Reduced Gene Expression of KCC2 Accelerates Axonal Regeneration and Reduces Motor Dysfunctions after Tibial Nerve Severance and Suturing.

Gamma-aminobutyric acid and glycine (GABA/Gly) are predominantly inhibitory neurotransmitters in the mature central nervous system; however, they mediate membrane potential depolarization during development. These differences in actions depend on intracellular Cl- concentrations ([Cl-]i), which are primarily regulated by potassium chloride cotransporter 2 (KCC2). After nerve injury, KCC2 expression markedly decreases and GABA/Gly mediate depolarization. Following nerve regeneration, KCC2 expression recovers and GABA/Gly become inhibitory, suggesting that KCC2 reduction and GABA/Gly excitation may be crucial for axonal regeneration. To directly clarify their involvement in regeneration, we analyzed recovery processes after tibial nerve severance and suturing between heterozygous KCC2 knockout mice (HT), whose KCC2 levels are halved, and their wild-type littermates (WT). Compared with WT mice, the sciatic functional index-indicating lower limb motor function-was significantly higher until 28 days after operation (D28) in HT mice. Furthermore, at D7, many neurofilament-positive fibers were elongated into the distal part of the sutured nerve in HT mice only, and myelinated axonal density was significantly higher at D21 and D28 in HT animals. Electron microscopy and galanin immunohistochemistry indicated a shorter nerve degeneration period in HT mice. Moreover, a less severe decrease in choline acetyltransferase was observed in HT mice. These results suggest that nerve degeneration and regeneration proceed more rapidly in HT mice, resulting in milder motor dysfunction. Via similar microglial activation, nerve surgery may reduce KCC2 levels more rapidly in HT mice, followed by earlier increased [Cl-]i and longer-lasting GABA/Gly excitation. Taken together, reduced KCC2 may accelerate nerve regeneration via GABA/Gly excitation.

Prohibitin 1 is essential to preserve mitochondria and myelin integrity in Schwann cells.

In peripheral nerves, Schwann cells form myelin and provide trophic support to axons. We previously showed that the mitochondrial protein prohibitin 2 can localize to the axon-Schwann-cell interface and is required for developmental myelination. Whether the homologous protein prohibitin 1 has a similar role, and whether prohibitins also play important roles in Schwann cell mitochondria is unknown. Here, we show that deletion of prohibitin 1 in Schwann cells minimally perturbs development, but later triggers a severe demyelinating peripheral neuropathy. Moreover, mitochondria are heavily affected by ablation of prohibitin 1 and demyelination occurs preferentially in cells with apparent mitochondrial loss. Furthermore, in response to mitochondrial damage, Schwann cells trigger the integrated stress response, but, contrary to what was previously suggested, this response is not detrimental in this context. These results identify a role for prohibitin 1 in myelin integrity and advance our understanding about the Schwann cell response to mitochondrial damage.

Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve.

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study of this movement in situ was only possible using radioisotopic pulse-labeling, which permits analysis of axonal transport in whole nerves with a temporal resolution of days and a spatial resolution of millimeters. To study neurofilament transport in situ with higher temporal and spatial resolution, we developed a hThy1-paGFP-NFM transgenic mouse that expresses neurofilament protein M tagged with photoactivatable GFP in neurons. Here we describe fluorescence photoactivation pulse-escape and pulse-spread methods to analyze neurofilament transport in single myelinated axons of tibial nerves from these mice ex vivo. Isolated nerve segments are maintained on the microscope stage by perfusion with oxygenated saline and imaged by spinning disk confocal fluorescence microscopy. Violet light is used to activate the fluorescence in a short axonal window. The fluorescence in the activated and flanking regions is analyzed over time, permitting the study of neurofilament transport with temporal and spatial resolution on the order of minutes and microns, respectively. Mathematical modeling can be used to extract kinetic parameters of neurofilament transport including the velocity, directional bias and pausing behavior from the resulting data. The pulse-escape and pulse-spread methods can also be adapted to visualize neurofilament transport in other nerves. With the development of additional transgenic mice, these methods could also be used to image and analyze the axonal transport of other cytoskeletal and cytosolic proteins in axons.

Diffusion of Radiodense Contrast Medium Following Perineural Injection of the Deep Branch of the Lateral Plantar Nerve Using Two Different Techniques in Horses: an In Vivo Study.

OBJECTIVES: This article evaluates and compares the diffusion pattern of radiopaque contrast medium following perineural analgesia of the deep branch of the lateral plantar nerve performed using two different techniques: weight-bearing or flexed. STUDY DESIGN: This was an in vivo experimental study. METHODS: Eight horses were enrolled. Perineural injection of the right and left deep branch lateral plantar nerves was performed with a weight-bearing or flexed technique, using radiopaque contrast medium (iohexol). Radiographic evaluation was performed after 5 (T5), 15 (T15) and 30 (T30) minutes. The diffusion of contrast medium was assessed independently by two blinded readers who analysed the extension of the main contrast medium bulk and the maximum diffusion of contrast medium in both proximal and distal directions. The effect of time and technique employed on contrast medium diffusion was assessed using two-way analysis of variance for repeated measures (p ≤ 0.05). RESULTS: There was no significant difference in the diffusion of the contrast medium between the two techniques at T15. However, at T30 the weight-bearing technique resulted in a significantly increased diffusion in the proximal direction (p = 0.02). In one case, belonging to the weight-bearing group, contrast medium was identified within the tarsal sheath. There was no evidence of contrast medium in the tarsometatarsal joint of any horse, regardless of the technique used. CONCLUSIONS: The two techniques resulted in a similar diffusion at T15. However, the use of a weight-bearing technique resulted in a significant increase in proximal contrast medium diffusion 30 minutes after injection.

Development and persistence of neuropathic pain through microglial activation and KCC2 decreasing after mouse tibial nerve injury.

Gamma-amino butyric acid (GABA) is an inhibitory neurotransmitter in the mature brain, but is excitatory during development and after motor nerve injury. This difference in GABAergic action depends on the intracellular chloride ion concentration ([Cl-]i), primarily regulated by potassium chloride co-transporter 2 (KCC2). To reveal precise processes of the neuropathic pain through changes in GABAergic action, we prepared tibial nerve ligation and severance models using male mice, and examined temporal relationships amongst changes in (1) the mechanical withdrawal threshold in the sural nerve area, (2) localization of the molecules involved in GABAergic transmission and its upstream signaling in the dorsal horn, and (3) histology of the tibial nerve. In the ligation model, tibial nerve degeneration disappeared by day 56, but mechanical allodynia, reduced KCC2 localization, and increased microglia density remained until day 90. Microglia density was higher in the tibial zone than the sural zone before day 21, but this result was inverted after day 28. In contrast, in the severance model, all above changes were detected until day 28, but were simultaneously and significantly recovered by day 90. These results suggested that in male mice, allodynia may be caused by reduced GABAergic synaptic inhibition, resulting from elevated [Cl-]i after the reduction of KCC2 by activated microglia. Furthermore, our results suggested that factors from degenerating nerve terminals may diffuse into the sural zone, whereby they induced the development of allodynia in the sural nerve area, while other factors in the sural zone may mediate persistent allodynia through the same pathway.

Vibration-induced depression in spinal loop excitability revisited.

KEY POINTS: While it has been well described that prolonged vibration locally applied to a muscle or its tendon (up to 1 h) decreases spinal loop excitability between homonymous Ia afferents and motoneurons, the involved mechanisms are not fully understood. By combining electrophysiological methods, this study aimed to provide new insights into the mechanisms involved in soleus decreased spinal excitability after prolonged local vibration. We report that prolonged vibration induces a decrease in motoneuron excitability rather than an increase in presynaptic mechanisms (as commonly hypothesized in the current literature). The present results may help to design appropriate clinical intervention and could reinforce the interest in vibration as a treatment for spastic patients who are characterized by spinal hyper-excitability responsible for spasms and long-lasting reflexes. ABSTRACT: The mechanisms that can explain the decreased spinal loop excitability in response to prolonged local vibration (LV), as assessed by the H-reflex, remain to be precisely determined. This study provides new insights into how prolonged Achilles' tendon LV (30 min, 100 Hz) acutely interacts with the spinal circuitry. The roles of presynaptic inhibition exerted on Ia afferents (Experiment A, n = 15), neurotransmitter release at the synapse level (Experiment B, n = 11) and motoneuron excitability (Experiment C, n = 11) were investigated in soleus. Modulation of presynaptic inhibition was assessed by conditioning the soleus H-reflex (tibial nerve electrical stimulation) with fibular nerve (D1 inhibition) and femoral nerve (heteronymous facilitation, HF) electrical stimulations. Potential vibration-induced changes in neurotransmitter depletion at the Ia afferent terminals was assessed through paired stimulations applied over the tibial nerve (HD). Intrinsic motoneuron excitability was assessed with thoracic motor evoked potentials (TMEPs) in response to electrical stimulation over the thoracic spine. Non-conditioned H-reflex was depressed by ∼60% after LV (P < 0.001), while D1 and HF H-reflexes increased by ∼75% after LV (P = 0.03 and 0.06, respectively). In Experiment B, HD remained unchanged after LV (P = 0.80). In Experiment C, TMEPs were reduced by ∼13% after LV (P = 0.01). Overall, presynaptic mechanisms do not seem to be involved in the depression of spinal excitability after LV. It rather seems to rely, at least in part, on a decrease in intrinsic motoneuron excitability. These results may have implications in reducing spinal hyper-excitability in spastic patients.

Viral vector delivery of follistatin enhances recovery of reinnervated muscle.

INTRODUCTION: Poor recovery following nerve repair is due to progressive temporal loss of muscle function. Follistatin (FS), a glycoprotein with anabolic properties, may enhance muscle recovery following reinnervation. METHODS: Seventy-two male Sprague-Dawley rats underwent temporary (3 or 6 month) denervation or sham denervation. After reinnervation, rats were administered adeno-associated viral vectors expressing FS deoxyribonucleic acid (isoform FS-317) injected into the target muscle or sham treatment. Final assessment included muscle function testing, muscle histomorphology, nerve histomorphology, and FS protein quantification. RESULTS: FS improved muscle mass and type IIB muscle fiber size, and increased G-ratios and mean axon diameter in the 6-month temporary denervation group (P < .05). Elevated FS protein levels were detected in treated muscle (P < .05). FS increased satellite cell counts following temporary denervation and repair (P < .05). DISCUSSION: FS treatment had anabolic, neurotrophic, and satellite cell stimulatory effects when administered following prolonged (6-month) temporary denervation and repair.

Muscle spindle reinnervation using transplanted embryonic dorsal root ganglion cells after peripheral nerve transection in rats.

OBJECTIVES: Muscle spindles are proprioceptive receptors in the skeletal muscle. Peripheral nerve injury results in a decreased number of muscle spindles and their morphologic deterioration. However, the muscle spindles recover when skeletal muscles are reinnervated with surgical procedures, such as nerve suture or nerve transfer. Morphological changes in muscle spindles by cell transplantation procedure have not been reported so far. Therefore, we hypothesized that transplantation of embryonic sensory neurons may improve sensory neurons in the skeletal muscle and reinnervate the muscle spindles. MATERIALS AND METHODS: We collected sensory neurons from dorsal root ganglions of 14-day-old rat embryos and prepared a rat model of peripheral nerve injury by performing sciatic nerve transection and allowing for a period of one week before which we performed the cell transplantations. Six months later, the morphological changes of muscle spindles in the cell transplantation group were compared with the naïve control and surgical control groups. RESULTS: Our results demonstrated that transplantation of embryonic dorsal root ganglion cells induced regeneration of sensory nerve fibre and reinnervation of muscle spindles in the skeletal muscle. Moreover, calbindin D-28k immunoreactivity in intrafusal muscle fibres was maintained for six months after denervation in the cell transplantation group, whereas it disappeared in the surgical control group. CONCLUSIONS: Cell transplantation therapies could serve as selective targets to modulate mechanosensory function in the skeletal muscle.

Morphological Study of Myelinated Fibers of the Sciatic Nerve in Mice after Space Flight and Readaptation to the Conditions of Earth Gravity.

We revealed a decrease in the thickness of the myelin sheath and myelin delamination in the tibial nerve of C57BL/6N mice after a 30-day flight aboard the biosatellite Bion-M1. The processes of myelin degeneration continued for seven days after return of the animals to Earth and adaptation to the conditions of natural gravity. Our data add to hypothesis on the role of neurogenic component in pathogenesis of hypogravity motor syndrome.

Remodeling of myelinated fibers and internal capillaries in distal peripheral nerves following aerobic exercise in aged rats.

The aim of this study was to determine whether aerobic exercise (AE) in old age contributes to improving the morphologies of myelinated fibers (MFs) in peripheral nerves as well as capillaries. Furthermore, we investigated whether such processes are associated with complementary activity of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) in the circulating blood and peripheral nerve tissue. Fourteen male Wistar rats (age: 95 wk) were randomly divided into moderate AE ( n = 8) and sedentary (SED; n = 6) groups. Rats in the AE group performed treadmill running for 1 h per day for 2 wk, following which the bilateral tibial nerves of the two groups were removed to examine MF and capillary structure. Levels of BDNF and VEGF in the serum and peripheral nerves were analyzed via enzyme-linked immunosorbent assay. Myelin thickness, axon diameter, and capillary luminal diameter were significantly larger in the AE group than in the SED group ( P < 0.0001). Levels of serum BDNF and VEGF were significantly lower and higher, respectively, in the AE group than in the SED group ( P < 0.001). Conversely, BDNF and VEGF levels in tibial nerve tissue were significantly higher, respectively, and lower in the AE group than in the SED group ( P < 0.001). In conclusion, our study indicates that regular AE induces enlargement of the capillaries and thickens the myelin in aged peripheral nerves, likely via a complementary process involving BDNF and VEGF. NEW & NOTEWORTHY Accumulating evidence indicates that age-related sarcopenia is accompanied by the degeneration of myelinated fibers (MFs) in peripheral nerves. Our study indicates that regular aerobic exercise contributes to increased thickness of the myelin surrounding MFs and enlargement of the capillaries, likely via a complementary process involving brain-derived neurotrophic factor and vascular endothelial growth factor. Our findings demonstrate that regular, moderate-intensity aerobic exercise may help to prevent and reverse peripheral nerve regression in older adults.

Analgesic effects of systemic fentanyl on cancer pain are mediated by not only central but also peripheral opioid receptors in mice.

Fentanyl is an opioid commonly prescribed for cancer pain. Using melanoma-bearing mice, we investigated whether peripheral action would contribute to fentanyl analgesia in cancer pain. Intravenous injection of fentanyl inhibited mechanical nociception in healthy mice, which was markedly inhibited by the opioid antagonist naloxone, but not naloxone methiodide, a peripherally acting opioid antagonist. Melanoma-bearing mice showed mechanical allodynia and spontaneous licking, a pain-related behavior, which were suppressed by intravenous and local injections of fentanyl. Both naloxone and naloxone methiodide inhibited the analgesic effect of intravenous fentanyl to the same degree. Electrophysiological analysis showed that melanoma growth increased the spontaneous and mechanical stimuli-evoked activity of the tibial nerve, which were inhibited by intravenous fentanyl. There was a greater expression of µ- opioid receptors in skin with a melanoma mass than in the contralateral normal skin. In addition, we found µ-opioid receptors in cultured melanoma cells. There was no difference between the number of µ-opioid receptors in the dorsal root ganglia and spinal cord of the melanoma-bearing and contralateral skin side. These results suggest that the analgesic effect of systemic fentanyl is produced via central and peripheral µ- opioid receptors in cancer pain, and cancer cells are a key site of peripheral action.

Role of cannabinoid receptor type 1 in tibial and pudendal neuromodulation of bladder overactivity in cats.

The role of cannabinoid type 1 (CB1) receptors in tibial and pudendal neuromodulation of bladder overactivity induced by intravesical infusion of 0.5% acetic acid (AA) was determined in α-chloralose anesthetized cats. AA irritation significantly (P < 0.01) reduced bladder capacity to 36.6 ± 4.8% of saline control capacity. Tibial nerve stimulation (TNS) at two or four times threshold (2T or 4T) intensity for inducing toe movement inhibited bladder overactivity and significantly (P < 0.01) increased bladder capacity to 69.2 ± 9.7 and 79.5 ± 7.2% of saline control, respectively. AM 251 (a CB1 receptor antagonist) administered intravenously at 0.03 or 0.1 mg/kg significantly (P < 0.05) reduced the inhibition induced by 2T or 4T TNS, respectively, without changing the prestimulation bladder capacity. However, intrathecal administration of AM 251 (0.03 mg) to L7 spinal segment had no effect on TNS inhibition. Pudendal nerve stimulation (PNS) also inhibited bladder overactivity induced by AA irritation, but AM 251 at 0.01-1 mg/kg iv had no effect on PNS inhibition or the prestimulation bladder capacity. These results indicate that CB1 receptors play an important role in tibial but not pudendal neuromodulation of bladder overactivity and the site of action is not within the lumbar L7 spinal cord. Identification of neurotransmitters involved in TNS or PNS inhibition of bladder overactivity is important for understanding the mechanisms of action underlying clinical application of neuromodulation therapies for bladder disorders.

Electrical muscle stimulation elevates intramuscular BDNF and GDNF mRNA following peripheral nerve injury and repair in rats.

Despite advances in surgery, patients with nerve injuries frequently have functional deficits. We previously demonstrated in a rat model that daily electrical muscle stimulation (EMS) following peripheral nerve injury and repair enhances reinnervation, detectable as early as two weeks post-injury. In this study, we explain the enhanced early reinnervation observed with electrical stimulation. In two groups of rats, the tibial nerve was transected and immediately repaired. Gastrocnemius muscles were implanted with intramuscular electrodes for sham or muscle stimulation. Muscles were stimulated daily, eliciting 600 contractions for one hour/day, repeated five days per week. Sixteen days following nerve injury, muscles were assessed for functional reinnervation by motor unit number estimation methods using electromyographic recording. In a separate cohort of rats, surgical and electrical stimulation procedures were identical but muscles and distal nerve stumps were harvested for molecular analysis. We observed that stimulated muscles had significantly higher motor unit number counts. Intramuscular levels of brain-derived and glial cell line-derived neurotrophic factor (BDNF and GDNF) mRNA were significantly upregulated in muscles that underwent daily electrical stimulation compared to those without stimulation. The corresponding levels of trophic factor mRNA within the distal stump were not different from one another, indicating that the intramuscular electrical stimulus does not modulate Schwann cell-derived trophic factor transcription. Stimulation over a three-month period maintained elevated muscle-derived GDNF but not BDNF mRNA. In conclusion, EMS elevates intramuscular trophic factor mRNA levels which may explain how EMS enhances neural regeneration following nerve injury.

Dynamic Changes in Local Protein Synthetic Machinery in Regenerating Central Nervous System Axons after Spinal Cord Injury.

Intra-axonal localization of mRNAs and protein synthesis machinery (PSM) endows neurons with the capacity to generate proteins locally, allowing precise spatiotemporal regulation of the axonal response to extracellular stimuli. A number of studies suggest that this local translation is a promising target to enhance the regenerative capacity of damaged axons. Using a model of central nervous system (CNS) axons regenerating into intraspinal peripheral nerve grafts (PNGs) we established that adult regenerating CNS axons contain several different mRNAs and protein synthetic machinery (PSM) components in vivo. After lower thoracic level spinal cord transection, ascending sensory axons regenerate into intraspinal PNGs but axon growth is stalled when they reach the distal end of the PNG (3 versus 7 weeks after grafting, resp.). By immunofluorescence with optical sectioning of axons by confocal microscopy, the total and phosphorylated forms of PSMs are significantly lower in stalled compared with actively regenerating axons. Reinjury of these stalled axons increased axonal localization of the PSM proteins, indicative of possible priming for a subcellular response to axotomy. These results suggest that axons downregulate protein synthetic capacity as they cease growing, yet they retain the ability to upregulate PSM after a second injury.

Changes in VGLUT1 and VGLUT2 expression in rat dorsal root ganglia and spinal cord following spared nerve injury.

Disturbance of glutamate homeostasis is a well-characterized mechanism of neuropathic pain. Vesicular glutamate transporters (VGLUTs) determine glutamate accumulation in synaptic vesicles and their roles in neuropathic pain have been suggested by gene-knockout studies. Here, we investigated the spatio-temporal changes in VGLUT expression during the development of neuropathic pain in wild-type rats. Spared nerve injury (SNI) induced mechanical allodynia from postoperative day 1 to at least day 14. Expression of VGLUT1 and VGLUT2 in dorsal root ganglia and spinal cord was examined by western blot analyses on different postoperative days. We observed that VGLUT2 were selectively upregulated in crude vesicle fractions from the ipsilateral lumbar enlargement on postoperative days 7 and 14, while VGLUT1 was transiently downregulated in ipsilateral DRG (day 4) and contralateral lumbar enlargement (day 1). Upregulation of VGLUT2 was not accompanied by alterations in vesicular expression of synaptotagmin or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Thus, VGLUTs expression, especially VGLUT2, is regulated following peripheral nerve injury. Temporal regulation of VGLUT2 expression in spinal cord may represent a novel presynaptic mechanism contributing to injury-induced glutamate imbalance and associated neuropathic pain.

Medial plantar nerve ligation as a novel model of neuropathic pain in mice: pharmacological and molecular characterization.

Peripheral neuropathic pain is a consequence of an injury/disease of the peripheral nerves. The mechanisms involved in its pathophysiology are not entirely understood. To better understand the mechanisms involved in the development of peripheral nerve injury-induced neuropathic pain, more experimental models are required. Here, we developed a novel peripheral neuropathic pain model in mice by using a minimally invasive surgery and medial plantar nerve ligation (MPNL). After MPNL, mechanical allodynia was established, and mice quickly recovered from the surgery without any significant motor impairment. MPNL causes an increased expression of ATF-3 in the sensory neurons. At 14 days after surgery, gabapentin was capable of reversing the mechanical allodynia, whereas anti-inflammatory drugs and opioids were ineffective. MPNL-induced neuropathic pain was mediated by glial cells activation and the production of TNF-α and IL-6 in the spinal cord. These results indicate MPNL as a reasonable animal model for the study of peripheral neuropathic pain, presenting analgesic pharmacological predictivity to clinically used drugs. The results also showed molecular phenotypic changes similar to other peripheral neuropathic pain models, with the advantage of a lack of motor impairment. These features indicate that MPNL might be more appropriate for the study of neuropathic pain than classical models.

Progression of motor axon dysfunction and ectopic Nav1.8 expression in a mouse model of Charcot-Marie-Tooth disease 1B.

Mice heterozygously deficient for the myelin protein P0 gene (P0+/-) develop a slowly progressing neuropathy modeling demyelinating Charcot-Marie-Tooth disease (CMT1B). The aim of the study was to investigate the long-term progression of motor dysfunction in P0+/- mice at 3, 7, 12 and 20months. By comparison with WT littermates, P0+/- showed a decreasing motor performance with age. This was associated with a progressive reduction in amplitude and increase in latency of the plantar compound muscle action potential (CMAP) evoked by stimulation of the tibial nerve at ankle. This progressive functional impairment was in contrast to the mild demyelinating neuropathy of the tibial nerve revealed by histology. "Threshold-tracking" studies showed impaired motor axon excitability in P0+/- from 3months. With time, there was a progressive reduction in threshold deviations during both depolarizing and hyperpolarizing threshold electrotonus associated with increasing resting I/V slope and increasing strength-duration time constant. These depolarizing features in excitability in P0+/- as well as the reduced CMAP amplitude were absent in P0+/- NaV1.8 knockouts, and could be acutely reversed by selective pharmacologic block of NaV1.8 in P0+/-. Mathematical modeling indicated an association of altered passive cable properties with a depolarizing shift in resting membrane potential and increase in the persistent Na(+) current in P0+/-. Our data suggest that ectopic NaV1.8 expression precipitates depolarizing conduction failure in CMT1B, and that motor axon dysfunction in demyelinating neuropathy is pharmacologically reversible.

Acupuncture plus low-frequency electrical stimulation (Acu-LFES) attenuates denervation-induced muscle atrophy.

Muscle wasting occurs in a variety of clinical situations, including denervation. There is no effective pharmacological treatment for muscle wasting. In this study, we used a tibial nerve denervation model to test acupuncture plus low-frequency electric stimulation (Acu-LFES) as a therapeutic strategy for muscle atrophy. Acupuncture needles were connected to an SDZ-II electronic acupuncture device delivering pulses at 20 Hz and 1 mA; the treatment was 15 min daily for 2 wk. Acu-LFES prevented soleus and plantaris muscle weight loss and increased muscle cross-sectional area in denervated mice. The abundances of Pax7, MyoD, myogenin, and embryonic myosin heavy chain were significantly increased by Acu-LFES in both normal and denervated muscle. The number of central nuclei was increased in Acu-LFES-treated muscle fibers. Phosphorylation of Akt was downregulated by denervation leading to a decline in muscle mass; however, Acu-LFES prevented the denervation-induced decline largely by upregulation of the IGF-1 signaling pathway. Acu-LFES reduced the abundance of muscle catabolic proteins forkhead O transcription factor and myostatin, contributing to the attenuated muscle atrophy. Acu-LFES stimulated the expression of macrophage markers (F4/80, IL-1b, and arginase-1) and inflammatory cytokines (IL-6, IFNγ, and TNFα) in normal and denervated muscle. Acu-LFES also stimulated production of the muscle-specific microRNAs miR-1 and miR-206. We conclude that Acu-LFES is effective in counteracting denervation-induced skeletal muscle atrophy and increasing muscle regeneration. Upregulation of IGF-1, downregulation of myostatin, and alteration of microRNAs contribute to the attenuation of muscle atrophy in denervated mice.

Sepsis induced denervation-like changes at the neuromuscular junction.

BACKGROUND: The aim of this study was to determine the functional and biochemical changes at the neuromuscular junction (NMJ) induced by sepsis. MATERIALS AND METHODS: Male Sprague-Dawley rats were divided into three groups as follows: control, denervation, and sepsis. The rats were subjected to cecal ligation and puncture (CLP) or tibias nerve transection. NMJ function and the area of end plates were assessed, and the protein level of acetylcholine receptors and axonal neuregulin-1 was evaluated on postoperative days 1, 7, and 14. RESULTS: In the control group, the amplitude of compound muscle action potential (CMAP) was 16.51 ± 2.53 mV. In the sepsis group, the amplitude of CMAP decreased, and duration was prolonged on postoperative days 7 and 14 (P < 0.01). Meanwhile, motor conduction velocity decreased significantly (P < 0.01). CMAP was lost in the denervation group. The twitch tension magnitude gradually declined (P < 0.05) in the sepsis group, although it could not be recorded after lesion. Sepsis and denervation upregulated the expression of γ-nicotinic acetylcholine receptor (nAChR) and α7-nAChR in muscle membrane, compared with those in normal NMJ (261.4 ± 26.5 µm(2)). The NMJ area decreased from 254.6 ± 23.8 µm(2) (1 d after CLP) to 275.4 ± 22.6 µm(2) (7 d after CLP) to 322.7 ± 34.4 µm(2) (14 d after CLP). The postsynaptic NMJ had more discrete fragments (3.84 ± 0.6) compared with the control group (2.13 ± 0.4; P < 0.01). After denervation, NMJ underwent fragmentation and the number of discrete fragments increased (5.57 ± 1.2; P < 0.01). NMJ area increased from 254.6 ± 23.8 µm(2) (1 d after CLP) to 275.4 ± 22.6 µm(2) (7 d after CLP) to 322.7 ± 34.4 µm(2) (14 d after CLP). Sepsis induced neuregulin-1 to decrease from 1 d up to 2 wk compared with the control group (P < 0.05). CONCLUSIONS: Chronic sepsis has a denervation-like effect on the NMJ, which was indicated by upregulation of heterogeneous nAChRs, the increased area of end plates, and demyelination of the motoneuron axon.

Paclitaxel inhibits mRNA transport in axons.

Paclitaxel is an integral component of solid tumor treatment. This chemotherapeutic agent provokes an often irreversible peripheral sensory neuropathy with pathological features of distal axonal degeneration. Current pathological concepts assume that polymerization of axonal microtubules and mitochondrial dysfunction contributes to the development of paclitaxel-induced peripheral neuropathy. The relationship, however, between microtubule stabilization, mitotoxicity and axonal degeneration is still not completely understood. To explore the function of axonal mitochondria we treated transgenic mice that harbor cyan fluorescent protein (CFP)-labeled neuronal mitochondria with repeated doses of paclitaxel and assessed neuropathic changes by nerve conduction and histological studies. In addition, mitochondrial content and morphology was determined by ex vivo imaging of axons containing CFP-labeled mitochondria. Using quantitative RT-PCR and fluorescence-labeled mRNA we determined axonal mRNA transport of nuclear encoded mitochondrial proteins. Prolonged treatment with high doses of paclitaxel-induced a predominant sensory neuropathy in mice. Although mitochondrial velocity in axons per se was not altered, we observed significant changes in mitochondrial morphology, suggesting that paclitaxel treatment impairs the dynamics of axonal mitochondria. These changes were caused by decreased levels of nuclear encoded mRNA, including the mitochondrial fusion/fission machinery. Moreover, impaired axonal mRNA transport in vitro resulted in mitochondrial dysfunction and subsequent axonal degeneration. Taken together, our experiments provide evidence that disrupted axonal transport of nuclear derived mRNA plays a crucial role in the pathogenesis of paclitaxel-induced sensory neuropathy.