Mitochondria Transplantation: Rescuing Innate Muscle Bioenergetic Impairment in a Model of Aging and Exercise Intolerance.

ABSTRACT: Arroum, T, Hish, GA, Burghardt, KJ, Ghamloush, M, Bazzi, B, Mrech, A, Morse, PT, Britton, SL, Koch, LG, McCully, JD, Hüttemann, M, and Malek, MH. Mitochondria transplantation: Rescuing innate muscle bioenergetic impairment in a model of aging and exercise intolerance. J Strength Cond Res 38(7): 1189-1199, 2024-Mitochondria, through oxidative phosphorylation, are crucial for energy production. Disease, genetic impairment, or deconditioning can harm muscle mitochondria, affecting energy production. Endurance training enhances mitochondrial function but assumes mobility. Individuals with limited mobility lack effective treatments for mitochondrial dysfunction because of disease or aging. Mitochondrial transplantation replaces native mitochondria that have been damaged with viable, respiration-competent mitochondria. Here, we used a rodent model selectively bred for low-capacity running (LCR), which exhibits innate mitochondrial dysfunction in the hind limb muscles. Hence, the purpose of this study was to use a distinct breed of rats (i.e., LCR) that display hereditary skeletal muscle mitochondrial dysfunction to evaluate the consequences of mitochondrial transplantation. We hypothesized that the transplantation of mitochondria would effectively alleviate mitochondrial dysfunction in the hind limb muscles of rats when compared with placebo injections. In addition, we hypothesized that rats receiving the mitochondrial transplantation would experience an improvement in their functional capacity, as evaluated through incremental treadmill testing. Twelve aged LCR male rats (18 months old) were randomized into 2 groups (placebo or mitochondrial transplantation). One LCR rat of the same age and sex was used as the donor to isolate mitochondria from the hindlimb muscles. Isolated mitochondria were injected into both hindlimb muscles (quadriceps femoris, tibialis anterior (TA), and gastrocnemius complex) of a subset LCR (n = 6; LCR-M) rats. The remaining LCR (n = 5; LCR-P) subset received a placebo injection containing only the vehicle without the isolated mitochondria. Four weeks after mitochondrial transplantation, rodents were euthanized and hindlimb muscles harvested. The results indicated a significant (p < 0.05) increase in mitochondrial markers for glycolytic (plantaris and TA) and mixed (quadricep femoris) muscles, but not oxidative muscle (soleus). Moreover, we found significant (p < 0.05) epigenetic changes (i.e., hypomethylation) at the global and site-specific levels for a key mitochondrial regulator (transcription factor A mitochondrial) between the placebo and mitochondrial transplantation groups. To our knowledge, this is the first study to examine the efficacy of mitochondrial transplantation in a rodent model of aging with congenital skeletal muscle dysfunction.

Modulation of mitochondrial function with near-infrared light reduces brain injury in a translational model of cardiac arrest.

BACKGROUND: Brain injury is a leading cause of morbidity and mortality in patients resuscitated from cardiac arrest. Mitochondrial dysfunction contributes to brain injury following cardiac arrest; therefore, therapies that limit mitochondrial dysfunction have the potential to improve neurological outcomes. Generation of reactive oxygen species (ROS) during ischemia-reperfusion injury in the brain is a critical component of mitochondrial injury and is dependent on hyperactivation of mitochondria following resuscitation. Our previous studies have provided evidence that modulating mitochondrial function with specific near-infrared light (NIR) wavelengths can reduce post-ischemic mitochondrial hyperactivity, thereby reducing brain injury during reperfusion in multiple small animal models. METHODS: Isolated porcine brain cytochrome c oxidase (COX) was used to investigate the mechanism of NIR-induced mitochondrial modulation. Cultured primary neurons from mice expressing mitoQC were utilized to explore the mitochondrial mechanisms related to protection with NIR following ischemia-reperfusion. Anesthetized pigs were used to optimize the delivery of NIR to the brain by measuring the penetration depth of NIR to deep brain structures and tissue heating. Finally, a model of out-of-hospital cardiac arrest with CPR in adult pigs was used to evaluate the translational potential of NIR as a noninvasive therapeutic approach to protect the brain after resuscitation. RESULTS: Molecular evaluation of enzyme activity during NIR irradiation demonstrated COX function was reduced in an intensity-dependent manner with a threshold of enzyme inhibition leading to a moderate reduction in activity without complete inhibition. Mechanistic interrogation in neurons demonstrated that mitochondrial swelling and upregulation of mitophagy were reduced with NIR treatment. NIR therapy in large animals is feasible, as NIR penetrates deep into the brain without substantial tissue heating. In a translational porcine model of CA/CPR, transcranial NIR treatment for two hours at the onset of return of spontaneous circulation (ROSC) demonstrated significantly improved neurological deficit scores and reduced histologic evidence of brain injury after resuscitation from cardiac arrest. CONCLUSIONS: NIR modulates mitochondrial function which improves mitochondrial dynamics and quality control following ischemia/reperfusion. Noninvasive modulation of mitochondria, achieved by transcranial treatment of the brain with NIR, mitigates post-cardiac arrest brain injury and improves neurologic functional outcomes.

Mitochondrial Transplantation's Role in Rodent Skeletal Muscle Bioenergetics: Recharging the Engine of Aging.

BACKGROUND: Mitochondria are the 'powerhouses of cells' and progressive mitochondrial dysfunction is a hallmark of aging in skeletal muscle. Although different forms of exercise modality appear to be beneficial to attenuate aging-induced mitochondrial dysfunction, it presupposes that the individual has a requisite level of mobility. Moreover, non-exercise alternatives (i.e., nutraceuticals or pharmacological agents) to improve skeletal muscle bioenergetics require time to be effective in the target tissue and have another limitation in that they act systemically and not locally where needed. Mitochondrial transplantation represents a novel directed therapy designed to enhance energy production of tissues impacted by defective mitochondria. To date, no studies have used mitochondrial transplantation as an intervention to attenuate aging-induced skeletal muscle mitochondrial dysfunction. The purpose of this investigation, therefore, was to determine whether mitochondrial transplantation can enhance skeletal muscle bioenergetics in an aging rodent model. We hypothesized that mitochondrial transplantation would result in sustained skeletal muscle bioenergetics leading to improved functional capacity. METHODS: Fifteen female mice (24 months old) were randomized into two groups (placebo or mitochondrial transplantation). Isolated mitochondria from a donor mouse of the same sex and age were transplanted into the hindlimb muscles of recipient mice (quadriceps femoris, tibialis anterior, and gastrocnemius complex). RESULTS: The results indicated significant increases (ranging between ~36% and ~65%) in basal cytochrome c oxidase and citrate synthase activity as well as ATP levels in mice receiving mitochondrial transplantation relative to the placebo. Moreover, there were significant increases (approx. two-fold) in protein expression of mitochondrial markers in both glycolytic and oxidative muscles. These enhancements in the muscle translated to significant improvements in exercise tolerance. CONCLUSIONS: This study provides initial evidence showing how mitochondrial transplantation can promote skeletal muscle bioenergetics in an aging rodent model.

Quantitative and Qualitative Behavioral Measurements to Assess Pain in Axolotls (Ambystoma mexicanum).

Effective pain relief in animals relies on the ability to discern pain and assess its severity. However, few objective measures exist to assess the presence and severity of pain in axolotls, and few resources are available regarding drugs and appropriate doses to provide pain relief in this species. This study evaluated behavioral tools for cageside pain assessment and validated a reproducible and reliable quantitative method to evaluate analgesic efficacy in axolotls. Animals were divided into control and treatment groups (n = 6 per group); treatment groups received buprenorphine through injection (50 mg/kg every 24 h for 48 h intracelomically) or butorphanol immersion (0.50 or 0.75 mg/L every 24 h for 48 h). Qualitative behavioral tests, adapted from other amphibian studies, included tapping on the home tank, directing water jets or physically touching specific anatomic points on the animal, and placing a novel object in the home tank. Quantitative methods used to produce noxious stimuli were the acetic acid test and von Frey aesthesiometers. Animals that were treated with analgesics did not demonstrate a significant difference compared with controls during behavioral assessment at 1, 6, 12, 25, 30, and 48 h after analgesia administration. The acetic acid test revealed a reproducible, concentration-dependent pain response. However, a significant difference in the AAT response was not observed between control and treated groups with the tested analgesics and doses.

Management of Multiple Protozoan Ectoparasites in a Research Colony of Axolotls (Ambystoma mexicanum).

Axolotls (Ambystoma mexicanum) from a research colony presented with multifocal, white chalky to gray skin lesions, a diffuse whitish to blue hue to the integument, and friable gill filaments. Skin scrapings and wet mounts revealed Chilodonella, Ichthyobodo, and a trichodinid species. The average overall burden (that is, all 3 species) per axolotl ranged from 0 to 25 parasites per 40 × field (p40f; mean ± 1 SD, 2.6 ± 5.5), with a prevalence of 12%, 60%, and 48%, respectively. Concurrent with husbandry modifications, axolotls were treated with an 8-h static immersion bath that contained 0.025 mL/L 37% formaldehyde. Chilodonella organisms were no longer observed after the initial treatment, and Ichthyobodo decreased from 2.4 ± 5.6 to 0.6 ± 1.8 organisms p40f. However, the average overall burden increased 4-fold to 10.5 ± 9.8 parasites p40f, and the trichodinid organisms increased 13-fold from 0.8 ± 2.3 to 10.4 ± 9.2 organisms p40f. A second treatment consisted of an 8-h immersion bath that contained 0.05 mL/L 37% formaldehyde on 2 consecutive days. A significant change was noted in the average overall burden of 0.5 ± 1.1 parasites p40f, a greater than 5- and 21-fold decrease from pretreatment and after the initial treatment, respectively. No significant change between the first and second treatment was observed for Ichthyobodo, with 0.6 ± 1.2 organisms p40f, but this number represented a significant decrease from pretreatment. After the second treatment, the trichodinid organism was detected in only one axolotl, with a low overall burden of 0.2 ± 0.4 organisms p40f and resulting in a significant decrease in the trichodinid count to 0.01 ± 0.04 organisms p40f. Treatment with formalin (37% formaldehyde), in conjunction with husbandry improvements, was effective in significantly reducing ectoparasite burden and eliminating clinical symptoms in axolotls but did not fully eliminate all protozoa.

Ulcerative Dermatitis in C57BL/6NCrl Mice on a Low-Fat or High-Fat Diet With or Without a Mineralized Red-Algae Supplement.

Ulcerative dermatitis (UD) is a spontaneous idiopathic disease that often affects C57BL/6 mice or mice on a C57BL/6 background. UD is characterized by intense pruritus and lesion formation, most commonly on the head or dorsal thorax. Self-trauma likely contributes to wound severity and delayed wound healing. Histologically, changes are nonspecific, consisting of ulceration with neutrophilic and mastocytic infiltration and epithelial hyperplasia and hyperkeratosis. Diet appears to have a profound effect on the development and progression of UD lesions. We investigated the incidence and severity of UD in C57BL/6NCrl mice on a high-fat western-style diet (HFWD) compared with a standard rodent chow. In addition, we examined the protective effects of dietary supplementation with a multimineral-rich product derived from marine red algae on UD in these 2 diet groups. HFWD-fed mice had an increased incidence of UD. In addition, mice on a HFWD had significantly more severe clinical and histologic lesions. Dietary mineral supplementation in mice on a HFWD decreased the histologic severity of lesions and reduced the incidence of UD in female mice in both diets. In conclusion, a high-fat western-style diet may potentiate UD in C57BL/6NCrl mice. Insufficient mineral supply and mineral imbalance may contribute to disease development. Mineral supplementation may be beneficial in the treatment of UD.

Effects of analgesic use on inflammation and hematology in a murine model of venous thrombosis.

Venous thrombosis (VT) is a significant cause of morbidity and mortality in humans. Surgical animal models are crucial in studies investigating the pathogenesis of this disease and evaluating VT therapies. Because inflammation is critical to both the development and resolution of VT, analgesic medications have the potential to adversely affect multiple parameters of interest in VT research. The objective of this study was to determine how several common analgesics affect key variables in a murine ligation model of deep vein thrombosis. Male C57BL/6 mice were randomly assigned to receive either local (bupivacaine) or systemic parenteral analgesia (buprenorphine, tramadol, or carprofen) or 0.9% NaCl (control). All mice underwent laparotomy and ligation of the inferior vena cava, and treatment was continued until euthanasia at 6 or 48 h after surgery. Analysis of harvested tissues and blood included: hematology, thrombus weight, serum and vein-wall cytokines (IL1ß, IL6, IL10, TNFα), soluble P-selectin, and vein-wall leukocyte infiltration. Compared with 0.9% NaCl, all of the analgesics affected multiple parameters important to VT research. Carprofen and tramadol affected the most parameters and should not be used in murine models of VT. Although they affected fewer parameters, a single dose of bupivacaine increased thrombus weight at 6 h, and buprenorphine was associated with reduced vein wall macrophages at 48 h. Although we cannot recommend the use of any of the evaluated analgesic dosages in this mouse model of VT, buprenorphine merits additional investigation to ensure the highest level of laboratory animal care and welfare.

Naturally occurring disseminated group B streptococcus infections in postnatal rats.

Group B Streptococcus (Streptococcus agalactiae, GBS) is a gram-positive commensal and occasional opportunistic pathogen of the human vaginal, respiratory, and intestinal tracts that can cause sepsis, pneumonia, or meningitis in human neonates, infants, and immunosuppressed persons. We report here on a spontaneous outbreak of postnatal GBS-associated disease in rats. Ten of 26 (38.5%) 21- to 24-d-old rat pups died or were euthanized due to a moribund state in a colony of rats transgenic for the human diphtheria toxin receptor on a Munich-Wistar-Frömter genetic background. Four pups had intralesional coccoid bacteria in various organs without accompanying inflammation. GBS was isolated from the liver of 2 of these pups and from skin abscesses in 3 littermates. A connection with the transgene could not be established. A treatment protocol was evaluated in the remaining breeding female rats. GBS is a potentially clinically significant spontaneous infection in various populations of research rats, with some features that resemble late-onset postnatal GBS infection in human infants.

Progression of ulcerative dermatitis lesions in C57BL/6Crl mice and the development of a scoring system for dermatitis lesions.

Ulcerative dermatitis (UD) is a common, spontaneous condition in mice with a C57BL/6 background. Although initial lesions may be mild, UD is a progressive disease that often results in ulcerations or debilitating fibrotic contractures. In addition, lesions typically are unresponsive to treatment. Euthanasia is often warranted in severe cases, thereby affecting study outcomes through the loss of research subjects. Because the clinical assessment of UD can be subjective, a quantitative scoring method and documentation of the likely time-frame of progression may be helpful in predicting when animals that develop dermatitis should be removed from a study. Such a system may also be helpful in quantitatively assessing success of various treatment strategies and be valuable to clinical laboratory animal veterinarians. In this 1.5-y, prospective cohort study, we followed 200 mice to monitor the development and course of UD. Mice were examined every 2 wk. A clinical sign (alopecia, pruritus, or peripheral lymphadenopathy) was not identified that predicted development of UD lesions in the subsequent 2-wk period. Once UD developed, pruritus, the character of the lesion (single or multiple crust, coalescing crust, erosion, or ulceration), and the size of the lesion were the only parameters that changed (increased) over the course of the disease. Pruritus was a factor in the rapid progression of UD lesions. We used these findings to develop a quantitative scoring system for the severity of UD. This enhanced understanding of the progression of UD and the quantitative scoring system will enhance the monitoring of UD.