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.

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.

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.

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.

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.

Adenosine A2A receptors activation facilitates neuromuscular transmission in the pre-symptomatic phase of the SOD1(G93A) ALS mice, but not in the symptomatic phase.

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease leading to motor neuron dysfunction resulting in impairment of neuromuscular transmission. A2A adenosine receptors have already been considered as a potential therapeutical target for ALS but their neuromodulatory role at the neuromuscular junction in ALS remains to be clarified. In the present work, we evaluated the effects of A2A receptors on neuromuscular transmission of an animal model of ALS: SOD1(G93A) mice either in the pre-symptomatic (4-6 weeks old) or in the symptomatic (12-14 weeks old) stage. Electrophysiological experiments were performed obtaining intracellular recordings in Mg2+ paralyzed phrenic nerve-hemidiaphragm preparations. Endplate potentials (EPPs), quantal content (q. c.) of EPPs, miniature endplate potentials (MEPPs) and giant miniature endplate potential (GMEPPs) were recorded. In the pre-symptomatic phase of the disease (4-6 weeks old mice), the selective A2A receptor agonist, CGS 21680, significantly enhanced (p<0.05 Unpaired t-test) the mean amplitude and q.c. of EPPs, and the frequency of MEPPs and GMEPPs at SOD1(G93A) neuromuscular junctions, the effect being of higher magnitude (p<0.05, Unpaired t-test) than age-matched control littermates. On the contrary, in symptomatic mice (12-14 weeks old), CGS 21680 was devoid of effect on both the amplitude and q.c. of EPPs and the frequency of MEPPs and GMEPPs (p<0.05 Paired t-test). The results herein reported clearly document that at the neuromuscular junction of SOD1(G93A) mice there is an exacerbation of A2A receptor-mediated excitatory effects at the pre-symptomatic phase, whereas in the symptomatic phase A2A receptor activation is absent. The results thus suggest that A2A receptors function changes with ALS progression.

HnRNP L and hnRNP LL antagonistically modulate PTB-mediated splicing suppression of CHRNA1 pre-mRNA.

CHRNA1 gene, encoding the muscle nicotinic acetylcholine receptor alpha subunit, harbors an inframe exon P3A. Inclusion of exon P3A disables assembly of the acetylcholine receptor subunits. A single nucleotide mutation in exon P3A identified in congenital myasthenic syndrome causes exclusive inclusion of exon P3A. The mutation gains a de novo binding affinity for a splicing enhancing RNA-binding protein, hnRNP LL, and displaces binding of a splicing suppressing RNA-binding protein, hnRNP L. The hnRNP L binds to another splicing repressor PTB through the proline-rich region and promotes PTB binding to the polypyrimidine tract upstream of exon P3A, whereas hnRNP LL lacking the proline-rich region cannot bind to PTB. Interaction of hnRNP L with PTB inhibits association of U2AF(65) and U1 snRNP with the upstream and downstream of P3A, respectively, which causes a defect in exon P3A definition. HnRNP L and hnRNP LL thus antagonistically modulate PTB-mediated splicing suppression of exon P3A.

Symptomatic giant Schmorl's node treated by a decompression procedure.

Schmorl's nodes (SN) are common lesions that represent disc material prolapse through the vertebral endplate, into the vertebral body with generally limited clinical significance. Rarely they have been related to the onset of back pain, usually self-limited. Recently giant variants of SN have been described, with very discrete clinical and imaging expression. Knowledge of the typical imaging appearance in both CT-scan and MRI may preclude the use of unnecessary diagnostic and treatment procedures. The authors present a case of symptomatic giant SN resembling these rare variants by its size and persistent pain, but also featuring distinct classic SN characteristics. Although not usually needed when the image is typical, a percutaneous biopsy was performed which allowed both the definitive diagnosis and, surprisingly, the almost immediate and complete resolution of the symptoms. Five years later the patient is pain free and a new MRI showed a significant decrease of the lesion size and no edema of the vertebral body.

Tyr682 in the Aß-precursor protein intracellular domain regulates synaptic connectivity, cholinergic function, and cognitive performance.

Processing of Aß-precursor protein (APP) plays an important role in Alzheimer's disease (AD) pathogenesis. The APP intracellular domain contains residues important in regulating APP function and processing, in particular the 682YENPTY687 motif. To dissect the functions of this sequence in vivo, we created an APP knock-in allele mutating Y682 to Gly (APP(YG/YG) mice). This mutation alters the processing of APP and TrkA signaling and leads to postnatal lethality and neuromuscular synapse defects when expressed on an APP-like protein 2 KO background. This evidence prompted us to characterize further the APP(YG/YG) mice. Here, we show that APP(YG/YG) mice develop aging-dependent decline in cognitive and neuromuscular functions, a progressive reduction in dendritic spines, cholinergic tone, and TrkA levels in brain regions governing cognitive and motor functions. These data are consistent with our previous findings linking NGF and APP signaling and suggest a causal relationship between altered synaptic connectivity, cholinergic tone depression and TrkA signaling deficit, and cognitive and neuromuscular decline in APP(YG/YG) mice. The profound deficits caused by the Y682 mutation underscore the biological importance of APP and indicate that APP(YG/YG) are a valuable mouse model to study APP functions in physiological and pathological processes.

Endplate structure and parameters of neuromuscular transmission in sporadic centronuclear myopathy associated with myasthenia.

Centronuclear myopathy is a pathologically diagnosed congenital myopathy. The disease genes encode proteins with membrane modulating properties (MTM1, DNM2, and BIN1) or alter excitation-contraction coupling (RYR1). Some patients also have myasthenic symptoms but electrodiagnostic and endplate studies in these are limited. A sporadic patient had fatigable weakness and a decremental EMG response. Analysis of centronuclear myopathy disease- and candidate-genes identified no mutations. Quantitative endplate electron microscopy studies revealed simplified postsynaptic regions, endplate remodeling with normal nerve terminal size, normal synaptic vesicle density, and mild acetylcholine receptor deficiency. The amplitude of the miniature endplate potential was decreased to 60% of normal. Quantal release by nerve impulse was reduced to 40% of normal due to a decreased number of releasable quanta. The safety margin of neuromuscular transmission is compromised by decreased quantal release by nerve impulse and by a reduced postsynaptic response to the released quanta.

Imaging the back pain patient.

Imaging is an integral part of the clinical examination of the patient with back pain; it is, however, often used excessively and without consideration of the underlying literature. The primary role of imaging is the identification of systemic disease as a cause of the back or limb pain; magnetic resonance imaging (MRI) excels at this. Systemic disease as a cause of back or limb pain is, however, rare. Most back and radiating limb pain is of benign nature, owing to degenerative phenomena. There is no role for imaging in the initial evaluation of the patient with back pain in the absence of signs or symptoms of systemic disease. When conservative care fails, imaging may be undertaken with due consideration of its risks: labeling the patient as suffering from a degenerative disease, cost, radiation exposure, and provoking unwarranted minimally invasive or surgical intervention. Imaging can well depict disc degeneration and disc herniation. Imaging can suggest the presence of discogenic pain, but the lack of a pathoanatomic gold standard obviates any definitive conclusions. The imaging natural history of disc herniation is resolution. There is very poor correlation between imaging findings of disc herniation and the clinical presentation or course. Psychosocial factors predict functional disability due to disc herniation better than imaging. Imaging with MRI, computed tomography (CT), or CT myelography can readily identify central canal, lateral recess, or foraminal compromise. Only when an imaging finding is concordant with the patient's pain pattern or neurologic deficit can causation be considered. The zygapophysial (facet) and sacroiliac joint are thought to be responsible for axial back pain, although with less frequency than the disc. Imaging findings of the structural changes of osteoarthritis do not correlate with pain production. Physiologic imaging, either with single-photon emission CT bone scan, heavily T2-weighted MRI sequences (short-tau inversion recovery), or gadolinium enhancement, can detect inflammation and are more predictive of an axial pain generator.

Reliability of a modified Modic classification of bone marrow changes in lumbar spine MRI.

OBJECTIVE: The purpose of this study was to determine the intra- and interobserver reliability of a modified Modic classification for bone marrow changes seen on lumbar spine magnetic resonance imaging (MRI), taking into consideration mixed signals. METHODS: Lumbar MRI scans from 94 patients with low back pain were assessed independently by 2 spine specialists (senior [senior1], junior) and a radiologist (senior2). One reviewer (senior1) assessed the MR images twice at a three-week interval for evaluation of intraobserver reliability. Senior2 and junior reviewers assessed the MR images once. Pure edema endplate signal changes were classified as Modic type I, and pure fatty endplate changes as Modic type II. A mixture of types I and II but predominantly edema signal changes was classified as Modic I-2 and a mixture of types I and II but predominantly fatty changes was classified as Modic II-1. RESULTS: The intraobserver agreement was excellent (weighted kappa 0.85). The interobserver agreement was moderate to substantial (weighted kappa range 0.56-0.74). Interobserver reliability depended on the experience of the observer, thus highlighting the importance of a learning curve. CONCLUSION: This study shows that the modified Modic classification is reliable and easy to apply for observers with different clinical experience. The inclusion of mixed marrow changes in the modified classification may have clinical and therapeutic implications.

[Histochemical changes of muscle fibers and motor end-plates of paravertebral muscles in scoliosis associated with syringomyelia].

OBJECTIVE: To study the histochemical changes of muscle fibers and motor end-plates of paravertebral muscles, and analyze their relationship with the etiology of scoliosis associated with syringomyelia as compared with adolescent idiopathic scoliosis (AIS) and non-scoliotic patients. METHODS: All the enrolled patients were divided into three groups: Group I consisted of 20 patients with scoliosis associated with syringomyelia, Group II included 16 patients with AIS, and Group III included 10 patients without scoliosis. Bilateral biopsy of paravertebral muscles was performed during scheduled spinal surgery. HE staining, nicotin-lateral biopsy of paravertebral muscles was performed during scheduled spinal surgery. HE staining, nicotinamide adenine dinucleotide hydrogen-tetrazolium reductase ( NADH-TR), and alpha-naphthyl acetate esterase staining techniques were used for histological evaluation. Neurogenic and myogenic pathological changes and changes of motor end-plates of paravertebral muscles were compared among these three groups. RESULTS: Neurogenic pathological changes of muscle fibers were found in 12 (60% ) patients in Group I but was not found in Group II and III. The numbers of both T0 type motor end-plates and pathological end-plates on the convex side were significantly larger than those on the concave side in Group I ( P < O. 05 ). In Group II , however, the numbers of both T0 type motor end-plates and pathological end-plates on the concave side were significantly larger than those on the convex side (P < 0.05). No significant difference was found between two sides in Group III. CONCLUSION: The histochemical changes of paravertebral muscles in patients with scoliosis and syringomyelia are different from those in AIS patients. It is suggested that a primary denervation of paravertebral muscles exist in scoliosis associated with syringomyelia, which may play a role in the pathogenesis of scoliosis.

ISSLS prize winner: A study of diffusion in human lumbar discs: a serial magnetic resonance imaging study documenting the influence of the endplate on diffusion in normal and degenerate discs.

STUDY DESIGN: An in vivo serial magnetic resonance imaging study of diffusion characteristics in human lumbar discs over 24 hours in healthy volunteers and patients with low back pain. OBJECTIVES: To document the temporal pattern of diffusion in normal human lumbar discs and to study the influence of the vascularity of bone and the status of endplate on diffusion in the normal and degenerate discs. SUMMARY OF BACKGROUND DATA: Diffusion is the only source of nutrition to the discs, but no firm data are available on pattern of diffusion in humans. More data on this important subject are required to improve our understanding of disc degeneration and to probe research possibilities for preventing the same. METHODS: The diffusion pattern over 24 hours following gadodiamide injection was studied in 150 discs (96 normal and 54 degenerate). Signal intensity values for three regions of interest in bone (i.e., vertebral body, subchondral bone, and endplate zone) and seven in the disc were calculated, and normal percentiles of diffusion were derived for these regions. Enhancement percentage for each time period, peak enhancement percentage for each region, and the time taken to achieve peak enhancement percentage (Tmax) were used to define and compare diffusion characteristics and plot a time-intensity curve to document the 24-hour temporal pattern. The correlation of blood flow of the bone as measured by peak enhancement percentage of vertebral body, the status of the endplate zone as measured by the peak enhancement percentage, and Tmax of the endplate zone were correlated with the diffusion of the disc.Univariate analysis of variance, multiple comparisons, appropriate tests for significance, and stepwise linear regression analysis were used for analysis of the data using SPSS software. RESULTS: In normal discs, a "diffusion march" from the vertebral body to the center of disc was noted with the SImax being observed at 5 min in the vertebral body and subchondral bone, at 2 hours in the endplate zone, and at 6 hours in the nucleus pulposus. A significant difference in mean peak enhancement percentage was observed between that of the body and the discs in those less than 10 years and those above the age of 20 years (P < 0.001). Alterations in endplate zone produced distinct magnetic resonance imaging signs of disturbance in diffusion, which offered a reliable noninvasive method of identifying endplate cartilage damage. Stepwise linear regression analysis showed that the significant variable influencing diffusion to the center of the nucleus pulposus of the total sample was peak enhancement percentage of endplate zone (R2 = 0.216; P < 0.001), that of degenerate discs was peak enhancement percentage of endplate zone (R2 = 0.322; P < 0.001), and that of normal discs (R2 = 0.324; P < 0.001) was age. CONCLUSIONS: Serial postcontrast magnetic resonance imaging studies offer a reliable method of assessing the diffusion of the discs and the functional status of the endplate cartilage. Endplate cartilage damage increases with age and produces considerable changes in diffusion. The present study has described reliable signs by which these damages can be identified in vivo. Aging and degeneration have been shown to be two separate processes by documenting clear-cut differences in diffusion. The present data encourage use of diffusion studies as a noninvasive method to assess the physiologic status of the disc and endplate and to study the effect of drugs, smoking, mechanical loading, exercises, etc. on the physiology of the disc.

Focal caspase activation underlies the endplate myopathy in slow-channel syndrome.

Slow-channel syndrome (SCS) is a progressive neuromuscular disorder caused by abnormal gating of mutant acetylcholine receptors (AChRs) in the neuromuscular junction (NMJ). The pathological hallmark is selective degeneration of the NMJ termed endplate myopathy. Endplate myopathy consists of a combination of ultrastructural abnormalities, including degenerating subsynaptic nuclei, mitochondria, and postsynaptic folds, caused by localized cation overload through mutant AChRs. Because some of these changes resemble those seen in programmed cell death, we evaluated SCS muscle for evidence of focal activation of apoptotic pathways. Using antisera specific for the activated forms of caspases, the family of cysteine proteases that underlies apoptosis, we demonstrated that active forms of initiator and effector caspases are selectively localized at the NMJ in SCS. In comparison with an electron microscopic assessment of the abnormalities seen in endplate myopathy, we found that activated caspases were present at between 15 and 57% of endplates, similar to the proportion of endplates with degenerating mitochondria or vacuoles. This greatly exceeds the number of NMJs exhibiting nuclear degeneration. These findings provide the first evidence supporting the view that caspase activation in human disease can play a prominent role in localized cellular degenerative processes without causing nuclear or cell death.

Delivery of an adenoviral vector to the crushed recurrent laryngeal nerve.

OBJECTIVES: Objectives were to create a model of recurrent laryngeal nerve injury for testing the efficacy of potential therapeutic viral gene therapy vectors and to demonstrate that remote injection of a viral vector does not cause significant additional neuronal injury. STUDY DESIGN: Animal model. METHODS: Rats were randomly assigned to three groups of 10 animals each. In group I, the recurrent laryngeal nerve was crushed. In group II, the nerve was crushed and then injected with an adenoviral vector containing no transgene. In group III, the nerve was identified but was not crushed. Rats were killed at 1 week, and their larynges and brainstems were cryosectioned in 15-microm sections. Laryngeal cryosections were processed for acetylcholine histochemical analysis (motor endplates) followed by neurofilament immunoperoxidase (nerve fibers). Percentage of nerve-endplate contact was determined and compared between groups. Fluorescent in situ hybridization was performed on brainstem sections from rats in group II to confirm the presence of virus. RESULTS: No significant difference in percentage of nerve-endplate contact exists between the two crushed-nerve groups (groups I and II) (P =.88). The difference between both crushed-nerve groups and the group with noncrushed nerves (group III) was highly significant (P <.0001). The presence of virus was confirmed in group II rats. CONCLUSIONS: Crush provides a significant measurable injury to the recurrent laryngeal nerve and may be used as a model to explore therapeutic interventions for nerve injury. The remote injection of viral vector did not cause significant additional neuronal injury. Remote delivery of viral vectors to the central nervous system holds promise in the treatment of recurrent laryngeal nerve injury and central nervous system diseases.

Effect of myasthenic immunoglobulin G on motor end-plate morphology.

This study was undertaken to clarify the role of complement in acetylcholine receptor loss and degeneration of the postsynaptic membrane in myasthenia gravis (MG). We examined the end-plate morphology in rats with passively transferred immunoglobulin G (IgG) from myasthenic patients and the effect of complement by treatment of the rats with cobra venom factor. We injected peroxidase-labeled alpha-BuTx (P-BuTx) into the extensor digitorum longus (EDL) muscle to label the motor end-plates. Three hours later, 100 mg of IgG from MG patients or healthy controls was injected into the tail vein. The EDL was removed 48 hours after the injection of IgG. The presence of macrophages and degeneration of the postsynaptic membrane were seen in 4 of 6 IgG samples from MG patients and a decrease in AChRs in the other 2 samples. These changes were reversed completely by treatment with cobra venom factor in all but one case in which the end-plates were severely degenerated. Injection of MG IgG only never induced end-plate morphology changes. The results suggest that complement has a critical role in degeneration of the postsynaptic membrane and AChR loss at the motor end-plates in the passively transferred model and probably in human MG.

End-plate gamma- and epsilon-subunit mRNA levels in AChR deficiency syndrome due to epsilon-subunit null mutations.

Acetylcholine receptor (AChR) deficiency is the most common of the congenital myasthenic syndromes (CMS). Typically, the number of AChRs, measured by alpha-bungarotoxin binding, is reduced to 10-30% of normal levels, the miniature end-plate potentials are correspondingly reduced, and there are morphological changes at the motor end-plates. The majority of these syndromes are due to either missense or frameshift mutations within the gene encoding the adult-specific epsilon-subunit. These are often null mutations, but some mutant epsilon-subunits can be incorporated, at low levels, into functional AChRs in transfected cell lines. It is not clear, therefore, whether upregulation of the mutant epsilon-subunit mRNA could generate sufficient AChR to support neuromuscular transmission, albeit at a reduced level. Conversely, it might be that the mutant epsilon-subunit transcripts are subject to mRNA surveillance and 'nonsense-mediated' loss, leading to reduced epsilon-subunit mRNA expression. In either case, it is thought that neuromuscular transmission may be provided partly or entirely by incorporation of the foetal-specific gamma-subunit into end-plate AChR. gamma-Subunit mRNA is expressed at low levels in normal human muscle, but might be upregulated in CMS. The study of mRNA levels for AChR subunits should improve our understanding of genotype-phenotype relationships in CMS. Here we have defined homozygous epsilon-subunit mutations in four unrelated families with AChR deficiency and studied the steady-state levels of mRNA for AChR subunits at the motor end-plates by in situ hybridization. Although we demonstrated that each mutation would lead to almost complete absence of surface adult AChR expression, we detected similar robust expression of alpha- and epsilon-subunit mRNAs at end-plates of patient and control muscles, suggesting that mRNA transcripts for the epsilon-subunit are neither upregulated nor degraded preferentially. Interestingly, we were unable to detect any increase in gamma-subunit mRNA expression at CMS end-plates. Transgenic mice lacking the epsilon-subunit die 2-3 months after birth, suggesting that alpha(2)betadelta(2) pentamers cannot sustain neuromuscular transmission. Therefore, we tentatively conclude that the persistent low level expression of the gamma-subunit, which is present in normal human muscles as well as in AChR deficiency syndromes, is sufficient to enable patients with epsilon-subunit null alleles to survive.

Efficacy of intervention strategies in a brachial plexus global avulsion model in the rat.

The treatment of brachial plexus avulsion lesions invariably involves the use of neurotization procedures. Although some of these therapeutic strategies have been used for the past 20 years to restore selective function to the injured extremity, the individual efficacy of these nerve transfers has not been measured objectively, thereby rendering the prognostication of outcomes for these techniques a major problem. Using a true global avulsion model, the present study compares outcomes of the various neurotization procedures for the first time. The strength of this experimental model is that each nerve transfer tested leads to a common terminal pathway involving a single target-namely, the biceps muscle. Thus, quantitative measurements of biceps restoration will provide strong clues to the power of axonal regrowth of that particular motor pool. This study also introduces the Terzis grooming test, a modified behavioral test that can be quantified and that can provide an overall functional scale in the assessment of outcome. Thirty-five Sprague-Dawley rats were divided into seven groups, with each group testing a different motor donor for biceps reinnervation. The ipsilateral brachial plexus was globally avulsed in all animals, with the exception of the ipsilateral C7 group, and the respective motor donor coapted in an end-to-end fashion to the musculocutaneous nerve. Functional outcomes were measured by the Terzis grooming test, electromyography, biceps muscle force measurements, motor end plate counts, and quantitative axonal morphometry. The values of the different parameters were expressed as a standard score on a common scale. The relative standings of each group on each parameter were compared. Superior outcome was observed in the phrenic, the hypoglossal, and the ipsilateral C7 groups.