Long-Lasting Impairments in Quadriceps Mitochondrial Health, Muscle Size, and Phenotypic Composition Are Present After Non-Invasive Anterior Cruciate Ligament Injury

Introduction: Despite rigorous rehabilitation aimed at restoring muscle health, anterior cruciate ligament (ACL) injury is often hallmarked by significant long-term quadriceps muscle weakness. Derangements in mitochondrial function are a common feature of various atrophying conditions, yet it is unclear to what extent mitochondria are involved in the detrimental sequela of quadriceps dysfunction after ACL injury. Using a preclinical, non-invasive ACL injury rodent model, our objective was to explore the direct effect of an isolated ACL injury on mitochondrial function, muscle atrophy, and muscle phenotypic transitions. Methods: A total of 40 male and female, Long Evans rats (16-week-old) were exposed to non-invasive ACL injury, while 8 additional rats served as controls. Rats were euthanized at 3, 7, 14, 28, and 56 days after ACL injury, and vastus lateralis muscles were extracted to measure the mitochondrial respiratory control ratio (RCR; state 3 respiration/state 4 respiration), mitochondrial reactive oxygen species (ROS) production, fiber cross sectional area (CSA), and fiber phenotyping. Alterations in mitochondrial function and ROS production were detected using two-way (sex:group) analyses of variance. To determine if mitochondrial characteristics were related to fiber atrophy, individual linear mixed effect models were run by sex. Results: Mitochondria-derived ROS increased from days 7 to 56 after ACL injury (30–100%, P \u3c 0.05), concomitant with a twofold reduction in RCR (P \u3c 0.05). Post-injury, male rats displayed decreases in fiber CSA (days 7, 14, 56; P \u3c 0.05), loss of IIa fibers (day 7; P \u3c 0.05), and an increase in IIb fibers (day 7; P \u3c 0.05), while females displayed no changes in CSA or phenotyping (P \u3e 0.05). Males displayed a positive relationship between state 3 respiration and CSA at days 14 and 56 (P \u3c 0.05), while females only displayed a similar trend at day 14 (P = 0.05). Conclusion: Long-lasting impairments in quadriceps mitochondrial health are present after ACL injury and play a key role in the dysregulation of quadriceps muscle size and composition. Our preclinical data indicate that using mitoprotective therapies may be a potential therapeutic strategy to mitigate alterations in muscle size and characteristic after ACL injury

Glucocortiocoid Treatment of MCMV Infected Newborn Mice Attenuates CNS Inflammation and Limits Deficits in Cerebellar Development

Infection of the developing fetus with human cytomegalovirus (HCMV) is a major cause of central nervous system disease in infants and children; however, mechanism(s) of disease associated with this intrauterine infection remain poorly understood. Utilizing a mouse model of HCMV infection of the developing CNS, we have shown that peripheral inoculation of newborn mice with murine CMV (MCMV) results in CNS infection and developmental abnormalities that recapitulate key features of the human infection. In this model, animals exhibit decreased granule neuron precursor cell (GNPC) proliferation and altered morphogenesis of the cerebellar cortex. Deficits in cerebellar cortical development are symmetric and global even though infection of the CNS results in a non-necrotizing encephalitis characterized by widely scattered foci of virus-infected cells with mononuclear cell infiltrates. These findings suggested that inflammation induced by MCMV infection could underlie deficits in CNS development. We investigated the contribution of host inflammatory responses to abnormal cerebellar development by modulating inflammatory responses in infected mice with glucocorticoids. Treatment of infected animals with glucocorticoids decreased activation of CNS mononuclear cells and expression of inflammatory cytokines (TNF-α, IFN-β and IFNγ) in the CNS while minimally impacting CNS virus replication. Glucocorticoid treatment also limited morphogenic abnormalities and normalized the expression of developmentally regulated genes within the cerebellum. Importantly, GNPC proliferation deficits were normalized in MCMV infected mice following glucocorticoid treatment. Our findings argue that host inflammatory responses to MCMV infection contribute to deficits in CNS development in MCMV infected mice and suggest that similar mechanisms of disease could be responsible for the abnormal CNS development in human infants infected in-utero with HCMV

Tumor Necrosis Factor Alpha-Induced Recruitment of Inflammatory Mononuclear Cells Leads to Inflammation and Altered Brain Development in Murine Cytomegalovirus-Infected Newborn Mice

Congenital human cytomegalovirus (HCMV) infection is a significant cause of abnormal neurodevelopment and long term neurological sequelae in infants and children. Resident cell populations of the developing brain have been suggested to be more susceptible to virus-induced cytopathology, a pathway thought to contribute to the clinical outcomes following intrauterine HCMV infection. However, recent findings in a newborn mouse model of this infection in the developing brain have indicated that elevated levels of proinflammatory mediators leading to mononuclear cell activation and recruitment could underlie the abnormal neurodevelopment. In this study, we demonstrated that treatment with TNFα neutralizing antibodies decreased the frequency of CD45+/Ly6C HI: /CD11b+/CCR2+ activated myeloid mononuclear cells (MMC) and the levels of proinflammatory cytokines in the blood and the brains of murine CMV infected mice. This treatment also normalized neurodevelopment in infected mice without significantly impacting the level of virus replication. These results indicate that TNFα is a major component of the inflammatory response associated with altered neurodevelopment that follows murine CMV infection of the developing brain and that a subset of peripheral blood myeloid mononuclear cells represent a key effector cell population in this model of virus-induced inflammatory disease of the developing brain. Congenital human cytomegalovirus (HCMV) is the most common viral infection of the developing human fetus and can result in neurodevelopmental sequelae. Mechanisms of disease leading to neurodevelopmental deficits in infected infants remain undefined but postulated pathways include loss of neuronal progenitor cells, damage to the developing vascular system of the brain, and altered cellular positioning. Direct virus-mediated cytopathic effects cannot explain the phenotypes of brain damage in most infected infants. Using a mouse model that recapitulates characteristics of the brain infection described in human infants, we have shown that TNFα plays a key role in brain inflammation, including recruitment of inflammatory mononuclear cells. Neutralization of TNFα normalized neurodevelopmental abnormalities in infected mice providing evidence that virus-induced inflammation is a major component of disease in the developing brain. These results suggest that interventions limiting inflammation associated with this infection could potentially improve the neurologic outcome of infants infected in-utero with HCMV