Ventral Horn Research Articles

MiR-137–3p rescue motoneuron death by targeting calpain-2

Brachial plexus root avulsion (BPRA) is a type of injury that leads to motor function loss as a result of motoneurons (MNs) degeneration. Here we identified that the reduced expression of rat miR-137–3p in the ventral horn of spinal cord was associated with MNs death. However, the pathophysiological role of miR-137–3p in root avulsion remains poorly understood. We demonstrated that the calcium-activated neutral protease-2 (calpain-2) was a direct target gene of miR-137–3p with miR-137–3p binding to the 3′-untranslated region of calpain-2. Silencing of calpain-2 suppressed the expression of neuronal nitric oxide synthase (nNOS), a primary source of nitric oxide (NO). After avulsion 2 weeks, up-regulation of miR-137–3p in the spinal cord reduced calpain-2 levels and nNOS expression inside spinal MNs, resulting in an amelioration of the MNs death. These events provide new insight into the mechanism by which upregulation of miR-137–3p can impair MN survival in the BPRA.

PTEN expression in astrocytic processes after spinal cord injury

The role of the Rho/ROCK/PTEN signaling pathway in the regulation of astrocyte function for consolidation/stabilization of the synapse has not been thoroughly studied. In this study, the expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in GFAP-positive astrocytic processes in the ventral horns (VH) of the rat spinal cord has been evaluated in the normal condition and in a delayed period (30 days) after dosed contusion spinal cord injury (SCI) in caudal thoracic segments. In intact rats and at 30 days post-injury (dpi), semi-quantitative immunohistochemical analysis showed that there is approximately 2 folds less synaptophysin reactivity in the motoneuron perikarya than outside the perikarya, i.e., on dendritic spines, in the VH area. At 30 dpi, the square occupied by synaptophysin reactivity on the motoneuron perikarya and dendritic spines decreased ~2.4 and ~2.1 folds, respectively. Western blotting of the postsynaptic density protein 95 (PSD95) showed a decreased amount in the area of injury of ~3 folds at 30 dpi. Expression of GFAP in the astrocytic processes around the synaptophysin spots (APAS) was less than in the astrocytic processes that were located at distance from the synapses (APFS) both in the intact and SCI groups. In the APAS, the expression level of PTEN increased significantly after SCI. In these astrocytic processes, the PTEN expression level was significantly higher than in the APFS for both the intact and SCI rats. In the intact spinal cord, different PTEN expression levels were detected both in APAS and APFS. This may be due to the varying degree of integration of PTEN in the membrane compartment of astrocyte stem processes and possibly the increased delivery of PTEN from the GFAP-positive stem into fine GFAP-negative peripheral processes. The observed shifts after SCI reflect the imbalance in the mechanisms of synaptic plasticity after injury. Thus, strategies that have been developed for the deletion or knockdown of the PTEN gene are quite promising.

(157) - Thermal stimulation alters human cervical spinal cord functional connectivity compared to rest: a functional magnetic resonance imaging study

The spinal cord is the first site of sensory processing in the central nervous system and has an active role in the modulation and transmission of neural signals traveling between the body and the brain. Disruption of this information flow can cause sensory, motor, and autonomic dysfunction, and the spinal cord has a part in the development and perpetuation of clinical pain conditions. Recent advancements in functional magnetic resonance imaging (fMRI) have made the in vivo examination of spinal cord function in humans possible. Most spinal cord fMRI studies to date have investigated spinal cord activity during sensory stimulation, motor tasks, and/or cognitive manipulations. Recently spinal cord fMRI has been extended to the investigation of resting state functional networks and the identification of distinct patterns of spinal cord functional connectivity. In this study, we further characterize the resting state cervical spinal cord functional network in healthy participants (n = 15, age = 28.1 ± 2.4 years) using high resolution imaging coupled with both seed-based functional connectivity analyses and graph theory-based metrics. Within spinal cord segment functional connectivity was identified between the left and right ventral horns (bilateral motor network), left and right dorsal horns (bilateral sensory network), and the ipsilateral ventral and dorsal horns (unilateral sensory-motor network). Functional connectivity between the spinal cord segments was less evident. In a subset of participants (n = 10, age = 28.4 ± 2.7 years), the pattern of functional connectivity and the topological properties of the cervical spinal cord functional network appear to be state-dependent. Thermal stimulation of the right ventrolateral forearm resulted in significant disruption of the bilateral sensory network, increased network global efficiency, and decreased network modularity compared to rest. The research was supported by the Redlich Pain Endowment and the following NIH grants: NIDA T32DA035165, NIDA K24DA029262, and NCCIH F32AT007800.

Intracerebroventricular administration of Cystatin C ameliorates disease in SOD1-linked amyotrophic lateral sclerosis mice.

Cystatin C (CysC) is a major protein component of Bunina bodies, which are a pathological hallmark observed in the remaining motor neurons of patients with amyotrophic lateral sclerosis (ALS). Dominant mutations in the SOD1 gene, encoding Cu/Zn superoxide dismutase (SOD1), are causative for a subset of inherited ALS cases. Our previous study showed that CysC exerts a neuroprotective effect against mutant SOD1‐mediated toxicity in vitro; however, in vivo evidence of the beneficial effects mediated by CysC remains obscure. Here we examined the therapeutic potential of recombinant human CysC in vivo using a mouse model of ALS in which the ALS‐linked mutated SOD1 gene is expressed (SOD1G93A mice). Intracerebroventricular administration of CysC during the early symptomatic SOD1G93A mice extended their survival times. Administered CysC was predominantly distributed in ventral horn neurons including motor neurons, and induced autophagy through AMP‐activated kinase activation to reduce the amount of insoluble mutant SOD1 species. Moreover, PGC‐1α, a disease modifier of ALS, was restored by CysC through AMP‐activated kinase activation. Finally, the administration of CysC also promoted aggregation of CysC in motor neurons, which is similar to Bunina bodies. Taken together, our findings suggest that CysC represents a promising therapeutic candidate for ALS.

Differential Spinal and Supraspinal Activation of Glia in a Rat Model of Morphine Tolerance

Development of tolerance is a well known pharmacological characteristic of opioids and a major clinical problem. In addition to the known neuronal mechanisms of opioid tolerance, activation of glia has emerged as a potentially significant new mechanism. We studied activation of microglia and astrocytes in morphine tolerance and opioid-induced hyperalgesia in rats using immunohistochemistry, flow cytometry and RNA sequencing in spinal- and supraspinal regions. Chronic morphine treatment that induced tolerance and hyperalgesia also increased immunoreactivity of spinal microglia in the dorsal and ventral horns. Flow cytometry demonstrated that morphine treatment increased the proportion of M2-polarized spinal microglia, but failed to impact the number or the proportion of M1-polarized microglia. In the transcriptome of microglial cells isolated from the spinal cord (SC), morphine treatment increased transcripts related to cell activation and defense response. In the studied brain regions, no activation of microglia or astrocytes was detected by immunohistochemistry, except for a decrease in the number of microglial cells in the substantia nigra. In flow cytometry, morphine caused a decrease in the number of microglial cells in the medulla, but otherwise no change was detected for the count or the proportion of M1- and M2-polarized microglia in the medulla or sensory cortex. No evidence for the activation of glia in the brain was seen. Our results suggest that glial activation associated with opioid tolerance and opioid-induced hyperalgesia occurs mainly at the spinal level. The transcriptome data suggest that the microglial activation pattern after chronic morphine treatment has similarities with that of neuropathic pain.

In vivo two-photon imaging of motoneurons and adjacent glia in the ventral spinal cord

BackgroundInteractions between motoneurons and glial cells are pivotal to regulate and maintain functional states and synaptic connectivity in the spinal cord. In vivo two-photon imaging of the nervous system provided novel and unexpected knowledge about structural and physiological changes in the grey matter of the forebrain and in the dorsal white matter of the spinal cord. New methodHere, we describe a novel experimental strategy to investigate the spinal grey matter, i.e. the ventral horn motoneurons and their adjacent glial cells by employing in vivo two-photon laser-scanning microscopy (2P-LSM) in anesthetized transgenic mice. ResultsAfter retrograde tracer labelling in transgenic mice with cell-specific expression of fluorescent proteins and surgical exposure of the lumbar intumescence groups of motoneurons could be visualized deeply localized in the ventral horn. In this region, morphological responses of microglial cells to ATP could be recorded for an hour. In addition, using in mice with expression of GCaMP3 in astrocytes, physiological Ca2+ signals could be recorded after local noradrenalin application. Comparison with existing methodsPrevious in vivo imaging protocols were restricted to the superficial dorsal white matter or upper layers of the dorsal horn. Here, we modified a multi-step procedure originally established for a root-crush injury. We adapted it to simultaneously visualize motoneurons and adjacent glial cells in living animals. ConclusionA modified surgery approach is presented to visualize fluorescently labelled motoneurons and glial cells at a depth of more than 200 μm in the grey matter ventral horn of the mouse spinal cord.

Excitatory and Inhibitory Neuronal Circuits in the Spinal Cord and Their Role in the Control of Motor Neuron Function and Degeneration.

The complex neuronal networks of the spinal cord coordinate a wide variety of motor functions, including walking, running, and voluntary and involuntary movements. This is accomplished by different groups of neurons, called center pattern generators, which control left-right alternation and flexor-extensor patterns. These spinal circuits, located in the ventral horns, are formed by several neuronal types, and the specific function of most of them has been identified by means of studies in vivo and in the isolated spinal cord of mice harboring genetically induced ablation of specific neuronal populations. These studies have shown that the coordinated activity of several interneuron types, mainly GABAergic and glycinergic inhibitory neurons, have a crucial role in the modulation of motor neurons activity that finally excites the corresponding muscles. A pharmacological experimental approach by administering in the spinal cord agonists and antagonists of glutamate, GABA, glycine, and acetylcholine receptors to alter their synaptic action has also produced important results, linking the deficits in the synaptic function with the resulting motor alterations. These results have also increased the knowledge of the mechanisms of motor neuron degeneration, which is characteristic of diseases such as amyotrophic lateral sclerosis, and therefore open the possibility of designing new strategies for the prevention and treatment of these diseases.

Immunohistochemical Expression of Synaptophysin in the Adult and Developing Cervical and Lumbar Enlargements of the Spinal Cord of Rabbit

Background: The principle findings of synaptophysin immunoreactivity (SynpIR) during the ontogeny of rabbit spinal cord are: At E14, SynpIR precedes in the entire marginal layer especially at the entrance zone of dorsal root and motor neurite outgrowth emerged from the basal plate. At E21, SynpIR is expressed in the motoneurons of ventral and lateral horns of mantle layer growing into the ventrolateral columns of marginal layer. Methods: We found intensely stained thick tracts and diffuse axons among proliferating neuroblasts of mantle layer. The peripheral parts of ventral horns were occupied with closely packed multipolar neurons from which long dendrites departed toward the surface of marginal layer. Results: At E28, pronounced SynpIR presented in the ventral grey horn while the white matter was faintly stained., meanwhile the dorsal horn was more cellular than ventral and lateral horns. Few intensively SynpIR fibers cross the dorsal and ventral commissures. In adult, profuse SynpIR appeared in the entire grey matter, and stained dendrites departed from neurons in the lateral laminae into the adjacent funiculi as finger-like projections. These projections did not reach the surface, so that the outer one-third to onefourth of the funiculi contained little or no SynpIR. In the periphery of ventral horns, we found large multipolar neurons with faintly stained cytoplasm. The white matter and the neuroepithelial cells surrounding the central canal were almost unstained. Conclusion: Synaptophysinis a reliable marker for fiber outgrowth and synapse formation in therabbit spinal cord, and its differential expression levels is specific and almost completed before birth.

Effects of Melatonin on the Behavioral Indices and Structural Characteristics of Cerebral and Spinal Neurons of Rats with Experimental Hemiparkinsonism

In adult Wistar rats, a model of experimental hemiparkinsonism (EHP) was induced by stereotaxic microinjection of 8.0 μg 6-hydroxydopamine (6-HODA) into the left forebrain lateral ascending bundle. In such animals, we studied behavioral indices in the open field and elevated plus maze tests and also structural changes in the cerebral substantia nigra and spinal L5 ventral horn neurons. Six groups of animals were formed (in each group, n = 8): intact rats (I), animals that obtained microinjections of saline into the above lateral bundle (sham-injected, ShI), two groups with weak and intense motor EHP manifestations (frequency of circulatory movements in the apomorphine test less than one and six and more per 1 min, groups HP<1 and HP≥6, respectively), and two groups of rats with weak and intense EHP manifestations, which obtained daily i.p. injections of 10 mg/kg melatonin during 18 days (groups HP<1+M and HP≥6+M). In the open-field test, we observed in groups HP<1 and HP≥6 significant suppression of horizontal motor activity (very intense in the latter of the above-mentioned groups) and also significant weakening of vertical activity (number of rearings). Changes in the number of defecation acts were less expressed. In the elevated plus maze, manifestations of a significantly increased anxiety level were observed in rats of the above two groups. In the substantia nigra of animals with EHP, a considerable part of the neurons demonstrated clear signs of negative structural modifications; the number of neurons with the normal structure was considerably smaller than in the control. Analogous negative morphological changes were found in the lumbar segment of the spinal cord. Course injections of melatonin into rats with EHP noticeable weakened negative shifts in both open-field behavioral indices and level of anxiety (according to estimates in the elevated plus maze). Under the action of melatonin, morphological characteristics of neurons and gliocytes in the substantia nigra and spinal cord of rats with EHP demonstrated appreciable normalization. The mechanisms underlying functionaland structural disorders in the CNS of animals after central microinjections of the 6-HODA neurotoxin and also neuroprotective effects of melatonin, which provide weakening of such disorders, are discussed.

Erythropoietin attenuates motor neuron programmed cell death in a burn animal model.

Burn-induced neuromuscular dysfunction may contribute to long-term morbidity; therefore, it is imperative to develop novel treatments. The present study investigated whether erythropoietin (EPO) administration attenuates burn-induced motor neuron apoptosis and neuroinflammatory response. To validate our hypothesis, a third-degree hind paw burn rat model was developed by bringing the paw into contact with a metal surface at 75°C for 10 s. A total of 24 male Sprague–Dawley rats were randomly assigned to four groups: Group A, sham-control; Group B, burn-induced; Group C, burn + single EPO dose (5000 IU/kg i.p. at D0); and Group D, burn + daily EPO dosage (3000 IU/kg/day i.p. at D0–D6). Two treatment regimens were used to evaluate single versus multiple doses treatment effects. Before sacrifice, blood samples were collected for hematological parameter examination. The histological analyses of microglia activation, iNOS, and COX-2 in the spinal cord ventral horn were performed at week 1 post-burn. In addition, we examined autophagy changes by biomarkers of LC3B and ATG5. The expression of BCL-2, BAX, cleaved caspase-3, phospho-AKT, and mTOR was assessed simultaneously through Western blotting. EPO administration after burn injury attenuated neuroinflammation through various mechanisms, including the reduction of microglia activity as well as iNOS and COX-2 expression in the spinal cord ventral horn. In addition, the expression of phospho-AKT, mTOR and apoptotic indicators, such as BAX, BCL-2, and cleaved caspase-3, was modulated. Furthermore, the activity of burn-induced autophagy in the spinal cord ventral horn characterized by the expression of autophagic biomarkers, LC3B and ATG5, was reduced after EPO administration. The present results indicate that EPO inhibits the AKT-mTOR pathway to attenuate burn-induced motor neuron programmed cell death and microglia activation. EPO can modulate neuroinflammation and programmed cell death and may be a therapeutic candidate for neuroprotection.

Reduction of microhemorrhages in the spinal cord of symptomatic ALS mice after intravenous human bone marrow stem cell transplantation accompanies repair of the blood-spinal cord barrier.

Blood-spinal cord barrier (BSCB) alterations, including capillary rupture, have been demonstrated in animal models of amyotrophic lateral sclerosis (ALS) and ALS patients. To date, treatment to restore BSCB in ALS is underexplored. Here, we evaluated whether intravenous transplantation of human bone marrow CD34+ (hBM34+) cells into symptomatic ALS mice leads to restoration of capillary integrity in the spinal cord as determined by detection of microhemorrhages. Three different doses of hBM34+ cells (5 × 104, 5 × 105 or 1 × 106) or media were intravenously injected into symptomatic G93A SOD1 mice at 13 weeks of age. Microhemorrhages were determined in the cervical and lumbar spinal cords of mice at 4 weeks post-treatment, as revealed by Perls’ Prussian blue staining for ferric iron. Numerous microhemorrhages were observed in the gray and white matter of the spinal cords in media-treated mice, with a greater number of capillary ruptures within the ventral horn of both segments. In cell-treated mice, microhemorrhage numbers in the cervical and lumbar spinal cords were inversely related to administered cell doses. In particular, the pervasive microvascular ruptures determined in the spinal cords in late symptomatic ALS mice were significantly decreased by the highest cell dose, suggestive of BSCB repair by grafted hBM34+ cells. The study results provide translational outcomes supporting transplantation of hBM34+ cells at an optimal dose as a potential therapeutic strategy for BSCB repair in ALS patients.

Differential Neuroprotective Effects of Interleukin-1 Receptor Antagonist on Spinal Cord Neurons after Excitotoxic Injury

Secondary damage following spinal cord injury (SCI) induces neuronal damage through inflammatory and excitotoxic pathways. We hypothesized that the interleukin-1 receptor antagonist (IL1RA) protects neuronal populations and suppresses apoptosis and gliosis after injury. Spinal cord slice cultures (SCSCs) were subjected to excitotoxic injury with N-methyl-<smlcap>D</smlcap>-aspartate (NMDA) and treated with IL1RA. Immunohistochemistry for neuronal nuclei (NeuN), MacII, glial fibrillary acidic protein, and TdT-mediated dUTP nick end labelling stains were used to evaluate neuronal survival, glial activation, and apoptosis. Treatment with IL1RA significantly reduced the number of apoptotic cells in both NMDA-lesioned and unlesioned cultures. Experimental injury with NMDA reduced the number of NeuN-positive ventral horn neurons, and IL1RA treatment counteracted this loss 1 day after injury. However, IL1RA had no effect on the number of presumable Renshaw cells, identified by their selective expression of the cholinergic nicotinic α₂-receptor subunit (Chrna2). Activated microglial cells were more numerous in NMDA-lesioned cultures 1 day after injury, and IL1RA significantly reduced their numbers. We conclude that IL1RA modulates neuronal apoptosis and microglial activation in excitotoxically injured SCSCs. Renshaw cells were more susceptible to excitotoxic injury than other neurons and were not rescued by IL1RA treatment. Modulation of IL-1-mediated pathways may thus be effective in reducing excitotoxically induced neuronal damage after SCI, however only in specific neuronal populations, such as ventral horn neurons. These findings motivate further investigations of the possibility to antagonize inflammatory pathways after SCI.

Spatial and Cellular Expression Patterns of Erythropoietin-Receptor and Erythropoietin during a 42-Day Post-Lesional Time Course after Graded Thoracic Spinal Cord Impact Lesions in the Rat.

Erythropoietin (Epo) exhibits promising neuroregenerative potential for spinal cord injury (SCI), and might be involved in other long-term sequelae, such as neuropathic pain development. The current studies investigated the time courses and spatial and cellular patterns of Epo and erythropoietin receptor (EpoR) expression along the spinal axis after graded SCI. Male Long Evans rats received 100 kdyn, 150 kdyn, and 200 kdyn thoracic (T9) contusions from an Infinite Horizon impactor. Sham controls received laminectomies. Anatomical and quantitative immunohistochemical analyses of the EpoR/Epo expression along the whole spinal axis were performed 7, 15, and 42 postoperative days (DPO) after the lesioning. Cellular expression was investigated by double- and triple-labeling for EpoR/Epo with cellular markers and proliferating cells in subgroups of 5-bromo-2-deoxyuridine pre-treated animals. Prolonged EpoR/Epo-expression was confirmed by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Quantified EpoR/Epo immunoreactivities in pain-related spinal cord regions and ventrolateral white matter (VLWM) were correlated with the mechanical sensitivity thresholds and locomotor function of the respective animals. EpoR and Epo were constitutively expressed in the ventral horn neurons and vascular and glial cells in the dorsal columns (DC) and the VLWM. After SCI, in addition to expression in the lesion core, EpoR/Epo immunoreactivities exhibited significant time- and lesion grade-dependent induction in the DC and VLWM along the spinal axis. EpoR and Epo immunoreactive cells were co-stained with markers for astroglial, neural precursor cell and vascular markers. In the VLWM, EpoR- and Epo-positive proliferating cells were co-stained with glial fibrillary acidic protein (GFAP) and nestin. The DC EpoR/Epo immunoreactivities exhibited linear relationships with the behavioral correlates of post-lesional chronic pain development at DPO 42. SCI leads to long-lasting multicellular EpoR/Epo induction beyond the lesion core in the spinal cord regions that are involved in central pain development and regenerative processes. Our studies provide a time frame to investigate the effects of Epo application on motor function or pain development, especially in the later time course after lesioning.

Decreased glutathione levels cause overt motor neuron degeneration in hSOD1WT over-expressing mice

Mutations in Cu/Zn-superoxide dismutase (SOD1) cause familial forms of amyotrophic lateral sclerosis (ALS), a fatal disorder characterized by the progressive loss of motor neurons. Several lines of evidence have shown that SOD1 mutations cause ALS through a gain of a toxic function that remains to be fully characterized. A significant share of our understanding of the mechanisms underlying the neurodegenerative process in ALS comes from the study of rodents over-expressing ALS-linked mutant hSOD1. These mutant hSOD1 models develop an ALS-like phenotype. On the other hand, hemizygous mice over-expressing wild-type hSOD1 at moderate levels (hSOD1WT, originally described as line N1029) do not develop paralysis or shortened life-span. To investigate if a decrease in antioxidant defenses could lead to the development of an ALS-like phenotype in hSOD1WT mice, we used knockout mice for the glutamate-cysteine ligase modifier subunit [GCLM(−/−)]. GCLM(−/−) mice are viable and fertile but display a 70–80% reduction in total glutathione levels. GCLM(−/−)/hSOD1WT mice developed overt motor symptoms (e.g. tremor, loss of extension reflex in hind-limbs, decreased grip strength and paralysis) characteristic of mice models over-expressing ALS-linked mutant hSOD1. In addition, GCLM(−/−)/hSOD1WT animals displayed shortened life span. An accelerated decrease in the number of large neurons in the ventral horn of the spinal cord and degeneration of spinal root axons was observed in symptomatic GCLM(−/−)/hSOD1WT mice when compared to age-matched GCLM(+/+)/hSOD1WT mice. Our results show that under conditions of chronic decrease in glutathione, moderate over-expression of wild-type SOD1 leads to overt motor neuron degeneration, which is similar to that induced by ALS-linked mutant hSOD1 over-expression.

Lumbar to sacral root rerouting to restore bladder function in a feline spinal cord injury model: Urodynamic and retrograde nerve tracing results from a pilot study.

Lumbar to sacral rerouting surgery can potentially allow voiding via a skin-central nervous system-bladder reflex pathway. Here, we assessed if this surgery was effective in treating neurogenic bladder dysfunction/sphincter in felines. Eight cats underwent spinal cord transection (SCT) at thoracic level 10/11. Unilateral L7 to S1 ventral root anastomosis was performed 1 month later in six cats. Two cats served as transection-only controls. Electrical and manual stimulation of L6-S1 dermatomes, and urodynamics were performed at 3, 5, 7, and 9/10 months post transection. At 9/10 months, cats were also evaluated by direct electrophysiological testing of anastomosed roots with urodynamics, then tissue collection and examination of the root anastomosis site and lumbosacral cord ventral horns for cells retrogradely labeled from tracer dye injected 2 weeks earlier into the bladder wall. At 9/10 months, four of six rerouted cats exhibited increased detrusor pressure provoked by cutaneous stimulation, one cat bilaterally. Two cats presented with a voiding stream after ipsilateral cutaneous stimulation at 7 and 9 months. All six rerouted animals showed regrowth of axons from the L7 ventral horn to the bladder, although some aberrant axonal regrowth was also observed. L7 to S1 ventral root rerouting below the level of SCT showed successful axonal regrowth to the bladder from the L7 spinal cord segment in all rerouted animals, and induced increased detrusor pressure response to cutaneous stimulation in a subset. This feasibility study paves the way for future animal studies for bladder reinnervation.

Mesenchymal Stem Cell-Derived Exosomes Reduce A1 Astrocytes via Downregulation of Phosphorylated NFκB P65 Subunit in Spinal Cord Injury

Background/Aims: Neurotoxic A1 astrocytes are induced by inflammation after spinal cord injury (SCI), and the inflammation-related Nuclear Factor Kappa B (NFκB) pathway may be related to A1-astrocyte activation. Mesenchymal stem cell (MSC) transplantation is a promising therapy for SCI, where transplanted MSCs exhibit anti-inflammatory effects by downregulating proinflammatory factors, such as Tumor Necrosis Factor (TNF)-α and NFκB. MSC-exosomes (MSC-exo) reportedly mimic the beneficial effects of MSCs. Therefore, in this study, we investigated whether MSCs and MSC-exo exert inhibitory effects on A1 astrocytes and are beneficial for recovery after SCI. Methods: The effects of MSC and MSC-exo on SCIinduced A1 astrocytes, and the potential mechanisms were investigated in vitro and in vivo using immunofluorescence and western blot. In addition, we assessed the histopathology, levels of proinflammatory cytokines and locomotor function to verify the effects of MSC and MSC-exo on SCI rats. Results: MSC or MSC-exo co-culture reduced the proportion of SCIinduced A1 astrocytes. Intravenously-injected MSC or MSC-exo after SCI significantly reduced the proportion of A1 astrocytes, the percentage of p65 positive nuclei in astrocytes, and the percentage of TUNEL-positive cells in the ventral horn. Additionally, we observed decreased lesion area and expression of TNFα, Interleukin (IL)-1α and IL-1β, elevated expression of Myelin Basic Protein (MBP), Synaptophysin (Syn) and Neuronal Nuclei (NeuN), and improved Basso, Beattie & Bresnahan (BBB) scores and inclined-plane-test angle. In vitro assay showed that MSC and MSC-exo reduced SCI-induced A1 astrocytes, probably via inhibiting the nuclear translocation of the NFκB p65. Conclusion: MSC and MSC-exo reduce SCI-induced A1 astrocytes, probably via inhibiting nuclear translocation of NFκB p65, and exert antiinflammatory and neuroprotective effects following SCI, with the therapeutic effect of MSCexo comparable with that of MSCs when applied intravenously.

Local Delivery of β-Elemene Improves Locomotor Functional Recovery by Alleviating Endoplasmic Reticulum Stress and Reducing Neuronal Apoptosis in Rats with Spinal Cord Injury

Background/Aims: Spinal cord injury (SCI) is a serious global problem that leads to permanent motor and sensory deficits. This study explores the anti-apoptotic and neuroprotective effects of the natural extract β-elemene in vitro and in a rat model of SCI. Methods: CCK-8 assay was used to evaluate cell viability and lactate dehydrogenase assay was used to evaluate cytotoxicity. A model of cell injury was established using cobalt chloride. Apoptosis was evaluated using a fluorescence-activated cell sorting assay of annexin V-FITC and propidium iodide staining. A rat SCI model was created via the modified Allen’s method and Basso, Beattie, and Bresnahan (BBB) scores were used to assess locomotor function. Inflammatory responses were assessed via enzyme-linked immunosorbent assay (ELISA). Apoptotic and surviving neurons in the ventral horn were respectively observed via terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and Nissl staining. Western blotting was used to measure protein expression. Results: β-elemene (20 μg/ml) promoted cell viability by activating phosphorylation of the PI3K-AKT-mTOR pathway. β-elemene reduced CoCl₂-induced cellular death and apoptosis by suppressing the expression levels of CHOP, cleaved-caspase 12, 78-kilodalton glucose-regulated protein, cleaved-caspase 3, and the Bax/Bcl-2 ratio. In the rat model of SCI, Nissl and TUNEL staining showed that β-elemene promoted motor neuron survival and reduced neuronal apoptosis in the spinal cord ventral horn. BBB scores showed that β-elemene significantly promoted locomotor behavioral recovery after SCI. In addition, β-elemene reduced the ELISA-detected secretion of interleukin (IL)-6 and IL-1β. Conclusion: β-elemene reduces neuronal apoptosis by alleviating endoplasmic reticulum stress in vitro and in vivo. In addition, β-elemene promotes locomotor function recovery and tissue repair in SCI rats. Thus, our study provides a novel encouraging strategy for the potential treatment of β-elemene in SCI patients.

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.

Sleep disordered breathing in motor neurone disease.

Motor neurone disease (MND) is a neurodegenerative disease defined by axonal loss and gliosis of upper and lower motor neurones in the motor cortex, lower brainstem nuclei and ventral horn of the spinal cord. MND is currently incurable and has a poor prognosis, with death typically occurring 3 to 5 years after disease onset. The disease is characterised by rapidly progressive weakness leading to paralysis, fasciculations, bulbar symptoms (including dysarthria and dysphagia) and respiratory compromise. Respiratory complications arise as a result of weakness of upper airway (pharyngeal and laryngeal) muscles and respiratory muscles (diaphragm, intercostal and accessory muscles) leading to respiratory failure. Due to early involvement of respiratory muscles in MND, sleep disordered breathing (SDB) occurs at a higher frequency than compared to the general population. SDB usually precedes daytime respiratory symptoms and chronic respiratory failure. It significantly impacts upon patients' quality of life and survival and its presence may predict prognosis. Managing SDB in MND with non-invasive ventilation (NIV) improves quality of life and survival. Early identification and management of SDB in MND patients is therefore crucial. This update will review assessments of respiratory muscle function, types of SDB and the effects of NIV in patients with MND.

Yellow Laser Stimulation at GV2 Acupoint Mitigates Apoptosis, Oxidative Stress, Inflammation, and Motor Deficit in Spinal Cord Injury Rats

Currently, the suppression of oxidative stress and inflammation is considered as the treatment targets of spinal cord injury due to their roles on the hindrance of recovery process. Since laser acupuncture decreased oxidative stress and enhanced the survival of neurons from oxidative stress damage and GV2 stimulation was selected as one stimulated acupoint in order to enhance the recovery of spinal cord injury, we hypothesized that laser acupuncture at GV2 should enhance the recovery of spinal cord injury. To test this hypothesis, male Wistar rats were induced spinal cord injury at T10 level and they were exposed to a 10 minute-stimulation at GV2 by yellow laser. Laser acupuncture was performed at 0.25 and 1, 2, 6, and 12 hours after spinal cord injury. Then, the stimulation was performed once daily for 7 days. Locomotor assessment was carried out on days 3 and 7 after injury. At the end of study period, the densities of polymorphonuclear of leukocyte, Bax, Caspase-3, Bcl-2, and BDNF positive stained cells in ventral horn of spinal cord were determined. Cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), and oxidative stress status was also assessed. The results showed that laser acupuncture at GV2 increased BBB score, gross motor score, and densities of Bcl-2 and BDNF positive stained cells but decreased density with polymorphonuclear leukocyte, the densities of Bax and Caspase-3 positive stained cells, COX-2 level, and oxidative stress status in ventral horn of the lesion spinal cord. The reduction of serum COX-2 was also decreased. Therefore, GV2 stimulation by yellow laser might enhance the recovery of spinal cord via the increase in BDNF and the decrease in inflammation, apoptosis, and oxidative stress status in the lesion spinal cord.