Voluntary wheel running behaviour as a tool to assess the severity in a mouse pancreatic cancer model

Laboratory animals frequently undergo routine experimental procedures such as handling, restraining and injections. However, as a known source of stress, these procedures potentially impact study outcome and data quality. In the present study, we, therefore, performed an evidence-based severity assessment of experimental procedures used in a pancreatic cancer model including surgical tumour induction and subsequent chemotherapeutic treatment via repeated intraperitoneal injections. Cancer cell injection into the pancreas was performed during a laparotomy under general anaesthesia. After a four-day recovery phase, mice received either drug treatment (galloflavin and metformin) or the respective vehicle substances via daily intraperitoneal injections. In addition to clinical scoring, an automated home-cage monitoring system was used to assess voluntary wheel running (VWR) behaviour as an indicator of impaired well-being. After surgery, slightly elevated clinical scores and minimal body weight reductions, but significantly decreased VWR behaviour were observed. During therapy, body weight declined in response to chemotherapy, but not after vehicle substance injection, while VWR activity was decreased in both cases. VWR behaviour differed between treatment groups and revealed altered nightly activity patterns. In summary, by monitoring VWR a high impact of repeated injections on the well-being of mice was revealed and substance effects on well-being were distinguishable. However, no differences in tumour growth between treatment groups were observed. This might be due to the severity of the procedures uncovered in this study, as exaggerated stress responses are potentially confounding factors in preclinical studies. Finally, VWR was a more sensitive indicator of impairment than clinical scoring in this model.

Treadmill Training Improves Aerobic Capacity in Aged Male Mice Compared to Voluntary Wheel Running

Preclinical exercise studies typically use two forms of exercise training protocols: 1) voluntary wheel running and 2) forced treadmill running. Previous work from our group clearly demonstrates that older (18-month-old) male mice do not voluntarily engage in wheel running, especially compared to younger males or female mice. Therefore, we implemented a forced exercise treadmill training protocol to determine if treadmill training was superior to wheel running in improving aerobic capacity in older male mice. Purpose To determine if a 3-week treadmill training protocol improved time to exhaustion (TTE) in older male mice. Methods 18-month-old male mice (n=5) were provided a running wheel in their individual cage for 2 weeks or underwent daily treadmill training (n=6) for 3 weeks with increasing speed/incline. At the end of the training period we assessed TTE. Results Older male mice that trained on the treadmill demonstrated higher TTE compared to wheel (1382 □ 32 seconds versus 500 □ 99 seconds, respectively). In addition, older male mice that trained on the treadmill improved on average ~8% in their TTE test. Conclusion A 3-week treadmill training protocol improves aerobic capacity in older male mice to a greater extent than voluntary wheel running. Ongoing experiments will utilize this training protocol to understand age-related declines in cardiorespiratory fitness, circadian rhythm, and to test exercise as an intervention in the aging population.

SYNAPTIC DYSFUNCTION UNDERLIES ALTERATIONS IN THE INITIATION OF GOAL-DIRECTED BEHAVIORS: IMPLICATIONS FOR HIV-1 ASSOCIATED APATHY

Individuals living with human immunodeficiency virus type 1 (HIV-1) exhibit an increased prevalence of neuropsychiatric comorbities (e.g., apathy) relative to their seronegative counterparts. Given the profound functional consequences associated with apathy, conceptualizing the multidimensional neuropsychiatric syndrome, and associated neural mechanisms, following chronic HIV-1 viral protein exposure remains a critical need. HIV-1 associated apathy was examined by quantifying goal-directed behaviors, indexed using voluntary wheel running, during the diurnal and nocturnal cycle. Apathetic behaviors in the HIV-1 Tg rat were characterized by a profound decrease in the number of running bouts during both the diurnal and nocturnal cycle, supporting a prominent deficit in the self-initiation of spontaneous behaviors. Additionally, HIV-1 Tg animals exhibited a decreased reinforcing efficacy of voluntary wheel running during the nocturnal cycle. Following the completion of voluntary wheel running, synaptic dysfunction in medium spiny neurons (MSNs) of the nucleus accumbens (NAc) was examined as a potential neural mechanism underlying HIV-1 associated apathy. HIV-1 Tg animals displayed prominent synaptic dysfunction in MSNs of the NAc, characterized by decreased synaptic connectivity and a population shift towards an immature dendritic spine phenotype relative to control animals. Synaptic dysfunction accounted for 42.0% to 68.5% of the variance in the number of running bouts affording a key neural mechanism underlying the self-initiation of spontaneous behaviors. The establishment of a fundamental neural mechanism underlying apathy affords a key target for the development of novel therapeutics and cure strategies for affective alterations associated with HIV-1.

Preconditioning by voluntary wheel running attenuates later neuropathic pain via Nrf2 antioxidant signaling in rats

Animal and human studies have shown that exercise prior to nerve injury prevents later chronic pain, but the mechanisms of such preconditioning remain elusive. Given that exercise acutely increases formation of free radicals, triggering antioxidant compensation, we hypothesized that voluntary running preconditioning would attenuate neuropathic pain by supporting redox homeostasis after sciatic nerve injury in male and female rats. We show that 6 weeks of voluntary wheel running suppresses neuropathic pain development induced by chronic constriction injury (CCI) across both sexes. This protection was associated with reduced nitrotyrosine immunoreactivity-a marker for peroxynitrite-at the sciatic nerve injury site. Our data suggest that prior voluntary wheel running does not reduce production of peroxynitrite precursors, as expression levels of inducible nitric oxide synthase and NADPH oxidase 2 were unchanged. Instead, voluntary wheel running increased superoxide scavenging by elevating expression of superoxide dismutases 1 and 2. Prevention of neuropathic pain was further associated with activation of the master transcriptional regulator of the antioxidant response, nuclear factor E2-related factor 2 (Nrf2). Six weeks of prior voluntary wheel running increased Nrf2 nuclear translocation at the sciatic nerve injury site; in contrast, 3 weeks of prior wheel running, which failed to prevent neuropathic pain, had no effect on Nrf2 nuclear translocation. The protective effects of prior voluntary wheel running were mediated by Nrf2, as suppression was abolished across both sexes when Nrf2 activation was blocked during the running phase. This study provides insight into the mechanisms by which physical activity may prevent neuropathic pain.

Voluntary Wheel Running Exercise Improves Aging-Induced Sarcopenia via Activation of Peroxisome Proliferator-Activated Receptor Gamma Coactivator-1α/Fibronectin Type III Domain-Containing Protein 5/Adenosine Monophosphate-Activated Protein Kinase Signaling Pathway

Purpose: In this study, the protective effect of voluntary wheel running exercise on muscle loss and muscle weakness in gastrocnemius of old rats was investigated. The association of voluntary wheel exercise with the peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α)/fibronectin type III domain-containing protein 5 (FNDC5)/adenosine monophosphate- activated protein kinase (AMPK) signaling pathway and vascular endothelial growth factor (VEGF) expression was also evaluated.Methods: Six-month-old and 22-month-old male rats were used for this experiment. The rats in voluntary wheel running exercise groups were performed wheel running for 2 months. Weight bearing test for walking strength, rotarod test for motor coordination and balance, hematoxylin and eosin (H&E) staining for histological changes in the muscle tissues, Western blot analysis for PGC-1α, FNDC5, AMPK, immunofluorescence for VEGF were conducted.Results: Decreased muscle mass, strength, and coordination due to aging were associated with a decrease in the PGC-1α/ FNDC5/AMPK signaling pathway in the gastrocnemius. Voluntary wheel running exercise enhanced VEGF expression by activating the PGC-1α/FNDC5/AMPK signaling pathway, then increased muscle mass, strength, and coordination.Conclusions: It has been suggested that voluntary wheel running exercise alleviates symptoms of urological diseases that are difficult to treat. Wheel running exercise is a good therapeutic strategy to prevent or treat aging-related sarcopenia.

Voluntary Wheel Running Partially Attenuates Early Life Stress-Induced Neuroimmune Measures in the Dura and Evoked Migraine-Like Behaviors in Female Mice

Migraine is a complex neurological disorder that affects three times more women than men and can be triggered by endogenous and exogenous factors. Stress is a common migraine trigger and exposure to early life stress increases the likelihood of developing chronic pain disorders later in life. Here, we used our neonatal maternal separation (NMS) model of early life stress to investigate whether female NMS mice have an increased susceptibility to evoked migraine-like behaviors and the potential therapeutic effect of voluntary wheel running. NMS was performed for 3 h/day during the first 3 weeks of life and initial observations were made at 12 weeks of age after voluntary wheel running (Exercise, -Ex) or sedentary behavior (-Sed) for 4 weeks. Mast cell degranulation rates were significantly higher in dura mater from NMS-Sed mice, compared to either naïve-Sed or NMS-Ex mice. Protease activated receptor 2 (PAR2) protein levels in the dura were significantly increased in NMS mice and a significant interaction of NMS and exercise was observed for transient receptor potential ankyrin 1 (TRPA1) protein levels in the dura. Behavioral assessments were performed on adult (>8 weeks of age) naïve and NMS mice that received free access to a running wheel beginning at 4 weeks of age. Facial grimace, paw mechanical withdrawal threshold, and light aversion were measured following direct application of inflammatory soup (IS) onto the dura or intraperitoneal (IP) nitroglycerin (NTG) injection. Dural IS resulted in a significant decrease in forepaw withdrawal threshold in all groups of mice, while exercise significantly increased grimace score across all groups. NTG significantly increased grimace score, particularly in exercised mice. A significant effect of NMS and a significant interaction effect of exercise and NMS were observed on hindpaw sensitivity following NTG injection. Significant light aversion was observed in NMS mice, regardless of exercise, following NTG. Finally, exercise significantly reduced calcitonin gene-related peptide (CGRP) protein level in the dura of NMS and naïve mice. Taken together, these findings suggest that while voluntary wheel running improved some measures in NMS mice that have been associated with increased migraine susceptibility, behavioral outcomes were not impacted or even worsened by exercise.

Endurance Exercise to Improve Physical Function in Adult and Older Mice: High Intensity Interval Training (HIIT) versus Voluntary Wheel Running (VWR)

With age comes a gradual decline in physical function and exercise capacity, concurrent with a progressive propensity for development of sarcopenia /(age-related loss of muscle mass and strength) and frailty (inability of body to thrive and maintain homeostasis). Prior research has demonstrated that exercise, while not a cure, can help mitigate sarcopenia/frailty and restore functional capacity. Reliable, validated, pre-clinical models are necessary to elucidate the underlying molecular mechanisms at the intersection of age, exercise, and functional decline. In this study, we hypothesized that endurance exercise programs mimicking typical human exercise protocols would improve physical function in both adult and older adult mice. Furthermore, our secondary hypothesis was that older mice would receive less benefit from a similar volume of exercise than adult mice. To test these hypotheses, we randomly assigned (with some selection criteria) male C57BL/6 mice either at adult ages during the study (n=24, designated 10m, aged 6 months to 10 months) or at older adult ages (n=18, designated 26m, aged 22 months to 26 months) to a 13-week program of voluntary wheel running (VWR, group termed RUN) or high intensity interval training (HIIT), with an additional 10m sedentary control (CONT). The functional aptitude of each mouse was determined pre- and post-training using our composite CFAB (comprehensive functional assessment battery) scoring system consisting of voluntary wheel running (volitional exercise and activity rate), treadmill (endurance), rotarod (overall motor function), grip meter (forelimb strength), and inverted cling (whole body strength/endurance). To measure sarcopenia, we tracked body mass and body composition changes with EchoMRI, and measured muscle wet mass post-training.