Slow progression of sciatic nerve degeneration and regeneration after loose ligation through microglial activation and decreased KCC2 levels in the mouse spinal cord ventral horn.

Peripheral nerve injury affects motor functions. To reveal the mechanisms underlying motor dysfunction and recovery after nerve compression, which have not been precisely examined, we investigated the temporal relationship among changes in motor function, nerve histopathology, and marker molecule expression in the spinal cord after loose ligation of the mouse sciatic nerve. After ligation, sciatic motor function suddenly declined, and axons gradually degenerated. During degeneration, galanin was localized in motor neuron cell bodies. Then, in the ventral horn, microglia were activated, and expression of choline acetyltransferase (ChAT), a synthetic enzyme of acetylcholine, and potassium chloride co-transporter 2 (KCC2), which shifts the action of γ-amino butyric acid (GABA) and glycine to inhibitory, decreased. Motor function recovery was insufficient although axonal regeneration was complete. ChAT levels gradually recovered during axonal regeneration. When regeneration was nearly complete, microglial activation declined, and KCC2 expression started to increase. The KCC2 level sufficiently recovered when axonal regeneration was complete, suggesting that the excitatory action of GABA/glycine may participate in axonal regeneration. Furthermore, these changes proceeded slower than those after severance, suggesting that loose ligation, compression, may mediate slower progression of degeneration and regeneration than severance, and these changes may cause the motor dysfunction and its recovery.

Orexin-A potentiates glycine currents by activating OX1R and IP3/Ca2+/PKC signaling pathways in spinal cord ventral horn neurons.

Orexin-A/B modulates multiple physical functions by activating their receptors (OX1R and OX2R), but its effects in the spinal cord motor control remain unknown. Using acute separation (by digestive enzyme) of cells and patch-clamp recordings, we aimed to investigate the effect and mechanisms of orexin-A on the glycine receptors in the spinal cord ventral horn neurons. Orexin-A potentiated the glycine currents by activating OX1R. In Ca2+-free extracellular solution, orexin-A still increased the glycine currents. While, the orexin-A-induced potentiation was blocked when Ca2+ was chelated by internal infusion of BAPTA, and the orexin-A effect was abolished by the IP3 receptor antagonists heparin and Xe-C. The PKC inhibitor Bis-IV nullified the orexin-A effect. In addition, orexin-A did not cause a further enhancement of the glycine currents after bath application of the PKC activator PMA. In conclusion, after OX1R is activated, a distinct IP3/Ca2+-dependent PKC signaling pathway, is likely responsible for the orexin-A potentiation on glycine currents in the spinal cord ventral horn neurons.

Histamine H3 Receptors Expressed in Ventral Horns Modulate Spinal Motor Output.

Motoneuron activity is modulated by histamine receptors. While H1 and H2 receptors have been widely explored, H3 histamine receptors (H3Rs) have not been sufficiently characterized. This paper targets the effects of the selective activation of H3Rs and their expression on the membranes of large ventral horn cells. The application of selective pharmacological agents to spinal cords isolated from neonatal rats was used to identify the presence of functional H3Rs on the membrane of physiologically identified lumbar motoneurons. Intra and extracellular recordings revealed that H3R agonist, α-methylhistamine, depolarized both single motoneurons and ventral roots, even in the presence of tetrodotoxin, an effect prevented by H3R antagonist, thioperamide. Finally, immunohistochemistry located the expression of H3Rs on a subpopulation of large cells in lamina IX. This study identifies H3Rs as a new exploitable pharmacological target against motor disturbances.

MiR-125a-5p Regulates Vitamin D Receptor Expression in a Mouse Model of Experimental Autoimmune Encephalomyelitis.

Multiple sclerosis (MS) is a chronic and incurable autoimmune neurodegenerative disease of the central nervous system. Although the symptoms of MS can be managed by vitamin D3 treatment alone, this condition cannot be completely eradicated. Thus, there might be unknown factors capable of regulating the vitamin D receptor (VDR). Genome-wide analysis showed that miRNAs were associated with VDRs. We sought to determine the role and mechanism of action of miRNA-125a-5p and VDRs in a model of MS, mice with experimental autoimmune encephalomyelitis (EAE), which was induced by myelin oligodendrocyte glycoprotein 35-55 peptides. EAE mice showed decreased mean body weight but increased mean clinical scores compared with vehicle or control mice. And inflammatory infiltration was found in the lumbosacral spinal cord of EAE mice. In addition, VDR expression was significantly lower while the expression of miR-125a-5p was markedly higher in the spinal ventral horn of EAE mice than in vehicle or control mice. Importantly, activation of VDRs by paricalcitol or inhibition of miR-125a-5p by its antagomir markedly decreased the mean clinical scores in EAE mice. Interestingly, VDR and miR-125a-5p were co-localized in the same neurons of the ventral horn. More importantly, inhibition of miR-125a-5p remarkably blocked the decrease of VDRs in EAE mice. These results support a critical role for miR-125a-5p in modulating VDR activity in EAE and suggest potential novel therapeutic interventions.

Upregulation of proteoglycans in the perilesion perimeter in ventral horns after spinal cord injury.

Spinal cord injury (SCI) results in pronounced focal tissue damage with subsequent formation of a glial scar that blocks axon regeneration and regrowth. Cellular changes and the composition of the extracellular matrix in regions distal from the injured area remain poorly characterized. In the present study, in the spinal cord distal to the damaged area (perilesion perimeter) there were minimal gross histological changes, but there were pronounced alterations in the extracellular proteoglycans even at 30 days after SCI. These abnormalities coincided with the appearance of reactive astrocytes and a reduction in main astrocytic glutamate transporter 1. Proteoglycan levels exhibited different kinetics and changes after SCI in areas near neuronal cell bodies and in areas distal from them. The results of the study suggest that SCI induces widespread changes in the spinal cord that may be responsible for neuronal dysfunction far from the damaged area and further aggravation of the SCI.

Teasing of Ventral Spinal Cord White Matter Fibers for the Analysis of Central Nervous System Nodes of Ranvier.

In the central nervous system, the formation of nodes of Ranvier, the short, unmyelinated regions of the axon where voltage-gated sodium channels that mediate saltatory conduction in myelinated nerves are concentrated, is orchestrated by oligodendrocytes, the myelinating cells of the CNS. While transmission electron microscopy remains the gold standard for the study of how the nodal region is organized, this approach is both technically demanding and time-consuming. The availability of antibodies that can be used to label paranodal myelin and the underlying axonal domains that are formed as a result of myelination allows for the precise analysis of the nodal region. In this chapter, we describe the method used to prepare teased fiber preparations of CNS white matter. Teased fiber preparations facilitate the rapid, quantitative analysis of a large number of nodes of Ranvier per animal compared to conventional histological approaches.

Pre-Injection of Small Interfering RNA (siRNA) Promotes c-Jun Gene Silencing and Decreases the Survival Rate of Axotomy-Injured Spinal Motoneurons in Adult Mice.

Brachial plexus injury is a common clinical peripheral nerve trauma. A series of genes in motoneurons were activated in the corresponding segments of the spinal cord after brachial plexus roots axotomy. The spatial and temporal expression of these genes directly affects the speed of motoneuron axon regeneration and precise target organ reinnervation. In a previous study, we observed the overexpression of c-Jun in motoneurons of the spinal cord ventral horn after brachial plexus injury in rats. However, the relevance of c-Jun expression with respect to the fate of axotomy-induced branchial plexus injury in adult mice remains unknown. In the present study, we explored the function of c-Jun in motoneuron recovery after axotomy. We pre-injected small interfering RNA (siRNA) to knockdown c-Jun expression in mice and examined the effects of the overexpression of c-Jun in motoneurons after the axotomy of the brachial plexus in vivo. Axotomy induced c-Jun overexpression in the ventral horn motoneurons of adult mice from 3 to 14 days after injury. In addition, the pre-injection of siRNA transiently inhibited c-Jun expression and decreased the survival rate of axotomy-injured motoneurons. These findings indicate that the axotomy-induced overexpression of c-Jun plays an important role in the survival of ventral horn motoneurons in adult mice. In addition, the pre-injection of c-Jun siRNA through the brachial plexus stem effectively adjusts c-Jun gene expression at the ipsilateral side.

Peripheral Nerve Injury-Induced Astrocyte Activation in Spinal Ventral Horn Contributes to Nerve Regeneration.

Accumulating evidences suggest that peripheral nerve injury (PNI) may initiate astrocytic responses in the central nervous system (CNS). However, the response of astrocytes in the spinal ventral horn and its potential role in nerve regeneration after PNI remain unclear. Herein, we firstly illustrated that astrocytes in the spinal ventral horn were dramatically activated in the early stage following sciatic nerve injury, and these profiles were eliminated in the chronic stage. Additionally, we found that the expression of neurotrophins, including brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and neurotrophin-3 (NT-3), also accompanied with astrocyte activation. In comparison with the irreversible transected subjects, astrocyte activation and the neurotrophic upregulation in the early stage were more drastic in case the transected nerve was rebridged immediately after injury. Furthermore, administering fluorocitrate to inhibit astrocyte activation resulted in decreased neurotrophin expression in the spinal ventral horn and delayed axonal regeneration in the nerve as well as motor function recovery. Overall, the present study indicates that peripheral nerve injury can initiate astrocyte activation accompanied with neurotrophin upregulation in the spinal ventral horn. The above responses mainly occur in the early stage of PNI and may contribute to nerve regeneration and motor function recovery.

Transcriptional Regulation of Notch1 Expression by Nkx6.1 in Neural Stem/Progenitor Cells during Ventral Spinal Cord Development.

Notch1 signaling plays a critical role in maintaining and determining neural stem/progenitor cell (NSPC) fate, yet the transcriptional mechanism controlling Notch1 specific expression in NSPCs remains incomplete. Here, we show transcription factor Nkx6.1 interacts with a cis-element (CR2, an evolutionarily conserved non-coding fragment in the second intron of Notch1 locus) and regulates the expression of Notch1 in ventral NSPCs of the developing spinal cord. We show that the Notch1 expression is modulated by the interaction of Nkx6.1 with a 139 bp enhancer sequence within CR2. Knockdown or overexpression of Nkx6.1 leads to down- or up-regulated Notch1 expression, respectively. In CR2-GFP transgenic mouse, GFP expression was found prominent in the ventricular zone and neural progenitor cells from embryonic day 9.5 to postnatal day 7. GFP+ cells were mainly neural progenitors for interneurons and not for motoneurons or glial cells. Moreover, GFP expression persisted in a subset of ependymal cells in the adult spinal cord, suggesting that CR2 is active in both embryonic and adult NSPCs. Together our data reveal a novel mechanism of Notch1 transcriptional regulation in the ventral spinal cord by Nkx6.1 via its binding with Notch1 enhancer CR2 during embryonic development.

Nonsynaptic glycine release is involved in the early KCC2 expression.

The cation-chloride co-transporters are important regulators of the cellular Cl(-) homeostasis. Among them the Na(+) -K(+) -2Cl(-) co-transporter (NKCC1) is responsible for intracellular chloride accumulation in most immature brain structures, whereas the K(+) -Cl(-) co-transporter (KCC2) extrudes chloride from mature neurons, ensuring chloride-mediated inhibitory effects of GABA/glycine. We have shown that both KCC2 and NKCC1 are expressed at early embryonic stages (E11.5) in the ventral spinal cord (SC). The mechanisms by which KCC2 is prematurely expressed are unknown. In this study, we found that chronically blocking glycine receptors (GlyR) by strychnine led to a loss of KCC2 expression, without affecting NKCC1 level. This effect was not dependent on the firing of Na(+) action potentials but was mimicked by a Ca(2+) -dependent PKC blocker. Blocking the vesicular release of neurotransmitters did not impinge on strychnine effect whereas blocking volume-sensitive outwardly rectifying (VSOR) chloride channels reproduced the GlyR blockade, suggesting that KCC2 is controlled by a glycine release from progenitor radial cells in immature ventral spinal networks. Finally, we showed that the strychnine treatment prevented the maturation of rhythmic spontaneous activity. Thereby, the GlyR-activation is a necessary developmental process for the expression of functional spinal motor networks. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 764-779, 2016.

Calbindin-D28K is increased in the ventral horn of spinal cord by neuroprotective factors for motor neurons.

Slow glutamate-mediated neuronal degeneration is implicated in the pathophysiology of motor neuron diseases such as amyotrophic lateral sclerosis (ALS). The calcium-binding proteins calbindin-D28K and parvalbumin have been reported to protect neurons against excitotoxic insults. Expression of calbindin-D28K is low in adult human motor neurons, and vulnerable motor neurons additionally may lack parvalbumin. Thus, it has been speculated that the lack of calcium-binding proteins may, in part, be responsible for early degeneration of the population of motor neurons most vulnerable in ALS. Using a rat organotypic spinal cord slice system, we examined whether the most potent neuroprotective factors for motor neurons can increase the expression of calbindin-D28K or parvalbumin proteins in the postnatal spinal cord. After 4 weeks of incubation of spinal cord slices with 1) glial cell line-derived neurotrophic factor (GDNF), 2) neurturin, 3) insulin-like growth factor I (IGF-I), or 4) pigment epithelium-derived factor (PEDF), the number of calbindin-D28K -immunopositive large neurons (>20 µm) in the ventral horn was higher under the first three conditions, but not after PEDF, compared with untreated controls. Under the same conditions, parvalbumin was not upregulated by any neuroprotective factor. The same calbindin increase was true of IGF-I and GDNF in a parallel glutamate toxicity model of motor neuron degeneration. Taken together with our previous reports from the same model, which showed that all these neurotrophic factors can potently protect motor neurons from slow glutamate injury, the data here suggest that upregulation of calbindin-D28K by some of these factors may be one mechanism by which motor neurons can be protected from glutamate-induced, calcium-mediated excitotoxicity.

Effects of adenosine monophosphate-activated kinase in the ventral horn of rabbit spinal cord after transient ischemia.

OBJECTIVE: To investigate the effect compound C, an adenosine monophosphate-activated kinase (AMPK) inhibitor, has on motor neurons of rabbit spinal cord after ischemia/reperfusion. DESIGN: Compound C (30 mg/kg) was administered intraperitoneally to rabbits 30 minutes before ischemia and the animals were sacrificed at 15 minutes after ischemia/reperfusion to measure lactate levels and at 72 hours after ischemia/reperfusion for morphological study. RESULTS: The administration of compound C did not produce any significant changes in physiological parameters such as pH, arterial blood gas (PaCO(2) and PaO(2)), and blood glucose in rabbit either at 10 minutes before ischemia or at 10 minutes after reperfusion. However, the administration of compound C did significantly ameliorate lactate acidosis at 15 minutes after reperfusion. In addition, the administration of compound C significantly improved the neurological scores of the rabbits and reduced the neuronal death seen in the ventral horn of their spinal cords at 72 hours after ischemia/reperfusion. CONCLUSIONS: Inhibition of AMPK can ameliorate the ischemia-induced neuronal death in the spinal cord via the reduction of early lactate acidosis.

Changes of 5-hydroxytryptamine and tryptophan hydroxylase expression in the ventral horn of spinal cord.

OBJECTIVE: To investigate changes of 5-hydroxytryptamine (5-HT) and its synthesis rate-limiting enzyme tryptophan hydroxylase (TPH) in the ventral horn of spinal cord after exercise-induced fatigue, and to further discuss the mechanism of exercise-induced central fatigue at spinal level. METHODS: Sixteen healthy adult Wistar rats were randomly divided into 2 groups: exercise-induced fatigue group and control group. Immunohistochemical staining for 5-HT and TPH in the ventral horn were performed and analyzed quantitatively. The mean optic densities of 5-HT and TPH positive fibers or terminals were measured by computerized image analyzer. RESULTS: Both 5-HT and TPH positive fibers/terminals decreased in the exercise-induced fatigue group. The immunohistochemical staining was weaker and the mean optic densities decreased obviously in the fatigue group compared with those in the control group (P< 0.05). CONCLUSION: 5-HT and TPH in the ventral horn of spinal cord might be involved in exercise-induced fatigue.