Cage Activity Research Articles

Activity in Group-Housed Home Cages of Mice as a Novel Preclinical Biomarker in Oncology Studies.

Improving experimental conditions in preclinical animal research is a major challenge, both scientifically and ethically. Automated digital ventilated cages (DVC®) offer the advantage of continuous monitoring of animal activity in their home-cage. The potential utility of this technology remains understudied and deserves investigation in the field of oncology. Using the DVC® platform, we sought to determine if the continuous assessment of locomotor activity of mice in their home cages can serve as useful digital readout in the monitoring of animals treated with the reference oncology compounds cisplatin and cyclophosphamide. SCID mice of 14 weeks of age were housed in DVC® cages in groups of four and followed with standard and digital examination before and after treatment over a 17-day total period. DVC® detected statistically significant effects of cisplatin on the activity of mice in the short and long term, as well as trends for cyclophosphamide. The activity differences between the vehicle- and chemotherapy-treated groups were especially marked during the nighttime, a period when animals are most active and staff are generally not available for regular checks. Standard clinical parameters, such as body weight change and clinical assessment during the day, provided additional and complementary information. The DVC® technology enabled the home cage monitoring of mice and non-invasive detection of animal activity disturbances. It can easily be integrated into a multimodal monitoring approach to better capture the different effects of oncology drugs on anti-tumor efficacy, toxicity, and safety and improve translation to clinical studies.

Amentoflavone attenuates oxidative stress and neuroinflammation induced by cerebral ischemia/reperfusion in rats by targeting HMGB1-mediated TLR4/NF-κB signaling pathway

Surveys indicated that stroke classified among the leading cause of death as well as combined death and disability worldwide resulting in a great loss for the global economy. The present study aims to evaluate the neuroprotective potential of the biflavonoid amentoflavone (AMNT) in alleviating cerebral ischemia/reperfusion (IR) injury in rats, and to elucidate the possible underlying mechanism of an experimental condition with similar circumstances to stroke. Cerebral ischemia was achieved through left common carotid artery occlusion for 60 min, followed by blood flow restoration. Sham-operated control rats subjected to the same surgical process except for brain IR. Rats were orally administered AMNT/ or vehicle for three days’ prior surgical operation, and for another three days after left brain IR. Rats of all groups were assessed for neurological deficits 24 h following brain IR. Each group was divided into two subgroups one for the rotarod testing and biochemical assessment while the other subgroup to perform the activity cage testing, histopathological study, immunohistochemistry, and gene expression analysis. AMNT enhanced brain levels of GSH and CAT activities, suppressed neuroinflammation via reducing the inflammatory cytokines in the serum, and enhanced brain contents of TBK1 and IFNβ. AMNT downregulated TLR4-/NF-κB signaling pathway as a result of the HMGB1 suppression. Moreover, AMNT blocked apoptotic cell death by suppressing the NF-κB signaling pathway and reducing the activation of caspase-3. These findings revealed that AMNT attenuates I/R–induced cerebral injury possibly by regulating the HMGB1-mediated TLR4/NF-kB pathway. Thus, AMNT could provide potential preventive and therapeutic option for cerebral stroke.

Somatostatin-expressing neurons in the ventral tegmental area innervate specific forebrain regions and are involved in stress response.

Expanding knowledge about the cellular composition of subcortical brain regions demonstrates large heterogeneity and differences from the cortical architecture. Recently, we described three subtypes of somatostatin-expressing (Sst) neurons in the mouse ventral tegmental area (VTA) and showed their local inhibitory action on the neighbouring dopaminergic neurons (Nagaeva et al., 2020). Here, we report that mouse Sst+ neurons especially from the anterolateral part of the VTA also project far outside the VTA and innervate forebrain regions that are mainly involved in the regulation of emotional behaviour, including the ventral pallidum (VP), lateral hypothalamus (LH), the medial part of the central amygdala (CeM), anterolateral division of the bed nucleus of stria terminalis (alBNST), and paraventricular thalamic nucleus (PVT). Deletion of these VTASst neurons in mice affected several behaviours, such as home cage activity, sensitization of locomotor activity to morphine, fear conditioning responses and reactions to the inescapable stress of forced swimming, often in a sex-dependent manner. Together, these data demonstrate that VTASst neurons have selective projection targets distinct from the main targets of VTA dopamine neurons. VTASst neurons are involved in the regulation of behaviours primarily associated with the stress response, making them a relevant addition to the efferent VTA pathways and stress-related neuronal network.Significance StatementThe ventral tegmental area (VTA) is involved in many reward- and aversion-related processes contributing to brain disorders, but the cellular composition and brain-wide connections of this subcortical brain region are still poorly known. We recently described a heterogeneous somatostatin-expressing (Sst) neuron population in the mouse VTA, and, here, we revealed that, in addition to being local interneurons, VTA Sst neurons send long-distance projections to specific forebrain regions. Deletion of these neurons altered behavioural reactions to acute stressors and to repeated morphine administration. The results add the VTA Sst neurons to the stress-related network of the brain and establish a possibility to find out the exact roles of these neurons acting as behaviourally relevant interneurons and/or projection neurons.

Effect of Hippocampal Overexpression of Dopamine Neurotrophic Factor (CDNF) on Behavior of Mice with Genetic Predisposition to Depressive-Like Behavior.

Cerebral dopamine neurotrophic factor (CDNF) is a promising agent for Parkinson's disease treatment. However, its role in regulation of non-motor behavior including various psychopathologies remains unclear. In this regard, the aim of the present work was to study effect of CDNF overexpression in hippocampus on behavior of the ASC mice (Antidepressant Sensitive Cataleptics) with genetic predisposition to depressive-like behavior. CDNF overexpression in the mouse hippocampal neurons was induced using an adeno-associated viral vector. Four weeks after stereotaxic injection of the AAV-CDNF construct into the dorsal hippocampus home cage activity, exploratory, anxious and depressive-like types of behavior, as well as spatial and associative learning were assessed. We found significant improvements in the dynamics of spatial learning in the Morris water maze in the CDNF-overexpressing animals. At the same time, no effect of CDNF was found on other types of behavior under study. Behavior of the experimental animals under home cage conditions did not differ from that in the control group, except for the decrease in the total amount of food eaten and slight increase in the number of sleep episodes during the light phase of the day. In the present study we also attempted to determine molecular basis for the above-mentioned changes through assessment of the gene expression pattern. We did not find significant changes in the mRNA level of key kinases genes involved in neuroplasticity and neuronal survival, as well as genes encoding receptors for the main neurotransmitter systems. However, the CDNF-overexpressing animals showed increased level of the spliced Xbp indicating activation of the Ire1α/Xbp-1 pathway traditionally associated with ER stress. Immunohistochemical analysis showed that CDNF was co-localized with the ER marker calreticulin. Thus, the effects of endogenous CDNF on behavior that we have found could be mediated by a specific molecular cascade, which emphasizes its difference from the classical neurotrophic factors.

Long-term behavioural alterations in mice following transient cerebral ischemia

Ischemic stroke is one of the leading causes of disability and mortality worldwide. Acute and chronic post-stroke changes have variable effects on the functional outcomes of the disease. Therefore, it is imperative to identify what daily activities are altered after stroke and to what extent, keeping in mind that ischemic stroke patients often have long-term post-stroke complications. Translational studies in stroke have also been challenging due to inconsistent study design of animal experiments. The objective of this study was to clarify whether and to what extent mouse behaviour was altered during a 6 months period after cerebral stroke. Experimental stroke was induced in mice by intraluminal filament insertion into the middle cerebral artery (fMCAo). Neurological deficits, recovery rate, motor performance, and circadian activity were evaluated following ischemia. We observed severe neurological deficits, motor impairments, and delay in the recovery rate of mice during the first 14 days after fMCAo. Aberrant circadian activity and distorted space map were seen in fMCAo mice starting one month after ischemia, similarly to altered new and familiar cage activity and sucrose preference using the IntelliCage, and was still evident 60- and 180- days following stroke in the voluntary running wheel using the PhenoMaster system. A preference towards ipsilateral side turns was observed in fMCAo mice both acutely and chronically after the stroke induction. Overall, our study shows the importance of determining time-dependent differences in the long-term post-stroke recovery (over 180 days after fMCAo) using multiple behavioural assessments.

Reambulation following hindlimb unloading attenuates disuse-induced changes in murine fracture healing.

Patients with bone and muscle loss from prolonged disuse have higher risk of falls and subsequent fragility fractures. In addition, fracture patients with continued disuse and/or delayed physical rehabilitation have worse clinical outcomes compared to individuals with immediate weight-bearing activity following diaphyseal fracture. However, the effects of prior disuse followed by physical reambulation on fracture healing cellular processes and adjacent bone and skeletal muscle recovery post-injury remains poorly defined. To bridge this knowledge gap and inform future treatment and rehabilitation strategies for fractures, a preclinical model of fracture healing with a history of prior unloading with and without reambulation was employed. First, skeletally mature male and female C57BL/6J mice (18 weeks) underwent hindlimb unloading by tail suspension (HLU) for 3 weeks to induce significant bone and muscle loss modeling enhanced bone fragility. Next, mice had their right femur fractured by open surgical dissection (stabilized with 24-gauge pin). The, mice were randomly assigned to continued HLU or allowed normal weight-bearing reambulation (HLU + R). Mice given normal cage activity throughout the experiment served as healthy age-matched controls. All mice were sacrificed 4-days (DPF4) or 14-days (DPF14) following fracture to assess healing and uninjured hindlimb musculoskeletal properties (6-10 mice per treatment/biological sex). We found that continued disuse following fracture lead to severely diminished uninjured hindlimb skeletal muscle mass (gastrocnemius and soleus) and femoral bone volume adjacent to the fracture site compared to healthy age-matched controls across mouse sexes. Furthermore, HLU led to significantly decreased periosteal expansion (DPF4) and osteochondral tissue formation by DPF14, and trends in increased osteoclastogenesis (DPF14) and decreased woven bone vascular area (DPF14). In contrast, immediate reambulation for 2 weeks after fracture, even following a period of prolonged disuse, was able to increase hindlimb skeletal tissue mass and increase osteochondral tissue formation, albeit not to healthy control levels, in both mouse sexes. Furthermore, reambulation attenuated osteoclast formation seen in woven bone tissue undergoing disuse. Our results suggest that weight-bearing skeletal loading in both sexes immediately following fracture may improve callus healing and prevent further fall risk by stimulating skeletal muscle anabolism and decreasing callus resorption compared to minimal or delayed rehabilitation regimens.

Recovery from FOLFOX chemotherapy-induced systemic and skeletal muscle metabolic dysfunction in mice.

FOLFOX (5-fluorouracil, leucovorin, oxaliplatin) chemotherapy is used to treat colorectal cancer and can acutely induce metabolic dysfunction. However, the lasting effects on systemic and skeletal muscle metabolism after treatment cessation are poorly understood. Therefore, we investigated the acute and lasting effects of FOLFOX chemotherapy on systemic and skeletal muscle metabolism in mice. Direct effects of FOLFOX in cultured myotubes were also investigated. Male C57BL/6J mice completed four cycles (acute) of FOLFOX or PBS. Subsets were allowed to recover for 4 wk or 10 wk. Comprehensive Laboratory Animal Monitoring System (CLAMS) metabolic measurements were performed for 5 days before study endpoint. C2C12 myotubes were treated with FOLFOX for 24 hr. Acute FOLFOX attenuated body mass and body fat accretion independent of food intake or cage activity. Acute FOLFOX decreased blood glucose, oxygen consumption (V̇o2), carbon dioxide production (V̇co2), energy expenditure, and carbohydrate (CHO) oxidation. Deficits in V̇o2 and energy expenditure remained at 10 wk. CHO oxidation remained disrupted at 4 wk but returned to control levels after 10 wk. Acute FOLFOX reduced muscle COXIV enzyme activity, AMPK(T172), ULK1(S555), and LC3BII protein expression. Muscle LC3BII/I ratio was associated with altered CHO oxidation (r = 0.75, P = 0.03). In vitro, FOLFOX suppressed myotube AMPK(T172), ULK1(S555), and autophagy flux. Recovery for 4 wk normalized skeletal muscle AMPK and ULK1 phosphorylation. Our results provide evidence that FOLFOX disrupts systemic metabolism, which is not readily recoverable after treatment cessation. FOLFOX effects on skeletal muscle metabolic signaling did recover. Further investigations are warranted to prevent and treat FOLFOX-induced metabolic toxicities that negatively impact survival and life quality of patients with cancer.NEW & NOTEWORTHY The present study demonstrates that FOLFOX chemotherapy induces long-lasting deficits in systemic metabolism. Interestingly, FOLFOX modestly suppressed skeletal muscle AMPK and autophagy signaling in vivo and in vitro. The FOLFOX-induced suppression of muscle metabolic signaling recovered after treatment cessation, independent of systemic metabolic dysfunction. Future research should investigate if activating AMPK during treatment can prevent long-term toxicities to improve health and quality of life of patients with cancer and survivors.

Effects of Na/K‐ATPase Inhibitor Marinobufagenin on Gene Expression and Cognition in a Mouse Model of Alzheimer’s Disease

AbstractBackgroundAlzheimer’s disease (AD) is characterized by brain amyloid‐β plaque formation, neuroinflammation and neurodegeneration, which lead to cognitive impairment (CI) and disruption of circadian rhythm. Bioactive steroidal Na/K‐ATPase inhibitor marinobufagenin (MBG) modulates neuroinflammation. In 16‐mo old double transgenic APPswe/PS1dE9 AD mice with advanced AD, treatment with MBG reduced mRNA expression of inflammatory markers. Here, we investigated whether treatment with MBG at early‐stage AD may impact AD manifestation in this AD mouse model.MethodsFive months old male AD mice (n = 15) and WT mice (n = 28) were administered MBG (100 µg/day/kg body weight) (AD‐MBG, n = 8; WT‐MBG, n = 14) or vehicle for control (AD‐C, n = 7; WT‐C, n = 14) via subcutaneous ALZET osmotic minipumps for 3 months. At 8‐mo of age, the mice underwent turn‐based discrimination learning in a water T‐maze (WTM) to assess procedural learning and 72‐hour home cage activity (HCA) analysis to assess circadian rhythm. The hippocampal inflammatory and AD mRNAs (by qPCR) and MBG levels (by immunoassay) were measured.ResultsMBG administration increased plasma MBG levels in WT and AD mice (Table 1). AD‐C mice exhibited greater home cage activity disruption vs. WT‐C (Fig.1a); in WTM, AD‐C required more trials to learn the turn‐based task than WT‐C (Fig. 1b). Though statistically insignificant, trends showed that MBG numerically worsened activity disruption by HCA and decreased ability to learn turn‐based task by WTM in WT mice, while numerically improving both measures in AD mice. During the reversal phase of WTM where response requirements were reversed, WT‐C mice performed significantly better than AD‐C mice, measured by the cumulative amount of time spent in the incorrect arm until reaching criterion performance (p = 0.038). Similar patterns of numerical improvement in AD‐MBG compared to AD‐C are shown (Fig. 1c). Hippocampal inflammatory and AD markers mRNAs were upregulated in AD‐C vs. WT‐C, and MBG treatment downregulated the expression of TNF and GFAP in AD mice (Table 1).ConclusionsBehavioral testing results illustrate CI and circadian impairment in 8‐mo old AD mice compared to WT. Numerical trends show a potential protective effect of MBG on AD CI likely via downregulation of hippocampal TNF and GFAP genes.0Supported by the NIH/NIA Intramural Research Program

Effect of Etifoxine on Locomotor Activity and Passive Learning in Rats with Diazepam-Induced Cognitive Deficit

Etifoxine is an anxiolytic drug with a dual mechanism of action. In contrast to conventional benzodiazepine anxiolytics, which induce cognitive dysfunction and myorelaxation, no memory impairment nor a decrease in motor activity is observed with etifoxine. This study aims to evaluate the effects of etifoxine on locomotor activity and passive learning in rats with diazepam-induced memory deficit. Male Wistar rats were treated intraperitoneally for 7 days with: (1) saline; (2) diazepam 2.5 mg/kg bw or (3) diazepam 2.5 mg/kg bw and etifoxine in a dose of 50 mg/kg bw. Activity cage test was used for evaluation of locomotor activity, and step-through and step-down tests were performed to study the passive learning. Etifoxine increased the number of horizontal movements on the 7th and 14th days of the experiment. The drug exhibits anti-amnesic effect in a model of diazepam-induced anterograde amnesia by enhancing long-term memory in passive learning tests. The data obtained suggest that etifoxine can reduce the benzodiazepine-induced cognitive deficit. Moreover, such a combination can alleviate the negative influence of benzodiazepines on locomotor activity. However, additional studies are necessary to translate these results into clinical practice.

Sarcolipin ablation decreases whole body VO2 in young female nice

The sarco(endo)plasmic reticulum calcium (Ca 2+ ) ATPase (SERCA) pump is a major regulator of cytosolic Ca 2+ in striated muscle. Sarcolipin (SLN) reduces SERCA-mediated Ca 2+ pumping efficiency and is a key regulator of muscle metabolism and contractile function. With SERCA activity comprising approximately 12% of whole-body metabolic rate, the inefficient SERCA Ca 2+ cycling promoted by SLN would be predicted to increase metabolic rate. Although SLN is involved in adaptive diet-induced thermogenesis and overexpression of SLN increases metabolic rate and protects against diet-induced obesity, we found previously that SLN ablation had no effect on whole body metabolic rate or body composition in male mice that were fed a standard chow diet. However, unpublished work from our lab indicates that SLN expression in mouse soleus is significantly higher in females compared with males. Therefore, the purpose of this study was to investigate the effects of SLN ablation on body composition and metabolic rate of female mice. Adult (4-6mo) female wild type (WT) and SLN knockout (KO) mice (n=4 per group) were used to collect anthropometric measures (adiposity and body weight) and were housed in a comprehensive lab animal monitoring system (CLAMS) over a 48-hour period to assess daily whole body VO2 and total cage activity. With SLN ablation, total whole body VO 2 (ml/kg/hr) was lower (p<0.05) in the KO females (3534 ± 95.1) compared to WT females (5080 ± 98.8), with no differences (p>0.05) found for total cage activity (counts) (WT, 14935.42 ± 1799.6 vs. KO, 11860.83 ± 679.48), body weight (grams) (WT, 20.97 ± 0.76 vs. KO, 22.18 ± 0.80), or adiposity (arbitrary units) (WT, 5.18 ± 1.71 vs. KO, 2.12 ± 0.36). These results showing that high levels of endogenous muscle SLN expression in female mice influence whole-body metabolism even under chow-fed conditions, demonstrate a potential important sex difference in whole body physiology and metabolism. Natural Sciences and Engineering Research Council of Canada (NSERC) [grant number 311922-05 (to A.R.T.)] 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.

Voluntary wheel running and lithium supplementation promotes fatigue resistance, fat oxidation, and improves insulin tolerance in D2 mdx mice

BACKGROUND: Duchenne muscular dystrophy (DMD) is an X-linked disorder characterized by progressive muscle wasting and premature death. Many DMD patients will also exhibit signs of metabolic dysfunction such as insulin resistance that predisposes them to type 2 diabetes and worsened cardiovascular disease. Most treatments for DMD aim to lower muscle inflammation and improve strength, however, none have addressed the complication of insulin resistance. The current standard of treatment is corticosteroids, which prolongs ambulation, however, chronic use increases susceptibility to obesity, insulin resistance, and type 2 diabetes. It is well-established that regular aerobic exercise enhances insulin sensitivity. Further, we have recently found that inhibiting glycogen synthase kinase 3 with lithium (Li), a known insulin mimetic, improves force production and fatigue resistance in wildtype (WT) mice. Here, we tested whether voluntary wheel running (VWR) and Li supplementation, together, could improve whole-body fatigue, metabolism, and insulin sensitivity in the DBA/2J (D2) mdx mouse model of DMD. METHODS: 5-week old male D2 WT and mdx mice were separated into four groups: WT, mdx sedentary (SED), mdx VWR, and mdx Li+VWR (n=11/group). The mdx VWR and mdx Li+VWR mice had unlimited access to a cagewheel, and mdx Li+VWR mice were given a low dose of Li (50 mg/kg/day) via their drinking water throughout the study. A treadmill time-to-fatigue test was performed to assess whole-body fatigue. Mice were housed for 48-hours in metabolic cages to measure energy expenditure. Glucose handling was measured using glucose and insulin tolerance tests. RESULTS: As expected, WT mice had ~2-fold greater total cage activity compared to all mdx groups. When examining cagewheel distance, we found that mdx Li+VWR mice ran half the total distance in kilometres than mdx VWR mice (39.1 ± 9.6 km vs. 74.8 ± 13.6 km). Despite this, VWR with and without Li improved whole-body fatigue as only mdx SED mice (22.8 ± 0.9 min) had a shorter time to exhaustion than WT mice (32.6 ± 0.9 min). All mdx groups had higher energy expenditure than WT mice, suggestive of a hypermetabolic phenotype. When examining the respiratory exchange ratio (RER) among mdx mice, mdxLi+VWR (0.87) mice had a lower RER than mdx SED mice (0.91), indicative of enhanced fat oxidation. Glucose tolerance was equally impaired among mdx groups compared to WT mice. However, insulin tolerance was notably improved in mdx VWR and mdx Li+VWR mice, with a greater improvement observed in the mdx Li+VWR mice. CONCLUSION: Despite running less than mdx VWR mice, mdx Li+VWR mice had enhanced fatigue resistance, fat utilization, and insulin sensitivity, suggesting Li may be a viable treatment to improve muscle and metabolic function in mdx mice. CIHR CRC (Tier II) to VAF 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.

EARLY PHYSICAL REHABILITATION FOLLOWING FRACTURE INCREASES OSTEOCHONDRAL CALLUS FORMATION IN A PRECLINICAL MODEL OF DISUSE

Patients with bone and muscle weakness from disuse have higher risk of fracture and worse post-injury mortality rates. The goal of this current study was to better inform post-fracture rehabilitation strategies by investigating if physical remobilization following disuse by hindlimb unloading improves osteochondral callus formation compared to continued disuse by hindlimb suspension (HLS). We hypothesized that continued HLS would impair callus bone and cartilage formation and that physical rehabilitation after HLS would increase callus properties.All animal procedures were approved by the VCU IACUC. Skeletally mature, male and female C57BL/6J mice (18 weeks) underwent HLS for 3 weeks. Mice then had their right femur fractured by open surgical dissection (stabilized with 24-gauge pin). Mice were then either randomly assigned to continued HLS or allow normal physical weight-bearing remobilization (HLS + R). Mice allowed normal cage activity throughout the experiment served as controls (GC). All mice were sacrificed 14-days following fracture with 4-8 mice (male and female) per treatment. Data analyzed by respective ANOVA with Tukey post-hoc (*p< 0.05; # p < 0.10)Male and female mice showed conserved and significant decreases in hindlimb callus bone formation from continued HLS versus HLS + R. Combining treatment groups regardless of mouse sex, histological analyses using staining on these same calluses demonstrated that HLS resulted in trends toward decreased cartilage cross-sectional area and increased osteoclast density in woven bone versus physically rehabilitated mice.In support of our hypothesis, physical remobilization increases callus bone formation following fracture compared to continued disuse potentially due to increased endochondral ossification and decreased bone resorption. In all, partial weight-bearing exercise immediately following fracture may improve callus healing compared to delayed rehabilitation regimens that are frequently used.

A Novel Device for Studying in Vivo Magnesium Degradation under Controllable Cyclic Load in the Rabbit Model

Magnesium is an important material for medical implants due to its special properties including biocompatibility, low density, high mechanical property, and biodegradability. The in vivo implantation experiment of magnesium is an important experimental method to obtain its degradation characteristics. In recent years, although in vitro studies have found that the degradation of magnesium under cyclic loading is different from static loading, there is the lack of in vivo experiment to verify the above in vitro results, while the degradation characteristic of magnesium under load is an important factor to be considered when magnesium is used as orthopedic implant. We designed a novel device to apply controllable cyclic load onto the magnesium material implanted in the rabbit tibia, containing loading chamber, motor and gear reduction mechanism, and Singlechip Computer, etc. The conducted experiments have shown that the devices were able to apply load onto the implanted magnesium in rabbits in vivo during unrestricted cage activity for at least 16 days. Likewise, no distortion or infections were observed. Results demonstrated that the novel device was able to produce alteration of the mechanical environment in vivo rabbit tibia, which showed that the load device is suitable for in vivo loading degradation characteristics testing of magnesium or magnesium alloys.

Sarm1 Regulates Circadian Rhythm Disorder in Alzheimer's Disease in Mice.

Sarm1 (Sterile alpha and TIR motif-containing 1) is a key protein that regulates neurodegenerative pathologies. Alzheimer's disease (AD) is highly associated with neurodegenerative lesions and biorhythmic disturbances. This study aims to decipher the role of Sarm1 in AD-induced circadian rhythm disturbances and AD progression. Open field and water maze tests were used to assess the cognitive function of mice. Thioflavin-S staining was used to assess amyloid-β (Aβ) plaque deposition in the hippocampus and cortex. Rhythmic waveform of home cage activity and temperature was recorded to evaluate circadian rhythm. Expression of clock molecules including Bmal1 and Per2 in the hippocampus were analyzed using western blot and real-time PCR. Further, HT22 cells with Sam1 knockout were treated with Aβ31-35 treatment to initiate circadian rhythm disorder in the cellular level to assess the changes in Bmal1 and Per2. Our data suggested that Sarm1 deficiency rescued cognitive disorder, decreased Aβ plaque deposition in the hippocampus and cortex, inhibited astrocyte activation, improved circadian rhythm, altered clock molecule expression in the cortex and hippocampus in APP/PS1 mice. Sarm1 attenuates circadian rhythm disturbances and reduces AD progression. These data support the potential use of Sarm1 as a therapeutic target to improve circadian rhythm to impede AD progression.

Brain Targeting by Intranasal Drug Delivery: Effect of Different Formulations of the Biflavone "Cupressuflavone" from Juniperus sabina L. on the Motor Activity of Rats.

The polar fractions of the Juniperus species are rich in bioflavonoid contents. Phytochemical study of the polar fraction of Juniperus sabina aerial parts resulted in the isolation of cupressuflavone (CPF) as the major component in addition to another two bioflavonoids, amentoflavone and robustaflavone. Biflavonoids have various biological activities, such as antioxidant, anti-inflammatory, antibacterial, antiviral, hypoglycemic, neuroprotective, and antipsychotic effects. Previous studies have shown that the metabolism and elimination of biflavonoids in rats are fast, and their oral bioavailability is very low. One of the methods to improve the bioavailability of drugs is to alter the route of administration. Recently, nose-to-brain drug delivery has emerged as a reliable method to bypass the blood-brain barrier and treat neurological disorders. To find the most effective CPF formulation for reaching the brain, three different CPF formulations (A, B and C) were prepared as self-emulsifying drug delivery systems (SEDDS). The formulations were administered via the intranasal (IN) route and their effect on the spontaneous motor activity in addition to motor coordination and balance of rats was observed using the activity cage and rotarod, respectively. Moreover, pharmacokinetic investigation was used to determine the blood concentrations of the best formulation after 12 h. of the IN dose. The results showed that formulations B and C, but not A, decreased the locomotor activity and balance of rats. Formula C at IN dose of 5 mg/kg expressed the strongest effect on the tested animals.

Intrinsic cardiorespiratory fitness modulates clinical and molecular response to caloric restriction

ObjectiveCaloric restriction (CR) is one extrinsic intervention that can improve metabolic health, and it shares many phenotypical parallels with intrinsic high cardiorespiratory fitness (CRF), including reduced adiposity, increased cardiometabolic health, and increased longevity. CRF is a highly heritable trait in humans and has been established in a genetic rat model selectively bred for high (HCR) and low (LCR) CRF, in which the HCR live longer and have reduced body weight compared to LCR. This study addresses whether the inherited high CRF phenotype occurs through similar mechanisms by which CR promotes health and longevity. MethodsWe compared HCR and LCR male rats fed ad libitum (AL) or calorically restricted (CR) for multiple physiological, metabolic, and molecular traits, including running capacity at 2, 8, and 12 months; per-hour metabolic cage activity over daily cycles at 6 and 12 months; and plasma lipidomics, liver and muscle transcriptomics, and body composition after 12 months of treatment. ResultsLCR-CR developed a physiological profile that mirrors the high-CRF phenotype in HCR-AL, including reduced adiposity and increased insulin sensitivity. HCR show higher spontaneous activity than LCR. Temporal modeling of hourly energy expenditure (EE) dynamics during the day, adjusted for body weight and hourly activity levels, suggest that CR has an EE-suppressing effect, and high-CRF has an EE-enhancing effect. Pathway analysis of gene transcripts indicates that HCR and LCR both show a response to CR that is similar in the muscle and different in the liver. ConclusionsCR provides LCR a health-associated positive effect on physiological parameters that strongly resemble HCR. Analysis of whole-body EE and transcriptomics suggests that HCR and LCR show line-dependent responses to CR that may be accreditable to difference in genetic makeup. The results do not preclude the possibility that CRF and CR pathways may converge.

Development of a New Model of Humeral Hemiarthroplasty in Rats

Purpose In vivo models are anatomically comparable to humans allowing to reproduce the patterns and progression of the disease and giving the opportunity to study the symptoms and responses to new treatments and materials. This study aimed to establish a valid and cost-effective in vivo rat model to assess the effects of implanted shoulder hemiarthroplasty materials on glenoid articular cartilage wear. Methods Eight adult male Wistar rats underwent right shoulder hemi-arthroplasty. A stainless steel metal bearing was used as a shoulder joint prosthesis. X-rays were performed one week after surgery to verify correct implant position. Additional X-rays were performed 30 and 60 days post-implantation. Animals were sacrificed 24 weeks after implantation. All specimens were evaluated with micro-CT for cartilage and bone wear characteristics as well as histologically for signs of osteoarthritis. Samples were compared to the non-operated shoulders. Results All animals recovered and resumed normal cage activity. All X-rays demonstrated correct implant positioning except for one in which the implant was displaced. Histologic evaluation demonstrated arthritic changes in the implanted shoulder. Decreased Trabecular thickness and Trabecular Spacing were documented among the implanted parties (p < .05). Bone Mineral Density and Tissue Mineral Density were reduced in the operated shoulder although not significantly (p = .07). Conclusions This study demonstrated significant glenoid cartilage wearing in the operated shoulder. Furthermore, the presence of an intra-articular hemiarthroplasty implant diminished underlying glenoid bone quality. This novel, in vivo-model will enable researchers to test implant materials and their effects on cartilage and bone tissue in a cost-effective reproducible rat model.

Beneficial Effect of Atomoxetine Treatment on Scopolamine-Induced Cognitive Impairments and Molecular Changes in Rats

Objective: Atomoxetine is a noradrenaline re-uptake inhibitory drug that is currently used in the pharmacological treatment of attention deficit/hyperactivity disorder in children, adolescents, and adults. Based on the findings that noradrenergic system is closely associated with learning and memory processes, dysfunction of this system might contribute to the pathophysiology of cognitive disorders. Therefore, atomoxetine, which alters the noradrenergic neurotransmission in the synaptic cleft, has a potential to modulate learning and memory functions in various areas of the central nervous system. In the present study, it was aimed to investigate the effects of atomoxetine treatment on the cognitive impairments in amnesic animals using behavioral and immunohistochemical methods. Method: Male Sprague-Dawley rats (adult, weighing 250-300 g) were used in the experiments. Atomoxetine at doses of 3 or 6 mg/kg (i.p.) was administered to the rats for 14 days. Experimental amnesia was induced in the animals by applying scopolamine (0.5 mg/kg, i.p.) injection. Following the treatment protocol, Morris water maze and passive avoidance tests were conducted to assess spatial and emotional learning and memory parameters of rats, respectively. Piracetam (300 mg/kg daily for 14 days) was used as reference drug in these experiments. In addition, motor performances of the animals were evaluated by activity cage and Rota-rod tests. Subsequent to behavioral tests, rats were euthanized and immunohistochemical analyses were performed to determine the alterations in the BDNF and NGF levels in the hippocampi of the animals. Results: In the Morris water maze test, amnesic rats found the hidden platform slower and spent less time in the target quadrant compared to the healthy controls. On the other hand, atomoxetine treatments significantly decreased the escape latencies, and also increased the target quadrant time values of the amnesic animals at both doses. Data obtained from the passive avoidance test showed that second transition latency values of the amnesic rats were significantly shortened with respect to the control animals; while atomoxetine treatment prolonged these shortened values significantly. It was observed that atomoxetine did not alter motor coordination or spontaneous locomotor activities of animals. Moreover, this drug increased the BDNF and NGF levels in the hippocampal sub-regions which were diminished by scopolamine administration. Conclusions: The obtained findings showed that atomoxetine treatment significantly improved the impaired learning and memory performances of the rats induced by scopolamine administration. Furthermore, anti-amnesic effects of this drug were concomitant with increased neurotrophic factors levels in the hippocampal brain regions. These results suggest a notable therapeutic potential of this drug in the treatment of dementia and several cognitive disorders; however, it should be underlined they are needed to be confirmed by well-designed clinical trials.

Protective Effect of Beta-Carotene against Myeloperoxidase- Mediated Oxidative Stress and Inflammation in Rat Ischemic Brain Injury.

Oxidative stress and inflammatory reaction play critical roles in ischemia/reperfusion (I/R) injury in the brain. β-carotene (βCAR) is a naturally occurring pigment present in fruits and vegetables that expresses antioxidant and anti-inflammatory activities. This study was conducted to investigate the involvement of Bcl2/Bax and NF-κB signaling pathways in the potential protective role of βCAR against brain injury in a middle cerebral artery occlusion (MCAO) rat model. A focal brain ischemia model was created for 2 h, followed by reperfusion. Rats were given 10 and 20 mg/kg of βCAR for 7 days orally before induction of ischemia, at the start of reperfusion, and 3 days after ischemia. Scores of neurological deficit were rated 24 h after induction of ischemia. Motor coordination and spontaneous coordinate activities were assessed using rotarod and activity cage, respectively. After 2 h of the last dose, the animals were killed and their brains were extracted for further examinations. The results of the study show that βCAR diminished the score of neurological deficits and ameliorated motor coordination, balance, and locomotor activity in the I/R control group. Further, βCAR resulted in diminution of malondialdehyde (MDA) and augmentation of reduced glutathione (GSH) contents, as well as the elevation of superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) enzyme activities in the brain homogenates of I/R rats. βCAR treatment significantly reduced nuclear factor kappa B (NF-κB) brain content and myeloperoxidase (MPO) activity and ameliorated the histological alterations in the brain tissues. βCAR significantly suppressed Bcl-2-associated X protein (Bax) and caspase-3 expression, as well as upregulated B-cell lymphoma-2 (Bcl-2) expression, suggesting a neuroprotective potential via downregulating NF-kB and protecting the rat brain against the I/R-associated apoptotic injury.

Skeletal Muscle Nuclei in Mice are not Post-mitotic.

The skeletal muscle research field generally accepts that nuclei in skeletal muscle fibers (ie, myonuclei) are post-mitotic and unable to proliferate. Because our deuterium oxide (D2O) labeling studies showed DNA synthesis in skeletal muscle tissue, we hypothesized that resident myonuclei can replicate in vivo. To test this hypothesis, we used a mouse model that temporally labeled myonuclei with GFP followed by D2O labeling during normal cage activity, functional overload, and with satellite cell ablation. During normal cage activity, we observed deuterium enrichment into myonuclear DNA in 7 out of 7 plantaris (PLA), 6 out of 6 tibialis anterior (TA), 5 out of 7 gastrocnemius (GAST), and 7 out of 7 quadriceps (QUAD). The average fractional synthesis rates (FSR) of DNA in myonuclei were: 0.0202±0.0093 in PLA, 0.0239±0.0040 in TA, 0.0076±0. 0058 in GAST, and 0.0138±0.0039 in QUAD, while there was no replication in myonuclei from EDL. These FSR values were largely reproduced in the overload and satellite cell ablation conditions, although there were higher synthesis rates in the overloaded PLA muscle. We further provided evidence that myonuclear replication is through endoreplication, which results in polyploidy. These novel findings contradict the dogma that skeletal muscle nuclei are post-mitotic and open potential avenues to harness the intrinsic replicative ability of myonuclei for muscle maintenance and growth.