Hindlimb Muscles Research Articles

Heterotopic Ossification of the Adductor Muscles in Bald Eagles (Haliaeetus leucocephalus)

Diseases of the musculature can be caused, for example, by alimentary or traumatic issues. In three bald eagles with painful lameness and/or a reduced range of the hip joint, a muscular disease was diagnosed via radiographic examination. The eagles were kept in a falconry and trained as demonstration birds, which were displayed on a gloved hand and flown in falconry education programs. The imaging revealed varying degrees of calcification of the adductors along the course of the puboischiofemoral muscle of the left hind limb in all cases. A histological examination of the affected musculature of one eagle confirmed the presence of a myopathy with muscle fiber atrophy, fibrosis, and metaplastic bone formation. Increased mechanical stress and injuries to the affected muscle groups are discussed as the cause of these alterations. The adductor muscles of the left leg of bald eagles in falconry husbandry appear to be particularly prone to muscular injuries and the development of heterotopic ossification. The healing process of such an injury with the stiffened musculature can lead to restrictions in the movement of the hip joint. Thus, the conscious and professional handling of the eagles is essential for maintaining the health of these birds.

Sucla2 Knock-Out in Skeletal Muscle Yields Mouse Model of Mitochondrial Myopathy With Muscle Type-Specific Phenotypes.

Pathogenic variants in subunits of succinyl-CoA synthetase (SCS) are associated with mitochondrial encephalomyopathy in humans. SCS catalyses the conversion of succinyl-CoA to succinate coupled with substrate-level phosphorylation of either ADP or GDP in the TCA cycle. This report presents a muscle-specific conditional knock-out (KO) mouse model of Sucla2, the ADP-specific beta subunit of SCS, generating a novel invivo model of mitochondrial myopathy. The mouse model was generated using the Cre-Lox system, with the human skeletal actin (HSA) promoter driving Cre-recombination of a CRISPR-Cas9-generated Sucla2 floxed allele within skeletal muscle. Inactivation of Sucla2 was validated using RT-qPCR and western blot, and both enzyme activity and serum metabolites were quantified by mass spectrometry. To characterize the model invivo, whole-body phenotyping was conducted, with mice undergoing a panel of strength and locomotor behavioural assays. Additionally, exvivo contractility experiments were performed on the soleus (SOL) and extensor digitorum longus (EDL) muscles. SOL and EDL cryosections were also subject to imaging analyses to assess muscle fibre-specific phenotypes. Molecular validation confirmed 68% reduction of Sucla2 transcript within the mutant skeletal muscle (p < 0.001) and 95% functionally reduced SUCLA2 protein (p < 0.0001). By 3 weeks of age, Sucla2 KO mice were 44% the size of controls by body weight (p < 0.0001). Mutant mice also exhibited 34%-40% reduced grip strength (p < 0.01) and reduced spontaneous exercise, spending about 88% less cumulative time on a running wheel (p < 0.0001). Contractile function was also perturbed in a muscle-specific manner; although no genotype-specific deficiencies were seen in EDL function, SUCLA2-deficient SOL muscles generated 40% less specific tetanic force (p < 0.0001), alongside slower contraction and relaxation rates (p < 0.001). Similarly, a SOL-specific threefold increase in mitochondria (p < 0.0001) was observed, with qualitatively increased staining for both COX and SDH, and the proportion of Type 1 myosin heavy chain expressing fibres within the SOL was nearly doubled (95% increase, p < 0.0001) in the Sucla2 KO mice compared with that in controls. SUCLA2 loss within murine skeletal muscle yields a model of SCS-deficient mitochondrial myopathy with reduced body weight, muscle weakness and exercise intolerance. Physiological and morphological analyses of hindlimb muscles showed remarkable differences in exvivo function and cellular consequences between the EDL and SOL muscles, with SOL muscles significantly more impacted by Sucla2 inactivation. This novel model will provide an invaluable tool for investigations of muscle-specific and fibre type-specific pathogenic mechanisms to better understand SCS-deficient myopathy.

Assessment of cardiac and skeletal muscle metabolites using 1H-MRS and chemical-shift encoded magnetic resonance imaging: Impact of diabetes, RAGE, and DIAPH1.

Diabetes affects metabolism and metabolite concentrations in multiple organs. Previous preclinical studies have shown that receptor for advanced glycation end products (RAGE, gene symbol Ager) and its cytoplasmic domain binding partner, Diaphanous-1 (DIAPH1), are key mediators of diabetic micro- and macro-vascular complications. In this study, we used 1H-Magnetic Resonance Spectroscopy (MRS) and chemical shift encoded (CSE) Magnetic Resonance Imaging (MRI) to investigate the metabolite and water-fat fraction in the heart and hind limb muscle in a murine model of type 1 diabetes (T1D) and to determine if the metabolite changes in the heart and hind limb are influenced by (a) deletion of Ager or Diaph1 and (b) pharmacological blockade of RAGE-DIAPH1 interaction in mice. Nine cohorts of male mice, with six mice per cohort, were used: wild type non-diabetic control mice (WT-NDM), WT-diabetic (WT-DM) mice, Ager knockout non-diabetic (RKO-NDM) and diabetic mice (RKO-DM), Diaph1 knockout non-diabetic (DKO-NDM), and diabetic mice (DKO-DM), WT-NDM mice treated with vehicle, WT-DM mice treated with vehicle, and WT-DM mice treated with RAGE229 (antagonist of RAGE-DIAPH1 interaction). A Point Resolved Spectroscopy (PRESS) sequence for 1H-MRS, and multi-echo gradient recalled echo (GRE) for CSE were employed. Triglycerides, and free fatty acids in the heart and hind limb obtained from MRS and MRI were compared to those measured using biochemical assays. Two-sided t-test, non-parametric Kruskal-Wallis Test, and one-way ANOVA were employed for statistical analysis. We report that the results were well-correlated with significant differences using MRI and biochemical assays between WT-NDM and WT-DM, as well as within the non-diabetic groups, and within the diabetic groups. Deletion of Ager or Diaph1, or treatment with RAGE229 attenuated diabetes-associated increases in triglycerides in the heart and hind limb in mice. These results suggest that the employment of 1H-MRS/MRI is a feasible non-invasive modality to monitor metabolic dysfunction in T1D and the metabolic consequences of interventions that block RAGE and DIAPH1.

Pedal Reaction Forces and Electromyography Responses Indicate Eccentric Contractions During Motorized Cycling in a Rat Model of Incomplete Spinal Cord Injury.

Motorized cycling (MC) is utilized as an alternative to traditional exercise in individuals who are unable to perform voluntary movements post-spinal cord injury. Although rodent models of MC often show more positive outcomes when compared with clinical studies, the cause of this difference is unknown. We postulate that biomechanical differences between rats and humans may contribute to this discrepancy. To begin to test this theory, we examined pedal reaction forces and electromyography (EMG) of hindlimb muscles as a function of cycle phase and cadence in a rat model of MC. We found that higher cadences (≥30 RPM) increased EMG and force, with higher forces observed in animals with contusion injuries as compared with transections. To further investigate the forces, we developed a technique to separate rhythmic (developed with the motion of the pedals) from nonrhythmic forces. Rhythmic forces resulted from induced eccentric muscle contractions that increased (amplitude and prevalence) at higher cadences, whereas nonrhythmic forces showed the opposite pattern. Our results suggest that muscle activity during MC in rats depends on the stretch reflex, which, in turn, depends on the rate of muscle lengthening that is modulated by cadence. Additionally, we provide a framework for understanding MC that may help translate results from rat models to clinical use in the future.

Hindlimb kinematics, kinetics, and muscle dynamics during sit-to-stand and sit-to-walk transitions in emus (Dromaius novaehollandiae).

Terrestrial animals not only need to walk and run but also lie prone to rest and then stand up. Sit-to-stand (STS) and sit-to-walk (STW) transitions are vital behaviours little studied in species other than humans so far, but likely impose biomechanical constraints on limb design because they involve near-maximal excursions of limb joints that should require large length changes and force production from muscles. By integrating data from experiments into musculoskeletal simulations, we analysed joint motions, ground reaction forces, and muscle dynamics during STS and STW in a large terrestrial, bipedal, and cursorial bird: the emu (Dromaius novaehollandiae, ∼30 kg). Simulation results suggest that in both STS and STW, emus operate near the functional limits (∼50 % of shortening/lengthening) of some of their hindlimb muscles, particularly in distal muscles with limited capacity for length change and leverage. Both movements involved high muscle activations (> 50 %) and force generation of the major joint extensor muscles early in the transition. STW required larger net joint moments and non-sagittal motions than STS, entailing greater demands for muscle capacity. Whilst our study involves multiple assumptions, our findings lay the groundwork for future studies to understand, for example, how tendon contributions may reduce excessive muscle demands, especially in the distal hindlimb. As the first investigation into how an avian species stands up, this study provides a foundational framework for future comparative studies investigating organismal morphofunctional specialisations and evolution, offering potential robotics and animal welfare applications.

Isolation of small extracellular vesicles from regenerating muscle tissue using tangential flow filtration and size exclusion chromatography

We have recently made the strikingly discovery that upon a muscle injury, Wnt7a is upregulated and secreted from new regenerating myofibers on the surface of exosomes to elicit its myogenerative response distally. Despite recent advances in extracellular vesicle (EVs) isolation from diverse tissues, there is still a lack of specific methodology to purify EVs from muscle tissue. To eliminate contamination with non-EV secreted proteins and cytoplasmic fragments, which are typically found when using classical methodology, such as ultracentrifugation, we adapted a protocol combining Tangential Flow Filtration (TFF) and Size Exclusion Chromatography (SEC). We found that this approach allows simultaneous purification of Wnt7a, bound to EVs (retentate fraction) and free non-EV Wnt7a (permeate fraction). Here we described this optimized protocol designed to specifically isolate EVs from hind limb muscle explants, without cross-contamination with other sources of non-EV bounded proteins. The first step of the protocol is to remove large EVs with sequential centrifugation. Extracellular vesicles are then concentrated and washed in exchange buffer by TFF. Lastly, SEC is performed to remove any soluble protein traces remaining after TFF. Overall, this procedure can be used to isolate EVs from conditioned media or biofluid that contains EVs derived from any cell type or tissue, improving reproducibility, efficiency, and purity of EVs preparations. Our purification protocol results in high purity EVs that maintain structural integrity and thus fully compatible with in vitro and in vivo bioactivity and analytic assays.

A Natural History Study of Hindlimb Physiology and Histopathology in a Heterozygous OPMD Mouse Model

ABSTRACTBackgroundOculopharyngeal muscular dystrophy (OPMD) is an adult‐onset autosomal dominant myopathy. OPMD is caused by a short alanine expansion in the gene encoding for poly(A) binding protein nuclear 1 (PABPN1) forming insoluble nuclear aggregates. OPMD patients are predominantly heterozygous, and the knock‐in Pabpn1+/A17 mouse, which expresses one copy of the expanded Pabpn1 gene under the PABPN1 promoter genetically, mimics OPMD. Insights into the A17/+ mouse model are necessary to evaluate its preclinical value and test therapeutics for OPMD. Here, we performed a natural disease history study for the A17/+ model.MethodsWe combined muscle force measurements of the tibialis anterior with magnetic resonance imaging (MRI) measurements of the calf muscles made in 4‐, 8‐ and 12‐month‐old wild‐type and A17/+ mice. These measures were complemented by muscle histopathology staining and image quantification to detect PABPN1 aggregates and to assess muscle wasting. Statistical significance between genotypes over the three time points was assessed using ANOVA or Student's t test.ResultsPABPN1 nuclear aggregates were found in the 12‐month‐old A17/+ mice at similar quantities of ~2% across hindlimb muscles. We did not observe changes in muscle strength of the tibialis anterior in A17/+ mice. MRI analyses of hindlimb muscles revealed no metabolic difference, no fatty infiltration and limited muscle atrophy between A17/+ and +/+ mice. The plantaris muscle in A17/+ showed 30% atrophy at 12 months of age, which was corroborated by a 30% myofiber shift in the myosin heavy chain −2A to −2B ratio. Histopathologic staining did not reveal muscle wasting in the hindlimb muscles.ConclusionsDespite the presence of PABPN1 insoluble aggregates in hindlimb muscles, muscle involvement in the 12‐month‐old A17/+ mice was limited. Our results query the usefulness of A17/+ hindlimb muscles for preclinical studies.

In dystrophic mdx hindlimb muscles where fibrosis is limited, versican haploinsufficiency transiently improves contractile function without reducing inflammation.

Versican is increased with inflammation and fibrosis, and is upregulated in Duchenne muscular dystrophy. In fibrotic diaphragm muscles from dystrophic mdx mice, genetic reduction of versican attenuated macrophage infiltration and improved contractile function. Versican is also implicated in myogenesis. Here, we investigated whether versican modulated mdx hindlimb muscle pathology, where inflammation and regeneration are increased but fibrosis is minimal. Immunohistochemistry and qRT-PCR were used to assess how fiber type and glucocorticoids (α-methylprednisolone) modify versican expression. To genetically reduce versican, female mdx and male versican haploinsufficient (hdf) mice were bred resulting in male mdx-hdf and mdx (control) pups. Versican expression, contractile function, and pathology were evaluated in hindlimb muscles. Versican immunoreactivity was greater in slow versus fast hindlimb muscles. Versican mRNA transcripts were reduced by α-methylprednisolone in soleus, but not in fast extensor digitorum longus, muscles. In juvenile (6-wk-old) mdx-hdf mice, versican expression was most robustly decreased in soleus muscles leading to improved force output and a modest reduction in fatiguability. These functional benefits were not accompanied by decreased inflammation. Muscle architecture, regeneration markers, and fiber type also did not differ between mdx-hdf mice and mdx littermates. Improvements in soleus contractile function were not retained in adult (20-wk-old) mdx-hdf mice. In conclusion, soleus muscles from juvenile mdx mice were most responsive to pharmacological or genetic approaches targeting versican; however, the benefits of versican reduction were limited due to low fibrosis. Preclinical matrix research in dystrophy should account for muscle phenotype (including age) and the interdependence between inflammation and fibrosis. NEW & NOTEWORTHY The proteoglycan versican is upregulated in muscular dystrophy. In fibrotic diaphragm muscles from mdx mice, versican reduction attenuated macrophage infiltration and improved performance. Here, in hindlimb muscles from 6- and 20-wk-old mdx mice, where pathology is mild, versican reduction did not decrease inflammation and contractile function improvements were limited to juvenile mice. In dystrophic mdx muscles, the association between versican and inflammation is mediated by fibrosis, demonstrating interdependence between the immune system and extracellular matrix.

Changes in intra- and interlimb reflexes from forelimb cutaneous afferents after staggered thoracic lateral hemisections during locomotion in cats.

In quadrupeds, such as cats, cutaneous afferents from the forepaw dorsum signal external perturbations and send inputs to spinal circuits to co-ordinate the activity in muscles of all four limbs. How these cutaneous reflex pathways from forelimb afferents are reorganized after an incomplete spinal cord injury is not clear. Using a staggered thoracic lateral hemisections paradigm, we investigated changes in intralimb and interlimb reflex pathways by electrically stimulating the left and right superficial radial nerves in seven adult cats and recording reflex responses in five forelimb and ten hindlimb muscles. After the first (right T5-T6) and second (left T10-T11) hemisections, forelimb-hindlimb co-ordination was altered and weakened. After the second hemisection, cats required balance assistance to perform quadrupedal locomotion. Short-, mid- and long-latency homonymous and crossed reflex responses in forelimb muscles and their phase modulation remained largely unaffected after staggered hemisections. The occurrence of homolateral and diagonal mid- and long-latency responses in hindlimb muscles evoked with left and right superficial radial nerve stimulation was significantly reduced at the first time point after the first hemisection, but partially recovered at the second time point with left superficial radial nerve stimulation. These responses were lost or reduced after the second hemisection. When present, all reflex responses, including homolateral and diagonal, maintained their phase-dependent modulation. Therefore, our results show a considerable loss in cutaneous reflex transmission from cervical to lumbar levels after incomplete spinal cord injury, albeit with preservation of phase modulation, probably affecting functional responses to external perturbations. KEY POINTS: Cutaneous afferent inputs co-ordinate muscle activity in the four limbs during locomotion when the forepaw dorsum contacts an obstacle. Thoracic spinal cord injury disrupts communication between spinal locomotor centres located at cervical and lumbar levels, impairing balance and limb co-ordination. We investigated cutaneous reflexes from forelimb afferents during quadrupedal locomotion by electrically stimulating the superficial radial nerve bilaterally, before and after staggered lateral thoracic hemisections in cats. We showed a loss/reduction of mid- and long-latency homolateral and diagonal reflex responses in hindlimb muscles early after the first hemisection that partially recovered with left superficial radial nerve stimulation, before being reduced after the second hemisection. Targeting cutaneous reflex pathways from forelimb afferents projecting to the four limbs could help develop therapeutic approaches aimed at restoring transmission in ascending and descending spinal pathways.

Age- and size-related changes in hind limb muscles in two baboon species (Papio anubis and P. papio).

Body size has an impact on all biological functions and analyzing how body size impacts functional traits such as locomotion is critical. Body size does not only vary across species but also during ontogeny. Indeed, juvenile animals are often at a competitive disadvantage due to their smaller absolute size. Consequently, understanding size- and age-related changes in the locomotor system is critical for our understanding of adult phenotypes. Here, we address this question by exploring growth of the hind limb muscles in two species of closely related baboons that differ in their ecology, the olive baboon, Papio Anubis, the Guinea baboon, and Papio papio. To do so, we dissected 40 P. anubis and 10 P. papio and measured the mass and physiological cross-sectional area (PCSA) of the hind limb muscles. Our results showed no sexual differences in size- or age-related growth patterns, but did show differences between species. Whereas the scaling of muscle mass and PCSA was largely isometric in P. anubis, allometric scaling was more common in P. papio. Despite these differences between species, the knee extensors and external rotators at the knee scaled with positive allometry in both species highlighting their important role during adult locomotion. Although life-history data for P. papio are scarce, we suggest that differences between species may be associated with differences in adult body size and age of locomotor independence between species.

EFFECTS OF SPINAL TRANSECTION AND LOCOMOTOR SPEED ON MUSCLE SYNERGIES OF THE CAT HINDLIMB.

It was suggested that during locomotion, the nervous system controls movement by activating groups of muscles, or muscle synergies. Analysis of muscle synergies can reveal the organization of spinal locomotor networks and how it depends on the state of the nervous system, such as before and after spinal cord injury, and on different locomotor conditions, including a change in speed. The goal of this study was to investigate the effects of spinal transection and locomotor speed on hindlimb muscle synergies and their time-dependent activity patterns in adult cats. EMG activities of 15 hindlimb muscles were recorded in 9 adult cats of either sex during tied-belt treadmill locomotion at speeds of 0.4, 0.7, and 1.0 m/s before and after recovery from a low thoracic spinal transection. We determined EMG burst groups using cluster analysis of EMG burst onset and offset times and muscle synergies using non-negative matrix factorization. We found five major EMG burst groups and five muscle synergies in each of six experimental conditions (2 states × 3 speeds). In each case, the synergies accounted for at least 90% of muscle EMG variance. Both spinal transection and locomotion speed modified subgroups of EMG burst groups and the composition and activation patterns of selected synergies. However, these changes did not modify the general organization of muscle synergies. Based on the obtained results, we propose an organization for a pattern formation network of a two-level central pattern generator that can be tested in neuromechanical simulations of spinal circuits controlling cat locomotion.

Multimodal three-dimensional characterization of murine skeletal muscle micro-scale elasticity, structure, and composition: Impact of dysferlinopathy, Duchenne muscular dystrophy, and age on three hind-limb muscles

Skeletal muscle tissue function is governed by the mechanical properties and organization of its components, including myofibers, extracellular matrix, and adipose tissue, which can be modified by the onset and progression of many disorders. This study used a novel combination of quantitative micro-elastography and clearing-enhanced three-dimensional (3D) microscopy to assess 3D micro-scale elasticity and micro-architecture of muscles from two muscular dystrophies: dysferlinopathy and Duchenne muscular dystrophy, using male BLA/J and mdx mice, respectively, and their wild-type (WT) controls. We examined three muscles with varying proportions of slow- and fast-twitch myofibers: the soleus (predominantly slow), extensor digitorum longus (EDL; fast), and quadriceps (mixed), from BLA/J and WTBLA/J mice aged 3, 10, and 24 months, and mdx and WTmdx mice aged 10 months. Both dysferlin deficiency and age reduced the elasticity and variability of elasticity of the soleus and quadriceps, but not EDL. Overall, the BLA/J soleus was 20% softer than WT and less mechanically heterogeneous (−14% in standard deviation of elasticity). The BLA/J quadriceps at 24 months was 72% softer than WT and less mechanically heterogeneous (−59% in standard deviation), with substantial adipose tissue accumulation. While mdx muscles did not differ quantitatively from WT, regional heterogeneity was evident in micro-scale elasticity and micro-architecture of quadriceps (e.g., 11.2 kPa in a region with marked pathology vs 3.8 kPa in a less affected area). These results demonstrate differing biomechanical changes in hind-limb muscles of two distinct muscular dystrophies, emphasizing the potential for this novel multimodal technique to identify important differences between various myopathies.

Protein kinase C epsilon contributes to chronic mechanoreflex sensitization in rats with heart failure.

We investigated second-messenger signalling components linked to the stimulation of Gq protein-coupled receptors (e.g. thromboxane A2 and bradykinin B2 receptors) on the sensory endings of thin fibre muscle afferents in the chronic mechanoreflex sensitization in rats with myocardial infarction-induced heart failure with reduced ejection fraction (HF-rEF). We hypothesized that injection of either the inositol 1,4,5-trisphosphate (IP3) receptor antagonist xestospongin C (5µg) or the PKCε translocation inhibitor PKCe141 (45µg) into the arterial supply of the hindlimb would reduce the increase in renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) evoked during 30s of 1Hz dynamic hindlimb muscle stretch in decerebrate, unanaesthetized HF-rEF rats but not sham-operated controls (SHAM). Ejection fraction was significantly reduced in HF-rEF (45 (19)%) compared to SHAM (80 (9)%; P<0.001) rats. In HF-rEF rats (n=3M/2F), IP3 receptor blockade had no effect on the peak ΔRSNA (pre: 99 (74)%; post: 133 (79)%; P=0.974) or peak ΔMAP response to stretch (peak ΔMAP: pre: 32 (14) mmHg; post: 36 (21) mmHg; P=0.719). Conversely, in another group of HF-rEF rats (n=4M/3F), the PKCε translocation inhibitor reduced the peak ΔRSNA (pre: 110 (77)%; post: 62 (58)%; P=0.029) and peak ΔMAP response to stretch (pre: 30 (20) mmHg; post: 17 (16) mmHg; P=0.048). In SHAM counterparts, neither drug affected the mechanoreflex responses. Our findings highlight PKCε, but not IP3 receptors, as a significant second-messenger in the chronic mechanoreflex sensitization in HF-rEF which may play a crucial role in the exaggerated sympathetic response to exercise in this patient population. KEY POINTS: Skeletal muscle contraction results in an exaggerated reflex increase in sympathetic nerve activity in heart failure patients with reduced ejection fraction (HF-rEF) compared to healthy individuals, contributing to increased cardiovascular risk and impaired tolerance for mild exercise. The exaggerated reflex sympathetic responses in HF-rEF may be attributed to a chronic sensitization of mechanically sensitive thin fibre muscle afferents mediated, at least in part, by stimulation of Gq protein-coupled thromboxane A2 and bradykinin B2 receptors on muscle afferent sensory endings. The specific Gq protein-linked signalling mechanisms that produce the chronic mechanoreflex sensitization in HF-rEF have not been investigated but may involve inositol 1,4,5-trisphosphate (IP3) receptors and/or protein kinase C epsilon (PKCε). Here we demonstrate that PKCε, but not IP3 receptors, within the sensory endings of thin fibre muscle afferents plays a role in the sensitization of mechanically sensitive thin fibre muscle afferents in rats with HF-rEF.

Differential effects of exercise and hormone treatment on spinal cord injury-induced changes in micturition and morphology of external urethral sphincter motoneurons.

Spinal cord injury (SCI) results in lesions that destroy tissue and spinal tracts, leading to deficits in locomotor and autonomic function. We have previously shown that after SCI, surviving motoneurons innervating hindlimb muscles exhibit extensive dendritic atrophy, which can be attenuated by treadmill training or treatment with gonadal hormones post-injury. We have also shown that following SCI, both exercise and treatment with gonadal hormones improve urinary function. Animals exercised with forced running wheel training show improved urinary function as measured by bladder cystometry and sphincter electromyography, and treatment with gonadal hormones improves voiding patterns as measured by metabolic cage testing. The objective of the current study was to examine the potential protective effects of exercise or hormone treatment on the structure and function of motoneurons innervating the external urethral sphincter (EUS) after contusive SCI. Gonadally intact young adult male rats received either a sham or a thoracic contusion injury. Immediately after injury, one cohort of animals was implanted with subcutaneous Silastic capsules filled with estradiol (E) and dihydrotestosterone (D) or left blank; continuous hormone treatment occurred for 4 weeks post-injury. A separate cohort of SCI-animals received either 12 weeks of forced wheel running exercise or no exercise treatment starting two weeks after injury. At the end of treatment, urinary void volume was measured using metabolic cages and EUS motoneurons were labeled with cholera toxin-conjugated horseradish peroxidase, allowing for assessment of dendritic morphology in three dimensions. Locomotor performance was improved in exercised animals after SCI. Void volumes increased after SCI in all animals; void volume was unaffected by treatment with exercise, but was dramatically improved by treatment with E + D. Similar to what we have previously reported for hindlimb motoneurons after SCI, dendritic length of EUS motoneurons was significantly decreased after SCI compared to sham animals. Exercise did not reverse injury-induced atrophy, however E + D treatment significantly protected dendritic length. These results suggest that some aspects of urinary dysfunction after SCI can be improved through treatment with gonadal hormones, potentially through their effects on EUS motoneurons. Moreover, a more comprehensive treatment regime that addresses multiple SCI-induced sequelae, i.e., locomotor and voiding deficits, would include both hormones and exercise.

Abstract 58: The 5-HT7 receptor contributes to increased hindquarter blood flow caused by skeletal muscle contraction

Exogenous serotonin (5-hydroxytryptamine, 5-HT) causes a reproducible decrease in blood pressure in normotensive and hypertensive rats. This is associated with dilation of arterioles in skeletal muscle. The 5-HT7 receptor KO rat strain allowed us to test the role of this receptor in both responses as well as the effects of endogenous 5-HT -- acting at 5-HT7 receptors -- on normal circulatory regulation. First, we hypothesized that the 5-HT7 receptor KO rat would have a higher hindquarter (HQ) vascular resistance (HQVR) than wildtype (WT) because of loss of this receptor. In rats anesthetized with isoflurane, instrumented with arterial catheters and flow probes on the terminal aorta, male KO rats had significantly higher resting HQVR [mm Hg/ml/min; KO (16.0±2.0) vs WT (10.8±0.6.0), p = 0.04] and lower HQ blood flow than normal Sprague-Dawley (SD) rats. No differences in resting HQVR or HQ flow were observed in female rats. Intravenous infusion of 5-HT (50 ug/kg/min) reduced systemic blood pressure and HQVR; these parameters were significantly attenuated (~56%) in male KO rats versus WT and SD animals. In females, falls in systemic blood pressure and HQVR were absent in both KO and WT rats. Second, as 5-HT has been implicated in the pathophysiology of heart failure (HF), a left anterior descending artery ligation model of HF was used to investigate the potential role of 5-HT7 regulation of HQ flow in HF-associated exercise intolerance. The ability of hindlimb muscle stimulation to increase blood flow using transdermal neuromuscular electrical stimulation was tested (NMES). M and F KO rats showed a reduced ability to increase HQ flow during muscle contraction vs WT (% over basal: M WT = 118±18 vs KO =14.6±7.1; F WT= 101±12 vs KO = 7.6±6) rats. This was observed in both intact and HF rats (in which flow changes were already impaired). Acute administration of the 5-HT7 receptor antagonist SB269970 abolished the ability of NMES to increase HQ flow in normal SD rats. Importantly, the microcirculatory capacity, visualized by Lectin-594, was not significantly different in the gastrocnemius muscle of WT (arbitrary fluorescence units/area: 946±40) vs KO (914±69). These data support that the 5-HT7 receptor, likely activated by endogenous 5-HT, is an important regulator of skeletal muscle vascular resistance. Thus, 5-HT may have a larger role in normal and pathophysiological regulation of the circulation than has previously been appreciated.

Springing into action: Comparing escape responses between bipedal and quadrupedal rodents.

Predation is a fundamental selective pressure on animal morphology, as morphology is directly linked with physical performance and evasion. Bipedal heteromyid rodents, which are characterized by unique morphological traits such as enlarged hindlimbs, appear to be more successful than sympatric quadrupedal rodents at escaping predators such as snakes and owls, but no studies have directly compared the escape performance of bipedal and quadrupedal rodents. We used simulated predator attacks to compare the evasive jumping ability of bipedal kangaroo rats (Dipodomys) to that of three quadrupedal rodent groups-pocket mice (Chaetodipus), woodrats (Neotoma), and ground squirrels (Otospermophilus). Jumping performance of pocket mice was remarkably similar to that of kangaroo rats, which may be driven by their shared anatomical features (such as enlarged hindlimb muscles) and facilitated by their relatively small body size. Woodrats and ground squirrels, in contrast, almost never jumped as a startle response, and they took longer to perform evasive escape maneuvers than the heteromyid species (kangaroo rats and pocket mice). Among the heteromyids, take-off velocity was the only jump performance metric that differed significantly between species. These results support the idea that bipedal body plans facilitate vertical leaping in larger-bodied rodents as a means of predator escape and that vertical leaping likely translates to better evasion success.

Macrophages modulate skeletal muscle wasting and recovery in acute lung injury in mice.

Skeletal muscle dysfunction in critical illnesses leaves survivors weak and functionally impaired. Macrophages infiltrate muscles; however, their functional role is unclear. We aim to examine muscle leukocyte composition and the effect of macrophages on muscle mass and function in the murine acute lung injury (ALI)-associated skeletal muscle wasting model. We performed flow cytometry of hindlimb muscle to identify myeloid cells pre-injury and time points up to 29 days after intratracheal lipopolysaccharide ALI. We evaluated muscle force and morphometrics after systemic and intramuscular clodronate-induced macrophage depletions between peak lung injury and recovery (day 5-6) versus vehicle control. Our results show muscle leukocytes changed over ALI course with day 3 neutrophil infiltration (130.5 ± 95.6cells/mg control to 236.3 ± 70.6cells/mg day 3) and increased day 10 monocyte abundance (5.0 ± 3.4%CD45+CD11b+ day 3 to 14.0 ± 2.6%CD45+CD11b+ day 10, p = 0.005). Although macrophage count did not significantly change, pro-inflammatory (27.0 ± 7.2% day 3 to 7.2 ± 3.8% day 10, p = 0.02) and anti-inflammatory (30.5 ± 11.1% day 3 to 52.7 ± 9.7% day 10, p = 0.09) surface marker expression changed over the course of ALI. Macrophage depletion following peak lung injury increased muscle mass and force generation. These data suggest muscle macrophages beyond peak lung injury limit or delay muscle recovery. Targeting macrophages could augment muscle recovery following lung injury.

Combining clinically common drugs with hindlimb stretching in spinal cord injured rodents.

Preclinical pilot study. To explore peripheral and central nociceptive mechanisms that contribute to muscle stretch-induced locomotor deficits following spinal cord injury. Kentucky Spinal Cord Injury Research Center, Louisville, KY, USA. Ten female Sprague-Dawley rats received moderate, 25 g/cm T10 contusion injuries and recovered for 4 weeks. Rats were divided into three groups: Morphine/Ibuprofen-treated, Acetaminophen/Baclofen-treated, and saline control. Each group received daily hindlimb muscle stretching during weeks 4, 5, 9, and 10 post-injury and drugs were administered with stretching during weeks 4 and 9 only. Locomotor function was assessed throughout the experiment using the BBB Open Field Locomotor Scale. Hindlimb responses including spasticity, writhing, and clonic-like vibrations during muscle stretching were classified and scored. Consistent with our previous studies, hindlimb muscle stretching caused significant deficits in locomotor recovery following spinal cord injury. Baclofen and Ibuprofen partially mitigated the stretching effect, but none of the drugs significantly prevented the drop in locomotor function during stretching. Interestingly, treatment with Baclofen or Ibuprofen significantly reduced hindlimb responses such as spasticity and writhing during stretching, while Morphine exacerbated clonic-like vibrations in response to stretching maneuvers. These findings suggest that stretching may inhibit locomotor recovery through combined mechanisms of peripheral inflammation and sensitization of nociceptive afferents. When combined with central sprouting and loss of descending controls after SCI, this results in exaggerated nociceptive input during stretching. The inability of the applied clinical drugs to mitigate the detrimental effects of stretching highlights the complexity of the stretching phenomenon and emphasizes the need for further investigation.

Intracerebroventricular insulin injection acutely normalizes the augmented exercise pressor reflex in male rats with type 2 diabetes mellitus.

The exercise pressor reflex (EPR) is exaggerated in type 2 diabetes mellitus (T2DM), but the underlying central nervous system aberrations have not been fully delineated. Stimulation of muscle afferents within working skeletal muscle activates the EPR, by sending information to neurons in the brainstem, where it is integrated and results in reflexively increased mean arterial pressure (MAP) and sympathetic nerve activity. Brain insulin is known to regulate neural activity within the brainstem. We hypothesize that brain insulin injection in T2DM rats attenuates the augmented EPR, and that T2DM is associated with decreased brain insulin. Using male Sprague-Dawley rats, T2DM and control rats were generated via an induction protocol with two low doses of streptozotocin (35 and 25mg/kg, i.p.) in combination with a 14-23-week high-fat diet or saline injections and a low-fat diet, respectively. After decerebration, MAP and renal sympathetic nerve activity (RSNA) were evaluated during EPR stimulation, evoked by electrically induced muscle contraction via ventral root stimulation, before and after (1 and 2h post) intracerebroventricular (i.c.v.) insulin microinjections (500mU, 50nl). i.c.v. insulin decreased peak MAP (ΔMAP Pre (36±14mmHg) vs. 1h (21±14mmHg) vs. 2h (11±6mmHg), P<0.05) and RSNA (ΔRSNA Pre (107.5±40%), vs. 1h (75.4±46%) vs. 2h (51±35%), P<0.05) responses in T2DM, but not controls. In T2DM rats, cerebrospinal fluid insulin was decreased (0.41±0.19 vs. 0.11±0.05ng/ml, control (n=14) vs. T2DM (n=4), P<0.01). The results demonstrated that insulin injections into the brain normalized the augmented EPR in brain hypoinsulinaemic T2DM rats, indicating that the EPR can be regulated by brain insulin. KEY POINTS: The reflexive increase in blood pressure and sympathetic nerve activity mediated by the autonomic nervous system during muscle contractions is also known as the exercise pressor reflex. The exercise pressor reflex is dangerously augmented in type 2 diabetes, in both rats and humans. In type 2 diabetic rats both cerebrospinal fluid insulin and phosphoinositide 3-kinase signalling within cardiovascular brainstem neurons decrease in parallel. Brain insulin injections decrease the magnitude of the reflexive pressor and sympathetic responses to hindlimb muscle contraction in type 2 diabetic rats. Partial correction of low insulin within the central nervous system in type 2 diabetes may treat aberrant exercise pressor reflex function.

Comparative muscle anatomy of the anuran pelvis and hindlimb in relation to locomotor mode.

Frogs have a highly conserved body plan, yet they employ a diverse array of locomotor modes, making them ideal organisms for investigating the relationships between morphology and locomotor function, in particular whether anatomical complexity is a prerequisite for functional complexity. We use diffusible iodine contrast-enhanced microCT (diceCT) imaging to digitally dissect the gross muscle anatomy of the pelvis and hindlimbs for 30 species of frogs representing five primary locomotor modes, including the first known detailed dissection for some of the world's smallest frogs, forming the largest digital comparative analysis of musculoskeletal structure in any vertebrate clade to date. By linking musculoskeletal dissections and phylogenetic comparative methods, we then quantify and compare relationships between anatomy and function across over 160 million years of anuran evolution. In summary, we have found that bone lengths and pelvic crest sizes are generally not reliable predictors of muscle sizes, which highlights important implications for future palaeontological studies. Our investigation also presents previously unreported differences in muscle anatomy between frogs specialising in different locomotor modes, including several of the smallest frog hindlimb muscles, which are extremely difficult to extract and measure using traditional approaches. Furthermore, we find evidence of many-to-one and one-to-many mapping of form to function across the phylogeny. Additionally, we perform the first quantitative analysis of how the degree of muscle separation can differ between frogs. We find evidence that phylogenetic history is the key contributing factor to muscle separation in the pelvis and thigh, while the separation of shank muscles is influenced more strongly by locomotor mode. Finally, our anatomical 3D reconstructions are published alongside this manuscript to contribute towards future research and serve as educational materials.