Alternatively, AGM1’s rapid pulmonary decline may have promoted end stage microhemorrhages. The timing of acute microhemorrhages in the disease process is unclear and warrants further investigation.

In addition to localized ischemic injury, all infected animals experienced variations in SpO2 that fluctuated between 89 and 99% but stayed below 95% for most over the study course (Fig. 5a). Correspondingly, blood carbon dioxide (CO2) ranged from 24 to 33 mEq/L, remaining above the physiological range for most of the study animals (Fig. 5b). While these levels are not immediately alarming, they may suggest mild hypoxemia and impaired gas exchange in the lungs. As such, chronic hypoxemia may contribute to impairment of the endothelium and/or neurovascular unit leading to increased vascular permeability. The brain requires aerobic metabolism of glucose for ATP production and any prolonged or intermittent reductions of blood O2 may contribute to localized CNS hypoxia and energy failure. Even minor reductions in oxygen may promote injury, particularly among neurons, which appear to have suffered the greatest insult in this study. In support of this notion, large regions of Purkinje cells, which are especially vulnerable to hypoxic insult13,14, as well as cells in their immediate proximity, appear degenerate or committed to undergoing apoptosis.

 

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Fig. 5: Reduced blood oxygen may contribute to brain hypoxia in SARS-CoV-2 infection.​

SARS-CoV-2 infection was associated with lower blood oxygen levels (a) and increased blood carbon dioxide (b). Yellow shading denotes a lower than physiological range of SpO2. HIF-1a expression appeared to be upregulated by cells comprising the vasculature and extended into the parenchyma in the context of infection. Expression was significantly greater than age-matched mock-infected control animals in (c) brainstem, *p = 0.0154 (95% CI = 0.06550–0.4895) control vs. infected animals and (d) basal ganglia, **p = 0.0016 (95%CI = 0.1149 to 0.3621) control vs. infected animals. Significant difference was not seen in (e) cerebellum (n = 4 biologically independent samples in the control group, and n = 8 biologically independent samples in the infected group). Statistics were performed with unpaired two-tailed t test, df = 10. Data are expressed as mean ± SEM. When separated by species, statistical significance is retained in the basal ganglia but not brainstem (Supplementary Data Fig. 7). Representative images show low HIF-1α expression in brainstem of mock-infected animals, RM5 (f) and AGM5 (h), as well as basal ganglia of RM6 (j) and AGM5 (l). In comparison, HIF-1a is upregulated in brain of infected animals. Representative images include brainstem of RM3 (g) and AGM4 (i) and basal ganglia of RM3 (k) and AGM1 (m). Immunohistochemical staining for HIF-1α was performed thrice on the brain regions investigated. Source data are provided as a Source Data file. Abbreviations: PI post-infection. O.D. optical density, %SpO2 blood oxygen saturation, AGM African green monkey, RM Rhesus macaque, C control, I infected. Scale bars = 50 µm.

 

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To assess brain tissue for evidence of hypoxia, we performed IHC against the oxygen-regulated alpha subunit of hypoxia inducible factor-1 (HIF-1a), which is upregulated and stabilized under hypoxic conditions. For this analysis, only basal ganglia, brainstem, and cerebellum were investigated because our earlier studies demonstrated these brain regions had the greatest injury/pathology. This study demonstrated marked upregulation of HIF-1a in brain of infected animals, as compared to mock-infected controls (Fig. 5f–m). Areas of intense positivity, suggestive of HIF-1a accumulation, were predominantly seen in and around blood vessels, which extended into the brain parenchyma in infection (Fig. 5g, i, k, m). Areas of HIF-1a positivity were noted in mock-infected animals but were less intense than that seen in brain of infected animals and/or did not extend appreciably into the parenchyma (Fig. 5f, h, j, l). Non-biased quantitation of HIF-1a intensity [optical density (OD)] around blood vessels, which excluded the blood vessel lumen, revealed a statistically significant increase in HIF-1a by cells comprising the vasculature and neighboring parenchymal cells of infected animals, as compared to controls, in brainstem (Fig. 5c, *p = 0.0154) and basal ganglia (Fig. 5d, **p = 0.0016) but not cerebellum (Fig. 5e, p = 0.0940). Our approach for quantifying HIF-1a expression around the vasculature, while excluding the blood vessel lumen, is shown in Supplementary Data Figs. 5 and 6. Statistical significance was only retained in the basal ganglia when stratified by species (RMs *p = 0.049, AGMs *p = 0.034; Supplementary Data Fig. 7).

Rare virus in brain-associated endothelium​

The potential for direct virus involvement in CNS pathology was explored through IHC and RNAscope analyses of all brain regions. Using an antibody against SARS-CoV-2 nucleocapsid protein (SARS-N), IHC studies revealed rare virus infection in brain that, when seen, appeared to be restricted to the vasculature (Fig. 6a). Sparse virus was detected most frequently within the basal ganglia, cerebellum, and/or brainstem and seen less often within the temporal, parietal, and occipital lobes (Table 2). This was verified further through in situ hybridization (ISH) analyses, employing RNAscope Technology with enhanced signal amplification. Using an anti-sense probe to the viral spike protein RNA (SARS-S), cytoplasmic positivity was seen in brain of infected animals but not in mock-infected controls (Fig. 6c–h; Supplementary Data Fig. 8). The specificity of the probe used in these studies is demonstrated in lung, which only showed positivity in the context of infection (Supplementary Data Fig. 8).

 

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Fig. 6: SARS-CoV-2 detection in the brain.​

Representative single-label IHC shows infrequent SARS-CoV-2 nucleocapsid (SARS-N) positivity in a cerebellar blood vessel of RM1 (a). Positivity was not detected in non-infected controls (AGM6 cerebellum b). SARS-CoV-2 spike (SARS-S) mRNA expression was assessed with in situ hybridization (RNAscope) but not seen in control animal tissue (RM6 cerebellum f-h). Rare positivity was seen in infection (AGM1 cerebellum c–e). RNAscope was performed 7 times on the brain regions investigated. A majority of brain tissue does not show any virus in infected animals which can be seen in the neighboring vessel (RM3 basal ganglia l–n) to a vessel with suggestive virus positivity (i–k). Endothelial cell infection is suggested by double fluorescent labeling of SARS-N with von Willebrand factor (vWF) (RM3 basal ganglia i–k). Merged images show colocalization of SARS-N (red; j) with vWF (green; i), indicated by white arrows. Blue color represents DAPI labeled cell nuclei. Immunohistochemical staining for SARS-N was performed 12 times on the brain regions investigated. SARS-CoV-2 RNA was detected in the different brain regions of infected animals via a CRISPR-based fluorescent method (o) where n = 3 repeats of independent samples. Dotted line indicates the cut-off value of positivity equal to 3.6 × 106 photoluminescence (PL) intensity. Data are expressed as mean ± SEM. Source data are provided as a Source Data file. Abbreviations: BG basal ganglia, CER cerebellum, BS brainstem, FL frontal lobe, CSF cerebrospinal fluid, BC blank control, arb. units arbitrary units, AGM African green monkey, RM Rhesus macaque. Scale bars = 50 µm (a and b), 20 µm (c–h), and 10 µm (i–n). Fluorescence images are at 100×.

 

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The single-label studies suggested SARS-CoV-2 infection in brain is limited to the brain vasculature and appeared to be restricted to endothelial cells. Suspected endothelial cell infection is supported by colocalization of SARS-N with von Willebrand factor (vWF; Fig. 6i–k). A blood vessel in close proximity to that shown in Fig. 6i–k but without detectable virus is included to demonstrate the specificity of the SARS-N antibody (Fig. 6l–n).

Using a highly sensitive CRISPR-based fluorescent detection system (CRISPR-FDS)15, virus was not identified in the cerebrospinal fluid (CSF) (Fig. 6o), consistent with most findings among human subjects, except in rare cases of encephalitis16,17,18. In contrast, this method detected limited viral RNA in whole brain, frozen at the time of necropsy, that was largely representative of our IHC/IF findings (Fig. 6o). Similar to our findings in fixed tissues, virus was more frequently observed in basal ganglia, cerebellum, and brainstem. CRISPR-FDS analysis also revealed viral RNA in the frontal lobe of one animal, AGM1, which was not convincingly seen by IHC/IF for this region in any study animal. This may reflect differences in sampling error that is inherently present in the two methods, where the amount of tissue used for the CRISPR-FDS studies is greater than that used in IHC, and/or extracerebral virus that may have been present in the blood vessel lumen.

Together, our findings demonstrate scarce SARS-CoV-2 infection in brain-associated endothelial cells in deep brain structures of NHPs, even in the absence of severe disease or overt neurological symptoms.