A suitable model to investigate acute neurological consequences of coronavirus infection

doi:10.21203/rs.3.rs-3014693/v1

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Research Article

A suitable model to investigate acute neurological consequences of coronavirus infection

https://doi.org/10.21203/rs.3.rs-3014693/v1

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Journal Publication

published 14 Oct, 2023

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Objective and design:

The present study aimed to investigate the neurochemical and behavioral effects of the acute consequences after coronavirus infection through a murine model.

Material:

Wild type C57 BL/6 mice were infected intranasally (i.n) with the murine coronavirus 3 (MHV-3).

Methods:

Mice were submitted to behavioral tests. Euthanasia was performed on the fifth day after infection (5 dpi), and the brain tissue was subjected to plaque assays for viral titration, synaptosome, ELISA, histopathological and immunohistochemical analysis.

Results:

Increased viral titers associated with mild histological changes, including signs of neuronal degeneration, were observed in the cerebral cortex of infected mice. Importantly, MHV-3 infection induced an increase in cortical levels of glutamate and calcium, as well as increased levels of pro-inflammatory cytokines (IL-6, IFN-γ) and reduced levels of neuroprotective mediators (BDNF and CX3CL1) in the mice brain, which is suggestive of excitotoxicity. Finally, behavioral analysis showed impaired motor, anhedonic and anxiety-like behaviors in animals infected with MHV-3.

Conclusions:

Overall, the data presented emulate many aspects of the acute neurological outcomes seen in patients with COVID-19. Therefore, this model may provide a preclinical platform to study acute neurological sequelae induced by coronavirus infection and test possible therapies.

MHV

SARS-CoV-2

acute neurological outcomes

glutamatergic levels

COVID-19

animal behavior

COVID-19 has resulted in more than 767 million cases and 6.9 million deaths worldwide [50]. Initially, COVID-19 was considered a respiratory disease, however soon after it has been observed that the disease affects several organs and tissues, including the central nervous system (CNS) [1].

The first neurological symptom resulting from COVID-19 is typically gustatory and/or olfactory dysfunction [2]. In the most severe cases, peripheral neurological conditions such as Guillain-Barré syndrome, as well as conditions that indicate CNS involvement such as stroke, epilepsy/seizures, encephalitis, and other have been reported [2]. The GCS-NeuroCOVID study, which involved more than 3,500 patients, found that 80% of them reported neurological manifestations during COVID-19, with encephalopathy being the most prevalent [72].

Viral genetic material, and SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) proteins have been identified in post-mortem brain tissue and cerebrospinal fluid (CSF) of patients affected by COVID-19 [3–7]. It is assumed that the olfactory nerve and olfactory bulb serve as a gateway to the CNS. Several viruses, including murine hepatitis coronavirus (MHV) [10, 11], have been shown to use this route to reach the CNS. SARS-CoV-2 RNA found in the CSF or CNS parenchyma of patients with COVID-19 may originate from the blood due to compromised blood-brain barrier (BBB) permeability [49], as well as from the migration of peripheral immune cells carrying the virus or viral fragments. COVID-19-related neurological symptoms are likely due to immune responses rather than a consequence of SARS-CoV-2 neurotropism [47]. The most accepted mechanism involved in the neurological and neuropsychiatric changes resulting from COVID-19 is the "cytokine storm", in which various mediators peripherally produced can reach the CNS, triggering neuroinflammation [9]. Thus, SARS-CoV-2 may induce a central loss of homeostasis both by direct action and by systemic inflammatory response [15]. Hypoxia resulting from SARS is also associated with disturbances in brain metabolism that can result in neurological manifestations [13].

Different mice strains of CoVs induce hepatic, respiratory, and enteric diseases that are followed by neurological alterations [12]. Given the inherent limits of human studies on the kinetics and mechanisms related to the disease [7], experimental models may provide a better understanding of the disease, and development of therapeutic strategies. Recently, our research group standardized a murine model to study the acute lung injury and systemic manifestation induced by a betacoronavirus, the MHV-3, whose natural host is the Mus musculus species [14, 16]. When inoculated intranasally (i.n.), infections induce a severe lung inflammation which peaks at day 3 post-infection followed by a subsequent viral dissemination and systemic disease manifestations which peak at day 5, when animals start to perish [14]. Recognizing the relationship between COVID-19 and the CNS, our objective was to investigate and characterize the effect of betacoronavirus infection in the CNS, using the i.n. route. We found that MHV-3 infected mice displayed important inflammatory, neurochemical, and behavioral changes. Overall, this model may serve as a platform for the study of possible acute neuropsychiatric alterations resulting from betacoronavirus infection, as well as to test the efficacy of potential therapeutic strategies.

2.1 Assay of cells, viruses, and plates. The cells used for viral propagation is L929 (ATCC CCL-1) were kept under a controlled atmosphere (37°C and 5% CO₂) in Dulbecco's modified Eagle's medium (DMEM) with high content glucose content. The MHV-3 strain was granted by Clarice Weis Arns and Ricardo Durães-Carvalho of the State University of Campinas [(UNICAMP, Brazil; GenBank accession no. no. MW620427.1; see reference 17)] and expanded in L929 cells. Viral titration was performed from brain homogenates (1:9 tissue for DMEM). Cells were grown in 24-well plates and each well with a confluent monolayer of cells was inoculated with 100 µL of this homogenate. These plates were gently rotated for 1 h (4 x 15 min) to ensure effective viral adsorption. After 1 h the samples were removed from the wells, the overlay medium was added (DMEM containing 0.8% carboxymethylcellulose, 2% FBS and 1% penicillin-streptomycin-glutamine) and the plates were placed for 48 h at 37° C and 5% CO₂. Subsequently, 10% neutral buffered formalin (NBF) was added for 1 h for cell fixation and 0.1% crystal violet was used for staining. The viral titer was expressed by counting the number of plaque forming units per gram of tissue (PFU/g tissue).

2.2 Mice. Animal experiments were carried out in WT C57BL/6, male and female, aged between 6 and 8 weeks, with approval from the Ethics Committee on Animal Experimentation of the UFMG (process nº 253/2020). The WT animals were obtained from the Central Animal Facility of UFMG maintained in micro-isolator cages at ABSL-2 under controlled conditions (24°C ± 2°C; on a 12-h light/12-h dark cycle), with ad libitum access to water and food.

2.3 MHV-3 infection. Mice were smoothly anesthetized (ketamine solution [50 mg/kg] and xylazine [5 mg/kg], [ip]) and then inoculated i.n with 30 µl of sterile saline (mock), with MHV-3 at a concentration of 3x 10³ PFU. Manifestations of morbidity, such as weight loss, dorsal arching, facial swelling, ruffled hair and prostration were monitored daily until the 5th dpi and the survival until the 14th dpi.

2.4 Sample. For sample collection, the simulated and infected animals were previously anesthetized (ketamine solution [80 mg/kg] and xylazine [10 mg/kg], ip) and after sedation they were euthanized by cervical dislocation, on the 5^th dpi. Cervical dislocation without anesthesia was used only in experiments in which drug interference in the analyses is proven. The brain was harvested, the left hemisphere was placed in 10% formalin buffer, and the other hemisphere was frozen. Other tissues were collected and frozen for further analysis.

2.5 Histopathological analysis. Brains from mock mice, infected with MHV-3 were removed and processed for hematoxylin and eosin (H&E) staining according to the following works [18, 19]. The histopathological score was adapted from the cited works and the analyzes performed by a pathologist in a blinded manner. The evaluation was performed in the cerebral cortex and in the hippocampus, following a point scale: 0, no lesion; 1 mild tissue injury and/or mild inflammation; 2 mild tissue injury and/or inflammation moderate; 3, definitive tissue damage (neuronal loss and parenchymal damage) and intense inflammation; 4, necrosis (complete loss of all tissue elements with the presence of cellular debris). Meningeal inflammation was assessed using a point scale (0 to 4) with 0 representing no inflammation and 1 to 4 corresponding to 1 to 4 layers affected by inflammation. The final score was the sum of the cerebral cortex and hippocampus scores plus the meningitis score, totaling 12 points.

2.6 Measurement of cytokine and chemokine concentration. Cerebral cortex homogenates were obtained by homogenizing frozen tissue in refrigerated cytokine extraction buffer (100 mM Tris [pH 7.4], 150 MM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X -100, 0.5% sodium deoxycholate and 1% protease inhibitor). This homogenate was subjected to centrifugation (14.000 g, 15 min, 4°C) and then the supernatant was collected for the determination of TNF-α, IFN-γ, IL-10, IL-6, BNDF and CX3CL1 using the immunosorbent assay system limited by mouse DuoSet enzymes (ELISA) (R&D Systems). According to the manufacturer, the sensitivity of the kit for each mediator: TNF-α (7,21 pg/mL), IFN-γ (2 pg/mL), IL-10 (5,22 pg/mL), IL-6 (1,8 pg/mL), BDNF (1,35 pg/mL) e CX3CL1 (0,32 ng/mL).

2.7 Immunohistochemistry IBA1, S100B, NeuN and cleaved Caspase-3. Sections of mouse cortex were evaluated for quantitative alteration of microglia, IBA1 (ionized calcium-binding adaptor molecule 1) antibody (PA5-21274, Invitrogen; 1:150), astrocytes (Anti-S100 beta antibody; ab41548, Abcam; 1:75), neurons (Anti-NeuN, MAB377, EMD Millipore; 1:200) and apoptosis (Anti-cleaved caspase-3; Asp175, Cell Signaling; 1:100,) according to the manufacturer's instructions (Vector Elite kit). The images presented in the article are representative of two experiments performed.

2.8 Transmission electron microscopy (TEM). For ultrastructural analyzes, we used the protocol described by Rodrigues et al. (2013) [see reference 24]. Briefly, mice were anesthetized with ketamine/xilazine and transcardially perfused with ice-cold modified Karnovsky fixative and maintained in this solution overnight at 4ºC. The brain was removed, and the cortex was isolated were washed with cacodylate buffer (0.1 M), cut into several pieces, post-fixed in reduced osmium (1% osmium tetroxide containing 1,6% potassium ferrocyanide) and contrasted en bloc with uranyl acetate (2% uranyl acetate in deionized water). The samples were then dehydrated through an ascending series of ethanol solutions and embedded in EPON resin. Blocks were sectioned (50 nm) and collected on 200 or 300 mesh copper grids 8 and contrasted with lead citrate. Sections were viewed with a Tecnai- G2-SpiritFEI/Quanta electron microscope (120 kV Philips) located at Microscopy Center – UFMG.

2.9 Purification of synaptosomes. Immediately after euthanasia, the cortex was removed and homogenized in a gradient solution containing: 320 mM sucrose, 0.25 mM dithiothreitol, 1 mM EDTA. Then, the homogenate was exposed to a low-speed centrifugation (1000 × g × 10 min). Synaptosomes were isolated from the supernatant by discontinuous Percoll density gradient centrifugation [according to reference 21]. Isolated nerve terminals were resuspended in Krebs-Ringer-HEPES (KRH) solution containing; 124 mM NaCl, 4 mM KCl, 1.2 mM MgSO4, 10 mM glucose, 25 mM HEPES, with pH 7.4 and without addition of CaCl₂, at a concentration of approximately 10 mg/ml. For measurement of glutamate release and intrasynaptosomal calcium concentration, aliquots of 30 μL were prepared and kept on ice until use.

2.10 Measurements of glutamate release. To measure continuous glutamate release, a fluorimetric assay was performed in a fluorimeter (Synergy TM2, Biotek®). Fluorescence emission was registered by using an excitation wavelength of 360 nm and emission of 450 nm. Glutamate release was measured indirectly by following the increase in the fluorescence due to the production of NADPH in the presence of glutamate dehydrogenase type II and NADP⁺. In brief, synaptosomes were incubated with 1Mm CaCl₂, 1mM NADP⁺ in KRH medium for 5 minutes. Glutamate dehydrogenase (50 units per well) was added to each well after 5 min. For depolarization, we used 33mM KCl. Calibration curve was achieved by the addition of glutamate (5 nM/μL) to the reaction medium. Glutamate levels were normalized to the total amount of protein per well.

2.11 Measurements of intrasynaptosomal free calcium concentration. To measure intrasynaptosomal free calcium concentration, synaptosomes were preincubated with 5 μmol/L of Fura-2 pentakis (acetoxymethyl) Ester (FURA2-AM) probe for 30 minutes at 35.5°C. Next, synaptosome was centrifuged (3000 x g × 60 s), resuspended in KRH without CaCl₂, and reincubated again during 30 min. After washout with KRH without CaCl₂ synaptosomes were immediately used for quantification of intracellular free calcium ([Ca²⁺]_i). Fluorescence was recorded with an excitation wavelength of 340/380nm and an emission of 510nm. CaCl₂ (1 mmol/L, final concentration) was added in synaptosomal suspension before reading and 33 mM of KCl was added to evoke calcium influx. Finally, we added 10% SDS to obtain Rmax and tris-EGTA (3 mol/L Tris, 400 mmol/L EGTA, pH 8.6) to obtain Rmin [as described in references 22 and 23].

2.12 Open Field Test. Locomotor activity and anxiety-like behavior were evaluated by the Open field test on the 3^rd,4^th and 5^th dpi [44]. Briefly, animals were videotaped by the Phenotyper apparatus (Noldus, Information Technology, Leesburg, VA, USA). There were four analysis cages in the room, which allowed for observation individually at the same time. Each cage (30x30 cm) contained a top unit with a digital video camera and infrared lights. Mice were placed in the center of the open field arena and were allowed to freely move and explore the environment for thirty minutes. Parameters such as locomotion activity and percentage of time spent at the center area (anxiety measure) were recorded and analyzed by a tracking software (EthoVision XT, Noldus Information Technology, Leesburg, VA, USA).

2.13 Marble Buried Test. Mice at 4^th day post-MHV-3 infection were tested in the Marble buried test, which evaluates compulsive-like behavior. The test was previously described [see reference 41]. Mice were tested in a rectangular cage (30x30x50cm) with 20cm of fresh beddings. 25 marbles were placed equidistant to each other. Briefly, the animal was placed in the middle of the cage and allowed to explore and bury for 30 minutes. At the end of the section, the animals were removed and measured the number of marbles buried. Only the balls buried by more than 2 cm were considered.

2.14 Forced Swim Test. Mice at 4^th day post-MHV-3 infection were tested in the forced swimming test, which evaluates the depressive-like behavior. The test was performed as previously described [see reference 43]. Mice were placed in a cylindrical tank (30 cm height x 20 cm diameters) and recorded for 6 minutes. The duration of immobility time was recorded during the last 4-min of the 6-min testing period, after a 2-min habituation period by the EthoVision XT (Noldus, Technology, Leesburg, VA, USA).

2.15 Elevated Plus Maze Test. Mice at 4^th day post-MHV-3 infection were tested in the elevated plus-maze test (EPM), which evaluates mice anxiety-like behavior. The EPM test was performed as previously described [see reference 47]. Mice were placed in the EPM apparatus consisting of two open and two closed arms, crossed in the middle perpendicularly to each other creating a center area. Briefly, mice were placed in the center of the apparatus and allowed to freely explore for 5 min. The apparatus was wiped clean with 70% alcohol and dried with paper towels between each trial. The percentage (%) of open arm entries and percentage (%) of time spent on the open arms (anxiety-like behavior measure) were recorded and analysed by EthoVision XT (Noldus, Technology, Leesburg, VA, USA).

2.16 Sucrose Preference Test. The sucrose preference test was employed to assess anhedonic-like behaviour in Mice at 3^rd ,4^th and 5^th day post-MHV-3 infection as described elsewhere [45]. Briefly, at day 1 and 2 after MHV infection, i.e. the first two days of the test, the animals were habituated to two bottles of freshwater. On the following three days, corresponding to the 3rd, 4th, and 5th-day post-MHV infection, the previous bottles were removed and replaced with a bottle filled with water and another one filled with 1% sucrose solution. The percentage of sucrose preference was calculated for each of the last three days of experiment.

2.17 Statistical analyses. All statistical analyses were performed, and all graphs were created in GraphPad Prism 8 (GraphPad Software). Data distribution was assessed by the Shapiro-Wilk test. Parametric comparisons between groups were performed using Student's t test. The Mann-Whitney test was used to assess differences between nonparametric data. To compare survival rates between groups the Longrank test was performed. Alterations body weight and clinical score of the groups were compared using two-way repeated measures ANOVA. Data were presented as the mean ± standard error of the mean (SEM). Differences with a P value < 0.05 were considered statistically significant.

3.1 Characterization of cellular and histopathological alterations in the brain of mice infected with MHV-3.

To evaluate potential brain alterations induced by the coronavirus, male and female C57BL/6J mice, 6 to 8 weeks of age, were infected i.n. with 10³ PFU of MHV-3 (Fig 1 a). Analyses were performed on the fifth day after infection (5^th dpi), i.e., at the peak of systemic manifestations as previously described [14]. Upon infection, MHV-3 replicates in the brain of female (Fig 1 b) and male mice (Fig S1 b). Histopathological analysis (H&E) of the cerebral cortex revealed the presence of hyperemic blood vessels, surrounded by few leukocytes, which translated to mild histopathological score in a similar manner in both sexes (Fig 1 c-d and S1 c-d). Subsequently, we investigated whether this mild CNS damage related to changes in the number of cells as well as in the processes of apoptosis. According to H&E data, no significant differences were found in the number of cells labeled for NeuN (Fig 1e), IBA1 (Fig 1 f), S100B (Fig 1 g), and caspase 3 (Fig S1 h) in the cerebral cortex of female MHV3-infected mice compared with mock animals. Similar results were found in male C57BL/6 mice (Fig S1). Overall, the presented data suggests that the mild histopathological damage induced by MHV-3 was not associated with neuronal or glial death.

3.2 Signs of neuronal degeneration were observed in the cerebral cortex of mice infected with MHV-3. To evaluate potential neurodegenerative signs caused by MHV-3 infection, we performed a qualitative ultrastructure analysis of female mouse cerebral cortex samples from infected and non-infected mice (Fig 2). Our data showed signs of degeneration in neuronal cell bodies. For example, Fig 2-a (red arrowhead) shows a dark degenerating neuron with a shrunk cell body, darker staining (increased electron density), Golgi and endoplasmic reticulum dilation (Fig 2-a’ arrows) and mitochondria with disrupted cristae (Fig 2-a’’ and c’’ arrows). We also observed typical neuronal cell bodies such as the profile shown in Fig 2b but with prominent and complex lipofuscin granules (Fig 2b’, c’ and C’’ arrowheads). Both in control and infected mice (in addition to the ones described above), there were neuronal cell bodies with typical morphological features such normal cell size, typical nucleus with normal chromatin (Fig 2d) and mitochondria with conserved cristae (Fig. 2d’). Eventually, we noticed occasional lipofuscin granules in control mice, such as the one on Fig 2d- blue arrowhead) but they were less complex than the ones observed in MHV-3 infected cerebral cortex.

3.3 MHV-3 infection induces glutamate release, intracellular calcium levels elevation and production of inflammatory mediators in the cortical brain of mice. Glutamate is an important excitatory neurotransmitter, and numerous studies have indicated that overstimulation of the glutamatergic system through activation of ionotropic glutamate receptors can lead to excitotoxicity, which may result in neuronal calcium overload and consequent neurodegeneration [19, 25, 26]. To investigate whether coronavirus has the potential to induce neurochemical changes in the cerebral cortex, we measured glutamate levels and intracellular calcium in cortical synaptosomes of female and male mice. Indeed, MHV-3 infection induced a greater release of glutamate, as well as an increase in intracellular calcium levels, in the cerebral cortex of infected animals of both sexes (Fig 3b-e).

Next, we evaluated the levels of certain molecules involved in the host response induced by infections, such as cytokines (interleukin-6 [IL-6], interferon-gamma [IFN-γ], tumor necrosis factor [TNF], interleukin-10 [IL-10], brain-derived neurotrophic factor [BDNF]), and the chemokine CX3CL1 (fractalkine) in distinct brain areas of mice infected with MHV-3 and their control littermates. Cytokines IL-6 and IFN-gamma showed an increase in the prefrontal cortex (PFC) of female mice infected with MHV-3 compared to mock animals (Fi. 4a and 4b). In male mice infected with MHV-3, there was an increase in IFN-gamma levels in the PFC, while IL-6 was not altered (Fig 4g and 4h). No significant differences were found in the concentration of TNF and IL-10 in both sexes (Fig 4c and 4j, respectively). Interestingly, female mice infected with MHV-3 had a reduction in BDNF and CX3CL1 levels in the PFC compared to control mice, while male mice responded to the infection with an increase in CX3CL1 levels and no significant changes in BDNF concentrations in the PFC compared to control littermates (Fig 4e-f and 4k-l, respectively). We measured the same mediators in the hippocampus and striatum of both sexes. Regarding the concentrations of IL-10, TNF, IFN-γ, and CX3CL1, there were no significant changes in these regions in either sex (Fig S2 and S3). IL-6, on the other hand, had its concentration increased in the hippocampus and striatum of females infected with MHV-3 when compared to their controls, while no change was observed in males (Fig S2 and S3). Levels of neurotrophic factor BDNF did not change in the hippocampus and striatum of infected female mice (Fig S2e and S2k) but were increased in the hippocampus of male MHV-3 mice (Fig S3e).

Overall, our results suggest that MHV-3 infection induces acute neurochemical alterations in the brain of mice, as demonstrated by increased levels of glutamate/calcium and changes in levels of inflammatory mediators, suggesting the occurrence of excitotoxicity. Furthermore, we observed that female mice seem to be more susceptible to MHV-3 infection and to the reduction of neuroprotective mediators compared to male mice.

3.4 MHV-3 infection promotes anxiety-and depressive-like behaviors alongside motor dysfunction in mice.

We evaluated whether MHV-3 infection is associated with acute behavioral changes in mice. No significant differences were observed in the total distance traveled in the Open Field Test (OFT) on days 3 (Fig 5b and S3a) and 4 (Fig 5c) post-infection. However, on day 5, when systemic disease symptoms peaked, MHV-3-infected female mice (Fig 5d) but not male mice (Fig S4b) showed a significant decrease in the total distance traveled compared to non-infected controls, indicating locomotor impairment in females. At 4 days post-infection, female infected mice displayed anxiety-like behavior, as indicated by a significant decrease in the percentage of time spent in the open arms of the elevated plus maze (EPM) (Fig 5e), which was not observed in male mice (Fig. S4d). Both female and male infected mice showed a depressive-like behavior in the Marble Burying test (MBT) at 4 days post-infection, as indicated by a significant reduction in the number of marbles buried (Fig 5f and S4d). In addition, infected male mice had an increase in immobility time in the Forced Swimming Test (FST) (Fig S4e), while no significant differences were found in immobility time in infected females (Fig 5g). Female mice infected with MHV-3 also showed an anhedonic-like behavior in the sucrose preference test (SPT) on days 3 (Fig 5h) and 5 (Fig 5i) post-infection. In summary, these results suggest that MHV-3 infection induces acute behavioral alterations in mice, especially in females, including locomotor impairment, anxiety-like behavior, depressive-like behavior, and anhedonic-like behavior.

COVID-19 has been associated with a wide range of clinical manifestations, including neurological and psychiatric alterations. In this study, we investigated whether MHV-3, can directly affect the CNS and serve as a suitable platform to study COVID-19-related neurobiological mechanisms and identify novel therapeutic targets. The main findings of this study are: (i) MHV-3 delivered intranasally is capable of infecting and replicating in the CNS, causing mild histopathological changes; (ii) MHV-3 infection results in increased glutamate release, intracellular calcium levels and pro-inflammatory mediators in the cortex; (iii) MHV-3 infection decreases the production of neuroprotective mediators such as BDNF and CX3CL1 in the brain; and (iv) MHV-3 infection induces anxiety-like, depressive-like behaviors, and motor dysfunction, especially in female mice.

Models that assess behavioral changes in the context of betacoronavirus infections in the CNS are still scarce. We have demonstrated the applicability of a murine betacoronavirus model as a platform to recapitulate the acute neurological and psychiatric symptoms observed in patients with COVID-19. All analyses were performed up to the 5th day post infection, i.e., at the peak of systemic disease [14]. First, the neurotropism of MHV-3 was confirmed by recovering viable virus particles in the brain of mice. In addition, histopathological analysis of the brain of MHV-3-infected mice revealed mild changes, such as dilated blood vessels in the meninges, an inflammatory infiltrate, and hyperemic vessels in the cerebral cortex. These findings support human studies showing that SARS-CoV-2 was detected in the post-mortem brain of half of COVID-19 patients along with mild neuropathological lesions [3]. Although many studies have not found SARS-CoV-2 in the brain, it is already known that the immune response triggered by the virus can culminate in a “cytokine storm”, which reaches and compromises the homeostasis of the CNS, as well as other tissues [9].

In addition, we conducted a quantitative analysis of neurons (NeuN), microglia/macrophage (IBA1), and astrocytes (S100B), as well as evaluated apoptosis (cleaved caspase 3). None of these markers were found to be altered in the cortex of infected animals when compared to mock. However, this finding does not necessarily indicate that glia and neurons were not affected. In fact, brain tissues from individuals who died with COVID-19 showed that microglia abundantly expressed the lysosomal marker CD68, which is a marker of phagocytic activity, especially in the olfactory bulb and cerebellum [74]. Boroujeni et al. (2021) also demonstrated that the inflammatory response to COVID-19 caused neuronal death in the cerebral cortex (post-mortem) of critically ill patients. NeuN quantification revealed a similar number of marker-positive neurons when we compared the infected and control groups. Nonetheless, the analysis of the ultrastructure of cerebral cortex samples from animals infected with MHV-3 revealed the presence of darkened neurons, known as Dark Neurons (DNs), indicating ongoing degeneration. Golgi complexes and dilated endoplasmic reticulum and mitochondria with ruptured cristae were also observed in these cases. Although the NeuN quantification showed no change, about 90–99% of DNs recover with time, while a small number of neurons die [76]. Electron microscopy studies have shown that these DNs have a low functional activity. However, the origin and mechanism of the emergence of these dark neurons remain undefined [76].

In the present study, we also measured the levels of the excitatory neurotransmitter glutamate and calcium (Ca²⁺) released at presynaptic terminals in the cerebral cortex. Our results showed that MHV-3 infection led to massive glutamate release and increased intracellular Ca²⁺ levels. Other viruses such as HIV, ZIKV, and H1N1 have been reported to impair glutamatergic transmission, which can result in excitotoxicity and impaired cell signaling [19, 51, 82, 83]. Additionally, HCoV-OC43, a human coronavirus, can infect human CNS neuronal and glial cells and activate neuroinflammatory mechanisms [52]. Infection of mice with HCoV-OC43 has been shown to induce neuronal dysfunction and decrease the expression of the glutamate transporter GLT-1 in astrocytes, potentially leading to increased central glutamate levels. We also observed an increase in IL-6 levels in the PFC of animals after MHV-3 infection. IL-6 can be produced within the CNS by various cell types and infiltrating inflammatory cells under neuroinflammatory conditions [54]. It has been reported that membrane depolarization is one of the primary mechanisms for the upregulation of IL-6 in neurons, which is dependent on glutamatergic activation of N-methyl-D-aspartate receptors (NMDA-R), an increase in Ca2⁺ levels, and activation of Ca2⁺/dependent kinases, such as calmodulin [55]. In a ZIKV infection model, we demonstrated that neuronal cell death could be prevented when infected animals were treated with a non-competitive inhibitor of NMDA-R. Neurodegeneration and microgliosis induced by the infection were also inhibited in vitro and in vivo [9, 83].

It is interesting to note that increased levels of IL-6 have been associated with anxiety and depressive symptoms [56, 57], and plays a role in neurogenesis [58]. Additionally, mounting evidence suggests that inflammation and alterations in glutamate neurotransmission may contribute to the pathophysiology of mood disorders [59]. There is evidence indicating that fractalkine has protective effects against glutamate-mediated excitotoxicity, once fractalkine increases BDNF levels and TrkB phosphorylation [60]. In our study, we observed a decrease in cortical levels of fractalkine and BDNF, which leads us to speculate that neuroprotection mechanisms are reduced, contributing to depressive-like behaviors in mice. While IFN-γ contributes to virus clearance [61], we observed a decrease in IFN-γ levels after MHV-3 infection. Interestingly, at low physiological glutamate concentrations, glutamate can directly activate naïve T cells via AMPA-R. However, when glutamate concentration markedly increases, this neurotransmitter usually inhibits T cell function [62].

In order to evaluate the behavioral consequences of MHV-3 infection, we conducted several specific tests. In the elevated plus maze test, we observed anxious-like behavior in the infected animals. This finding is consistent with a previous study on DENV-3 encephalitis, which showed that infected mice also displayed anxiety-like behavior and increased mRNA expression of pro-inflammatory cytokines in the hippocampus associated with neuronal loss [77]. BDNF has been shown to have an antidepressant function in the PFC [66] and hippocampus [67]. In addition, another study demonstrated that administration of BDNF into the hippocampus of rats reduced anxiety-like behavior in the elevated plus maze test [68], suggesting that the reduction in BDNF levels may be associated with the development of anxiety disorders. Moreover, impaired neuron-microglia communication, specifically the CX3CL1/CX3CR1 pathways, has been increasingly linked to the development of psychiatric conditions [69].

Studies performed in transgenic mice (K18-hACE2) that overexpress human ACE-2 and are infected with SARS-CoV-2 strongly support the acute CNS impairments observed in our MHV-3 model. Kumari and colleagues (2021) reported that intranasal inoculation of SARS-CoV-2 in K18-hACE2 mice was associated with neuroinvasion and encephalitis. These findings were corroborated by Oladunni et al. (2020), who demonstrated that intranasal infection of K18-hACE2 mice resulted in viable viral titers in various organs, including the nostril, lungs, and brain. Moreover, the infection was linked to local and systemic chemokine storm, with increased levels of CXCL-2, CXCL-10, and CCL-3 in the brain of SARS-CoV-2-infected mice. Finally, another recent study showed that primary neurons isolated from K18-hACE2 mice are susceptible to SARS-CoV-2 infection, and that the infection upregulates the expression of genes involved in innate immunity and inflammation, including IFN-α, ISG-15, CXCL-10, CCL-2, IL-6, and TNF, in the neurons [85].

According to Andrade et al. [14], there is no gender difference in the lung disease caused by MHV-3 in mice. This finding is supported by Oladunni et al. (2020) who found no significant difference in chemokine levels in the lung between male and female K18-hACE2 transgenic-infected mice, except for CXCL-10 at early time points of infection, which was significantly higher in female K18- hACE2 mice. However, our results indicate significant gender differences in the central nervous system (CNS) of MHV-3 infected mice, particularly in terms of the inflammatory response. Female mice infected with MHV-3 had increased levels of IL-6 in the analyzed brain regions (PFC, hippocampus, and striatum), while this cytokine did not change in male mice. On the other hand, infected male mice showed an increase in CX3CL1 in the PFC and BDNF in the hippocampus compared to the control group, while females infected had reduced levels of these mediators in the PFC. Previous studies have also reported the role of BDNF in the pathogenesis of depression, and its action is directly related to the brain region [65].

The Brazilian COVID Registry Project, a multicenter study that collected data from 39 Brazilian hospitals in 17 cities, observed that patients admitted with COVID-19 and clinically diagnosed with neurological syndromes had a higher incidence of septic shock, ICU admission and death compared to controls [70]. Furthermore, the study separated neurological manifestations presented at hospital admission according to incidence by sex and age group and found that women were more likely to have headaches and less likely to have seizures [71]. In MHV-3 infection in females there was a reduction of BDNF and CX3CL1, which may result in behavioral changes and loss of protection mechanisms. On the other hand, infected males show an increase in CX3CL1 and maintain BDNF levels, which may help to mitigate possible behavioral repercussions.

The MHV-3 model, like other models used to study the neurological effects of COVID-19, has several limitations. Firstly, it is not suitable for investigating the viral entry step, as SARS-CoV-2 uses a different receptor, ACE-2, while MHV-3 enters the cell through the CEACAM-1 adhesion molecule. This limits the usefulness of the model in studying drugs that target viral entry. Additionally, MHV-3 model is very acute and lethal, with mice succumbing to the infection within a short period of time (around 6–7 dpi). As a result, it may be challenging to detect tissue damage in refractory organs such as the brain and heart [86] using regular techniques. However, despite these limitations, the MHV-3 model can still be valuable in mimicking a severe betacoronavirus infection and may serve as a useful platform for further studies on acute neuropsychiatric changes caused by COVID-19, as well as testing potential novel therapeutic strategies.

Acknowledgments: This work was supported by grants from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior –CAPES/Brazil (Projeto: CAPES - Programa: 9951 - Programa Estratégico Emergencial de Prevenção e Combate a Surtos, Endemias, Epidemias e Pandemias AUX 0641/2020 - Processo 88881.507175 /2020 -01). This work also received support from the National Institute of Science and Technology in Dengue and Host -Microorganism Interaction (INCT em Dengue), sponsored by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; Brazil) (Processo CNPQ: 465425 /2014 - 3) and the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG; Brazil) (Processo FAPEMIG: 25036). Also, FAPEMIG process APQ 02281-18. We also acknowledge Ilma Marçal de Souza, Rosemeire Oliveira and Tânia Colina for their technical assistance.

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A suitable model to investigate acute neurological consequences of coronavirus infection

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