Neuropsychiatric sequelae in an experimental model of post-COVID syndrome in mice, 2025, Pimenta et al

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Neuropsychiatric sequelae in an experimental model of post-COVID syndrome in mice

Spoiler: Authors

Jordane Clarisse Pimenta a 1, Vinícius Amorim Beltrami a 1, Bruna da Silva Oliveira a 1, Celso Martins Queiroz-Junior a, Jéssica Barsalini a, Danielle Cunha Teixeira a, Luiz Pedro de Souza-Costa d, Anna Luiza Diniz Lima b, Caroline Amaral Machado a, Bárbara Zuccolotto Schneider Guimarães Parreira a, Felipe Rocha da Silva Santos d, Pedro Augusto Carvalho Costa f, Larisse de Souza Barbosa Lacerda a, Matheus Rodrigues Gonçalves e, Ian de Meira Chaves a, Manoela Gonzaga Gontijo Couto a, Victor Rodrigues de Melo Costa e, Natália Ribeiro Cabacinha Nóbrega a, Bárbara Luísa Silva a, Talita Fonseca a, Filipe Resende a, Natália Teixeira Wnuk a, Fernanda Martins Marim i, Felipe Emanuel Oliveira Rocha a, Hanna L. Umezu f, Gabriel Campolina-Silva a g, Ana Cláudia dos Santos Pereira Andrade a h, Renato Santana de Aguiar i, Guilherme Mattos Jardim Costa a, Pedro Pires Goulart Guimarães f, Glauber Santos Ferreira Silva f, Milene Alvarenga Rachid j, Luciene Bruno Vieira b, Vanessa Pinho a, Antônio Lúcio Teixeira c, Mauro Martins Teixeira d, Aline Silva Miranda a, Vivian Vasconcelos Costa a

Highlights
• MHV-A59 inoculation induces a self-limited lung disease that mimics acute COVID-19.
• MHV-A59 infection emulates Post-COVID neuropsychiatric symptoms in female mice.
• Neuropsychiatric changes in mice are dependent on female sex-hormones.
• Ovariectomy prevents neuropsychiatry sequelae induced by MHV-A59 infection.

Abstract
The global impact of the COVID-19 pandemic has been unprecedented, and presently, the world is facing a new challenge known as post-COVID syndrome (PCS). Current estimates suggest that more than 100 million people are grappling with PCS, encompassing several manifestations, including pulmonary, musculoskeletal, metabolic, and neuropsychiatric sequelae (cognitive and behavioral). The mechanisms underlying PCS remain unclear.

The present study aimed to: (i) comprehensively characterize the acute effects of pulmonary inoculation of the betacoronavirus MHV-A59 in immunocompetent mice at clinical, cellular, and molecular levels; (ii) examine potential acute and long-term pulmonary, musculoskeletal, and neuropsychiatric sequelae induced by the betacoronavirus MHV-A59; and to (iii) assess sex-specific differences.

Male and female C57Bl/6 mice were initially inoculated with varying viral titers (3x103 to 3x105 PFU/30 μL) of the betacoronavirus MHV-A59 via the intranasal route to define the highest inoculum capable of inducing disease without causing mortality. Further experiments were conducted with the 3x104 PFU inoculum.

Mice exhibited an altered neutrophil/lymphocyte ratio in the blood in the 2nd and 5th day post-infection (dpi). Marked lung lesions were characterized by hyperplasia of the alveolar walls, infiltration of polymorphonuclear leukocytes (PMN) and mononuclear leukocytes, hemorrhage, increased concentrations of CCL2, CCL3, CCL5, and CXCL1 chemokines, as well as high viral titers until the 5th dpi.

While these lung inflammatory signs resolved, other manifestations were observed up to the 60 dpi, including mild brain lesions with gliosis and hyperemic blood vessels, neuromuscular dysfunctions, anhedonic-like behavior, deficits in spatial working memory, and short-term aversive memory. These musculoskeletal and neuropsychiatric complications were exclusive to female mice and prevented after ovariectomy.

In summary, our study describes for the first time a novel sex-dependent model of PCS focused on neuropsychiatric and musculoskeletal disorders. This model provides a unique platform for future investigations regarding the effects of acute therapeutic interventions on the long-term sequelae unleashed by betacoronavirus infection.

Web | Brain, Behavior, and Immunity | Paywall

 

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This is testing infecting mice with MHV-A59:

Wild-type mice have shown resistance to SARS-CoV-2 infection (Dinnon et al., 2020; Gu et al., 2020). To study coronavirus infections in mice, researchers commonly use betacoronaviruses such as Murine Hepatitis Viruses (MHV-1, MHV-3, MHV-A59, and MHV-S strains), which naturally infect mice. They are associated with pulmonary infection and disease, effectively mimicking several aspects of human coronavirus infections (Andrade et al., 2021a; De Albuquerque et al., 2006; Yang et al., 2014).

The section about sex differences in long-term behavioral effects:

3.3. MHV-A59-infected mice display behavioral and cognitive alterations in a time and sex-dependent manner

Following the characterization of lung effects induced by MHV-A59, we explored potential neuropsychiatric sequelae associated with post- COVID syndrome (Xu et al., 2022). We analyzed behavioral and cognitive changes in male and female mice infected with MHV-A59 (Fig. 3A). In the open field test, no significant difference was found at 5 dpi (peak of lung disease) regardless of sex. Meanwhile, female MHV-A59 mice exhibited a significant decrease in spontaneous locomotor activity at 16 dpi. Notably, female mice exhibited complete recovery at 28 dpi, as evidenced by the total distance traveled in the open field. MHV-A59 infection did not result in locomotor activity impairment in male mice (Fig. 3B-D).

As olfactory loss is a core symptom of COVID-19 (Harapan and Yoo, 2021), we investigated olfactory discrimination memory at early and later time points after MHV-A59 infection. Male-infected mice had no disturbance in the olfactory discrimination memory, while at 6 dpi female-infected mice presented a significant dysfunction compared to mock groups (Fig. 3E). Olfactory discrimination memory dysfunction was completely resolved by 38 dpi (Fig. 3F). However, from this test alone we cannot state that infected female mice exhibit anosmia. Neuromuscular dysfunction is also an important symptom related to COVID-19 (Rossi et al., 2023). At 16 dpi, only female MHV-A59-infected mice displayed a significant decrease in the forelimb and all limbs grip force compared with mock controls (Fig. 3G). Grip strength impairments were not observed for forelimbs and all limbs at 28 dpi regardless of sex (Fig. 3H).

Male MHV-A59-infected mice displayed an anhedonic-like behavior, as indicated by a significant decrease in the percentage of sucrose preference at 17 dpi (Fig. 3I). A reduction in the percentage of sucrose preference was also observed in infected female mice (Fig. 3I), however it did not reach statistical significance (p = 0.056). Interestingly, female animals presented a decrease in the number of buried marbles at 34 dpi, also suggesting an anhedonic-like behavior (Fig. 3J).

Importantly, only female mice showed significant cognitive dysfunctions at 60 dpi MHV-A59 infection. There was a significant decrease in the percentage of spontaneous alternations in the Y maze, indicating an impairment in spatial working memory. No significant differences were found between MHV-A59 infected male mice and mock controls (Fig. 3K). Regarding aversive memory, mock and infected mice displayed similar step-down latency in the training session regardless of sex (Fig. 3L). MHV-A59 female mice showed impairment in short-term but not long-term aversive memory compared with mocks, as indicated by a decrease in the step-down latency 1.5 h but not 24 h after the training session. No significant changes in aversive memory were observed in male mice after MHV-A59 infection (Fig. 3L-N).

Figure 3:

Spoiler: Fig 3 Caption

Fig. 3. MHV-A59 induces behavioral and cognitive alterations in a time and sex dependent manner. (A) Experimental design. Total distance traveled in the open field test at (B) 5-, (C) 16-, and (D) 28-dpi (n = 13–18). Olfactory discrimination time (E) 6- and (F) 38-dpi (n = 7–15). Force in newtons (N) in 2 or 4 paws grip force test at (G) 16- and (H) 28-dpi (n = 10–16). (I) Sucrose preference test at 17-dpi (n = 10–12). (J) Marble burying test at 34-dpi (n = 13–18). (K) Spontaneous alternation in Y-maze test at 60-dpi (n = 7–8). (L) Latency in training session, (M) short-term aversive memory, and (N) long-term aversive memory, in step-down inhibitory avoidance test at 60-dpi (n = 10–16). Significance was determined by Student’s t-test to data that passed Shapiro-Wilk test and Mann–Whitney test to data that did not pass Shapiro-Wilk test *p < 0.05.

Looking at the figures for what stands out most clearly for sex differences:

• Decreased olfactory discrimination in females at 6 days post infection (dpi) and still but less so at 38 dpi (3E,F)
• Decreased marble burying in females at 34 dpi (3J)
• Decreased spontaneous alternations in females at 60 dpi (3K)
• Decreased short-term (3M) but not long-term aversive memory (3N) in females at 60 dpi (how well do they remember being shocked).

Comparing to the recent preprint about a similar study but with SARS-CoV-2: Mechanisms of sex differences in acute and long COVID sequelae in mice, 2025, Liu et al.

• Little difference between sexes for odor discrimination:

In the social odor cue test, both infected males and females were impaired in their ability to distinguish social odors as compared with their mock-infected counterparts 7-84 dpi

• The other study also found decreased marble burying in females but not males, in this case at 42 and 84 dpi:

Infected females buried fewer marbles in the marble burying test as compared with either infected males or mock infected females, suggesting some alteration in compulsive-like behavior following infection, consistent with hamsters (Extended Data Fig. 3M).

• Also found decreased spontaneous alternations in females:

As early as 7 dpi (Extended Data Fig. 3B) and continuing through 42 and 84 dpi (Fig. 3B), infected females had lower percentages of correct spontaneous alternation than either males or mock-infected females.

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The main long-term female-associated behavioral findings that are consistent, in my opinion, appear to be worse memory (based on maze spontaneous alternations and short-term aversive memory), and decreased marble-burying. The interpretation of marble-burying seems to not be straightforward, as the other paper referred to the finding as an alteration in compulsive behavior, while this paper says it suggests anhedonic behavior.

Edit: typo

 

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Increases in CD4, CD8, and NK cells that express IFN gamma at 8 dpi in females:

At 5 dpi, female infected mice showed an increased number of T CD4+ lymphocytes in the lungs (Fig. 2F). At 8 dpi, a pro-inflammatory subset of these cells, characterized by elevated levels of IFN-γ, accumulated in female infected mice but not in males (Fig. 2G). The T CD4-+ lymphocytes were characterized by high levels of the cell proliferation marker Ki67+ (Fig. 2H). Both female and male MHV-A59-infected mice exhibited an accumulation of T CD8+ lymphocytes in lung tissue (Fig. 2I). However, only female infected mice demonstrated an increase in the IFN-γ+ secreting CD8+ T lymphocyte subset in the lungs (Fig. 2J). While the overall number of pulmonary NK cells remained consistent between female and male mice throughout the disease (data not shown), a subpopulation of NK cells with high IFN-γ levels increased solely in the lung tissue of female infected mice (Fig. 2K). Protective humoral immunity against SARS-CoV-2 is a crucial determinant in COVID-19, correlating with clinical outcomes (Carrillo et al., 2021).

Notably, males exhibited prolonged IgM plasma levels compared to females, whereas females demonstrated faster IgG production compared to male infected mice (Fig. 2L-M).

males exhibited increased chemokine production, while females demonstrated higher T lymphocyte activation.

Fig 4:

Spoiler: Fig 4 Caption

Fig. 2. Intranasal infection with MHV-A59 promotes differential accumulation of pulmonary chemokines and leukocytes. (A) Z Score representation of pulmonary levels of CXCL1, CCL2, CCL3, CCL5 measured by ELISA assay. Significance was determined by Two-way ANOVA and Dunnet’s multiple comparison test *p < 0.01 (n = 4–6). Number of pulmonary leukocytes (B), neutrophils (C), dendritic cells (D), IL-10+ dendritic cells (E), CD4+ T lymphocytes (F), IFN-γ+ CD4+ T lymphocytes (G), Ki67+ CD4+ T lymphocytes (H), CD8+ T lymphocytes (I), IFN-γ+ CD8+ T lymphocytes (J), and IFN-γ+ NK cells (K), assessed by flow cytometry. Significance was determined by Two-way ANOVA and Dunnet’s multiple comparison test to analyze the differences between mock controls and infected mice of respective sex at different time points. Significance was determined by Two-way ANOVA and the Šídák multiple comparison test to analyze the differences between the sexes at the respective infection time points. *p < 0.05 to data that passed Shapiro-Wilk test and *p < 0.01 to data that did not pass Shapiro-Wilk test (n = 5–6). Plasma levels of IgM (L) and IgG (M). Significance was determined by Two-way ANOVA and Dunnet’s multiple comparison test to analyze the differences between mock controls and infected mice of respective sex at different time points. Significance was determined by Two-way ANOVA and the Šídák multiple comparison test to analyze the differences between the sexes at the respective infection time points *p < 0.05 (n = 6–8).

 

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Investigating effects on the brain

MHV-A59 RNA viral copies were found in the brains of infected animals from 2- to 8-dpi, in both sexes. However, the replicative virus was not detected by plaque assay (data not shown). The highest numbers of virus RNA copies were detected on days 5 and 8 dpi, with females having notably more virus at 5 dpi compared to males (Fig. 4B).

Brain viral load higher in females at 5 dpi, but gone from both sexes by 16 dpi.


Increased calcium and glutamate in the hippocampus in females but not males, but no changes in these chemicals in cortex:

A potential mechanism associated with brain damage is neuronal excitotoxicity (Verma et al., 2022). To examine this hypothesis, we assessed intracellular calcium and glutamate levels in isolated nerve terminals, specifically synaptosomes isolated from the hippocampus (Fig. 4C and D, respectively) and cortex (Fig. S3A-B, respectively) of both female and male mice at 5- and 30-dpi. Female, but not male mice, exhibited higher levels in intrasynaptosomal calcium concentrations (Fig. 4C) and an increase in hippocampal glutamate levels (Fig. 4D) after MHV-A59 infection. As for the cortex, no changes were found in glutamate and calcium levels in both sexes (Fig. S3A-B).

They talk about sex differences in microglial and astrocyte activation:

Histopathological analysis of the cerebral cortex revealed discrete changes in both sexes, such as the presence of leukocytes surrounding some hyperemic vessels (Fig. S3C-D). Immunohistochemical assays showed an increase in the number of IBA-1+ (microglia/macrophages) cells in the brain cortex after MHV-A59 infection in both sexes (Fig. 4E). In males, this increase was detected from the 2nd to the 60th-dpi when compared to the mock group, while females showed an increase from the 5th-dpi onwards (Fig. 4E). Noteworthy, at 8-dpi, females showed higher numbers of IBA1+ cells when compared to males (Fig. 4E). IBA-1 labeling was also performed in the hippocampus and there was a similar profile of IBA-1+ cells in both sexes. Relative to their respective mocks, there was an increase in cells labeled for IBA-1 from 2- to 60-dpi (Fig. 4F). The number of S100B+ astrocytes increased in the brain cortex of female mice after the 2nd-dpi onwards when compared to the mock group. In males, this increase was only observed on the 2nd-dpi (Fig. 4G). Significant difference was observed between females and males at 5 and 16-dpi, with females showing higher numbers than males (Fig. 4G). A similar pattern was detected in the hippocampus (Fig. 4H). Females showed a significant increase in S100B+ cells from 2- to 16-dpi when compared to their control group, while no increase was observed in males. When comparing the sexes, females exhibited higher numbers of hippocampus S100B+ cells at 5-, 8-, and 16-dpi than males (Fig. 4H).

Increased S100B+ astrocytes in the hippocampus up to 16 dpi in females seems to be the most pronounced difference from males (Fig 4H).

 

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More brain findings. Just picking out the figures that visually look the most like there's a sex difference.

The number of neutrophils increased in the brains of females at 8-dpi, but not in males (Fig. 5B).

The expression of iNOS in this population of activated microglia was upregulated in females and was significantly higher when compared to the male group (Fig. 5D).

MHV-A59 infection also prompted a lymphocytic response in brain tissue, which was prominent in infected female mice since they exhibited higher numbers of T CD4+ lymphocytes at 5-dpi than their control group and males at the same time point (Fig. 5E).

Additionally, at 5-dpi, there was an accumulation of a pro-inflammatory subpopulation of T CD4+ lymphocytes in the brain of infected female mice, expressing CD69+, which is a marker of early activation, Ki67+, a marker of cell proliferation, aside from high levels of IFN-γ (Fig. 5F-H).

In line with the leukocyte infiltration, MHV-A59 induced an increase in CX3CL1 in the prefrontal cortex (PFC) of females at 2- and 5-dpi when compared to the mock group, while in males, such increase occurred only at 2-dpi. Furthermore, this increase was significantly more pronounced in females (Fig. 5M).

Females also showed a reduction in BDNF levels [in the prefrontal cortex] at the 8th and 16th − dpi in the PFC, while males showed this reduction only at 2 dpi. At the 30th − dpi, there was a significant difference between the sexes, with males exhibiting lower levels of BDNF than females (Fig. 5N).

male mice infected with MHV-A59 showed an increase in IL-6 levels in the PFC at 30- and 60-dpi, while infected females showed no alterations (Fig. 5O).

 

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Removing the ovaries of the mice before infection prevented the decrease in marble burying at 34 dpi. It also reduced the spatial memory problem at 60 dpi, but it looks like it didn't completely eliminate it.

Importantly, ovariectomy also prevented the MHV-A59-associated anhedonia-like behavior and spatial working memory deficit at 34- and 60-dpi, respectively (Fig. 6I-J).

Accordingly, calcium concentration in the hippocampus of SHAM-infected animals increased relative to their control group at 30 dpi, while OVX-infected mice exhibited no significant alterations at the same time point (Fig. 6L). In contrast, glutamate release increased similarly in the hippocampus of both SHAM and OVX-infected groups (Fig. 6M). Accordingly, OVX prevented the mild histopathological damage induced by MHV-A59 in brain tissue compared with the SHAM-infected group at the corresponding time point of infection (Fig. 6N).

It looks like calcium and glutamate levels in hippocampus were not as high 30 dpi in mice with ovariectomies.