Number Of Neuromuscular Junctions Research Articles

Porcine Acellular Nerve-Derived Hydrogel Improves Outcomes of Direct Muscle Neurotization in Rats.

Background: The ability to reinnervate a muscle in the absence of a viable nerve stump is a challenging clinical scenario. Direct muscle neurotization (DMN) is an approach to overcome this obstacle; however, success depends on the formation of new muscle endplates, a process, which is often limited due to lack of appropriate axonal pathfinding cues. Objective: This study explored the use of a porcine nerve extracellular matrix hydrogel as a neuroinductive interface between nerve and muscle in a rat DMN model. The goal of the study was to establish whether such hydrogel can be used to improve neuromuscular function in this model. Materials and Methods: A common peroneal nerve-to-gastrocnemius model of DMN was developed. Animals were survived for 2 or 8 weeks following DMN with or without the addition of the hydrogel at the site of neurotization. Longitudinal postural thrust, terminal electrophysiology, and muscle weight assessments were performed to qualify and quantify neuromuscular function. Histological assessments were made to qualify the host response at the DMN site, and to quantify neuromuscular junctions (NMJs) and muscle fiber diameter. Results: The hydrogel-treated group showed a 132% increase in postural thrust at 8 weeks compared with that of the DMN alone group. This was accompanied by an 80% increase in the number of NMJs at 2 weeks, and 26% increase in mean muscle fiber diameter at 8 weeks. Conclusions: These results suggest that a nerve-derived hydrogel may improve the neuromuscular outcome following DNM.

Alignment of Skeletal Muscle Cells Facilitates Acetylcholine Receptor Clustering and Neuromuscular Junction Formation with Co-Cultured Human iPSC-Derived Motor Neurons

In vitro neuromuscular junction (NMJ) models are powerful tools for studying neuromuscular disorders. Although linearly patterned culture surfaces have been reported to be useful for the formation of in vitro NMJ models using mouse motor neuron (MNs) and skeletal muscle (SkM) myotubes, it is unclear how the linearly patterned culture surface increases acetylcholine receptor (AChR) clustering, one of the steps in the process of NMJ formation, and whether this increases the in vitro NMJ formation efficiency of co-cultured human MNs and SkM myotubes. In this study, we investigated the effects of a linearly patterned culture surface on AChR clustering in myotubes and examined the possible mechanism of the increase in AChR clustering using gene expression analysis, as well as the effects of the patterned surface on the efficiency of NMJ formation between co-cultured human SkM myotubes and human iPSC-derived MNs. Our results suggest that better differentiation of myotubes on the patterned surface, compared to the flat surface, induced gene expression of integrin α7 and AChR ε-subunit, thereby increasing AChR clustering. Furthermore, we found that the number of NMJs between human SkM cells and MNs increased upon co-culture on the linearly patterned surface, suggesting the usefulness of the patterned surface for creating in vitro human NMJ models.

A Functional Human-on-a-Chip Autoimmune Disease Model of Myasthenia Gravis for Development of Therapeutics.

Myasthenia gravis (MG) is a chronic and progressive neuromuscular disease where autoantibodies target essential proteins such as the nicotinic acetylcholine receptor (nAChR) at the neuromuscular junction (NMJ) causing muscle fatigue and weakness. Autoantibodies directed against nAChRs are proposed to work by three main pathological mechanisms of receptor disruption: blocking, receptor internalization, and downregulation. Current in vivo models using experimental autoimmune animal models fail to recapitulate the disease pathology and are limited in clinical translatability due to disproportionate disease severity and high animal death rates. The development of a highly sensitive antibody assay that mimics human disease pathology is desirable for clinical advancement and therapeutic development. To address this lack of relevant models, an NMJ platform derived from human iPSC differentiated motoneurons and primary skeletal muscle was used to investigate the ability of an anti-nAChR antibody to induce clinically relevant MG pathology in the serum-free, spatially organized, functionally mature NMJ platform. Treatment of the NMJ model with the anti-nAChR antibody revealed decreasing NMJ stability as measured by the number of NMJs before and after the synchrony stimulation protocol. This decrease in NMJ stability was dose-dependent over a concentration range of 0.01–20 μg/mL. Immunocytochemical (ICC) analysis was used to distinguish between pathological mechanisms of antibody-mediated receptor disruption including blocking, receptor internalization and downregulation. Antibody treatment also activated the complement cascade as indicated by complement protein 3 deposition near the nAChRs. Additionally, complement cascade activation significantly altered other readouts of NMJ function including the NMJ fidelity parameter as measured by the number of muscle contractions missed in response to increasing motoneuron stimulation frequencies. This synchrony readout mimics the clinical phenotype of neurological blocking that results in failure of muscle contractions despite motoneuron stimulations. Taken together, these data indicate the establishment of a relevant disease model of MG that mimics reduction of functional nAChRs at the NMJ, decreased NMJ stability, complement activation and blocking of neuromuscular transmission. This system is the first functional human in vitro model of MG to be used to simulate three potential disease mechanisms as well as to establish a preclinical platform for evaluation of disease modifying treatments (etiology).

Regenerating Urethral Striated Muscle by CRISPRi/dCas9-KRAB-Mediated Myostatin Silencing for Obesity-Associated Stress Urinary Incontinence.

Overweight females are prone to obesity-associated stress urinary incontinence (OA-SUI), and there are no definitive medical therapies for this common urologic condition. This study was designed to test the hypothesis that regenerative therapy to restore urethral striated muscle (stM) and pelvic floor muscles might represent a valuable therapeutic approach. For the in vitro experiment, single-guide RNAs targeting myostatin (MSTN) were used for CRISPRi/dCas9-Kruppel associated box (KRAB)-mediated gene silencing. For the in vivo experiment, a total of 14 female lean ZUC-Leprfa 186 and 14 fatty ZUC-Leprfa 185 rats were used as control and CRISPRi-MSTN treated groups, respectively. The results indicated that lentivirus-mediated expression of MSTN CRISPRi/dCas9-KRAB caused sustained downregulation of MSTN in rat L6 myoblast cells and significantly enhanced myogenesis in vitro. In vivo, the urethral sphincter injection of lentiviral-MSTN sgRNA and lentiviral-dCas9-KRAB significantly increased the leak point pressure, the thickness of the stM layer, the ratio of stM to smooth muscle, and the number of neuromuscular junctions. Downregulation of MSTN with CRISPRi/dCas9-KRAB-mediated gene silencing significantly enhanced myogenesis in vitro and in vivo. It also improved urethral continence in the OA-SUI rat model.

Remnant neuromuscular junctions in denervated muscles contribute to functional recovery in delayed peripheral nerve repair.

Schwann cell proliferation in peripheral nerve injury (PNI) enhances axonal regeneration compared to central nerve injury. However, even in PNI, long-term nerve damage without repair induces degeneration of neuromuscular junctions (NMJs), and muscle atrophy results in irreversible dysfunction. The peripheral regeneration of motor axons depends on the duration of skeletal muscle denervation. To overcome this difficulty in nerve regeneration, detailed mechanisms should be determined for not only Schwann cells but also NMJ degeneration after PNI and regeneration after nerve repair. Here, we examined motor axon denervation in the tibialis anterior muscle after peroneal nerve transection in thy1-YFP mice and regeneration with nerve reconstruction using allografts. The number of NMJs in the tibialis anterior muscle was maintained up to 4 weeks and then decreased at 6 weeks after injury. In contrast, the number of Schwann cells showed a stepwise decline and then reached a plateau at 6 weeks after injury. For regeneration, we reconstructed the degenerated nerve with an allograft at 4 and 6 weeks after injury, and evaluated functional and histological outcomes for 10 to 12 weeks after grafting. A higher number of pretzel-shaped NMJs in the tibialis anterior muscle and better functional recovery were observed in mice with a 4-week delay in surgery than in those with a 6-week delay. Nerve repair within 4 weeks after PNI is necessary for successful recovery in mice. Prevention of synaptic acetylcholine receptor degeneration may play a key role in peripheral nerve regeneration. All animal experiments were approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University on 5 July 2017, 30 March 2018, and 15 May 2019 (A2017-311C, A2018-297A, and A2019-248A), respectively.

The effects of microenergy acoustic pulses on an animal model of obesity-associated stress urinary incontinence. Part 1: Functional and histologic studies.

Obesity is a strong independent risk factor for urinary incontinence. Effective therapeutic approaches for obesity-associated stress urinary incontinence (OA-SUI) are lacking as the mechanisms remain unclear. The aim of our study is to explore the impacts of microenergy acoustic pulse (MAP) therapy on urethral and pelvic floor muscle structure and function in female lean and fatty rats. A total 24 Zucker fatty (ZF) and 24 Zucker lean (ZL) female 24-week-old rats were grouped into four groups: ZL control, ZLMAP, ZF control, and ZFMAP. For MAP treatment, 500 pulses were delivered at an energy level of 0.033 mJ/mm 2 and a frequency of 3 Hz and were applied twice a week for 4 weeks. After a 1-week washout, all rats underwent conscious cystometry and leak-point pressure (LPP) measurements followed by ex vivo organ-bath assay and histological study. ZF rats had lower LPP as compared to ZL rats, and MAP treatment significantly improved LPP in ZF rats (P < .05). Impaired muscle contractile activity (MCA) in organ-bath study was noted in ZF rats. MAP treatment significantly increased MCA in ZF rats (P < .05) and also increased the thickness of the striated muscle layer and the number of neuromuscular junctions (NMJs). In situ, MAP activated muscle satellite cells significantly (P < .05). Obesity impairs the function of both the urethral sphincter and the pelvic floor and leads to atrophy and distortion of the striated muscle in obese female rats. These issues contribute to OA-SUI. MAP improves continence by stimulating muscle regeneration and nerve innervation as well as by activating satellite cells.

Maternal protein restriction during pregnancy and lactation affects the development of muscle fibers and neuromuscular junctions in rats.

A balanced maternal diet is a determining factor in normal fetal development. The objective of this study was to evaluate the effects of maternal protein restriction during pregnancy and lactation on muscle fiber and neuromuscular junction (NMJ) morphology of rat offspring at 21 days of age. Wistar rats were divided into a control group (CG), offspring of mothers fed a normal protein diet (17%), and a restricted group (RG), offspring of mothers fed a low-protein diet (6%). After a period of lactation, the animals were euthanized, and soleus muscles were obtained from pups for analysis. The soleus muscles of the RG exhibited an increase of 133% in the number of fibers and of 79% in the amount of nuclei. Moreover, the number of NMJs was lower in the restricted group than in the CG. Maternal protein restriction alters the normal development of the neuromuscular system. Muscle Nerve 55: 109-115, 2017.

Simultaneous reversal of age‐related declines in muscle health and cognition with transplantation of preconditioned mesenchymal stem cells (1163.2)

Mechanical strain can potently influence paracrine factor release from skeletal muscle‐derived mesenchymal stem cells (mMSCs). Since the MSC secretome has the potential to impact both local and distant tissue health, we assessed the extent to which transplanted mMSCs influence the local muscle environment as well as brain plasticity and behavior. mMSCs isolated from young muscle were preconditioned (10% multiaxial strain, 5 hrs) and immediately transplanted into the muscle of 24 month old mice. Mice receiving saline or non‐strained mMSCs served as controls. In addition to measuring indices of muscle health, the dentate gyrus of the hippocampus was evaluated for immature neurons one week post‐injection. Two weeks after transplantation, cognitive performance was measured by water maze, rotarod, and contextual fear conditioning. Although satellite cell number and fiber size were unchanged, vessel size and number of neuromuscular junctions were greater in muscle injected with strained mMSCs versus controls resulting in increased muscle function (P &lt; 0.05). A significant increase in the number of immature neurons (P &lt; 0.05) and a trend towards increased cognitive function on the water maze test was observed in these mice. Overall, mMSC preconditioning prior to transplantation may provide a novel method to prevent or reverse age‐related impairments in muscle strength, neuroplasticity, and cognitive function.Grant Funding Source: Supported by Ellison Medical Foundation (AG‐NS‐0547‐09), Abbott Laboraties (Abbott CNLM ZA68)

The impact of nimodipine administration combined with nerve‐muscle pedicle implantation on long‐term denervated rat thyroarytenoid muscle

To evaluate the impact of nimodipineon reinnervation of the long-term denervated rat thyroarytenoid (TA) muscle following nerve-muscle pedicle flap (NMP) implantation. Quantitative histologic and physiologic assessments. Using 120 Wistar rats, we performed NMP implantation at different times after transection of the left recurrent laryngeal nerve (RLN). Sixty animals received nimodipine treatment (NIMO [+] group), and the remaining 60 animals received no nimodipine treatment (NIMO [-] group). As a control, an additional 28 animals were subjected only to transection of the left RLN (DNV group). Subgroups were assigned based on the period after RLN transection (0 [immediate], 8, 16, and 32 weeks). In the DNV group, we assessed histologically the muscle area, axons, neuromuscular junctions (NMJs), and myosin heavy chains (MyHC) type IIA and IIB in the TA muscle. In the NIMO (-) and NIMO (+) groups, histologic assessments and evoked electromyography were performed on the TA muscle at 10 weeks post-NMP implantation. In 8-week interval subgroups, the muscle fiber area and the number of NMJs in the NIMO (+) group were significantly greater than in the NIMO (-) group (P < 0.05, each). In the 0-week and 32-week interval subgroups, the muscle fiber subtype changed significantly, from IIA to IIB (P < 0.01 and P < 0.05, respectively); and, at all time-points the muscle fiber area, number of NMJs, and action potentials in the TA muscle tended to be greater in the NIMO (+) group than in the NIMO (-) group. Nimodipine expedited the effects of NMP implantation on reinnervation of the long-term denervated TA muscle. Laryngoscope, 2012.

Nerve-muscle pedicle implantation in the denervated thyroarytenoid muscle of aged rats

Conclusion: Nerve-muscle pedicle (NMP) implantation was effective in the recovery of atrophic changes in the denervated thyroarytenoid (TA) muscle in aged rats. Objectives: To evaluate the effects of NMP implantation on the denervated TA muscle in aged rats. Methods: Wistar rats aged 20 months and 8 weeks were respectively divided into two groups in which the left recurrent laryngeal nerve (RLN) was transected without (aged/young DNV group) or with (aged/young NMP group) NMP implantation. The aged DNV and NMP groups were further divided into two subgroups, based on the period after RLN transection (10 or 20 weeks). In the DNV groups, we assessed the area of muscle and the number of neuromuscular junctions (NMJs) histologically. In the NMP groups, we performed electromyography and histological assessments. For electromyography, we stimulated the transferred nerve and evaluated the muscle action potentials (MAPs) of the TA muscle. Results: The muscle areas in the aged NMP groups were significantly larger than those in the aged DNV groups. More NMJs were found in aged NMP groups compared with aged DNV groups. MAPs were seen in all NMP animals. No significant differences were observed between the aged and young NMP groups in histological and physiological assessments.

Nerve-muscle pedicle implantation facilitates re-innervation of long-term denervated thyroarytenoid muscle in rats

Conclusions: Nerve-muscle pedicle (NMP) implantation was effective in the recovery from atrophic changes in long-term denervated thyroarytenoid (TA) muscle. Re-innervation occurred via the transferred nerve. However, the effectiveness of the NMP method may decline with increasing duration of denervation. Objectives: To evaluate the effects of NMP implantation on long-term denervated rat TA muscle. Materials and methods: Wistar rats (n=105) were divided into two groups in which the left recurrent laryngeal nerve (RLN) was transected without (DNV group) or with (NMP group) subsequent NMP implantation, and subgroups of each group were formed depending on the period after RLN transection (immediate to 48 weeks). In the DNV subgroups, we histologically assessed the area of muscle and the number of neuromuscular junctions. In the NMP subgroups, we performed electromyographic, videolaryngoscopic, and histologic assessments. The muscle area and muscle action potentials were evaluated by comparing the treated and untreated sides. The ratio of the number of nerve terminals to that of acetylcholine receptors was also assessed. Results: The TA muscle area was significantly larger in most of the NMP subgroups compared with the DNV subgroups. Muscle action potentials were present in all NMP animals. All histologic and physiologic assessments revealed degradation as the denervation period in the five NMP subgroups.

Hyperinnervation of Mesenteric Arteries in Spontaneously Hypertensive Rats by Sympathetic but Not Primary Afferent Axons

Hypertrophy of the perivascular plexus is thought to play a role in the development of hypertension in spontaneously hypertensive rats (SHR). However, it is not known whether the sympathetic varicosities are more numerous or larger, or form more neurovascular junctions. Further, a parallel hypertrophy of primary afferent terminals around the vessels might modulate any effects of hypertrophied sympathetic terminals. We have investigated the perivascular plexus around second-order mesenteric arteries of SHR and Wistar-Kyoto (WKY) rats by electron microscopy. Noradrenergic terminals were identified by the presence of small granular vesicles after chromaffin fixation, and substance P (SP+) afferent axons were identified by immunohistochemistry. The numbers of noradrenergic axon and varicosity profiles were higher (48 and 25%, respectively) in SHR than in WKY rats, and the majority lay closer to the medio-adventitial border. In contrast, there was no difference in the numbers of SP+ axons. Sympathetic and SP+ varicosities were indistinguishable in size, shape, vesicle content and mitochondrion content between each other and between the strains. However, both the number of neuromuscular junctions and the proportion of varicosities that formed them in SHR arteries were more than double those in WKY vessels. The data clearly show that hyperinnervation in SHR is specific for noradrenergic axons.

Insulin growth factor‐1 decreases muscle atrophy following denervation

Despite modern microsurgical techniques for nerve repair, functional outcome following proximal injury is often unsatisfactory because irreversible muscle atrophy may develop before reinnervation occurs. Because insulin growth factor-1 (IGF-1) has been shown to improve muscle regeneration after injury, and may have a role in muscle preservation following denervation, the purpose of this investigation was to evaluate the histological, immunohistochemical, and electrophysiological differences between normal, denervated, and IGF-1-injected denervated muscle over an 8-week period. Denervated mice gastrocnemius muscles demonstrated a decrease in muscle weight, a decrease in myofiber diameter, an absence of muscle regeneration, an early increase in the number of neuromuscular junctions (NMJs), and a decrease in fast-twitch and maximum tetanic strength as compared to normal muscle up to 8 weeks following denervation. IGF-1-injected denervated muscle, on the other hand, sustained muscle diameter and muscle weight, maintained a smaller number of NMJs, and relatively sustained fast-twitch and maximum tetanic strength as compared to normal muscle over 8 weeks. These data suggest that IGF-1 may help prevent muscle atrophy and secondary functional compromise after denervation.