Mouse Adapted SARS-CoV-2 (MA10) Viral Infection Induces Neuroinflammation in Standard Laboratory Mice

Increasing evidence suggests that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection impacts neurological function both acutely and chronically, even in the absence of pronounced respiratory distress. Developing clinically relevant laboratory mouse models of the neuropathogenesis of SARS-CoV-2 infection is an important step toward elucidating the underlying mechanisms of SARS-CoV-2-induced neurological dysfunction. Although various transgenic models and viral delivery methods have been used to study the infection potential of SARS-CoV-2 in mice, the use of commonly available laboratory mice would facilitate the study of SARS-CoV-2 neuropathology. Herein we show neuroinflammatory profiles of immunologically intact mice, C57BL/6J and BALB/c, as well as immunodeficient (Rag2−/−) mice, to a mouse-adapted strain of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2 (MA10)). Our findings indicate that brain IL-6 levels are significantly higher in BALB/c male mice infected with SARS-CoV-2 MA10. Additionally, blood-brain barrier integrity, as measured by the vascular tight junction protein claudin-5, was reduced by SARS-CoV-2 MA10 infection in all three strains. Brain glial fibrillary acidic protein (GFAP) mRNA was also elevated in male C57BL/6J infected mice compared with the mock group. Lastly, immune-vascular effects of SARS-CoV-2 (MA10), as measured by H&E scores, demonstrate an increase in perivascular lymphocyte cuffing (PLC) at 30 days post-infection among infected female BALB/c mice with a significant increase in PLC over time only in SARS-CoV-2 MA10) infected mice. Our study is the first to demonstrate that SARS-CoV-2 (MA10) infection induces neuroinflammation in laboratory mice and could be used as a novel model to study SARS-CoV-2-mediated cerebrovascular pathology.


Introduction
Since its emergence in late 2019, SARS-CoV-2 has been a significant source of morbidity and mortality worldwide. It is now known that infection can cause a wide range of mild to severe symptoms, especially in patients with significant comorbidities such as chronic lung disease. Importantly, those with cerebrovascular diseases, including brain ischemia and vascular dementia, are more susceptible to SARS-CoV-2 systemic infection [1][2][3][4], suggesting

Mice and Ethics Statement
Male 10-12-week-old mice were obtained from The Jackson Laboratory. Rag2 −/− mice on a C57BL/6 background have a disruption of the recombination activating gene 2 (Rag2) and fail to produce mature T or B lymphocytes [19]. Mice were housed in the animal facility at Tulane University School of Medicine. The Institutional Animal Care and Use Committee of Tulane University reviewed and approved all procedures for sample handling, inactivation, and removal from a BSL3 containment (permit number 4267). Oneyear-old female BALB/cAnNHsd mice (strain 047) (herein referred to as "BALB/c" female mice throughout the manuscript) were obtained from Envigo and were housed at the University of North Carolina at Chapel Hill.

SARS-CoV-2 Infection
Male mice were inoculated with either mock or SARS-CoV-2 (MA10) strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, BEI Resources, NR-55329) in a total volume of 50 µL via intranasal administration by ABSL3-trained staff with a dose of 1 × 10 5 TCID 50 /mouse to induce viral infection in these animals. Infected mice were observed daily for changes in body weight and clinical signs of illness. After 3 days (3 dpi), the mice were euthanized by CO 2 asphyxiation followed by cervical dislocation. The lungs and brains were collected. The right hemisphere was harvested for histology and immunofluorescence, and the left hemisphere for gene expression analysis. One-year-old female BALB/c mice were intranasally infected with 10 3 PFU SARS-CoV-2 (MA10) under BSL3 conditions at the University of North Carolina at Chapel Hill. Whole brains were harvested for histology and immunofluorescence.

Gene Expression
Tissues were homogenized in Trizol Lysis Reagent, and RNA was extracted from the left hemisphere of the brain according to RNA extraction kit manufacturer instructions (RNeasy Plus Mini Kit; Qiagen). RNA was converted to cDNA using iScript reverse transcriptase master mix (Bio-Rad, Hercules, CA, USA). Gene expression was carried out with QuantStudio 3 Real-Time PCR Systems (Life Technologies, Carlsbad, CA, USA) using TaqMan PCR Master Mix and premixed primers/probe sets (Thermo Fisher Scientific, Waltham, MA, USA) specific for Il-1β (Mm00434228_m1), IL-6 (Mm00446190_m1), Ocel1 (Mm01349279_m1), Cldn5 (Mm00727012_s1), GFAP (Mm01253033_m1), Tnf-α (Mm00443258_m1), Ccl2 (Mm00441242_m1), Cxcl10 (Mm00445235_m1), and Gapdh (Mm99999915_g1, Control gene) (Life Technologies) for gene expression [20]. Data was analyzed comparing control to SARS-CoV-2 infected mice and are presented as a fold change of control. Subgenomic mRNA encoding the N gene (sgm-N) was quantified using a published assay [20]. The viral copy numbers from the lung samples are represented as copies/100 ng of RNA [21].

Histology
The tissue of one-year-old female BALB/c mice intranasally infected with 10 3 PFU of mouse-adapted SARS-CoV-2 (MA10) or mock-infected [16] were stored in 10% phosphatebuffered formalin fixative and processed for paraffin embedding. After paraffin embedding, paraffin sections (5 µm in thickness) were used for hematoxylin and eosin (H&E) stains to identify morphological changes in brains. Slides were scanned with a digital slide scanner (Zeiss Axio Scan; Zeiss, White Plains, NY, USA). Representative photomicrographs at 20× magnification were acquired from whole scanned coronal sections using the Aperio Image Scope software (version 12.3.2.8013, Leica, Buffalo Grove, IL, USA). The number of perivascular cuffing sites was counted from all the mice in each group (uninfected control n = 5; SARS-CoV-2 (MA10)-infected n = 5) and was quantified by a pathological score. Whole specimens were examined by using Aperio Image Scope software to establish a histopathological score in each case.

SARS-CoV-2 Immunofluorescence
Immunofluorescent analysis was conducted on the brains of one-year-old female BALB/c mice intranasally infected with 10 3 PFU of mouse-adapted SARS-CoV-2 MA10 or mock infection (detailed experimental protocol as previously described [16]). Paraffin sections (5 µm in thickness) were used, and slides were deparaffinized in xylene and rehydrated through an ethanol series, followed by heat-induced antigen retrieval with high pH antigen unmasking EDTA solution. The slides were washed with PBS with 0.3% Triton X-100 and blocked with 5% normal goat serum for 1 h. Primary antibodies include anti-Iba1, Rabbit (1:1000, Cat# 019-19741, FUJIFILM Wako Pure Chem Corp), and glial fibrillary acidic protein (GFAP) Polyclonal Antibody (1:1000, Cat# PA5-16291, Invitrogen) incubation was achieved at room temperature for 1 h. Slides were then washed, and the primary antibody was detected following 60 min incubation in an appropriate secondary antibody tagged with Alexa Fluor fluorochromes (1:1000) in normal goat serum. After washing in PBS, mounting media with DAPI was used to label the nuclei.

Statistics
Statistical tests were performed using GraphPad Prism, 9.3.1 version (GraphPad Software, San Diego, CA). Data are presented as mean ± SEM. Significant differences were designated using omnibus one-way ANOVA and, when significant, followed up with twogroup planned comparisons selected a priori to probe specific hypothesis-driven questions (saline vs SARS-CoV-2MA10). Statistical significance was taken at the p < 0.05 level.

Cytokine and Chemokine Responses in the Brains of Mouse Adapted Strain of SARS-CoV-2 (MA10) Infected Laboratory Mice
C57BL/6J and BALB/c are the most commonly used background strains for the majority of transgenic mice [22]. The mRNA expression of TNF-α (Figure 2A

Claudin-5 mRNA Expression Is Decreased, and GFAP Expression Increased in the Brains of Mouse Adapted Strain of SARS-CoV2 (MA10) Infected Mice
Claudin-5 expression as a measure of blood-brain barrier integrity was significantly lower in brains SARS-CoV-2 (MA10) infected C57BL/6J and Rag2 −/− ( Figure 3A), BALB/c ( Figure 3C), male mice compared with mock-treated groups. There is a downward trend in occludin mRNA in BALB/c mice following infection ( Figure 3D). Additionally, GFAP expression as a measure of resident immune response was significantly higher in the brains of SARS-CoV-2 (MA10) infected C57BL/6J mice ( Figure 3B).  1-year-old female BALB/c mice were inoculated intranasally with either saline (black bar) or SARS-CoV-2 (MA10) (1 × 10 3 PFU/mL) (red bar) as described previously [16,23]. A previous study reported that there is no detectable virus found in the brains of the infected BALB/c mice at the time of peak lung titer 2 days post-infection (2 dpi) [16]. At 2 dpi, the mice were euthanized, and the whole brain was harvested and fixed in 10% phosphate-buffered formalin, followed by embedding in paraffin and sectioning at 4 um thickness. Sequential sections were stained with Iba-1 by immunofluorescence. Representative images of Iba-1 positive microglial cells (Iba1 staining, green) with DAPI as nuclear counterstaining showed that Iba1 levels are highly induced in the cortical region of brains of SARS-CoV-2 (MA10) infected female BALB/c mice ( Figure 4A). Quantification of Iba-1 positive microglial cells was significantly higher in SARS-CoV-2 (MA10) infected brains compared with mock infection ( Figure 4B). SARS-CoV-2 (MA10) infection showed a trend towards increased GFAP expression in the hippocampus of 12-week old male BALB/c mice after 3 days of infection compared to mock-treated mice ( Figure 4C,D).

SARS-CoV-2 (MA10) Infection Significantly Increases Iba-1 Positive Microglial Cells in Cortical Region of Brain of 1-Year Old Female BALB/c Mice
1-year-old female BALB/c mice were inoculated intranasally with either saline (black bar) or SARS-CoV-2 (MA10) (1 × 10 3 PFU/mL) (red bar) as described previously [16,23]. A previous study reported that there is no detectable virus found in the brains of the infected BALB/c mice at the time of peak lung titer 2 days post-infection (2 dpi) [16]. At 2 dpi, the mice were euthanized, and the whole brain was harvested and fixed in 10% phosphatebuffered formalin, followed by embedding in paraffin and sectioning at 4 um thickness. Sequential sections were stained with Iba-1 by immunofluorescence. Representative images of Iba-1 positive microglial cells (Iba1 staining, green) with DAPI as nuclear counterstaining showed that Iba1 levels are highly induced in the cortical region of brains of SARS-CoV-2 (MA10) infected female BALB/c mice ( Figure 4A). Quantification of Iba-1 positive microglial cells was significantly higher in SARS-CoV-2 (MA10) infected brains compared with mock infection ( Figure 4B). SARS-CoV-2 (MA10) infection showed a trend towards increased GFAP expression in the hippocampus of 12-week old male BALB/c mice after 3 days of infection compared to mock-treated mice ( Figure 4C,D).

Discussion
Suitable animal models for the study of SARS-CoV-2 pathophysiology are important tools in the development of novel therapeutics and in understanding fundamental biological mechanisms of how COVID-19 may affect neurological function over time. As the full

Discussion
Suitable animal models for the study of SARS-CoV-2 pathophysiology are important tools in the development of novel therapeutics and in understanding fundamental biological mechanisms of how COVID-19 may affect neurological function over time. As the full extent of the neuropathological changes induced by SARS-CoV-2 is currently unknown, with limited availability of relevant animal models, we sought to characterize the pathological changes induced in the brain after acute and chronic infection with a novel mouse-adapted SARS-CoV-2 (MA10) in the standard laboratory or immunocompromised mice. As this model has been shown to recapitulate disease course and severity in mice comparable to that in humans [16,24], we analyzed key markers in the brain relevant to immune and vascular function after successful viral inoculation.
The study demonstrated that SARS-CoV-2 (MA10) infection did not cause any mortality in 10-week-old C57BL/6, BALB/c, and Rag2 −/− mice, although there is an increase in pulmonary viral load and increased weight loss in C57BL/6, BALB/c and Rag2 −/− mice. Consistently, we have previously shown elevated pulmonary viral load in BALB/c mice following MA10 infection [16]. However, Leist et al. reported that SARS-CoV-2 (MA10) infection causes mortality in BALB/c and C57BL/6 mice in an age-dependent manner [16]. Quantitative real-time PCR analysis of key inflammatory mediators demonstrated that SARS-CoV-2 (MA10) infection elevated cytokine IL-6 in BLAB/c and chemokine Ccl2 expression in Rag2 −/− mice in comparison with mock-treated mice. Rag2 −/− mice have smaller lymphoid organs (mature B or T lymphocytes), lack functional B and T cells, and display a stunted adaptive immune response [18]. As Ccl2 acts to recruit immune cells to sites of inflammation after injury [25], this suggests that SARS-CoV-2 acts independently of antigen recognition produce these effects. In addition, SARS-CoV-2 (MA10) infection increased blood-brain barrier permeability, evidenced by reduced claudin 5 mRNA expression in C57BL/6, BALB/c, and Rag2 −/− mice and increased GFAP mRNA expression indicating astrogliosis, which broadly supports the hypothesis that SARS-CoV-2 induces a neuroinflammatory phenotype as found in previous studies using the hACE2 over-expressing K18 mouse model [26][27][28], rendering the brain susceptible to infection-induced dysfunction.
In our previous study, we observed that 1-year-old mice were highly susceptible to SARS-CoV-2 MA10, with high morbidity and nearly 100% mortality when infected with 10 4 and 10 5 PFU [16]. This might be due to SARS-CoV-2 and other emerging human coronaviruses exhibiting an age-dependent increase in disease severity [16]. Hence we aimed to determine whether increased age had any bearing on the effects on the brain of COVID infection. We observed SARS-CoV-2 (MA10) infection-induced neuroinflammation in one-year-old female BALB/c mice as shown by increased ionized calcium-binding adapter molecule 1 (Iba-1) immunoreactivity, demonstrating elevated microglial activation even at 2 days post-infection. Elevated pulmonary inflammation and tissue damage were already reported in BALB/c mice following SARS-CoV-2 (MA10) infection [16]. Further, SARS-CoV-2 (MA10) elevated perivascular accumulation of lymphocytes within the brain was consistent with an immune and inflammatory response typical of viral infection of BALB/c mice [29][30][31].
Although COVID-19-associated neurovascular manifestations are of major concern among the scientific community, only limited studies have been conducted to date in this regard. Substantial evidence of the neurological manifestation of SARS-CoV-2 infection in non-human primates has been reported recently by our group [32]. Consistent with our findings, elevated neuroinflammation, microglial activation, and perivascular cuffing were observed in the brains of aged non-human primates. In addition, Kaufer et al. demonstrated that SARS-CoV-2 infection augments microglial activation in olfactory bulbs following acute infection in Syrian golden hamsters [33]. They also found that SARS-CoV-2 infectioninduced neurodegeneration 14 dpi, as demonstrated by increased hyperphosphorylated tau and alpha-synuclein in cortical neurons, underlines the long COVID-19 neurological manifestation [33].
In summary, our findings demonstrate that SARS-CoV-2 (MA10) infection induces neuropathology in common laboratory mice, although it is primarily a pulmonary infection. These observations also suggest that laboratory mice infected with SARS-CoV-2 (MA10) recapitulate multiple aspects of SARS-CoV-2 mediated neuropathogenesis ( Figure 6) found in humans and may serve as a suitable model to study the neurological manifestation of SARS-CoV-2 infection. Hence, the SARS-CoV-2 (MA10) infected mouse model can be used to understand neuroinflammation and associated blood-brain barrier disruption by SARS-CoV-2 infection that may have a negative impact on cognitive function. Being an initial study, our evaluations are limited to a few inflammatory mediators and histological evaluations and warrant further cross-sectional studies on pathophysiological mechanisms of SARS-CoV-2 (MA10) associated neurovascular damage. In addition, long-term experimental studies are needed to assess the consequences of SARS-CoV-2 (MA10) infection on functional neurovascular outcomes. Future studies with this model may allow for the testing of therapeutic compounds against SARS-CoV-2.  Institutional Review Board Statement: The animal study was approved by the Institutional Animal Care and Use Committee at Tulane University.