Neuroprotective macromolecular methylprednisolone prodrug nanomedicine prevents glucocorticoid-induced muscle atrophy and osteoporosis in a rat model of spinal cord injury.

To address the adverse side effects associated with systemic high-dose methylprednisolone (MP) therapy for acute spinal cord injury (SCI), we have developed a N-2-hydroxypropyl methacrylamide copolymer-based MP prodrug nanomedicine (Nano-MP). Intravenous Nano-MP selectively targeted to the inflamed SCI lesion and significantly improved neuroprotection and functional recovery after acute SCI. In the present study, we comprehensively assessed the potential adverse side effects associated with the treatment in the SCI rat models, including reduced body weight and food intake, impaired glucose metabolism, and reduced musculoskeletal mass and integrity. In contrast to free MP treatment, intravenous Nano-MP after acute SCI not only offered superior neuroprotection and functional recovery but also significantly mitigated or even eliminated the aforementioned adverse side effects. The superior safety features of Nano-MP observed in this study further confirmed the clinical translational potential of Nano-MP as a highly promising drug candidate for better clinical management of patients with acute SCI.

Targeted-delivery of nanomedicine-enabled methylprednisolone to injured spinal cord promotes neuroprotection and functional recovery after acute spinal cord injury in rats.

To date, no therapy has been proven to be efficacious in fully restoring neurological functions after spinal cord injury (SCI). Systemic high-dose methylprednisolone (MP) improves neurological recovery after acute SCI in both animal and human. MP therapy remains controversial due to its modest effect on functional recovery and significant adverse effects. To overcome the limitation of MP therapy, we have developed a N-(2-hydroxypropyl) methacrylamide copolymer-based MP prodrug nanomedicine (Nano-MP) that can selectively deliver MP to the SCI lesion when administered systemically in a rat model of acute SCI. Our in vivo data reveal that Nano-MP is significantly more effective than free MP in attenuating secondary injuries and neuronal apoptosis. Nano-MP is superior to free MP in improving functional recovery after acute SCI in rats. These data support Nano-MP as a promising neurotherapeutic candidate, which may provide potent neuroprotection and accelerate functional recovery with improved safety for patients with acute SCI.

Septin 7 interacts with Numb to preserve sarcomere structural organization and muscle contractile function.

Here, we investigated the mechanisms by which aging-related reductions of the levels of Numb in skeletal muscle fibers contribute to loss of muscle strength and power, two critical features of sarcopenia. Numb is an adaptor protein best known for its critical roles in development, including asymmetric cell division, cell-type specification, and termination of intracellular signaling. Numb expression is reduced in old humans and mice. We previously showed that, in mouse skeletal muscle fibers, Numb is localized to sarcomeres where it is concentrated near triads; conditional inactivation of Numb and a closely related protein Numb-like (Numbl) in mouse myofibers caused weakness, disorganization of sarcomeres, and smaller mitochondria with impaired function. Here, we found that a single knockout of Numb in myofibers causes reduction in tetanic force comparable to a double Numb, Numbl knockout. We found by proteomics analysis of protein complexes isolated from C2C12 myotubes by immunoprecipitation using antibodies against Numb that Septin 7 is a potential Numb-binding partner. Septin 7 is a member of the family of GTP-binding proteins that organize into filaments, sheets, and rings, and is considered part of the cytoskeleton. Immunofluorescence evaluation revealed a partial overlap of staining for Numb and Septin 7 in myofibers. Conditional, inducible knockouts of Numb led to disorganization of Septin 7 staining in myofibers. These findings indicate that Septin 7 is a Numb-binding partner and suggest that interactions between Numb and Septin 7 are critical for structural organization of the sarcomere and muscle contractile function.

Transcutaneous Spinal Cord Stimulation to Stabilize Seated Systolic Blood Pressure in Persons With Chronic Spinal Cord Injury: Protocol Development.

Transcutaneous spinal cord stimulation (tSCS) is an emerging therapeutic strategy to target spinal autonomic circuitry to normalize and stabilize blood pressure (BP) in hypotensive persons living with chronic spinal cord injury (SCI). Our aim is to describe our current methodological approach to identify individual tSCS parameters that result in the maintenance of seated systolic blood pressure (SBP) within a pre-defined target range. The parent study is a prospective, randomized clinical trial in which eligible participants will undergo multiple mapping sessions to optimize tSCS parameter settings to promote stable SBP within a target range of 110-120 mm Hg for males and 100-120 mm Hg for females. Parameter mapping includes cathode electrode placement site (T7/8, T9/10, T11/12, and L1/2), stimulation frequency (30, 60 Hz), current amplitudes (0-120 mA), waveform (mono- and biphasic), pulse width (1000 µs), and use of carrier frequency (0, 10 kHz). Each participant will undergo up to 10 mapping sessions involving different electrode placement sites and parameter settings. BP will be continuously monitored throughout each mapping session. Stimulation amplitude (mA) will be increased at intervals of between 2 and 10 mA until one of the following occurs: 1) seated SBP reaches the target range; 2) tSCS intensity reaches 120 mA; or 3) the participant requests to stop. Secondary outcomes recorded include 1) symptoms related to autonomic dysreflexia and orthostatic hypotension, 2) Likert pain scale, and 3) skin appearance after removal of the tSCS electrode. Clinical Trials Registration: NCT05180227.

Conserved and species-specific transcriptional responses to daily programmed resistance exercise in rat and mouse.

Mice are often used in gain or loss of function studies to understand how genes regulate metabolism and adaptation to exercise in skeletal muscle. Once-daily resistance training with electrical nerve stimulation produces hypertrophy of the dorsiflexors in rat, but not in mouse. Using implantable pulse generators, we assessed the acute transcriptional response (1-h post-exercise) after 2, 10, and 20 days of training in free-living mice and rats using identical nerve stimulation paradigms. RNA sequencing revealed strong concordance in the timecourse of many transcriptional responses in the tibialis anterior muscles of both species including responses related to "stress responses/immediate-early genes, and "collagen homeostasis," "ribosomal subunits," "autophagy," and "focal adhesion." However, pathways associated with energy metabolism including "carbon metabolism," "oxidative phosphorylation," "mitochondrial translation," "propanoate metabolism," and "valine, leucine, and isoleucine degradation" were oppositely regulated between species. These pathways were suppressed in the rat but upregulated in the mouse. Our transcriptional analysis suggests that although many pathways associated with growth show remarkable similarities between species, the absence of an actual growth response in the mouse may be because the mouse prioritizes energy metabolism, specifically the replenishment of fuel stores and intermediate metabolites.

A spatially specified systems pharmacology therapy for axonal recovery after injury.

There are no known drugs or drug combinations that promote substantial central nervous system axonal regeneration after injury. We used systems pharmacology approaches to model pathways underlying axonal growth and identify a four-drug combination that regulates multiple subcellular processes in the cell body and axons using the optic nerve crush model in rats. We intravitreally injected agonists HU-210 (cannabinoid receptor-1) and IL-6 (interleukin 6 receptor) to stimulate retinal ganglion cells for axonal growth. We applied, in gel foam at the site of nerve injury, Taxol to stabilize growing microtubules, and activated protein C to clear the debris field since computational models predicted that this drug combination regulating two subcellular processes at the growth cone produces synergistic growth. Physiologically, drug treatment restored or preserved pattern electroretinograms and some of the animals had detectable visual evoked potentials in the brain and behavioral optokinetic responses. Morphology experiments show that the four-drug combination protects axons or promotes axonal regrowth to the optic chiasm and beyond. We conclude that spatially targeted drug treatment is therapeutically relevant and can restore limited functional recovery.

Spinal cord injury regulates circular RNA expression in axons.

Introduction: Neurons transport mRNA and translational machinery to axons for local translation. After spinal cord injury (SCI), de novo translation is assumed to enable neurorepair. Knowledge of the identity of axonal mRNAs that participate in neurorepair after SCI is limited. We sought to identify and understand how axonal RNAs play a role in axonal regeneration. Methods: We obtained preparations enriched in axonal mRNAs from control and SCI rats by digesting spinal cord tissue with cold-active protease (CAP). The digested samples were then centrifuged to obtain a supernatant that was used to identify mRNA expression. We identified differentially expressed genes (DEGS) after SCI and mapped them to various biological processes. We validated the DEGs by RT-qPCR and RNA-scope. Results: The supernatant fraction was highly enriched for mRNA from axons. Using Gene Ontology, the second most significant pathway for all DEGs was axonogenesis. Among the DEGs was Rims2, which is predominately a circular RNA (circRNA) in the CNS. We show that Rims2 RNA within spinal cord axons is circular. We found an additional 200 putative circRNAs in the axonal-enriched fraction. Knockdown in primary rat cortical neurons of the RNA editing enzyme ADAR1, which inhibits formation of circRNAs, significantly increased axonal outgrowth and increased the expression of circRims2. Using Rims2 as a prototype we used Circular RNA Interactome to predict miRNAs that bind to circRims2 also bind to the 3'UTR of GAP-43, PTEN or CREB1, all known regulators of axonal outgrowth. Axonally-translated GAP-43 supports axonal elongation and we detect GAP-43 mRNA in the rat axons by RNAscope. Discussion: By enriching for axonal RNA, we detect SCI induced DEGs, including circRNA such as Rims2. Ablation of ADAR1, the enzyme that regulates circRNA formation, promotes axonal outgrowth of cortical neurons. We developed a pathway model using Circular RNA Interactome that indicates that Rims2 through miRNAs can regulate the axonal translation GAP-43 to regulate axonal regeneration. We conclude that axonal regulatory pathways will play a role in neurorepair.

Boldine Alters Serum Lipidomic Signatures after Acute Spinal Cord Transection in Male Mice.

Traumatic spinal cord injury (SCI) results in wide-ranging cellular and systemic dysfunction in the acute and chronic time frames after the injury. Chronic SCI has well-described secondary medical consequences while acute SCI has unique metabolic challenges as a result of physical trauma, in-patient recovery and other post-operative outcomes. Here, we used high resolution mass spectrometry approaches to describe the circulating lipidomic and metabolomic signatures using blood serum from mice 7 d after a complete SCI. Additionally, we probed whether the aporphine alkaloid, boldine, was able to prevent SCI-induced changes observed using these 'omics platforms'. We found that SCI resulted in large-scale changes to the circulating lipidome but minimal changes in the metabolome, with boldine able to reverse or attenuate SCI-induced changes in the abundance of 50 lipids. Multiomic integration using xMWAS demonstrated unique network structures and community memberships across the groups.

Synaptojanin 1 Modulates Functional Recovery After Incomplete Spinal Cord Injury in Male Apolipoprotein E Epsilon 4 Mice.

Apolipoprotein E epsilon 4 (ApoE4) is the second most common variant of ApoE, being present in ∼14% of the population. Clinical reports identify ApoE4 as a genetic risk factor for poor outcomes after traumatic spinal cord injury (SCI) and spinal cord diseases such as cervical myelopathy. To date, there is no intervention to promote recovery of function after SCI/spinal cord diseases that is specifically targeted at ApoE4-associated impairment. Studies in the human and mouse brain link ApoE4 to elevated levels of synaptojanin 1 (synj1), a lipid phosphatase that degrades phosphoinositol 4,5-bisphosphate (PIP2) into inositol 4-monophosphate. Synj1 regulates rearrangements of the cytoskeleton as well as endocytosis and trafficking of synaptic vesicles. We report here that, as compared to ApoE3 mice, levels of synj1 messenger RNA and protein were elevated in spinal cords of healthy ApoE4 mice associated with lower PIP2 levels. Using a moderate-severity model of contusion SCI in mice, we found that genetic reduction of synj1 improved locomotor function recovery at 14 days after SCI in ApoE4 mice without altering spared white matter. Genetic reduction of synj1 did not alter locomotor recovery of ApoE3 mice after SCI. Bulk RNA sequencing revealed that at 14 days after SCI in ApoE4 mice, genetic reduction of synj1 upregulated genes involved in glutaminergic synaptic transmission just above and below the lesion. Overall, our findings provide evidence for a link between synj1 to poor outcomes after SCI in ApoE4 mice, up to 14 days post-injury, through mechanisms that may involve the function of excitatory glutaminergic neurons.

Muscle-restricted Nox4 knockout partially corrects muscle contractility following spinal cord injury in mice.

Spinal cord injury (SCI) results in severe atrophy of skeletal muscle in paralyzed regions, and a decrease in the force generated by muscle per unit of cross-sectional area. Oxidation of skeletal muscle ryanodine 1 receptors (RyR1) reduces contractile force due to reduced binding of calstabin 1 to RyR1 together with altered gating of RyR1. One cause of RyR1 oxidation is NADPH oxidase 4 (Nox4). We have previously shown that in rats, RyR1 was oxidized and bound less calstabin 1 at 56 days after spinal cord injury (SCI) by transection. Here, we used a conditional knock-out mouse model of Nox4 in muscle to investigate the role of Nox4 in reduced muscle specific force after SCI. Peak twitch force in control mice after SCI was reduced by 42% compared to sham-operated controls but was increased by approximately 43% in SCI Nox4 conditional KO mice compared to SCI controls although it remained less than that for sham-operated controls. Unlike what observed in rats, after SCI the expression of Nox4 was not increased in gastrocnemius muscle and binding of calstabin 1 to RyR1 was not reduced in this muscle. The results suggest a link between Nox4 expression in muscle tissue and reduction in muscle twitch force, however further studies are needed to understand the mechanistic basis for this linkage.

SEPTIN 7 INTERACTS WITH NUMB TO PRESERVE SARCOMERE STRUCTURAL ORGANIZATION AND MUSCLE CONTRACTILE FUNCTION.

Here, we investigated mechanisms by which aging-related reductions of the levels of Numb in skeletal muscle fibers contribute to loss of muscle strength and power, two critical features of sarcopenia. Numb is an adaptor protein best known for its critical roles in development including asymmetric cell division, cell-type specification and termination of intracellular signaling. Numb expression is reduced in old humans and mice. We previously showed that, in mouse skeletal muscle fibers, Numb is localized to sarcomeres where it is concentrated near triads; conditional inactivation of Numb and a closely related protein Numb-like (NumbL) in mouse myofibers caused weakness, disorganization of sarcomeres and smaller mitochondria with impaired function. Here, we found that a single knockout of Numb in myofibers causes reduction in tetanic force comparable to a double Numb, NumbL knockout. We found by proteomics analysis of protein complexes isolated from C2C12 myotubes by immunoprecipitation using antibodies against Numb, that Septin 7 is a potential Numb binding partner. Septin 7 is a member of the family of GTP-binding proteins that organize into filaments, sheets and rings, and is considered part of the cytoskeleton. Immunofluorescence evaluation revealed a partial overlap of staining for Numb and Septin 7 in myofibers. Conditional, inducible knockouts of Numb led to disorganization of Septin 7 staining in myofibers. These findings indicate that Septin 7 is a Numb binding partner and suggest that interactions between Numb and Septin 7 are critical for structural organization of the sarcomere and muscle contractile function.

Boldine modulates glial transcription and functional recovery in a murine model of contusion spinal cord injury.

Membrane channels such as those formed by connexins (Cx) and P2X7 receptors (P2X7R) are permeable to calcium ions and other small molecules such as adenosine triphosphate (ATP) and glutamate. Release of ATP and glutamate through these channels is a key mechanism driving tissue response to traumas such as spinal cord injury (SCI). Boldine, an alkaloid isolated from the Chilean boldo tree, blocks both Cx and Panx1 hemichannels (HCs). To test if boldine could improve function after SCI, boldine or vehicle was administered to treat mice with a moderate severity contusion-induced SCI. Boldine led to greater spared white matter and increased locomotor function as determined by the Basso Mouse Scale and horizontal ladder rung walk tests. Boldine treatment reduced immunostaining for markers of activated microglia (Iba1) and astrocytic (GFAP) markers while increasing that for axon growth and neuroplasticity (GAP-43). Cell culture studies demonstrated that boldine blocked glial HC, specifically Cx26 and Cx30, in cultured astrocytes and blocked calcium entry through activated P2X7R. RT-qPCR studies showed that boldine treatment reduced expression of the chemokine Ccl2, cytokine IL-6 and microglial gene CD68, while increasing expression of the neurotransmission genes Snap25 and Grin2b, and Gap-43. Bulk RNA sequencing revealed that boldine modulated a large number of genes involved in neurotransmission in spinal cord tissue just caudal from the lesion epicenter at 14 days after SCI. Numbers of genes regulated by boldine was much lower at 28 days after injury. These results indicate that boldine treatment ameliorates injury and spares tissue to increase locomotor function.

Assessing the impact of boldine on the gastrocnemius using multiomics profiling at 7 and 28 days post-complete spinal cord injury in young male mice.

Spinal cord injury (SCI) results in rapid muscle loss. Exogenous molecular interventions to slow muscle atrophy after SCI have been relatively ineffective and require the search for novel therapeutic targets. Connexin hemichannels (CxHCs) allow nonselective passage of small molecules into and out of the cell. Boldine, a CxHC-inhibiting aporphine found in the boldo tree (Peumus boldus), has shown promising preclinical results in slowing atrophy during sepsis and restoring muscle function in dysferlinopathy. We administered 50 mg/kg/day of boldine to spinal cord transected mice beginning 3 days post-injury. Tissue was collected 7 and 28 days post-SCI and the gastrocnemius was used for multiomics profiling. Boldine did not prevent body or muscle mass loss but attenuated SCI-induced changes in the abundance of the amino acids proline, phenylalanine, leucine and isoleucine, as well as glucose, 7 days post-SCI. SCI resulted in the differential expression of ∼7,700 and ∼2,000 genes at 7 and 28 days, respectively, compared with Sham controls. Pathway enrichment of these genes highlighted ribosome biogenesis at 7 days and translation and oxidative phosphorylation at both timepoints. Boldine altered the expression of ∼150 genes at 7 days and ∼110 genes at 28 days post-SCI. Pathway enrichment of these genes indicated a potential role for boldine in suppressing protein ubiquitination and degradation at the 7-day timepoint. Methylation analyses showed minimal differences between groups. Taken together, boldine is not an efficacious therapy to preserve body and muscle mass after complete SCI, though it attenuated some SCI-induced changes across the metabolome and transcriptome.NEW & NOTEWORTHY This is the first study to describe the multiome of skeletal muscle paralyzed by a spinal cord injury (SCI) in mice across the acute and subacute timeframe after injury. We show large-scale changes in the metabolome and transcriptome at 7 days post-injury compared with 28 days. Furthermore, we show that the alkaloid boldine was able to prevent SCI-induced changes in muscle glucose and free amino acid levels at 7 days, but not 28 days, after SCI.

Abaloparatide prevents immobilization-induced cortical but not trabecular bone loss after spinal cord injury.

Spinal cord injury (SCI) causes severe and resistant sublesional disuse bone loss. Abaloparatide, a modified parathyroid hormone related peptide, is an FDA approved drug for treatment of severe osteoporosis with potent anabolic activity. The effects of abaloparatide on SCI-induced bone loss remain undefined. Thus, female mice underwent sham or severe contusion thoracic SCI causing hindlimb paralysis. Mice then received subcutaneous injection of vehicle or 20 µg/kg/day abaloparatide for 35 days. Micro-computed tomography (micro-CT) analysis of the distal and midshaft femoral regions of the SCI-vehicle mice revealed reduced trabecular fractional bone volume (56%), thickness (75%), and cortical thickness (80%) compared to sham-vehicle controls. Treatment with abaloparatide did not prevent SCI-induced changes in trabecular or cortical bone. However, histomorphometry evaluation of the SCI-abaloparatide mice demonstrated that abaloparatide treatment increased osteoblast (241%) and osteoclast (247%) numbers and the mineral apposition rate (131%) compared to SCI-vehicle animals. In another independent experiment, treatment with 80 µg/kg/day abaloparatide significantly attenuated SCI-induced loss in cortical bone thickness (93%) when compared to SCI-vehicle mice (79%) but did not prevent SCI-induced trabecular bone loss or elevation in cortical porosity. Biochemical analysis of the bone marrow supernatants of the femurs showed that SCI-abaloparatide animals had 2.3-fold increase in procollagen type I N-terminal propeptide, a bone formation marker than SCI-vehicle animals. SCI groups had 70% higher levels of cross-linked C-telopeptide of type I collagen, a bone resorption marker, than sham-vehicle mice. These findings suggest that abaloparatide protects the cortical bone against the deleterious effects of SCI by promoting bone formation.

Denervation Atrophy of Skeletal Muscle is Not Influenced by Numb Levels in Mice.

Skeletal muscle undergoes rapid and extensive atrophy following nerve transection though the underlying mechanisms remain incompletely understood. We previously showed transiently elevated Notch 1 signaling in denervated skeletal muscle that was abrogated by administration of nandrolone (an anabolic steroid) combined with replacement doses of testosterone. Numb is an adaptor molecule present in myogenic precursors and skeletal muscle fibers that is vital for normal tissue repair after muscle injury and for skeletal muscle contractile function. It is unclear whether the increase in Notch signaling observed in denervated muscle contributes to denervation and whether expression of Numb in myofibers slows denervation atrophy. To address these questions, the degree of denervation atrophy, Notch signaling, and Numb expression was studied over time after denervation in C57B6J mice treated with nandrolone, nandrolone plus testosterone or vehicle. Nandrolone increased Numb expression and reduced Notch signaling. Neither nandrolone alone nor nandrolone plus testosterone changed the rate of denervation atrophy. We next compared rates of denervation atrophy between mice with conditional, tamoxifen-inducible knockout of Numb in myofibers and genetically identical mice treated with vehicle. Numb cKO had no effect on denervation atrophy in this model. Taken together, the data indicate that loss of Numb in myofibers does not alter the course of denervation atrophy and that upregulation of Numb and blunting of the denervation-atrophy induced activation of Notch do not change the course of denervation atrophy.

Boldine modulates glial transcription and functional recovery in a murine model of contusion spinal cord injury.

Membrane channels such as connexins (Cx), pannexins (Panx) and P2X 7 receptors (P2X 7 R) are permeable to calcium ions and other small molecules such as ATP and glutamate. Release of ATP and glutamate through these channels is a key mechanism driving tissue response to traumas such as spinal cord injury (SCI). Boldine, an alkaloid isolated from the Chilean boldo tree, blocks both Cx hemichannels (HC) and Panx. To test if boldine could improve function after SCI, boldine or vehicle was administered to treat mice with a moderate severity contusion-induced SCI. Boldine led to greater spared white matter and increased locomotor function as determined by the Basso Mouse Scale and horizontal ladder rung walk tests. Boldine treatment reduced immunostaining for markers of activated microglia (Iba1) and astrocytic (GFAP) markers while increasing that for axon growth and neuroplasticity (GAP-43). Cell culture studies demonstrated that boldine blocked glial HC, specifically Cx26 and Cx30, in cultured astrocytes and blocked calcium entry through activated P2X 7 R. RT-qPCR studies showed that boldine treatment reduced expression of the chemokine Ccl2, cytokine IL-6 and microglial gene CD68, while increasing expression of the neurotransmission genes Snap25 and Grin2b, and Gap-43. Bulk RNA sequencing (of the spinal cord revealed that boldine modulated a large number of genes involved in neurotransmission in in spinal cord tissue just below the lesion epicenter at 14 days after SCI. Numbers of genes regulated by boldine was much lower at 28 days after injury. These results indicate that boldine treatment ameliorates injury and spares tissue to increase locomotor function.

SS-31 does not prevent or reduce muscle atrophy 7 days after a 65 kdyne contusion spinal cord injury in young male mice.

Spinal cord injury (SCI) leads to major reductions in function, independent living, and quality of life. Disuse and paralysis from SCI leads to rapid muscle atrophy, with chronic muscle loss likely playing a role in the development of the secondary metabolic disorders often seen in those with SCI. Muscle disuse is associated with mitochondrial dysfunction. Previous evidence has suggested targeting the mitochondria with the tetrapeptide SS-31 is beneficial for muscle health in preclinical models that lead to mitochondrial dysfunction, such as cast immobilization or burn injury. We gave young male mice a sham (n = 8) or 65 kdyne thoracic contusion SCI with (n = 9) or without (n = 9) daily administration of 5.0 mg/kg SS-31. Hindlimb muscle mass and muscle bundle respiration were measured at 7 days post-SCI and molecular targets were investigated using immunoblotting, RT-qPCR, and metabolomics. SS-31 did not preserve body mass or hindlimb muscle mass 7 days post-SCI. SS-31 had no effect on soleus or plantaris muscle bundle respiration. SCI was associated with elevated levels of protein carbonylation, led to reduced protein expression of activated DRP1 and reductions in markers of mitochondrial fusion. SS-31 administration did result in reduced total DRP1 expression, as well as greater expression of inhibited DRP1. Gene expression of proinflammatory cytokines and their receptors were largely stable across groups, although SS-31 treatment led to greater mRNA expression of IL1B, TNF, and TNFRSF12A. In summation, SS-31 was not an efficacious treatment acutely after a moderate thoracic contusion SCI in young male mice.

Notch, Numb and Numb-like responses to exercise-induced muscle damage in human skeletal muscle.

NEW FINDINGS: What is the central question of this study? Do Notch, Numb and Numb-like expression change in human skeletal muscle after exercise-induced muscle damage? What are the main finding and its importance? Notch gene expression trends toward an increase in response to an acute bout of exercise-induced muscle damage, while Numb and Numb-like expression does not change. These results suggest that human skeletal muscle response to exercise-induced muscle damage is dynamic and may differ from Drosophila and rodent models. Furthermore, the timing of muscle biopsies, training status and muscle damage protocols should be considered. ABSTRACT: This investigation examined changes in the gene and protein expression of Notch, Numb and Numb-like (Numbl) in human skeletal muscle after an acute bout of eccentric exercise-induced muscle damage. Twelve recreationally active male subjects participated in this study. These individuals completed seven sets of 10 repetitions of eccentric leg extension at 120% of one-repetition max with 2 min of rest period between sets. Four muscle biopsies of the vastus lateralis were collected: before exercise (Pre), and 3 h, 2 days and 5 days post-muscle damage. Biopsy samples were used to probe Notch, Numb and Numbl utilizing western blot and RT-qPCR techniques. The results were analysed using a one-way repeated-measures ANOVA. Notch1 mRNA expression trended toward a significant increase from Pre to 2 days post-muscle damage from baseline measures (P = 0.087), while Numb (P = 0.804) and Numbl (P = 0.480) expression was unaltered post-muscle damage. There were no significant differences in protein expression post-muscle damage for any of the proteins. These results suggest that exercise-induced muscle damage, via eccentric exercise, slightly elevates Notch1 mRNA expression.

RNA-sequencing Reveals a Gene Expression Signature in Skeletal Muscle of a Mouse Model of Age-associated Postoperative Functional Decline.

This study aimed to characterize the effects of laparotomy on postoperative physical function and skeletal muscle gene expression in male C57BL/6N mice at 3, 20, and 24 months of age to investigate late-life vulnerability and resiliency to acute surgical stress. Pre and postoperative physical functioning was assessed by forelimb grip strength on postoperative day (POD) 1 and 3 and motor coordination on POD 2 and 4. Laparotomy-induced an age-associated postoperative decline in forelimb grip strength that was the greatest in the oldest mice. While motor coordination declined with increasing age at baseline, it was unaffected by laparotomy. Baseline physical function as stratified by motor coordination performance (low functioning vs high functioning) in 24-month-old mice did not differentially affect postlaparotomy reduction in grip strength. RNA sequencing of soleus muscles showed that laparotomy-induced age-associated differential gene expression and canonical pathway activation with the greatest effects in the youngest mice. Examples of such age-associated, metabolically important pathways that were only activated in the youngest mice after laparotomy included oxidative phosphorylation and NRF2-mediated oxidative stress response. Analysis of lipid mediators in serum and gastrocnemius muscle showed alterations in profiles during aging and confirmed an association between such changes and functional status in gastrocnemius muscle. These findings demonstrate a mouse model of laparotomy which recapitulated some features of postoperative skeletal muscle decline in older adults, and identified age-associated, laparotomy-induced molecular signatures in skeletal muscles. Future research can build upon this model to study molecular mechanisms of late-life vulnerability and resiliency to acute surgical stress.