Focal Application of Neurotrophic Factors Augments Outcomes of Nerve-Muscle-Endplate Grafting Technique for Limb Muscle Reinnervation.

BACKGROUND: We have developed a novel muscle reinnervation technique called "nerve-muscle-endplate grafting (NMEG) in the native motor zone (NMZ)." This study aimed to augment the outcomes of the NMEG-NMZ (NN) by focal application of exogenous neurotrophic factors (ENFs) for limb reinnervation. METHODS: Adult rats were used to conduct NN plus ENF (NN/ENF) and autologous nerve grafting (ANG, technique control). The nerve innervating the left tibialis anterior (TA) muscle was resected and the denervated TA was immediately treated with NN/ENF or ANG. For NN procedure, an NMEG pedicle was taken from the lateral gastrocnemius muscle and transferred to the NMZ of the denervated TA. For ANG, the nerve gap was bridged with sural nerve. Three months after treatment, the extent of functional and neuromuscular recovery was assessed by measuring static toe spread, maximal muscle force, wet muscle weight, regenerated axons, and innervated motor endplates (MEPs). RESULTS: NN/ENF resulted in 90% muscle force recovery of the treated TA, which is far superior to ANG (46%) and NN alone (79%) as reported elsewhere. Toe spread recovered up to 89 and 49% of the control for the NN/ENF and ANG groups, respectively. The average wet muscle weight was 87 and 52% of the control for muscles treated with NN/ENF and ANG, respectively. The mean number of the regenerated axons was 88% of the control for the muscles treated with NN/ENF, which was significantly larger than that for the ANG-repaired muscles (39%). The average percentage of the innervated MEPs in the NN/ENF-treated TA (89%) was higher compared with that in the ANG-repaired TA (48%). CONCLUSION: ENF enhances nerve regeneration and MEP reinnervation that further augment outcomes of NN. The NN technique could be an alternative option to treat denervated or paralyzed limb muscles caused by traumatic nerve injuries or lesions.

Toxic effects of subacute exposure to acrylamide on motor endplates of the gastrocnemius in rats.

Acrylamide (ACR) is a recognized toxin that is known to induce neurotoxicity in humans and experimental animals. This study aimed to investigate the toxic effects of subacute exposure of the motor endplate (MEP) of the gastrocnemius in rats to ACR. All rats were randomly divided into control, 9, 18, and 36 mg/kg ACR groups, and ACR was administered by gastric gavage for 21 days. The behavioral tests were performed weekly. On the 22nd day, the wet weight of the gastrocnemius was measured. The changes in muscle fiber structure, nerve endings, and MEP in the gastrocnemius were examined by hematoxylin-eosin (HE) and gold chloride staining. Acetylcholinesterase (AChE) content in the gastrocnemius was detected by AChE staining. The expression of AChE and calcitonin gene-related peptide was detected by immunohistochemistry and western blot. Rats exposed to ACR showed a significant increase in gait scores and hind limb splay distance compared with the control group, and the wet weight of the gastrocnemius was reduced, HE staining showed that the muscle fiber structure of the gastrocnemius became thin and the arrangement was dense with nuclear aggregation, gold chloride staining showed that nerve branches decreased and became thin, nerve fibers became short and light, the number of MEPs was decreased, the staining became light, and the structure was not clear. AChE staining showed that the number of MEPs was significantly reduced after exposure to ACR, the shape became small, and the AChE content decreased in a dose-dependent manner. Immunohistochemistry and western blot analysis results of the expression levels of AChE and CGRP showed a decreasing trend as compared to the control group with increasing ACR exposure dose. The reduction in protein levels may be the mechanism by which ACR has a toxic effect on the MEP in the gastrocnemius of rats.

Intervertebral disc and endplate cells response to IL-1ß inflammatory cell priming and identification of molecular targets of tissue degeneration.

Inflammation represents an important factor leading to metabolic imbalance within the intervertebral disc (IVD), conducive to degenerative changes. Therefore, a thorough knowledge of the IVD and endplate (EP) cell behaviour in such pathological environments is essential when designing regenerative therapeutic strategies. The present study aimed at assessing the molecular response of the IVD constitutive nucleus pulposus (NPCs)-, annulus fibrosus (AFCs)- and endplate (EPCs)-derived cells to interleukin (IL)-1ß treatment, through large-scale, high-throughput microarray and protein analysis, identifying the differentially expressed genes and released proteins. Overall, the inflammatory stimulus downregulated stemness genes while upregulating pro-inflammatory, pro-angiogenic and catabolic genes, including matrix metalloproteases, which were not balanced by a concomitant upregulation of their inhibitors. Upregulation of anti-inflammatory and anabolic tumour necrosis factor inducible gene 6 protein (TNFAIP6), of IL-1 receptor antagonist (IL-1Ra) (at gene and protein levels) and of trophic insulin-like growth factor 1 (IGF1) was also observed in all cell types; IGF1 particularly in AFCs. An overall inhibitory effect of tumour necrosis factor alpha (TNFα) signal was observed in all cell types; however, EPCs showed the strongest anti-inflammatory behaviour. AFCs and EPCs shared the ability to limit the activation of the signalling mediated by specific chemokines. AFCs showed a slightly senescent attitude, with a downregulation of genes related to DNA repair or pro-mitosis. Results allowed for the identification of specific molecular targets in IVD and EP cells that respond to an inflammatory environment. Such targets can be either silenced (when pathological targets) or stimulated to counteract the inflammation.

Herbal complex 'Buyang Huanwu Tang' improves motor endplate function of denervated-dependent skeletal muscle atrophy in rat.

Denervated-dependent skeletal muscle atrophy is a disease induced by skeletal muscle associated peripheral neuro-disconnection. Its specific molecular mechanisms remain unknown. The treating for denervated-dependent skeletal muscle atrophy is applied with an herbal complex Buyang Huanwu Tang used in traditional Chinese medicine and subjected to the established denervated-dependent skeletal muscle atrophy in rat models, and the therapeutic effects and associated mechanisms were evaluated in the pathogenesis of denervated-dependent skeletal muscle atrophy. Denervated-dependent skeletal muscle atrophy in rats was established and randomly divided into eight groups, including Normal control, Model, Positive control, Model + Buyang Huanwu Tang, Model + astragalus extracts, Model + Buyang Huanwu Tang-astragalus, Buyang Huanwu Tang + LY294002, and astragalus extract + LY294002 group. Hematoxylin-eosin staining and quantitative RT-PCR (qRT-PCR) assay were used to examine the inflammatory response of muscle tissues. Quantitative RT-PCR and Western blotting assay were utilized to analyze mRNA and protein expression. Immunohistochemistry assay was used to detect molecule expression in anterior cervical muscle tissues. Motor endplate activity was examined using the wholemount acetylcholinesterase staining method. The wet mass ratio of anterior cervical muscle was measured. The results indicated that Buyang Huanwu Tang treatment significantly alleviated inflammatory response, enhanced acetylcholinesterase activity, and motor endplate functions, and promoted wet mass of anterior cervical muscle compared to denervated-dependent skeletal muscle atrophy rat models (P < 0.05). Buyang Huanwu Tang regulated molecules of PI3K/PKB/GSK3ß/FOXO1 signaling pathway. Buyang Huanwu Tang significantly reduced muscle atrophy F-box protein, MuFR-1, Bax and caspase 9 expression, significantly enhanced Bcl-2 expression, and remarkably increased element-binding protein and vascular endothelial growth factor levels, compared to Model group (P < 0.05). Buyang Huanwu Tang suppressed caspase 9 and caspase 3 activity and associated apoptosis. Moreover, PI3K specific blocker, LY294002, significantly inhibited the effects of Buyang Huanwu Tang on the above molecule expression (P < 0.05). In conclusion, Buyang Huanwu Tang improved motor endplate functions of denervated-dependent skeletal muscle atrophy rat model through suppressing mitochondria-mediated apoptosis and activating PI3K/PKB/FOXO1 signaling pathway.

Intervertebral disc and endplate cell characterisation highlights annulus fibrosus cells as the most promising for tissue-specific disc degeneration therapy.

Degenerative processes of the intervertebral disc (IVD) and cartilaginous endplate lead to chronic spine pathologies. Several studies speculated on the intrinsic regenerative capacity of degenerated IVD related to the presence of local mesenchymal progenitors. However, a complete characterisation of the resident IVD cell populations, particularly that isolated from the endplate, is lacking. The purpose of the present study was to characterise the gene expression profiles of human nucleus pulposus (NPCs), annulus fibrosus (AFCs) and endplate (EPCs) cells, setting the basis for future studies aimed at identifying the most promising cells for regenerative purposes. Cells isolated from NP, AF and EP were analysed after in vitro expansion for their stemness ability, immunophenotype and gene profiles by large-scale microarray analysis. The three cell populations shared a similar clonogenic, adipogenic and osteogenic potential, as well as an immunophenotype with a pattern resembling that of mesenchymal stem cells. NPCs maintained the greatest chondrogenic potential and shared with EPCs the loss of proliferation capability during expansion. The largest number of selectively highly expressed stemness, chondrogenic/tissue-specific and surface genes was found in AFCs, thus representing the most promising source of tissue-specific expanded cells for the treatment of IVD degeneration.

Biomechanical Comparison of 1-Level Corpectomy and 2-Level Discectomy for Cervical Spondylotic Myelopathy: A Finite Element Analysis.

BACKGROUND Anterior cervical discectomy and fusion (ACDF) and anterior cervical corpectomy and fusion (ACCF) are effective treatments for cervical spondylotic myelopathy (CSM), but it is unclear which is better. In this study, we compared the biomechanical properties of 2-level ACDF and 1-level ACCF. MATERIAL AND METHODS An intact C3-C7 cervical spine model was developed and validated, then ACDF and ACCF simulation models were developed. We imposed 1.0 Nm moments and displacement-controlled loading on the C3 superior endplate. The range of motions (ROMs) of surgical and adjacent segments and von Mises stresses on endplates, fixation systems, bone-screw interfaces, and bone grafts were recorded. RESULTS ACDF and ACCF significantly reduced the surgical segmental ROMs to the same extent. ACCF induced much lower stress peaks in the fixation system and bone-screw interfaces and higher stress peaks on the bone graft. ACDF induced much lower stress peaks on the C4 inferior endplate and equivalent stress on the C6 superior endplate. There was no difference in the ROMs of surgical and adjacent segments and the intradiscal stress of adjacent levels between ACDF and ACCF. CONCLUSIONS Both ACDF and ACCF can provide satisfactory spinal stability. ACDF may be beneficial for subsidence resistance due to the lower stress peaks on the endplate. The ACCF may perform better in long-term stability and bone fusion owing to the lower stress peaks in the fixation system and bone-screw interfaces, and higher stress peaks in the bone graft.

Examination of the human motor endplate after brachial plexus injury with two-photon microscopy.

INTRODUCTION: After traumatic nerve injury, neuromuscular junction remodeling plays a key role in determining functional outcomes. Immunohistochemical analyses of denervated muscle biopsies may provide valuable prognostic data regarding clinical outcomes to supplement electrodiagnostic studies. METHODS: We performed biopsies on nonfunctioning deltoid muscles in two patients after gunshot wounds and visualized the neuromuscular junctions using two-photon microscopy with immunohistochemistry. RESULTS: Although the nerves in both patients showed evidence of acute Wallerian degeneration, some of the motor endplates were intact but exhibited significantly decreased surface area and volume. Both patients exhibited substantial recovery of motor function over several weeks postinjury. DISCUSSION: Two-photon microscopic assessment of neuromuscular junction integrity and motor endplate morphometry in muscle biopsies provided evidence of partial sparing of muscle innervation. This finding supported the clinical judgment that eventual recovery would occur. With further study, this technique may help to guide operative decisionmaking after traumatic nerve injuries.

Sensory innervation in porous endplates by Netrin-1 from osteoclasts mediates PGE2-induced spinal hypersensitivity in mice.

Spinal pain is a major clinical problem, however, its origins and underlying mechanisms remain unclear. Here we report that in mice, osteoclasts induce sensory innervation in the porous endplates which contributes to spinal hypersensitivity in mice. Sensory innervation of the porous areas of sclerotic endplates in mice was confirmed. Lumbar spine instability (LSI), or aging, induces spinal hypersensitivity in mice. In these conditions, we show that there are elevated levels of PGE2 which activate sensory nerves, leading to sodium influx through Nav 1.8 channels. We show that knockout of PGE2 receptor 4 in sensory nerves significantly reduces spinal hypersensitivity. Inhibition of osteoclast formation by knockout Rankl in the osteocytes significantly inhibits LSI-induced porosity of endplates, sensory innervation, and spinal hypersensitivity. Knockout of Netrin-1 in osteoclasts abrogates sensory innervation into porous endplates and spinal hypersensitivity. These findings suggest that osteoclast-initiated porosity of endplates and sensory innervation are potential therapeutic targets for spinal pain.

Slow-channel myasthenia due to novel mutation in M2 domain of AChR delta subunit.

OBJECTIVE: To characterize the molecular and phenotypic basis of a severe slow-channel congenital myasthenic syndrome (SCCMS). METHODS: Intracellular and single-channel recordings from patient endplates; alpha-bungarotoxin binding studies; direct sequencing of AChR genes; microsatellite analysis; kinetic analysis of AChR activation; homology modeling of adult human AChR structure. RESULTS: Among 24 variants reported to cause SCCMS only two appear in the AChR δ-subunit. We here report a 16-year-old patient harboring a novel δL273F mutation (δL294F in HGVS nomenclature) in the second transmembrane domain (M2) of the AChR δ subunit. Kinetic analyses with ACh and the weak agonist choline indicate that δL273F prolongs the channel opening bursts 9.4-fold due to a 75-fold increase in channel gating efficiency, whereas a previously identified εL269F mutation (εL289F in HGVS nomenclature) at an equivalent location in the AChR ε-subunit prolongs channel opening bursts 4.4-fold due to a 30-fold increase in gating efficiency. Structural modeling of AChR predicts that inter-helical hydrophobic interactions between the mutant residue in the δ and ε subunit and nearby M2 domain residues in neighboring α subunits contribute to structural stability of the open relative to the closed channel states. INTERPRETATION: The greater increase in gating efficiency by δL273F than by εL269F explains why δL273F has more severe clinical effects. Both δL273F and εL269F impair channel gating by disrupting hydrophobic interactions with neighboring α-subunits. Differences in the extent of impairment of channel gating in δ and ε mutant receptors suggest unequal contributions of ε/α and δ/α subunit pairs to gating efficiency.

What is Normal? Neuromuscular junction reinnervation after nerve injury.

INTRODUCTION: In this study we present a reproducible technique to assess motor recovery after nerve injury via neuromuscular junction (NMJ) immunostaining and electrodiagnostic testing. METHODS: Wild-type mice underwent sciatic nerve transection with repair. Hindlimb muscles were collected for microscopy up to 30 weeks after injury. Immunostaining was used to assess axons (NF200), Schwann cells (S100), and motor endplates (α-bungarotoxin). Compound motor action potential (CMAP) amplitude was used to assess tibialis anterior (TA) function. RESULTS: One week after injury, nearly all (98.0%) endplates were denervated. At 8 weeks, endplates were either partially (28.3%) or fully (71.7%) reinnervated. At 16 weeks, NMJ reinnervation reached 87.3%. CMAP amplitude was 83% of naive mice at 16 weeks and correlated with percentage of fully reinnervated NMJs. Morphological differences were noted between injured and noninjured NMJs. DISCUSSION: We present a reproducible method for evaluating NMJ reinnervation. Electrodiagnostic data summarize NMJ recovery. Characterization of wild-type reinnervation provides important data for consideration in experimental design and interpretation.

mTORC1 and PKB/Akt control the muscle response to denervation by regulating autophagy and HDAC4.

Loss of innervation of skeletal muscle is a determinant event in several muscle diseases. Although several effectors have been identified, the pathways controlling the integrated muscle response to denervation remain largely unknown. Here, we demonstrate that PKB/Akt and mTORC1 play important roles in regulating muscle homeostasis and maintaining neuromuscular endplates after nerve injury. To allow dynamic changes in autophagy, mTORC1 activation must be tightly balanced following denervation. Acutely activating or inhibiting mTORC1 impairs autophagy regulation and alters homeostasis in denervated muscle. Importantly, PKB/Akt inhibition, conferred by sustained mTORC1 activation, abrogates denervation-induced synaptic remodeling and causes neuromuscular endplate degeneration. We establish that PKB/Akt activation promotes the nuclear import of HDAC4 and is thereby required for epigenetic changes and synaptic gene up-regulation upon denervation. Hence, our study unveils yet-unknown functions of PKB/Akt-mTORC1 signaling in the muscle response to nerve injury, with important implications for neuromuscular integrity in various pathological conditions.

LINGO-1 shRNA Loaded by Pluronic F-127 Promotes Functional Recovery After Ventral Root Avulsion.

Spinal root avulsion typically leads to massive motoneuron death and severe functional deficits of the target muscles. Multiple pathological factors such as severe neuron loss, induction of inhibitory molecules, and insufficient regeneration are responsible for the poor functional recovery. Leucine-rich repeat and immunoglobulin-like domain-containing Nogo receptor-interacting protein 1 (LINGO-1), a central nervous system (CNS)-specific transmembrane protein that is selectively expressed on neurons and oligodendrocytes, serves as a potent negative mediator of axonal regeneration and myelination in CNS injuries and diseases. Although accumulating evidence has demonstrated improvement in axonal regeneration and neurological functions by LINGO-1 antagonism in CNS damage, the possible effects of LINGO-1 in spinal root avulsion remain undiscovered. In this study, a LINGO-1 knockdown strategy using lentiviral vectors encoding LINGO-1 short hairpin interfering RNA (shRNA) delivered by the Pluronic F-127 (PF-127) hydrogel was described after brachial plexus avulsion (BPA). We provide evidence that following BPA and immediate reimplantation, transplantation of LINGO-1 shRNA lentiviral vectors encapsulated by PF-127 rescued the injured motoneurons, enhanced axonal outgrowth and myelination, rebuilt motor endplates, facilitated the reinnervation of terminal muscles, improved angiogenesis, and promoted recovery of avulsed forelimbs. Altogether, these data suggest that delivery of LINGO-1 shRNA by a gel scaffold is a potential therapeutic approach for root avulsion. Impact Statement In this study, we attempted transplantation of lentivirus (LV)/leucine-rich repeat and immunoglobulin-like domain-containing Nogo receptor-interacting protein 1 (LINGO-1)-short hairpin interfering RNA (shRNA) encapsulated by the Pluronic F-127 (PF-127) hydrogel into a brachial plexus avulsion (BPA)-reimplantation model. We found that administration of LV/LINGO-1 shRNA facilitates neuron survival and axonal regeneration, attenuates muscle atrophy and motor endplate (MEP) loss, enhances neovascularization, and promotes functional recovery in BPA rats. Co-transplantation of LV/LINGO-1 shRNA and gel reinforces the survival-promoting effect, axonal outgrowth, and angiogenesis in comparison with LV/LINGO-1 shRNA application alone. Our research provides evidence that LV /LINGO-1 shRNA delivered by PF-127 represents a new treatment strategy for BPA repair.

The Sagittal Thoracic Modifier Component of the Lenke Classification System in Moderate and Severe Adolescent Idiopathic Scoliosis: Reliability and Reproducibility of Measurements Among 5 Observers.

STUDY DESIGN: A prospective radiographic study. SUMMARY OF BACKGROUND DATA: As the importance of the spinal sagittal profile becomes increasingly evident, there is a need to ensure that the measuring methods used to evaluate thoracic kyphosis (TK) are both accurate and reproducible. OBJECTIVE: The purpose of the following study was to determine the intraobserver and interobserver variability of measurements of the sagittal profile in moderate and severe thoracic scoliosis. METHODS: Five experienced Faculty Spine surgeons independently reviewed thirty standing long 30-inch cassette lateral radiographs of preoperative moderate and severe curves ≥50 degrees of adolescent idiopathic scoliosis (AIS) patients on 2 different occasions. The parameters measured were the vertebral endplate clarity and measurability of the sagittal angle from D5 to D12 and categories of thoracic sagittal modifier. κ statistics and Intraclass Correlation Coefficient (ICC) were used for analysis. RESULTS: The interobserver percentage of agreement for the Sagittal modifier was 58% in both trials. The mean κ coefficient value was only moderate 0.43 (range, 0.14-0.66) for both trials. The number of the vertebral endplates that were difficult to identify was 201 of 300 measurements (67%). There was a predominance of difficulty to identify vertebral endplate clarity in all curve types. CONCLUSIONS: The results of this study yielded poor to moderate interobserver reliability of the thoracic sagittal profile component of the Lenke classification system in moderate and severe AIS. This was attributed to the difficulty in identification of the vertebral endplates. The current standard lateral radiographs routinely used in AIS patients have inherent difficulties and limitations to visualize, identify, and analyze the thoracic endplates in moderate and severe curves.

Neuromuscular Junction Morphology and Gene Dysregulation in the Wobbler Model of Spinal Neurodegeneration.

Amyotrophic lateral sclerosis (ALS) is the most common adult-onset neuromuscular disease for which there is currently no effective treatment. The progression of ALS includes loss of motor neurons controlling the voluntary muscles, with much of this loss occurring at the neuromuscular junction. In an effort to better understand changes at the neuromuscular junction, we utilized the wobbler mouse model of motor neuron loss. We examined biceps and end plate morphologies and monitored selected factors involved in end plate function. Structural volumes were determined from 3D reconstructions that were generated for the end plates. Wobbler mice exhibited size reductions of both the muscle fibers and the end plates within the biceps, and we found that the end plate volumes were the most sensitive indicator of the degeneration. Concurrently, we found increases in calcitonin gene-related peptide (CGRP) and its receptor in wobbler biceps and spinal cord. We also found increases in gene expression of two acetylcholine receptors within the wobbler biceps, which may be a result of altered CGRP/CALCRL (calcitonin receptor-like receptor) expression.

Length of Lumbar Interbody Cage Using Radiological Measurements of Chinese Endplates and the Apophyseal Ring.

OBJECTIVE: To radiologically measure the parameters of endplates and the apophyseal ring and to suggest an applicable length for a lumbar interbody cage for Chinese patients. METHODS: Twenty-four volunteers were enrolled to undergo lumbar computed tomography (CT). On the endplate plane, the diameters of the endplates (L1-S1) were measured along the axis of the cage with different lumbar interbody fusion procedures. Whereas the mid-oblique diameter (mid-OD) and maximum oblique diameter (max-OD) were defined as the minimal and maximal diameters of the endplates in transforaminal lumbar interbody fusion (TLIF), side-sagittal diameter (side-SD), mid-sagittal diameter (mid-SD), and transverse diameter (TD) represented the diameters of endplates in posterior lumbar interbody fusion (PLIF), anterior lumbar interbody fusion (ALIF); and oblique lateral interbody fusion (OLIF)/extreme lateral interbody fusion (XLIF), /direct lateral interbody fusion (DLIF), respectively. R1-R10 were the widths of the apophyseal ring covered by diameters at both ends. We used the proposed formula to calculate the cage length: 1) minimal length of TLIF cage = mid-OD - ½ (R1 + R2), 2) maximal length of TLIF cage = max-OD - ½ (R3 + R4), 3) length of PLIF cage = side-SD - ½ (R5 + R6), 4) length of OLIF/XLIF/DLIF cage = TD - ½ (R7 + R8), and 5) length of ALIF cage = mid-SD - ½ (R9 + R10). RESULTS: The lengths of the TLIF cage were more than 30 mm for men and 26 mm for women in L1/2-L4/5, with a large range in L5-S1. For PLIF, the lengths were 28 to 30 mm for men and 24 to 26 mm for women in L1/2-L4/5, with 26 mm and 22 mm, respectively, in L5-S1. For the OLIF/XLIF/DLIF cage, the lengths were 38 mm in L1/2 and 41 to 43 mm in L2/3-L4/5 for men and 35 mm in L1/2-L2/3 and 38 mm in L3/4-L4/5 for women. The ALIF cage lengths were 27 mm in L1/2 and L5-S1 and 29 mm in L2/3-L4/5 for men and 23 mm in L1/2 and L5-S1 and 25 mm in L2/3-L4/5 for women. CONCLUSIONS: The choice of an appropriate length for a lumbar interbody cage should be based on the procedure and fusion level, which can match the endplates anatomically. The size of the lumbar interbody cage is affected by many factors, and a simple calculation may not be clinically relevant.

Immunohistochemical Detection of Motor Endplates in the Long-Term Denervated Muscle.

BACKGROUND: We have demonstrated that the native motor zone (NMZ) within a muscle is an ideal target for performing nerve-muscle-endplate band grafting (NMEG) to restore motor function of a denervated muscle. This study was designed to determine spatiotemporal alterations of the myofibers, motor endplates (MEPs), and axons in the NMZ of long-term denervated muscles for exploring if NMEG-NMZ technique would have the potential for delayed reinnervation. METHODS: Sternomastoid (SM) muscles of adult female Sprague-Dawley rats (n = 21) were experimentally denervated and denervation-induced changes in muscle weight, myofiber size, MEPs, and intramuscular nerve axons were evaluated histomorphometrically and immunohistochemically at the end of 3, 6, and 9 months after denervation. The values obtained from the ipsilateral normal side served as control. RESULTS: The denervated SM muscles exhibited a progressive reduction in muscle weight (38%, 31%, and 19% of the control) and fiber diameter (52%, 40%, and 28% of the control) for 3-, 6-, and 9-month denervation, respectively. The denervated MEPs were still detectable even 9 months after denervation. The mean number of the denervated MEPs was 79%, 65%, and 43% of the control in the 3-, 6-, and 9-month denervated SM, respectively. Degenerated axons in the denervated muscles became fragmented. CONCLUSIONS: Persistence of MEPs in the long-term denervated SM suggests that some surgeries targeting the MEPs such as NMEG-NMZ technique should be effective for delayed reinnervation. However, more work is needed to develop strategies for preservation of muscle mass and MEPs after denervation.

Leptin regulates disc cartilage endplate degeneration and ossification through activation of the MAPK-ERK signalling pathway in vivo and in vitro.

Recent findings demonstrate that leptin plays a significant role in chondrocyte and osteoblast differentiation. However, the mechanisms by which leptin acts on cartilage endplate (CEP) cells to give rise to calcification are still unclear. The aim of this study was to evaluate the effects of leptin that induced mineralization of CEP cells in vitro and in vivo. We constructed a rat model of lumbar disc degeneration and determined that leptin was highly expressed in the presence of CEP calcification. Rat CEP cells treated with or without leptin were used for in vitro analysis using RT-PCR and Western blotting to examine the expression of osteocalcin (OCN) and runt-related transcription factor 2 (Runx2). Both OCN and Runx2 expression levels were significantly increased in a dose- and time-dependent manner. Leptin activated ERK1/2 and STAT3 phosphorylation in a time-dependent manner. Inhibition of phosphorylated ERK1/2 using targeted siRNA suppressed leptin-induced OCN and Runx2 expression and blocked the formation of mineralized nodules in CEP cells. We further demonstrated that exogenous leptin induced matrix mineralization of CEP cells in vivo. We suggest that leptin promotes the osteoblastic differentiation of CEP cells via the MAPK/ERK signal transduction pathway and may be used to investigate the mechanisms of disc degeneration.

A genome-wide analysis of the gene expression profiles and alternative splicing events during the hypoxia-regulated osteogenic differentiation of human cartilage endplate-derived stem cells.

It has been hypothesized that intervertebral disc degeneration is initiated by degeneration of the cartilage endplate (CEP), which is characterized by cartilage ossification. CEP­derived stem cells (CESCs), with the potential for chondro­osteogenic differentiation, may be responsible for the balance between chondrification and ossification in the CEP. The CEP remains in an avascular and hypoxic microenvironment; the present study observed that hypoxia was able to markedly inhibit the osteogenic differentiation of CESCs. This tissue­specific CESC differentiation in response to a hypoxic microenvironment was physiologically important for the prevention of ossification in the CEP. In order to study the hypoxia­regulated mechanisms underlying osteogenic differentiation of CESCs, a Human Transcriptome Array 2.0 was used to detect differentially expressed genes (DEGs) and alternatively spliced genes (ASGs) during the osteogenic differentiation of CESCs under hypoxia, compared with those induced under normoxia. High­throughput analysis of DEGs and ASGs demonstrated that genes in the complement pathway were enriched, which may be a potential mechanism underlying hypoxia inhibition of CESCs osteogenesis. The results of the present study may provide a basis for future mechanistic studies regarding gene expression levels and alternative splicing events during the hypoxia­regulated inhibition of osteogenesis, which may be helpful in identifying targets for CEP degeneration therapy.

Postnatal Development of Spasticity Following Transgene Insertion in the Mouse ßIV Spectrin Gene (SPTBN4).

BACKGROUND: The L25 mouse line was generated by random genomic insertion of a lens-specific transgene. Inbreeding of L25 hemizygotes revealed an unanticipated spastic phenotype in the hind limbs. OBJECTIVE: The goals were to characterize the motor phenotype in the L25 mice and to map the transgene insert site within the mouse genome. METHODS: Six pairs of L25+/- mice were repeatedly mated. Beginning at weaning, all progeny were inspected for body weight and motor signs twice weekly until they displayed predefined ethical criteria for termination. The transgene insert site was determined by whole genome sequencing. Western blotting was used to compare the expression levels of beta-IV spectrin protein in the brain. RESULTS: Matings of hemizygous L25+/- × L25+/- mice yielded 20% (29/148) affected weanlings, identified by an abnormal retraction of the hind limbs when lifted by the tail, and a fine tremor. Affected mice were less mobile and grew more slowly than wild-type littermates. All affected mice required termination due to >15% loss of body weight (50% survival age 92 days). At the endpoint, mice showed varying degrees of spastic paresis or spastic paralysis localised to the hind limbs. Motor endplates remained fully innervated. Genome sequencing confirmed that the transgene was inserted in the locus of ßIV spectrin of L25 mice. Western blotting indicated that this random insertion had greatly reduced the expression of ßIV spectrin protein in the affected L25 mice. CONCLUSIONS: The results confirm the importance of ßIV spectrin for maintaining central motor pathway control of the hind limbs, and provide a developmental time course for the phenotype.

KLF7-transfected Schwann cell graft transplantation promotes sciatic nerve regeneration.

Our former study demonstrated that Krüppel-like Factor 7 (KLF7) is a transcription factor that stimulates axonal regeneration after peripheral nerve injury. Currently, we used a gene therapy approach to overexpress KLF7 in Schwann cells (SCs) and assessed whether KLF7-transfected SCs graft could promote sciatic nerve regeneration. SCs were transfected by adeno-associated virus 2 (AAV2)-KLF7 in vitro. Mice were allografted by an acellular nerve (ANA) with either an injection of DMEM (ANA group), SCs (ANA+SCs group) or AAV2-KLF7-transfected SCs (ANA+KLF7-SCs group) to assess repair of a sciatic nerve gap. The results indicate that KLF7 overexpression promoted the proliferation of both transfected SCs and native SCs. The neurite length of the dorsal root ganglia (DRG) explants was enhanced. Several beneficial effects were detected in the ANA+KLF7-SCs group including an increase in the compound action potential amplitude, sciatic function index score, enhanced expression of PKH26-labeling transplant SCs, peripheral myelin protein 0, neurofilaments, S-100, and myelinated regeneration nerve. Additionally, HRP-labeled motoneurons in the spinal cord, CTB-labeled sensory neurons in the DRG, motor endplate density and the weight ratios of target muscles were increased by the treatment while thermal hyperalgesia was diminished. Finally, expression of KLF7, NGF, GAP43, TrkA and TrkB were enhanced in the grafted SCs, which may indicate that several signal pathways may be involved in conferring the beneficial effects from KLF7 overexpression. We concluded that KLF7-overexpressing SCs promoted axonal regeneration of the peripheral nerve and enhanced myelination, which collectively proved KLF-SCs as a novel therapeutic strategy for injured nerves.