Neurogenic Atrophy Research Articles

Identification and Characterization TSSK6 Activating Co‐chaperone (TSACC) in Skeletal Muscle

Skeletal muscle atrophy is a physiological condition that is caused by a range of conditions, including immobilization, denervation, spinal cord injury, and aging. To better characterize the molecular genetic events of neurogenic atrophy, a previous study isolated the gastrocnemius muscle from mice following 3 and 14 days of sciatic nerve transection. The gene expression profile in the denervated muscle tissue was then analyzed by microarray and compared to control muscle tissue to identify novel neurogenic atrophy‐induced genes. The microarray data revealed for the first time that TSSK6 Activating Co‐chaperone (TSACC) is expressed in skeletal muscle and is significantly induced in response to denervation. To confirm that TSACC is expressed in muscle, the predicted TSACC cDNA was cloned from cultured muscle cells, as was a novel TSACC splice variant that contains an additional exon. Quantitative PCR (qPCR) was performed to assess TSACC expression levels of the full‐length and novel splice variants in proliferating and differentiated muscle cells. The results demonstrate that TSACC expression levels are relatively low in proliferating myoblasts but show significantly elevated expression in differentiated myotubes. In addition, characterization of the transcriptional regulation of TSACC was assessed by fusing fragments of the proximal promoter located immediately upstream of the start of transcription with a reporter gene. The reporter plasmids were then transfected into C2C12 mouse muscle cells in combination with myogenic regulatory factor (MRF) expression plasmids, which resulted in significant activation of reporter gene activity. Interestingly, there are several predicted E‐box elements in the proximal promoter region of the TSACC gene further suggesting that TSACC may be regulated by muscle‐specific transcription factors. To identify a possible role for TSACC in skeletal muscle, sub‐cellular localization was assessed by fusing TSACC with GFP and expressing the fusion protein in cultured muscle cells, revealing that TSACC is localized predominantly to the cytoplasm. The discovery that TSACC is induced in response to neurogenic atrophy helps further our understanding of the molecular genetic events of muscle wasting and may eventually lead to the identification of new therapeutic targets for the treatment of muscle atrophy.

Denervating the pelvic suspensory ligaments of horses causes morphological and histological changes in the ligaments.

To examine changes occurring in normal pelvic suspensory ligaments (SLs) of horses after denervating these ligaments and to investigate the effect chronic inflammation might have on these changes. 10 horses. The SL of 1 randomly selected pelvic limb of each of 5 horses was injected with collagenase to induce desmitis, and 42 days later, the proximal aspect of both pelvic SLs were denervated. The SLs were harvested 120 days after being denervated, and the morphological and histological characteristics of each collagenase-injected, denervated SL were compared with those of the contralateral, non-injected, denervated SL. All denervated SLs were compared with non-denervated pelvic SLs harvested from 5 horses similar in weight and age. The mean width and the cross-sectional area of the musculature of all denervated SLs were significantly less than that of the non-denervated ligaments. The mean thickness of collagenase-injected denervated ligaments, but not that of the non-injected denervated ligaments, was significantly less than that of the non-denervated ligaments. Histological abnormalities typical of neurogenic muscular atrophy were observed in all denervated ligaments. Loss of motor neuronal input to the proximal aspect of the SL of the pelvic limb of horses causes neurogenic atrophy of the musculature in that portion of the ligament. Denervating a SL of a pelvic limb may weaken the ligament, increasing its risk of injury. Chronic inflammation of the SL before neurectomy may exacerbate atrophy of the musculature after neurectomy.

Identification and Characterization of Calcium Binding and Coiled Coil Domain 1 (Calcoco1) in Skeletal Muscle

Skeletal muscle atrophy results in loss of muscle mass and reduced strength and is caused by disparate physiological conditions including aging, cancer, corticosteroid use, and denervation. To better understand the molecular genetic events that lead to muscle atrophy, a previous study isolated skeletal muscle from mice following 3 and 14 days of denervation. Gene expression was then analyzed by microarray and compared to control muscle to identify novel, atrophy‐induced genes. The microarray revealed that Calcium Binding and Coiled Coil Domain 1 (Calcoco1) is expressed in skeletal muscle and induced in response to denervation. To confirm that Calcoco1 is expressed in skeletal muscle, the cDNA was successfully amplified and cloned from cultured C2C12 cells. Moreover, quantitative PCR was conducted using RNA isolated from proliferating and differentiated muscle cells to determine the expression profile of Calcoco1 at the transcriptional level. We observed moderate activation of Calcoco1 through myoblast proliferation and early differentiation, followed by a robust increase in activation by the later stages of myotube differentiation. These results were mirrored at the protein level where it was observed that Calcoco1 is expressed during early proliferation and increases as muscle cell differentiation progresses. To elucidate a functional role for Calcoco1 in muscle, we transfected cultured muscle cells with a Calcoco1 expression plasmid and then harvested the cells at timepoints ranging from proliferation through late differentiation. The cell lysates were probed by Western blot for markers of muscle cell differentiation, including Myosin Heavy Chain (MyHC) and myogenin, which both show significant repression in response to Calcoco1 overexpression. The Calcoco1 protein is predicted to contain both a calcium binding domain and a coiled‐coiled domain, suggesting that it may play a role in protein‐protein interactions or as a putative transcription factor. To investigate these possibilities, we sought to determine the sub‐cellular localization of Calcoco1 in muscle cells by fusing Calcoco1 cDNA to Green Fluorescent Protein (GFP) and expressing it in muscle cells. Visualization by confocal microscopy revealed clear nuclear‐exclusion of Calcoco1 protein in unchallenged myoblast cells, suggesting that it may not participate directly in gene regulation. The discovery that Calcoco1 is expressed in skeletal muscle and is induced in response to neurogenic atrophy helps further our understanding of the molecular and cellular events of muscle wasting and may eventually contribute to the identification of new therapeutic targets for the treatment and of muscle atrophy.

Brachial plexus avulsion in cat: Case report

The aim of this paper is to report a case of brachial plexus avulsion (BPA) in a domestic cat, treated at the Polyclinic Veterinary School of the University Center of Valença – UNIFAA. The brachial plexus is a branch of nerves that emerge from the spinal cord and innervate the thoracic limb. Avulsion in this plexus occurs when the nerves are frayed, by abduction or violent traction of the foreleg, caused in trauma that often happens to domestic cats (Felis catus). The most evident clinical signs are inability to extend the elbow, drag the back of the limb on the ground, difficulty in supporting weight, neurogenic muscle atrophy, absence or decrease in muscle tone and segmental reflexes, with the use of an Elizabethan collar and the fixation of a bracing around the involved limb. Horner's Syndrome is another common clinical sign in brachial plexus injuries, due to damage to the nerve roots of the spinal segments T2 and T3. The diagnosis of BPA is made through the patient's history, anamnesis, neurological physical examination, clinical signs, electrodiagnosis and complementary exams, the biggest obstacle being to discover the extension and exact location of the lesion. Treatment can be conservative if there is improvement in the clinical picture after three weeks of treatment, surgical if the animal is showing signs of pain and self-mutilation, or neurotization by transposition and microsurgical anastomosis. The prognosis will depend on the location, extension and type of injury. She was seen at the Polyclinic Veterinary of the UNIFAA, a cat, with no defined breed, weighing 5.2 kg. The tutor reported that he found her inside the engine compartment of a car. On physical examination, the patient did not show any pain response to the tests performed, nor proprioception in the right thoracic limb (MTD). After neurological examination, it could be identified that the animal had Horner's Syndrome and spinal cord injury between C6-T2 due to brachial plexus avulsion. Conservative treatment was instituted until ambulation improved, and then high MTD amputation surgery was indicated. It is concluded that this report can contribute to the literature as a source of study in anatomy, neurology, surgical technique, diagnosis and treatment for brachial plexus avulsions in cats.

Supraphysiological activation of TAK1 promotes skeletal muscle growth and mitigates neurogenic atrophy

Skeletal muscle mass is regulated through coordinated activation of multiple signaling pathways. TAK1 signalosome has been found to be activated in various conditions of muscle atrophy and hypertrophy. However, the role and mechanisms by which TAK1 regulates skeletal muscle mass remain less understood. Here, we demonstrate that supraphysiological activation of TAK1 in skeletal muscle of adult mice stimulates translational machinery, protein synthesis, and myofiber growth. TAK1 causes phosphorylation of elongation initiation factor 4E (eIF4E) independent of mTOR. Inactivation of TAK1 disrupts neuromuscular junction morphology and causes deregulation of Smad signaling. Using genetic approaches, we demonstrate that TAK1 prevents excessive loss of muscle mass during denervation. TAK1 favors the nuclear translocation of Smad4 and cytoplasmic retention of Smad6. TAK1 is also required for the phosphorylation of eIF4E in denervated skeletal muscle. Collectively, our results demonstrate that TAK1 supports skeletal muscle growth and prevents neurogenic muscle atrophy in adult mice.

Scavenging mitochondrial hydrogen peroxide by peroxiredoxin 3 overexpression attenuates contractile dysfunction and muscle atrophy in a murine model of accelerated sarcopenia.

Age‐related muscle atrophy and weakness, or sarcopenia, are significant contributors to compromised health and quality of life in the elderly. While the mechanisms driving this pathology are not fully defined, reactive oxygen species, neuromuscular junction (NMJ) disruption, and loss of innervation are important risk factors. The goal of this study is to determine the impact of mitochondrial hydrogen peroxide on neurogenic atrophy and contractile dysfunction. Mice with muscle‐specific overexpression of the mitochondrial H2O2 scavenger peroxiredoxin3 (mPRDX3) were crossed to Sod1KO mice, an established mouse model of sarcopenia, to determine whether reduced mitochondrial H2O2 can prevent or delay the redox‐dependent sarcopenia. Basal rates of H2O2 generation were elevated in isolated muscle mitochondria from Sod1KO, but normalized by mPRDX3 overexpression. The mPRDX3 overexpression prevented the declines in maximum mitochondrial oxygen consumption rate and calcium retention capacity in Sod1KO. Muscle atrophy in Sod1KO was mitigated by ~20% by mPRDX3 overexpression, which was associated with an increase in myofiber cross‐sectional area. With direct muscle stimulation, maximum isometric specific force was reduced by ~20% in Sod1KO mice, and mPRDX3 overexpression preserved specific force at wild‐type levels. The force deficit with nerve stimulation was exacerbated in Sod1KO compared to direct muscle stimulation, suggesting NMJ disruption in Sod1KO. Notably, this defect was not resolved by overexpression of mPRDX3. Our findings demonstrate that muscle‐specific PRDX3 overexpression reduces mitochondrial H2O2 generation, improves mitochondrial function, and mitigates loss of muscle quantity and quality, despite persisting NMJ impairment in a murine model of redox‐dependent sarcopenia.

M6A demethylase ALKBH5 drives denervation‐induced muscle atrophy by targeting HDAC4 to activate FoxO3 signalling

BackgroundSkeletal muscle atrophy is a common clinical manifestation of various neurotrauma and neurological diseases. In addition to the treatment of primary neuropathies, it is a clinical condition that should be investigated. FoxO3 activation is an indispensable mechanism in denervation‐induced muscle atrophy; however, upstream factors that control FoxO3 expression and activity have not been fully elucidated. N6‐methyladenosine (m6A) methylation is a novel mode of epitranscriptional gene regulation that affects several cellular processes. However, the biological significance of m6A modification in FoxO3‐dependent atrophy is unknown.MethodsWe performed gain‐of‐function and loss‐of‐function experiments and used denervation‐induced muscle atrophy mouse model to evaluate the effects of m6A modification on muscle mass control and FoxO3 activation. m6A‐sequencing and mass spectrometry analyses were used to establish whether histone deacetylase 4 (HDAC4) is a mediator of m6A demethylase ALKBH5 regulation of FoxO3. A series of cellular and molecular biological experiments (western blot, immunoprecipitation, half‐life assay, m6A‐MeRIP‐qPCR, and luciferase reporter assays among others) were performed to investigate regulatory relationships among ALKBH5, HDAC4, and FoxO3.ResultsIn skeletal muscles, denervation was associated with a 20.7–31.9% decrease in m6A levels (P < 0.01) and a 35.6–115.2% increase in demethylase ALKBH5 protein levels (P < 0.05). Overexpressed ALKBH5 reduced m6A levels, activated FoxO3 signalling, and induced excess loss in muscle wet weight (−10.3% for innervation and −11.4% for denervation, P < 0.05) as well as a decrease in myofibre cross‐sectional areas (−35.8% for innervation and −33.3% for denervation, P < 0.05) during innervation and denervation. Specific deletion of Alkbh5 in the skeletal muscles prevented FoxO3 activation and protected mice from denervation‐induced muscle atrophy, as evidenced by increased muscle mass (+16.0%, P < 0.05), size (+50.0%, P < 0.05) and MyHC expression (+32.6%, P < 0.05). Mechanistically, HDAC4 was established to be a crucial central mediator for ALKBH5 in enhancing FoxO3 signalling in denervated muscles. ALKBH5 demethylates and stabilizes Hdac4 mRNA. HDAC4 interacts with and deacetylates FoxO3, resulting in a significant increase in FoxO3 expression (+61.3–82.5%, P < 0.01) and activity (+51.6–122.0%, P < 0.001).ConclusionsOur findings elucidate on the roles and mechanisms of ALKBH5‐mediated m6A demethylation in the control of muscle mass during denervation and activation of FoxO3 signalling by targeting HDAC4. These results suggest that ALKBH5 is a potential therapeutic target for neurogenic muscle atrophy.

Survey of patients with advanced-stage Okinawa-type neurogenic muscular atrophy (hereditary motor and sensory neuropathy with proximal dominant involvement: HMSN-P)

We conducted a survey of 16 Japanese patients (9 males, 7 females) aged 48-70 years in the advanced-stage Okinawa-type neurogenic muscular atrophy (i.e. hereditary motor and sensory neuropathy with proximal dominant involvement: HMSN-P) by a questionnaire asking the patients' disease name notification, acceptance, and expectations for treatment. In amyotrophic lateral sclerosis (ALS), since symptoms such as four-limb motor weakness and respiratory disorder are serious, patients are notified of the disease name at each progression stage. Individuals with HMSN-P exhibit ALS-like severe motor paralysis, but HMSN-P shows autosomal dominant inheritance, and progresses slowly (over >30 years). Many of the present patients who had one parent with the disease were able to predict what their diagnosis would be. However, several patients stated that they could not sleep for several months due to the shock of the diagnosis and their concern about how to explain to their children that the disease is hereditary. All patients in the advanced stage of HMSN-P progress to severe proximal dominant quadriplegia and ultimately need auxiliary tools such as a wheelchair. New developments toward a specific HMSN-P treatment are expected, with methods such as nucleic acid medicine.

Gait phenotype in Batten disease: A marker of disease progression.

Gait impairment and its etiologic correlate has not previously been subject of special attention in Batten disease. In the present review, the clinical picture of gait phenotype during Batten disease course accompanied by descriptions of the known concomitant patho-anatomical changes is presented. In CLN1 a non-rhythmic gait is seen around 1-1½ years of age. Shortly after, postural hypotonia and exaggerated tendon reflexes develop. The disease reaches a burnt-out stage during the third year of age and subsequently the children are almost without voluntary movements. The existing literature indicates that gait phenotype in CLN1 is caused by early involvement of the spinal interneurons followed by impact of the cortex and the cortico-spinal tracts. The earliest walking abnormality in children with CLN2 is a clumsy, ataxic, and spastic gait, which is in accordance with the existing imaging and histologic studies showing early involvement of the cerebellum and the cortico-spinal pathways. In CLN3, a reduction in walking speed is present at the age of 7-8 years. It occurs simultaneously with a reduction in the white matter microstructure and brain connectivity networks. Functional impairment of the basal ganglia contributing to a parkinsonian gait phenotype occurs in the mid-teens. In the late teens and early twenties involvement of the peripheral nerves, neurogenic musculoskeletal atrophy, loss of tendon reflexes and postural control are seen. The progressively impaired gait function in Batten disease is related to timing of damage of distinct areas of the nervous system depending on subtype and is a powerful marker of disease progression.

Comparison between normal and reverse orientation of graft in functional and histomorphological outcomes after autologous nerve grafting: An experimental study in the mouse model.

Autologous nerve grafting has been considered the gold standard for the treatment of irreparable nerve gaps. However, the choice of effective proximodistal orientation of autografts (normal or reversed) is controversial. Therefore, we compared functional and histological outcomes between normal and reversed orientations of autografts in a mouse sciatic nerve model. Thirty C57BL/6J mice weighing 20-25 g were assigned to the donor, normally oriented autograft, and reverse-oriented autograft groups (n= 10 per group). A 10-mm section of the sciatic nerve was harvested from a donor mouse. Half the harvested nerve was grafted onto an irreparable gap in a recipient mouse using either a normal or reversed orientation. The sciatic functional index (SFI) was measured biweekly for up to 12 weeks postoperatively. Morphological analysis was performed using immunofluorescence staining for neurofilament (NF) and myelin protein zero (P0) in cross-sectional and whole-mount nerve preparations in 12 weeks postoperatively. Additionally, morphological analysis of the tibialis anterior muscle was performed using hematoxylin and eosin staining. NF or P0-expressing axons were counted and cross-sectional area (CSA) and minimum Feret's diameter of myofibers were measured. The SFI recovered gradually up to 12 weeks after autografting, but there were no significant differences in the SFI between the normal and reversed orientations. The number of NF-expressing axons in center of graft was significantly higher in the normal orientation than in the reversed orientation (P < .05). However, there were no significant differences in the number and mean intensity of P0-expressing axons between the orientations. The CSA of myofibers was significantly larger in the normal orientation than in the reversed orientation (P < .05). Normally oriented autografts promote axonal regrowth and prevent neurogenic muscular atrophy compared with reverse-oriented autografts. However, despite these positive histomorphometric effects, the proximodistal orientation of the autograft does not affect functional outcomes.

MyD88‐mediated signaling intercedes in neurogenic muscle atrophy through multiple mechanisms

Skeletal muscle atrophy is a debilitating complication of many chronic disease states and disuse conditions including denervation. However, molecular and signaling mechanisms of muscle wasting remain less understood. Here, we demonstrate that the levels of several toll-like receptors (TLRs) and their downstream signaling adaptor, myeloid differentiation primary response 88 (MyD88), are induced in skeletal muscle of mice in response to sciatic nerve denervation. Muscle-specific ablation of MyD88 mitigates denervation-induced skeletal muscle atrophy in mice. Targeted ablation of MyD88 suppresses the components of ubiquitin-proteasome system, autophagy, and FOXO transcription factors in skeletal muscle during denervation. We also found that specific inhibition of MyD88 reduces the activation of canonical nuclear factor-kappa (NF-κB) pathway and expression of receptors for inflammatory cytokines in denervated muscle. In contrast, inhibition of MyD88 stimulates the activation of non-canonical NF-κB signaling in denervated skeletal muscle. Ablation of MyD88 also inhibits the denervation-induced increase in phosphorylation of AMPK without having any effect on the phosphorylation of mTOR. Moreover, targeted ablation of MyD88 inhibits the activation of a few components of the unfolded protein response (UPR) pathways, especially X-box protein 1 (XBP1). Importantly, myofiber-specific ablation of XBP1 mitigates denervation-induced skeletal muscle atrophy in mice. Collectively, our experiments suggest that TLR-MyD88 signaling mediates skeletal muscle wasting during denervation potentially through the activation of canonical NF-κB signaling, AMPK and UPR pathways.

WS3.4. Electrodiagnostic Approach to Motor Neuron Disease: Split Hand in ALS

Amyotrophic lateral sclerosis (ALS) is characterized by involvement of both upper and lower motor neurons. Electrophysiologic approach includes EMG (denervation potentials and fasciculation potentials) and nerve conduction studies (NCS; low CMAP, decreased motor unit number, and axonal hyperexcitability on nerve excitability testing) for lower motor neuron involvement, and transcranial magnetic stimulation with threshold tracking (decreased short- latency intracortical inhibition) for upper motoneuronal hyperexcitability. Of these, routine NCS can detect the “split hand” pattern, which is a very important indicator for diagnosis, because this symptom has been recognized as being specific to ALS; the thenar and first dorsal interosseous (FDI) muscles are preferentially affected. In contrast, the hypothenar muscle (abductor digiti minimi, ADM) is relatively preserved. FDI and ADM muscles are innervated by the same C8/Th1 spinal segments and the same ulnar nerve. The split hand indicates a potentially specific pathological mechanism, suggesting hyperexcitability of both upper and lower motor neuron/axons. Along with this symptom, wide-spread fasciculations are prominent clinical features in patients with ALS, specifically seen in ALS among disorders with neurogenic muscle atrophy. Fasciculations frequently arise from the motor nerve terminals, and the hyperexcitability of motor axons appears to constitute the pathophysiology of the disease. Neurophysiologic investigations of the upper and lower motor neurons/axons have shown increased excitability. The altered excitability is supposed to relate to motor neuron death.

GADD45A is a protective modifier of neurogenic skeletal muscle atrophy.

Neurogenic muscle atrophy is the loss of skeletal muscle mass and function that occurs with nerve injury and in denervating diseases, such as amyotrophic lateral sclerosis. Aside from prompt restoration of innervation and exercise where feasible, there are currently no effective strategies for maintaining skeletal muscle mass in the setting of denervation. We conducted a longitudinal analysis of gene expression changes occurring in atrophying skeletal muscle and identified growth arrest and DNA damage-inducible A (Gadd45a) as a gene that shows one of the earliest and most sustained increases in expression in skeletal muscle after denervation. We evaluated the role of this induction using genetic mouse models and found that mice lacking GADD45A showed accelerated and exacerbated neurogenic muscle atrophy, as well as loss of fiber type identity. Our genetic analyses demonstrate that, rather than directly contributing to muscle atrophy as proposed in earlier studies, GADD45A induction likely represents a protective negative feedback response to denervation. Establishing the downstream effectors that mediate this protective effect and the pathways they participate in may yield new opportunities to modify the course of muscle atrophy.

HDAC4 Knockdown Alleviates Denervation-Induced Muscle Atrophy by Inhibiting Myogenin-Dependent Atrogene Activation.

Denervation can activate the catabolic pathway in skeletal muscle and lead to progressive skeletal muscle atrophy. At present, there is no effective treatment for muscle atrophy. Histone deacetylase 4 (HDAC4) has recently been found to be closely related to muscle atrophy, but the underlying mechanism of HDAC4 in denervation-induced muscle atrophy have not been described clearly yet. In this study, we found that the expression of HDAC4 increased significantly in denervated skeletal muscle. HDAC4 inhibition can effectively diminish denervation-induced muscle atrophy, reduce the expression of muscle specific E3 ubiquitin ligase (MuRF1 and MAFbx) and autophagy related proteins (Atg7, LC3B, PINK1 and BNIP3), inhibit the transformation of type I fibers to type II fibers, and enhance the expression of SIRT1 and PGC-1 α. Transcriptome sequencing and bioinformatics analysis was performed and suggested that HDAC4 may be involved in denervation-induced muscle atrophy by regulating the response to denervation involved in the regulation of muscle adaptation, cell division, cell cycle, apoptotic process, skeletal muscle atrophy, and cell differentiation. STRING analysis showed that HDAC4 may be involved in the process of muscle atrophy by directly regulating myogenin (MYOG), cell cycle inhibitor p21 (CDKN1A) and salt induced kinase 1 (SIK1). MYOG was significantly increased in denervated skeletal muscle, and MYOG inhibition could significantly alleviate denervation-induced muscle atrophy, accompanied by the decreased MuRF1 and MAFbx. MYOG overexpression could reduce the protective effect of HDAC4 inhibition on denervation-induced muscle atrophy, as evidenced by the decreased muscle mass and cross-sectional area of muscle fibers, and the increased mitophagy. Taken together, HDAC4 inhibition can alleviate denervation-induced muscle atrophy by reducing MYOG expression, and HDAC4 is also directly related to CDKN1A and SIK1 in skeletal muscle, which suggests that HDAC4 inhibitors may be a potential drug for the treatment of neurogenic muscle atrophy. These results not only enrich the molecular regulation mechanism of denervation-induced muscle atrophy, but also provide the experimental basis for HDAC4-MYOG axis as a new target for the prevention and treatment of muscular atrophy.

Ultrasonographic and Histological Correlation after Experimental Reconstruction of a Volumetric Muscle Loss Injury with Adipose Tissue.

Different types of scaffolds are used to reconstruct muscle volume loss injuries. In this experimental study, we correlated ultrasound observations with histological findings in a muscle volume loss injury reconstructed with autologous adipose tissue. The outcome is compared with decellularized and porous matrix implants. Autologous adipose tissue, decellularized matrix, and a porous collagen matrix were implanted in volumetric muscle loss (VML) injuries generated on the anterior tibial muscles of Wistar rats. Sixty days after implantation, ultrasound findings were compared with histological and histomorphometric analysis. The muscles with an autologous adipose tissue implant exhibited an ultrasound pattern that was quite similar to that of the regenerative control muscles. From a histological point of view, the defects had been occupied by newly formed muscle tissue with certain structural abnormalities that would explain the differences between the ultrasound patterns of the normal control muscles and the regenerated ones. While the decellularized muscle matrix implant resulted in fibrosis and an inflammatory response, the porous collagen matrix implant was replaced by regenerative muscle fibers with neurogenic atrophy and fibrosis. In both cases, the ultrasound images reflected echogenic, echotextural, and vascular changes compatible with the histological findings of failed muscle regeneration. The ultrasound analysis confirmed the histological findings observed in the VML injuries reconstructed by autologous adipose tissue implantation. Ultrasound can be a useful tool for evaluating the structure of muscles reconstructed through tissue engineering.

The Potential Repurposing Effect 4‐Aminopyridine in Nerve and Muscle Injury‐induced Bone Loss

Muscle injuries are one of the most common injuries occurring in physical and sporting activities, and account for 10-55% of all injuries. Peripheral nerve injury (PNI) itself accounts for 3-5% of all injuries in trauma patients, and muscle injuries can be concurrent with PNI because of their close proximity. Importantly, the peripheral nervous system is critically involved in bone metabolism, bone mineralization, and bone remodeling, and PNI results in substantial bone loss. While first aid for muscle injuries follows the conservative RICE (Rest, Ice, Compression and Elevation) principle, there is no medical treatment available after the acute phase except anti-inflammatory medications and rehabilitation. The poor healing process in damaged muscle, prolonged muscle disuse, and the lack of therapeutic strategies affect the performance of daily activities and predispose to further risk of muscle injury and bone loss. Therefore, there is an unmet need to develop therapeutic strategies that can help the recovery of damaged muscle and nerve, and limit bone loss. Recently we demonstrated the potential repurposing effect of 4-aminopyridine (4-AP), a potassium channel blocker, on neurogenic muscle atrophy and functional recovery after PNI in mice. However, it is unknown whether these beneficial effects of 4-AP are present in traumatic muscle injury and muscle injury-induced bone loss. This study was thus designed to evaluate the potential therapeutic effect of 4-AP in traumatic muscle healing and PNI-induced bone loss. Standardized crush injury was performed on right sciatic nerve, biceps femoris (BF) muscle (innervated by sciatic nerve), and quadriceps femoris (QF) muscle (innervated by femoral nerve) of adult male mice (6 animals/group). Post-surgical animals were randomly assigned to normal saline and 4-AP. 4-AP (40 µg, intraperitoneal) was given daily for 21 days, and the functional recovery after injury was assessed by sciatic function index (SFI), sensation by von Frey test (VFT), and grip strength test. After 21 days, the muscles at the injury/peri-injury site were processed for histomorphometry and tibial bone density was analyzed using DEXA scanning. We found that 4-AP significantly enhanced SFI, VFT, and hind limb paw grip strength from post-injury day 7, improved BF muscle morphology, cross-sectional area, and minimum ferret diameter, and totally reversed trauma-induced muscle fiber type composition changes (type-1 vs. type-2). 4-AP-induced muscle effects were also associated with a significantly increased number of regenerating stem cells (Pax7+) and proliferating cells (Ki67+) compared with saline group. Of note, 4-AP-induced muscle effects were either less pronounced or nonsignificant in QF muscle. Most importantly, 4-AP treatment significantly reduced PNI-induced bone loss by enhancing bone mineral density and bone mineral content. In conclusion, these interesting findings, for the first time, demonstrate the potential therapeutic effect of 4-AP on the recovery of traumatic muscle and bone loss after mixed muscle and nerve injury.

Unacylated ghrelin ameliorates muscle atrophy and contractile dysfunction in age‐associated loss of muscle mass and function

Sarcopenia, the progressive loss of muscle mass and dysfunction, universally affects the elderly and is closely associated with frailty and reduced quality of life. Despite the inevitable consequences of sarcopenia and its relevance to healthspan, no pharmacological therapies are currently available. Ghrelin is a gut-released hormone that increases appetite and body weight through acylation (acyl ghrelin, AG), which binds to its receptors (GHSR1a) in the brain. Growing studies demonstrate that AG and “unacyl” ghrelin (UnAG) both have direct impacts on proliferation independent of GHSR1a. Neuronal precursor cells and myoblast incubated with UnAG increases proliferation and protein synthesis rate by promoting differentiation and fusion. Thus, we hypothesized that UnAG ameliorates neurogenic atrophy and contractile dysfunction in aged skeletal muscle. Young (4 mo.) and old (18 mo.) female mice were treated with saline or UnAG. UnAG (100 µg/kg/day) via osmotic pump for 9-10 months. We assessed in vitro contractile properties of isolated extensor digitorum longus (EDL). UnAG treatment significantly increased terminal serum UnAG levels in the old animals. In contrast to previous studies using AG, UnAG did not affect food consumption or body weight. Muscle mass of gastrocnemius and quadriceps were ~20-30% decreased in old mice, but UnAG ameliorated the atrophy by 15-30% for both muscles. Specific force (force per cross-sectional are) of isolated EDL muscle was diminished by ~30% in Sod1KO, but UnAG restored the force deficit by ~80% in the KO mice. We find that upregulation of protein kinase A (PKA) and hyper-phosphorylation of FoxO3a signaling pathways may contribute to the protection. RNA-seq analysis further revealed that UnAG shifted mRNA expression of 366 genes towards younger states that are differentially regulated in old mice. Our data show a direct role of UnAG on sarcopenia independent of changes of food consumption or body weight, implicating UnAG as a potential therapeutic strategy that can enhance quality of life and healthspan of rapidly growing elderly population.

Autologous Hematopoietic Stem Cell Transplantation for Chronic Inflammatory Demyelinating Polyradiculoneuropathy.

Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) refractory to conventional therapy can lead to marked disability and represents a therapeutic challenge. To report five cases of treatment-refractory disabling CIDP treated with autologous hematopoietic stem cell transplantation (AHSCT). This was a retrospective cohort study from a tertiary care referral center for both neuromuscular disease and AHSCT. Patients with CIDP treated with AHSCT between 2008 and 2020 were included. All patients had major persistent and disabling neuropathic deficits despite combinations of intensive immunosuppressive therapy. The primary outcome measures were: Medical Research Council sum score, Overall Neuropathy Limitations Scale and requirement for ongoing CIDP immunotherapy after transplantation. We also analyzed safety outcomes by documenting all severe AHSCT-related complications. Five patients with refractory CIDP underwent AHSCT. Three were classified as manifesting a typical syndrome, two were classified as the multifocal Lewis Sumner variant. The mean age at time of CIDP diagnosis was 33.4 years (range 24-46 years), with a median delay of 46 months (range 21-135 months) between diagnosis and AHSCT. The median follow-up period was 41 months. All five patients were able to wean off CIDP-related immunotherapy. Marked improvements in Medical Research Council scale and overall Neuropathy Limitations Scale were noted in 4/5 patients. One patient with longstanding neurogenic atrophy showed no improvement in disability scales. There were no treatment-related deaths or critical illnesses. AHSCT can achieve marked sustained clinical improvement of refractory CIDP and may allow for weaning off long-term complex immunotherapies.

Molecular pathophysiology of human MICU1 deficiency.

MICU1 encodes the gatekeeper of the mitochondrial Ca2+ uniporter, MICU1 and biallelic loss-of-function mutations cause a complex, neuromuscular disorder in children. Although the role of the protein is well understood, the precise molecular pathophysiology leading to this neuropaediatric phenotype has not been fully elucidated. Here we aimed to obtain novel insights into MICU1 pathophysiology. Molecular genetic studies along with proteomic profiling, electron-, light- and Coherent anti-Stokes Raman scattering microscopy and immuno-based studies of protein abundances and Ca2+ transport studies were employed to examine the pathophysiology of MICU1 deficiency in humans. We describe two patients carrying MICU1 mutations, two nonsense (c.52C>T; p.(Arg18*) and c.553C>T; p.(Arg185*)) and an intragenic exon 2-deletion presenting with ataxia, developmental delay and early onset myopathy, clinodactyly, attention deficits, insomnia and impaired cognitive pain perception. Muscle biopsies revealed signs of dystrophy and neurogenic atrophy, severe mitochondrial perturbations, altered Golgi structure, vacuoles and altered lipid homeostasis. Comparative mitochondrial Ca2+ transport and proteomic studies on lymphoblastoid cells revealed that the [Ca2+ ] threshold and the cooperative activation of mitochondrial Ca2+ uptake were lost in MICU1-deficient cells and that 39 proteins were altered in abundance. Several of those proteins are linked to mitochondrial dysfunction and/or perturbed Ca2+ homeostasis, also impacting on regular cytoskeleton (affecting Spectrin) and Golgi architecture, as well as cellular survival mechanisms. Our findings (i) link dysregulation of mitochondrial Ca2+ uptake with muscle pathology (including perturbed lipid homeostasis and ER-Golgi morphology), (ii) support the concept of a functional interplay of ER-Golgi and mitochondria in lipid homeostasis and (iii) reveal the vulnerability of the cellular proteome as part of the MICU1-related pathophysiology.

Implications of the neuroanatomy of the equine thoracolumbar vertebral column with regional anaesthesia and complications following desmotomy of the interspinous ligament.

Impinging/overriding dorsal spinous processes (DSPs) of the thoracolumbar vertebrae are a common cause of poor performance in horses. In the last five decades, numerous surgical treatments have been reported on, from transverse transection of the affected DSPs, and endoscopic resection of the affected DSPs, to transection of the interspinous ligament. Until recently, cosmetic outcomes have been reported as good to excellent in studies. However, a previously unreported complication of neurogenic atrophy of the contralateral epaxial muscle following desmotomy of the interspinous ligament has been recently reported. The authors hypothesised that this was because of a more lateral approach than previously described, resulting in the scissors being too far across midline and transecting a nerve in the region. Considering this finding, we have reviewed the literature on the neuroanatomy of the thoracolumbar region in the horse. Literature on the neuroanatomy of the horse is lacking when compared with that of humans and companion animals, with most of the work extrapolated from companion animals. Based on the current literature, we hypothesise that transection of an intermediate branch of the dorsal spinal nerve supplying the m. longissimus is potentially the cause of the post-operative neurogenic atrophy. The lack of detailed knowledge of the neural anatomy of the equine back has resulted in the role of local anaesthesia in localising pain in the equine back being poorly understood. The wide variation in techniques used for localising back pain may explain why some horses suffering from poor performance or an abnormal gait because of back pain improve to local anaesthesia of the back while others do not. This review article highlights a lack of anatomical knowledge regarding the equine thoracolumbar region in the literature despite diagnostic local anaesthesia, medication, and surgery in this area being relatively common.