The emergence of life-threatening zoonotic diseases caused by betacoronavirus, including the ongoing COVID-19 pandemic, has highlighted the need for developing preclinical models mirroring respiratory and systemic pathophysiological manifestations seen in infected humans. Here, we showed that C57BL/6J wild-type mice intranasally inoculated with the murine betacoronavirus MHV-3 develop a robust inflammatory response leading to acute lung injuries, including alveolar edema, hemorrhage, and fibrin thrombi. Although such histopathological changes seemed to resolve as the infection advanced, they efficiently impaired the respiratory function, as the infected mice displayed restricted lung distention and increased respiratory frequency and ventilation. Following respiratory manifestation, the MHV-3 infection became systemic and a high virus burden could be detected in multiple organs alongside with morphological changes. The systemic manifestation of MHV-3 infection was also marked by a sharp drop in the number of circulating platelets and lymphocytes, besides the augmented concentration of the pro-inflammatory cytokines IL-1β, IL-6, IL-12, IFN-γ, and TNF, thereby mirroring some clinical features observed in moderate and severe cases of COVID-19. Importantly, both respiratory and systemic changes triggered by MHV-3 infection were greatly prevented by blocking TNF signaling, either via genetic or pharmacologic approaches. In line, TNF blockage also diminished the infection-mediated release of pro-inflammatory cytokines and virus replication of human epithelial lung cells infected with SARS-CoV-2. Collectively, results show that MHV-3 respiratory infection leads to a large range of clinical manifestations in mice and may constitute an attractive, lower cost, biosafety level-2 in vivo platform for evaluating the respiratory and multi-organ involvement of betacoronavirus infections. Importance Mouse models have long been used as valuable in vivo platforms to investigate the pathogenesis of viral infections and effective countermeasures. The natural resistance of mice to the novel betacoronavirus SARS-CoV-2, the causative agent of COVID-19, has launched a race towards the characterization of SARS-CoV-2 infection in other animals (e.g. hamsters, cats, ferrets, bats, and monkeys) as well as the adaptation of the mouse model, by either modifying the host or the virus. In the present study, we utilized the natural pathogen of mice MHV as a prototype to model betacoronavirus-induced acute lung injure and multi—organ involvement under biosafety level 2 condition. We showed that C57BL/6J mice intranasally inoculated with MHV-3 develops a severe disease which includes acute lung damage and respiratory distress preceding systemic inflammation and death. Accordingly, the proposed animal model may provide a useful tool for studies regarding betacoronavirus respiratory infection and related diseases.

Study objectives Exposure to postnatal chronic intermittent hypoxia (pCIH), as experienced in sleep-disordered breathing, is a risk factor for developing cardiorespiratory diseases in adulthood. pCIH causes respiratory instability and motor dysfunction that persist until adult life. In this study, we investigated the impact of pCIH on the sympathetic control of arterial pressure in rats. Methods and Results Neonate male Holtzman rats (P0-1) were exposed to pCIH (6% O2 for 30 s, every 10 min, 8 h/day) during their first 10-15 days of life, while control animals were maintained under normoxia. In early adult life (P25-40), freely behaving pCIH animals (n=13) showed higher baseline arterial pressure levels linked to augmented sympathetic-mediated variability than control animals (n=12, P<0.05). Using decerebrated in situ preparations, we found that juvenile pCIH rats exhibited a two-fold increase in thoracic sympathetic nerve activity (n=14) and elevated firing frequency of ventromedullary presympathetic neurons (n=7) compared to control rats (n=6-7, P<0.05). This pCIH-induced sympathetic dysregulation was associated with increased HIF-1α (hypoxic inducible factor) mRNA expression in catecholaminergic pre-sympathetic neurons (n=5,P<0.05). At older age (P90-99), pCIH rats displayed higher arterial pressure levels and larger depressor responses to ganglionic blockade (n=6-8, P<0.05), confirming the sympathetic overactivity state. Conclusions pCIH facilitates the vasoconstrictor sympathetic drive by mechanisms associated with enhanced firing activity and HIF-1α expression in ventromedullary pre-sympathetic neurons. This excessive sympathetic activity persists until adulthood resulting in high blood pressure levels and variability, which contribute to developing cardiovascular diseases.

Periods of apnoea, commonly observed in prematures and newborns, are an important risk factor for the development of cardiorespiratory diseases in adulthood. In the present study, we evaluated changes in pulmonary ventilation and respiratory motor pattern in juvenile and adult rats exposed to postnatal chronic intermittent hypoxia (pCIH). Newborn male Holtzman rats (P1) were submitted to pCIH (6% O 2 for 30 s, every 9 min, 8 h a day (09.30-17.30 h)) during their first 10 days of life, while control animals were maintained under normoxic conditions (20.8% O 2). Thereafter, animals of both groups were maintained under normoxia until the experiments. Unanaesthetized juvenile pCIH rats (n = 27) exhibited elevated tidal volume and respiratory irregularities (P < 0.05) compared to control rats (n = 7). Decerebrate, arterially perfused in situ preparations of juvenile pCIH rats (n = 11) displayed augmented phrenic nerve (PN) burst amplitude and reduced central vagus nerve activity in comparison to controls (n = 10). At adulthood, pCIH rats (n = 5) showed enhanced tidal volume (P < 0.05) and increased respiratory variability compared to the control group (n = 5). The pCIH-induced changes in ventilation and respiratory motor outputs were prevented by treatment with the DNA methyltransferase inhibitor decitabine (1 mg kg −1 , I.P.) during the exposure to pCIH. Our data demonstrate that pCIH in rats impacts, in a persistent way, control of the respiratory pattern, increasing PN activity to the diaphragm and reducing the vagal-related activity to laryngeal muscles, which, respectively, may contribute to improve resting pulmonary ventilation and predispose to collapse of the upper airways during quiet breathing.

Epilepsy is a neurological disease characterized by the recurrence of seizures. Seizures are associated with respiratory suppression and apneas that can increase the risk of death. The highest prevalence of seizures occurs during childhood, however, respiratory changes related to seizures during development are unknown. It is also unknown whether genetic susceptibility to epilepsy itself induces respiratory changes. Thus, the objective of this study is to test the hypothesis that animals genetically susceptible to epilepsy (Wistar Audiogenic Rats, WAR) present respiratory alterations in the neonatal period, defined as presence of apneas and attenuation of ventilatory responses to hypercapnia and hypoxia. Specifically, we hypothesize that the respiratory alterations in these animals precede the occurrence of seizures. For this study, we used WAR and Wistar rats (males and females) in three developmental windows P1-3 (P, postnatal days), P12-14 and P21-23, and in adulthood (>60 days old). Pulmonary ventilation in room air and during hypercapnia (7%CO2) or hypoxia (10%O2) was measured by pressure (P1-3) or whole-body plethysmography (P12-14, P21- 23 and adults). Our results showed that under room air conditions, WAR animals had a higher number of apneas at P1-3 (9.60±3.0 event/15min) compared to the Wistar group (4.55±0.7 event/15min), but not at the other ages studied. The ventilatory response to hypercapnia was not significantly different between the Wistar and WAR groups during development (P1-3: 185.1±12.9 vs 170.5±8; P12-14: 174.7±8.7 vs 159.3±11.2; P21-23: 229.6±16.7 vs 221.5±25.0 VE% at baseline; Wistar vs WAR, respectively). Moreover, the ventilatory response to hypercapnia was attenuated in adult WAR animals only after seizure induced by acoustic stimuli (210±30 vs 150±30 VE% at baseline, Pre-Acoustic Stimuli vs Post Acoustic Stimuli, respectively, P<0.05). The ventilatory response to hypoxia of WAR rats was lower only at P1-3 (127.0±7.8%VE at baseline) when compared to Wistar (168.8±14.6%, P<0.05), but not at P12-14 (127.2±7.4 vs 124.0±6.4) and P21-23 (170.0±18.5 vs 175.6±16.5; Wistar vs WAR, respectively). Overall, the results suggest that genetic susceptibility to epilepsy confers respiratory alterations in early life (P1-3), such as higher occurrence of apneas and attenuated VE response to hypoxia. These alterations disappear during development. Moreover, no difference in CO2 chemoreflex was observed in WAR across ages, suggesting that the impairment of hypercapnic ventilatory responses depends on audiogenic seizure Acknowledgments: financial support; FAPEMIG and CNPq This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.