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Mitogen-activated protein kinases, including c-Jun NH2-terminal kinase (JNK), play an important role in the development and function of a large variety of tissues. The skeletal phenotype of JNK1 and JNK2 double-knockout (dKO) mice (JNK1 fl/fl Col2-Cre/JNK2 -/- ) and control genotypes were analyzed at different embryonic and postnatal stages. JNK1/2 dKO mice displayed a severe scoliotic phenotype beginning during development that was grossly apparent around weaning age. Alcian blue staining at embryonic day 17.5 showed abnormal fusion of the posterior spinal elements. In adult mice, fusion of vertebral bodies and of spinous and transverse processes was noted by micro-computed tomography, Alcian blue/Alizarin red staining, and histology. The long bones developed normally, and histologic sections of growth plate and articular cartilage revealed no significant abnormalities. Histologic sections of the vertebral column at embryonic days 15.5 and 17.5 revealed an abnormal organization of the annulus fibrosus in the dKOs, with chondrocyte-like cells and fusion of dorsal processes. Spinal sections in 10-week-old dKO mice showed replacement of intervertebral disk structures (annulus fibrosus and nucleus pulposus) by cartilage and bone tissues, with cells staining for markers of hypertrophic chondrocytes, including collagen X and runt-related transcription factor 2. These findings demonstrate a requirement for both JNK1 and JNK2 in the normal development of the axial skeleton. Loss of JNK signaling results in abnormal endochondral bone formation and subsequent severe scoliosis., (Copyright © 2019 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Introduction: A variety of methods have been used to study inflammatory changes in the acutely injured spinal cord. Recently novel multiplex assays have been used in an attempt to overcome limitations in numbers of available targets studied in a single experiment. Other technical challenges in developing pre-clinical rodent models to investigate biomarkers in cerebrospinal fluid (CSF) include relatively small volumes of sample and low concentrations of target proteins. The primary objective of this study was to characterize the inflammatory profile present in CSF at a subacute time point in a clinically relevant rodent model of traumatic spinal cord injury (SCI). Our other aim was to test a microarray proteomics platform specifically for this application., Methods: A 34 cytokine sandwich ELISA microarray was used to study inflammatory changes in CSF samples taken 12 days post-cervical SCI in adult rats. The difference between the median foreground signal and the median background signal was measured. Bonferroni and Benjamini-Hochburg multiple testing corrections were applied to limit the False Discovery Rate (FDR), and a linear mixed model was used to account for repeated measures in the array., Results: We report a novel subacute SCI biomarker, elevated levels of matrix metalloproteinase-8 protein in CSF, and discuss application of statistical models designed for multiplex testing., Conclusions: Major advantages of this assay over conventional methods include high-throughput format, good sensitivity, and reduced sample consumption. This method can be useful for creating comprehensive inflammatory profiles, and biomarkers can be used in the clinic to assess injury severity and to objectively grade response to therapy.

Background: Cervical spondylotic myelopathy (CSM), in part, results from degeneration of the posterior longitudinal ligament (PLL), which mechanically compresses the spinal cord. Much research was done on the ossification of PLL, but not concerning the non-ossifying degeneration of cervical PLL. The degeneration of cervical PLL may be related to inflammation. The aim of this study was to elucidate the pathological features of the PLL and the role of cyclooxygenase 2 (COX-2) in the degeneration of the PLL in CSM., Methods: A total of 23 PLL specimens were collected during surgery from patients with CSM for the histological and immunohistochemical (type II collagen and Ki-67) study. For the control group 14 cervical PLL autopsy specimens were investigated in the same manner. mRNA expression of COX-2 was quantitatively measured by real-time reverse transcription-polymerase chain reaction (RT-PCR) from 18 PLL specimens of patients with CSM and 18 PLL specimens of autopsy cases. Immunohistochemistry was used to evaluate the cellular location of COX-2 in PLL., Results: A distinct amount of fibrotic area, chondrometaplastic tissue and calcification were found in the PLL of the patient group, compared with the control group. Type II collagen was apparent around chondrometaplastic cells. Ki-67 positive reaction was less than 5%. A COX-2 positive reaction was found in 9 of the patient specimens (39.1%) in which the COX-2 was released from vascular endothelial cells in the PLL. However, such reactions were not found in the control group. Real-time PCR showed that the mRNA expression level of COX-2 in the patient group was significantly higher than that in the control group (P < 0.01)., Conclusions: Chondrometaplastic tissue producing type II collagen was identified as the most predominant pathological feature in the degenerative PLL. The higher expression of COX-2 might be related to degeneration of the PLL in CSM.

Corticospinal tract (CST) connections to spinal interneurons are conserved across species. We identified spinal interneuronal populations targeted by the CST in the cervical enlargement of the cat during development. We focused on the periods before and after laminar refinement of the CST terminations, between weeks 5 and 7. We used immunohistochemistry of choline acetyltransferase (ChAT), calbindin, calretinin, and parvalbumin to mark interneurons. We first compared interneuron marker distribution before and after CST refinement. ChAT interneurons increased, while calbindin interneurons decreased during this period. No significant changes were noted in parvalbumin and calretinin. We next used anterograde labeling to determine whether the CST targets different interneuron populations before and after the refinement period. Before refinement, the CST terminated sparsely where calbindin interneurons were located and spared ChAT interneurons. After refinement, the CST no longer terminated in calbindin-expressing areas but did so where ChAT interneurons were located. Remarkably, early CST terminations were dense where ChAT interneurons later increased in numbers. Finally, we determined whether corticospinal system activity was necessary for the ChAT and calbindin changes. We unilaterally inactivated M1 between weeks 5 and 7 by muscimol infusion. Inactivation resulted in a distribution of calbindin and ChAT in spinal gray matter regions where the CST terminates that resembled the immature more than mature pattern. Our results show that the CST plays a crucial role in restructuring spinal motor circuits during development, possibly through trophic support, and provide strong evidence for the importance of connections with key spinal interneuron populations in development of motor control functions.

Upregulation of extracellular chondroitin sulfate proteoglycans (CSPGs) after CNS injuries contributes to the impediment of functional recovery by restricting both axonal regeneration and synaptic plasticity. In the present study, the effect of degrading CSPGs with the application of the bacterial enzyme chondroitinase ABC (chABC) into the cuneate nucleus of rats partially denervated of forepaw dorsal column axons was examined. A dorsal column transection between the C6-C7 dorsal root entry zones was followed immediately by an ipsilateral brainstem injection of either chABC or a bacterial-derived control enzyme [penicillinase (P-ase)] and then subsequently (1 week later) followed with a second brainstem enzyme injection and cholera toxin B subunit (CTB) tracer injection into the ipsilateral forepaw digits and pads. After 1 additional week, the rats underwent electrophysiological receptive field mapping of the cuneate nucleus and/or anatomical evaluation. Examination of the brainstems of rats from each group revealed that CSPGs had been reduced after chABC treatment. Importantly, in the chABC-treated rats (but not in the P-ase controls), a significantly greater area of the cuneate nucleus was occupied by physiologically active CTB traced forepaw afferents that had been spared by the initial cord lesion. These results demonstrate, for the first time, a functional change directly linked to anatomical evidence of sprouting by spinal cord afferents after chABC treatment.

Background: Left ventricular hypertrabeculation (LVHT)/non-compaction, a rare myocardial abnormality and frequently associated with neuromuscular disorders, has not been reported in primary myoadenylate-deaminase (MAD) deficiency (MADD)., Case Report: In a 53-year-old man with easy fatigability and myalgia since boyhood, primary MADD was diagnosed based upon slightly, but recurrently elevated creatine-kinase, absent staining for MAD on muscle biopsy, markedly reduced MAD activity in the muscle homogenate, and the C34T mutation within exon 2 of the AMPD1 gene. An ambulatory ECG showed nocturnal sinus bradycardia and echocardiography thickening of the interventricular septum (20 mm) and the posterior wall (16 mm) and additionally LVHT. Cardiac MRI confirmed myocardial thickening and LVHT. Myocardial thickening and LVHT were regarded as causally related to the C34T mutation., Conclusion: Cardiac involvement in MADD may manifest as left ventricular myocardial thickening and LVHT. MADD should be included in the list of neuromuscular disorders which are associated with LVHT.

Objective: To study the effect of Qishejingkang recipe (QSJKR) on activity of alkaline phosphatase (ALP) in osteophyte formation of degenerated cervical vertebrae., Methods: A model of degenerated cervical vertebrae in the rabbit was established through resection of the cervical supraspinous and interspinous ligaments from the cervical spine elicited by surgical intervention. The sites of osteophyte was observed with a light microscope. The activity of ALP of the annuls fibrosis and nucleus pulpous, end-plate in each cervical spine was assayed with biochemical method respectively., Results: Osteophyte come from end-plate, the activity of ALP in the degenerated end-plate in QSJKR group declined significantly compared with model group., Conclusion: QSJKR could decrease the activity of ALP in end-plate of degenerated cervical vertebrae and it might inhibit the formation of osteophyte.

Nitration of neurofilament (NF) has been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). Evidence of such includes elevated 3-nitrotyrosine levels in spinal cord tissue and localized nitrotyrosine immunoreactivity with neurofilamentous aggregates in cortical and spinal motor neurons. To determine if neuronal nitric oxide synthase (nNOS) and inducible NOS (iNOS) are the sources of nitric oxide in sporadic ALS (sALS), particularly through over-expression of the enzyme, steady-state mRNA levels of these isoforms were studied by in situ hybridization. Paraffin-embedded, archival cervical spinal cord tissues from 7 sALS and 6 control cases were used. 35S-labeled riboprobes were generated from partial cDNAs. Immunohistochemistry was utilized to confirm results of iNOS hybridization. We observed that nNOS mRNA was constitutively expressed in cervical spinal motor neurons. However, iNOS mRNA and iNOS immunoreactivity was not observed in ALS or control motor neurons. Our observations suggest that the source of nitric oxide is the endogenous nNOS. Together with the results from other immunohistochemical studies, we further hypothesize a possible role of translational deregulation of nNOS in sALS.

Study Design: Herniated cervical disc specimens were obtained from patients undergoing surgical discectomy for persistent radiculopathy and cultured in vitro to determine whether various biochemical agents were being produced., Objectives: Our hypothesis is that biochemical mediators of inflammation and tissue degradation play a role in cervical intervertebral disc degeneration and in the pathophysiology of cervical radiculopathy., Summary of Background Data: Neck pain with or without radiculopathy is a common clinical problem, but the etiology of neck pain and the exact pathophysiology of radiculopathy remain uncertain. We have previously reported the production of various biochemical agents by herniated lumbar disc specimens in vitro. Because of a lack of such studies in the literature with respect to the cervical spine, the purpose of this study was to determine whether similar biochemical agents of inflammation and tissue degradation were being produced by herniated cervical disc specimens., Methods: Eighteen herniated cervical discs were obtained from 15 patients undergoing anterior disc surgery. The specimens were cultured and incubated for 72 hours, and the media were subsequently collected for biochemical analysis. Biochemical assays for matrix metalloproteinases, nitric oxide, prostaglandin E2, and a variety of cytokines were performed. As a control group, six cervical discs specimens were obtained from three patients undergoing anterior surgery for traumatic burst fractures, and similar biochemical analyses were performed., Results: The culture media from the herniated cervical disc specimens showed increased levels of matrix metalloproteinase activity compared with the control discs. Similarly, the levels of nitric oxide, prostaglandin E2, and interleukin-6 were significantly higher in the herniated disc specimens compared with the control discs. Interleukin-1 alpha, interleukin-1 beta, tumor necrosis factor-alpha, interleukin-1 receptor antagonist protein, and substance P were not detected in the culture media of the herniated or control discs., Conclusions: Herniated cervical disc specimens were making spontaneously increased amounts of matrix metalloproteinases, nitric oxide, prostaglandin E2, and interleukin-6. These results were similar to those obtained in herniated lumbar disc specimens that we have previously reported. These products may be intimately involved in the biochemistry of disc degeneration and the pathophysiology of radiculopathy.