ABSTRACT
Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may impact neurological function acutely or chronically, even in the absence of severe respiratory illness. Developing clinically relevant laboratory models to understand the neuropathogenesis of SARS-CoV-2 infection is an important step towards unravelling this neurologic consequence. We hypothesize that mouse adapted SARS-CoV-2 viral infection will induce neuroinflammation in immuno-competent C57BL/6J (10 weeks old male) as well as immunodeficient RAG2-/- (10 weeks old male) and BALB/c (1 year old female) mice. Method(s): All three mouse strains were inoculated intranasally with a dose of 1x103 PFU/mouse (50 microL) of either mock or the mouse-adapted (MA)10 strain of SARS-CoV-2 (BEI resource, NR-55329). Mice were euthanized on day 2, 3, 7, 15 or 30 post infection and brain samples processed for qRT-PCR, immunofluorescence, and H&E analysis. Result(s): SARS-CoV-2 MA10 resulted in a significantly higher (p < 0.05) mRNA expression for chemokine ligand 2 (CCL2) and lower (p < 0.05) mRNA expression for the blood-brain barrier component Claudin-5 in RAG2-/- and WT mice when compared to mock infection. Also, SARS-CoV-2 MA10 infection increased microglial expression in 1 year old female BALB/c mice after 2 days of infection, compared to mock infected group. At 30 days post infection, MA10 infected BALB/c mice had a higher perivascular lymphocyte cuffing in the brain. Conclusion(s): This study demonstrates that the mouse-adapted MA10 strain of SARS-CoV-2 can induce a neuroinflammatory state in the brain and more so in immunodeficient and aging mouse models. These mouse models will enable the investigation of the long-term neurological effects of SARS-CoV-2 infection. Copyright © 2022 the Alzheimer's Association.
ABSTRACT
Patients with significant cerebrovascular comorbidities (e.g. brain ischemia, vascular dementia) are more affected and are more likely to have worsened post-acute neurologic sequelae after SARSCoV-2 infection. This may be due to viral invasion and propagation in brain endothelial cells (BECs) and disruption of the blood-brain barrier (BBB). Viral spike protein used to bind and infect host cells encodes an arginine-glycine-aspartic acid (RGD) motif that it may use to bind integrins cell receptors that play an important role in cerebrovascular integrity. Therefore, integrins may represent an acute and post-acute SARS-CoV-2 therapeutic target. However, the interplay between vascular dysregulation, integrin function, and COVID-19 is unclear. As we have previously demonstrated that activation of the integrin α5 plays a key role in BBB breakdown, stroke injury, OGD/R, SARS-CoV-2 infection, and its inhibition with the clinically validated peptide ATN-161 is therapeutic in these conditions, we hypothesize that SARS-CoV-2 alters BEC α5 integrin (and associated tight junction protein) expression as a means of infecting and altering cerebrovascular integrity, and this can be prevented by ATN-161. Methods: Mouse BECs (bEnd3) were inoculated with heat-inactivated SARS-CoV-2 (Isolate USAWA1/2020) or delta variant of SARS-CoV-2 spike protein for 24 h then later exposed to hypoxia for 6h to model the effects of in vivo pulmonary infection. Cells were pretreated with ATN-161 (1, 5, and 10μM) 1h before SARS-CoV-2 challenge and during hypoxia. α5 and claudin-5 proteins were analyzed by immunoblotting. Results: Both SARS-CoV-2 inoculations induced integrin α5 and decreased claudin-5 expression (delta > SARS-CoV-2) in a dose-dependent fashion, although higher doses of SARS-CoV-2 (2.5 and 5 μg) had no effect on these proteins. SARS-CoV-2 spike protein challenge at 0.5 μg followed by hypoxia resulted in increased α5 and decreased claudin-5 expression in either hypoxia or SARSCoV-2+hypoxia combination. ATN-161 (10μM) pretreatment inhibited SARS-CoV-2+hypoxia-induced α5 upregulation and restored claudin-5 loss. In addition to its demonstrated anti-viral effects, ATN161 may be an important therapy for SARS-CoV-2-mediated cerebrovascular injury.