Angeli's salt and spinal motor neuron injury

Nitroxyl anion or its conjugate acid (NO_-/HNO) and nitric oxide (NO) may both have pro-oxidative and cytotoxic properties. Superoxide dismutase (SOD) enzyme has been shown to convert reversibly HNO to NO. Mutations found in the SOD enzyme in some familial amyotrophic lateral sclerosis (ALS) patients affect redox properties of the SOD enzyme in a manner, which may affect the equilibrium between NO and HNO. Therefore, we studied the effects of HNO releasing compound, Angeli's salt (AS), on both motor and sensory functions after intrathecal administration in the lumbar spinal cord of a male rat. These functions were measured by rotarod, spontaneous activity, paw- and tail-flick tests. In addition, we compared the effect of AS to NO releasing papanonoate, old AS solution and sulphononoate in the motor performance test. The effect of intrathecal delivery of AS on the markers of the spinal cord injury and oxidative/nitrosative stress were further studied.

Results: Freshly prepared AS (5 or 10 μmol), but not papanonoate, caused a marked decrease in the rotarod performance 3-7 days after the intrathecal administration. The peak motor deficiency was noted 3 days after AS (5 μmol) delivery. Old, degraded, AS solution and nitrous oxide releasing sulphononoate did not decrease motor performance in the rotarod test. AS did not affect the sensory stimulus evoked responses as measured by the paw-flick and tail-flick tests. Immunohistological examination revealed that AS caused injury related changes in the expression of glial fibrillary acidic protein (GFAP), fibroblast growth factor (FGF-2) and laminins in the spinal cord. Moreover, AS increased nitrotyrosine immunoreactivity in the spinal motor neurons.

Therefore, we conclude that AS, but not NO releasing papanonoate, causes motor neuron injury but does not affect the function of sensory nerves in behavioural tests.     

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6 weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2 days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.     

鞘内注射NOS反义寡核苷酸抑制吗啡戒断大鼠脊髓和脑干NMDA1A受体mRNA表达的影响

运用逆转录－多聚酶联反应（RT－PCR）、鞘内注射和反义技术,研究脊髓水平一氧化氮（NO）对大鼠吗啡戒断反应和脊髓及脑干NMDA1A受体mRNA(NMDA1AR mRNA)表达的影响。结果表明,鞘内注射NOS反义寡核苷酸能明显减轻吗啡戒断反应,且脑型NOS（nNOS）反义寡苷酸的作用强于内皮型NOS(eNOS）反义寡核苷酸,吗啡依赖大鼠脊髓和脑干NMDA1AR mRNA表达增加,纳洛酮催促戒断,使其进一步增加;鞘内注射nNOS反义寡核苷酸,能明显抑制吗啡戒断大鼠脊髓和脑干NMDA1AR mRNA表达的增加;eNOS反义寡核苷酸也可抑制吗戒断大鼠脊髓NMDA1AR mRNA表达的增加,但作用弱于nNOS反义寡核苷酸,对脑干NMDA1AR mRNA表达无明显影响,上述结果提示：脊髓水平NO参与介导吗啡戒断反庆和NMDA受体表达的调控。     

Topical application of dynorphin A (1-17) antiserum attenuates trauma induced alterations in spinal cord evoked potentials,microvascular permeability disturbances,edema formation and cell injury: an experimental study in the rat using electrophysiological and morphological approaches

Summary.  Dynorphin is a neuropeptide that is present in high quantities in the dorsal horn of the spinal cord. The peptide is actively involved in pain processing pathways. However, its involvement in spinal cord injury is not well known. Alteration in dynorphin immunoreactivity occurs following a focal trauma to the rat spinal cord. Infusion of dynorphin into the intrathecal space of the cord results in ischemia, cell damage and abnormal motor function. Antibodies to dynorphin when injected into the intrathecal space of the spinal cord following trauma improve motor recovery, reduce edema and cell changes. However, influence of dynorphin on trauma induced alteration in spinal cord bioelectrical activity is still not known. Spinal cord evoked potentials (SCEP) are good indicator of spinal cord pathology following trauma. Therefore, in present investigation, influence of dynorphin antibodies on trauma induced changes in SCEP were examined in our rat model. In addition, spinal cord edema formation, microvascular permeability disturbances and cell injury were also investigated. Our results show that topical application of dynorphin antiserum (1 : 200) two min before injury markedly attenuated the SCEP changes immediately after injury. In the antiserum treated animals, a significant reduction in the microvascular permeability, edema formation and cell injury was observed in the traumatised spinal cord. These observations suggest that (i) dynorphin is involved in the altered bioelectrical activity of the spinal cord following trauma, (ii) the peptide actively participates in the pathophysiological processes of cell injury in the spinal cord trauma, and (iii) the dynorphin antiserum has potential therapeutic value for the treatment of spinal cord injuries. Received July 3, 2001 Accepted August 6, 2001 Published online July 31, 2002     

NO参与介导吗啡戒断大鼠脊髓神经元敏感化

运用Fos免疫组织化学、NADPH－d组织化学、F/NADPH－d双标、鞘内注射和反义寡核苷酸技术,观察吗啡戒断大鼠脊髓神经元活动变化及NO在其中的作用,结果发现：非吗啡依赖大鼠急性应用纳洛酮和吗啡依赖大鼠脊髓水平Fos-LI和NADPH－d阳性神经元表达与对照组相比无明显变化,二者也无Fos／NADPH－d双标神经元表达;吗啡依赖纳洛酮催促戒断大鼠脊髓Fos-LI、NADPH－d阳性神经元、纤维和终末表达明显增加,且出现Fos／NADPH－d双标神经元表达。Fos-LI和Fos／NADPH－d双标神经元呈现双侧脊髓全层分布,NADPH－d阳性神经元、纤维和终末主要位于双侧脊髓背角浅层。鞘内注射NOS抑制剂L－NA和nNOS反义寡核苷酸均明显降低吗啡依赖大鼠纳洛酮催促戒断症状评分,减少吗啡戒断大鼠脊髓Fos-LI表达。上述结果提示：NO参与介导吗啡戒断大鼠脊髓神经元敏感化。     

Bilateral movement training promotes axonal remodeling of the corticospinal tract and recovery of motor function following traumatic brain injury in mice

Traumatic brain injury (TBI) results in severe motor function impairment, and subsequent recovery is often incomplete. Rehabilitative training is considered to promote restoration of the injured neural network, thus facilitating functional recovery. However, no studies have assessed the effect of such trainings in the context of neural rewiring. Here, we investigated the effects of two types of rehabilitative training on corticospinal tract (CST) plasticity and motor recovery in mice. We injured the unilateral motor cortex with contusion, which induced hemiparesis on the contralesional side. After the injury, mice performed either a single pellet-reaching task (simple repetitive training) or a rotarod task (bilateral movement training). Multiple behavioral tests were then used to assess forelimb motor function recovery: staircase, ladder walk, capellini handling, single pellet, and rotarod tests. The TBI+rotarod group performed most forelimb motor tasks (staircase, ladder walk, and capellini handling tests) better than the TBI-only group did. In contrast, the TBI+reaching group did not perform better except in the single pellet test. After the injury, the contralateral CST, labeled by biotinylated dextran amine, formed sprouting fibers into the denervated side of the cervical spinal cord. The number of these fibers was significantly higher in the TBI+rotarod group, whereas it did not increase in the TBI+reaching group. These results indicate that bilateral movement training effectively promotes axonal rewiring and motor function recovery, whereas the effect of simple repetitive training is limited.     

Human Motor Neurons Generated from Neural Stem Cells Delay Clinical Onset and Prolong Life in ALS Mouse Model

Amyotrophic lateral sclerosis (ALS) is the most common adult onset motor neuron disease. The etiology and pathogenic mechanisms of the disease remain unknown, and there is no effective treatment. Here we show that intrathecal transplantation of human motor neurons derived from neural stem cells (NSCs) in spinal cord of the SOD1G93A mouse ALS model delayed disease onset and extended life span of the animals. When HB1.F3.Olig2 (F3.Olig2) cells, stable immortalized human NSCs encoding the human Olig2 gene, were treated with sonic hedgehog (Shh) protein for 5–7 days, the cells expressed motor neuron cell type-specific phenotypes Hb9, Isl-1 and choline acetyltransferase (ChAT). These F3.Olig2-Shh human motor neurons were transplanted intrathecally in L5–L6 spinal cord of SOD1G93A mice, and at 4 weeks post-transplantation, transplanted F3.Olig2-Shh motor neurons expressing the neuronal phenotype markers NF, MAP2, Hb9, and ChAT were found in the ventral horn of the spinal cord. Onset of clinical signs in ALS mice with F3.Olig2-Shh motor neuron implants was delayed for 7 days and life span of animals was significantly extended by 20 days. Our results indicate that this treatment modality of intrathecal transplantation of human motor neurons derived from NSCs might be of value in the treatment of ALS patients without significant adverse effects.     

Antinociceptive effects in mice after intrathecal injection of a substance P receptor antagonist, Spantide: lack of 'neurotoxic' action

The present investigation was undertaken to determine the antinociceptive potency and possible neurotoxic effects of a substance P (SP) receptor antagonist, [D-Arg,D-Trp,Leu]SP (Spantide), after intrathecal injection in mice. After the nociceptive tests had been carried out, the animals were sacrificed and the spinal cords were investigated for histopathological changes, since such have been reported previously to occur in rats. It was found that the reaction latency in the tail-flick test increased in the dose range 0-10 micrograms. The effect was maximal at 10 and 45 min after 10 micrograms Spantide, and somewhat lower when 5 micrograms was used. None of the animals showed the complete motor impairment reported previously to occur after intrathecal administration in rats. In some of the mice we observed a slight rigidity in the hind-legs. At histopathological examination, it was found that Spantide produced no histological changes indicative of 'neurotoxic' effects. In agreement with this, the immunohistochemical evaluation, using calcitonin gene-related peptide (CGRP) as a marker for motoneurons and central branches of primary sensory neurons, did not provide evidence that the intrathecal injection of 10 micrograms Spantide produced any effects when compared to vehicle-injected animals. In conclusion, the present results demonstrate an antinociceptive effect of Spantide when injected intrathecally in mice, and that this occurred without any signs of toxic reactions in spinal cord as previously has been reported for the rat.     

Safety and possible outcome assessment of autologous Schwann cell and bone marrow mesenchymal stromal cell co-transplantation for treatment of patients with chronic spinal cord injury

Background aimsCell replacement therapy has become a promising issue that has raised much hope in the regeneration of central nervous system injury. Evidence indicates that successful functional recovery in patients with spinal cord injury will not simply emphasize a single therapeutic strategy. Therefore, many recent studies have used combination strategies for spinal cord regeneration.MethodsWe assessed the safety and feasibility of a bone marrow mesenchymal stromal cell and Schwann cell combination for the treatment of patients with chronic spinal cord injury. Eight subjects who received a complete traumatic spinal cord injury (American Spinal Injury Association [ASIA] classification A) enrolled in this study. The patients received this autologous combination of cells directly into the injury site. The mean duration of follow-up was approximately 24 months.ResultsNo magnetic resonance imaging evidence of neoplastic tissue overgrowth, syringomyelia or psuedomeningocele in any of the patients was seen during the study. There was no deterioration in sensory or motor function in any of the patients during the course of the study. Three patients had negligible improvement in ASIA sensory scale. No motor score improvement and no change in ASIA classification was seen. The patients had widely subjective changes in the course of the study such as urination and defecation sensation and more stability and trunk equilibrium in the sitting position.ConclusionsThere were no adverse findings at least 2 years after autologous transplantation of Schwann cell and mesenchymal stromal cell combination into the injured spinal cord. It appears that the use of this combination of cells is safe for clinical application to spinal cord regeneration.     

Cholinergic receptor alterations in the cerebral cortex of spinal cord injured rat

Many areas of the cerebral cortex process sensory information or coordinate motor output necessary for control of movement. Disturbances in cortical cholinergic system can affect locomotor coordination. Spinal cord injury causes severe motor impairment and disturbances in cholinergic signalling can aggravate the situation. Considering the impact of cortical cholinergic firing in locomotion, we focussed the study in understanding the cholinergic alterations in cerebral cortex during spinal cord injury. The gene expression of key enzymes in cholinergic pathway - acetylcholine esterase and choline acetyl transferase showed significant upregulation in the cerebral cortex of spinal cord injured group compared to control with the fold increase in expression of acetylcholine esterase prominently higher than cholineacetyl transferase. The decreased muscarinic receptor density and reduced immunostaining of muscarinic receptor subtypes along with down regulated gene expression of muscarinic M1 and M3 receptor subtypes accounts for dysfunction of metabotropic acetylcholine receptors in spinal cord injury group. Ionotropic acetylcholine receptor alterations were evident from the decreased gene expression of alpha 7 nicotinic receptors and reduced immunostaining of alpha 7 nicotinic receptors in confocal imaging. Our data pin points the disturbances in cortical cholinergic function due to spinal cord injury; which can augment the locomotor deficits. This can be taken into account while devising a proper therapeutic approach to manage spinal cord injury.     

Xanthine oxidase converts nitric oxide to nitroxyl that inactivates the enzyme

Xanthine oxidase (XO) was found to convert nitric oxide (NO* ) released from spermine-NONOate to nitroxyl (HNO), the one-electron reduction product of NO*, in the presence of its substrate hypoxanthine under anaerobic conditions. Under these conditions, XO lost its activity. Upon aerobic incubation of XO with its substrate, neither conversion of NO* to HNO nor inactivation of the enzyme was observed. Angeli's salt (an HNO generator) or synthetic peroxynitrite inactivated XO at low concentrations, whereas high concentrations of diethylamine-NONOate (an NO* donor) and SIN-1 (which generates peroxynitrite by releasing both NO* and superoxide) were required to inactivate XO. These results suggest that HNO generated by XO under anaerobic conditions inactivates XO. As both XO and NO* synthase are activated and/or induced in ischemia-reperfusion injury, HNO formed by XO may contribute to pathogenesis by exerting its potent oxidation activity against a variety of biological compounds.     

脐带间充质干细胞移植治疗脊髓损伤的临床研究

目的:探讨脐带间充质干细胞移植治疗脊髓损伤的疗效及安全性。方法:40例脊髓损伤患者给予脐带间充质干细胞移植治疗,移植方法采用静脉输注联合腰穿鞘内注射的方法。术后随访1年余定期观察患者临床症状及各项指标的变化并进行综合分析。移植过程中为促进干细胞的生长和分化,根据患者病情及身体状况给予相应的康复功能锻炼。结果:与入院时比较,脐带间充质干细胞移植治疗3、6、12个月后,不完全性脊髓损伤患者针刺觉评分、轻触觉评分、运动评分均有明显改善(P<0.05或0.01),完全性脊髓损伤患者针刺觉评分、轻触觉评分、运动评分均无明显变化(P>0.05),两组残损分级均无明显改善(P>0.05)。移植后各项生化指标正常,未出现严重的并发症和明显的不良反应。结论:脐带间充质干细胞移植治疗脊髓损伤近期疗效明显,可以改善患者的临床症状,提高患者的生存质量,是一种值得借鉴的治疗方法。     

Angeli's salt induces neurotoxicity in dopaminergic neurons in vivo and in vitro

In this study, we investigated the hypothesis that the pro-oxidative properties of Angeli's salt (AS), a nitroxyl anion (HNO/NO -) releasing compound, cause neurotoxicity in dopaminergic neurons. The pro-oxidative properties were demonstrated in vitro by measuring hydroxylation products of salicylate and peroxidation of lipids under various redox conditions. AS (0-1000 μM) released high amounts of hydroxylating species in a concentration dependent manner. AS also increased lipid peroxidation in brain homogenates at concentrations below 100 μM, while inhibiting it at 1000 μM concentration. The AS induced pro-oxidative effects were completely suppressed by copper (II), which converts nitroxyl anion to nitric oxide, as well as by a potent nitroxyl anion scavenger glutathione. Neurotoxicity towards dopaminergic neurons was tested in rat nigrostriatal dopaminergic system in vivo and by using primary mesencephalic dopaminergic neuronal cultures in vitro . Intranigral infusion of AS (0-400 nmol) caused neurotoxicity reflected as a dose dependent decrease of striatal dopamine seven days after treatment. The effect of the 100 nmol dose was more pronounced when measured 50 days after the infusion. Neurotoxicity was also confirmed as a decrease of tyrosine hydroxylase positive neurons in the substantia nigra. Neither sulphononoate, a close structural analog of AS, nor sodiumnitrite caused changes in striatal dopamine, thus reflecting lack of neurotoxicity. In primary dopaminergic neuronal cultures AS reduced [ 3 H] dopamine uptake with concentrations over 200 μM confirming neurotoxicity. In line with the quite low efficacy to increase lipid peroxidation in vitro , infusion of AS into substantia nigra did not cause increased formation of fluorescent products of lipid peroxidation. These results support the hypothesis that AS derived species oxidize critical thiol groups, rather than membrane lipids, potentially leading to protein oxidation/dysfunction and demonstrated neurotoxicity. These findings may have pathophysiological relevance in case of excess formation of nitroxyl anion.     

Intrathecal Administration of Tempol Reduces Chronic Constriction Injury-Induced Neuropathic Pain in Rats by Increasing SOD Activity and Inhibiting NGF Expression

We investigate the antinociceptive effect of intrathecal and intraperitoneal tempol administration in a rat model of chronic constriction injury (CCI)-induced neuropathic pain and explore the underlying antinociceptive mechanisms of tempol. Rats were randomly assigned to four groups (n = 8 per group): sham group, CCI group, Tem1 group (intrathecal injection of tempol), and Tem2 group (intraperitoneal injection of tempol). Neuropathic pain was induced by CCI of the sciatic nerve. Tempol was intrathecally or intraperitoneally administered daily for 7 days beginning on postoperative day one. The mechanical withdrawal threshold and thermal withdrawal latency were tested on preoperative day 3 and postoperative days 1, 3, 5, 7, 10, 14, and 21. Structural changes were examined by hematoxylin and eosin staining, toluidine blue staining, and electron microscopy. Malondialdehyde (MDA) and superoxide dismutase (SOD) levels were determined using the thiobarbituric acid and nitroblue tetrazolium methods, respectively. Nerve growth factor (NGF) expression levels were determined by immunohistochemistry and Western blot. Intrathecal, but not intraperitoneal, injection of tempol produced a persistent antinociceptive effect. Intraperitoneal injection of tempol did not result in high enough concentration of tempol in the cerebrospinal fluid. Intrathecal, but not intraperitoneal, injection of tempol inhibited CCI-induced structural damage in the spinal cord reduced MDA levels, and increased SOD activities in the spinal cord. Furthermore, intrathecal, but not intraperitoneal, injection of tempol further downregulated the expression of NGF in the spinal cord following CCI, and this effect was blocked by p38MAPK inhibitor. Intrathecal injection of tempol produces antinociceptive effects and reduces CCI-induced structural damage in the spinal cord by increasing SOD activities and downregulating the expression of NGF via the p38MAPK pathway. Intraperitoneal administration of tempol does not exhibit antinociceptive effects.     

Protein Oxidative Damage in a Transgenic Mouse Model of Familial Amyotrophic Lateral Sclerosis

Abstract: The Gly⁹³→Ala mutation in the Cu,Zn superoxide dismutase (Cu,Zn-SOD) gene (SOD1) found in some familial amyotrophic lateral sclerosis (FALS) patients has been shown to result in an aberrant increase in hydroxyl radical production by the mutant enzyme that may cause oxidative injury to spinal motor neurons. In the present study, we analyzed the extent of oxidative injury to lumbar and cervical spinal cord proteins in transgenic FALS mice that overexpress the SOD1 mutation [TgN(SOD1-G93A)G1H] in comparison with nontransgenic mice. Total protein oxidation was examined by spectrophotometric measurement of tissue protein carbonyl content by the dinitrophenylhydrazine (DNPH) assay. Four ages were investigated: 30 (pre-motor neuron pathology and clinical disease), 60 (after initiation of pathology, but pre-disease), 100 (～50% loss of motor neurons and function), and 120 (near complete hindlimb paralysis) days. Protein carbonyl content in 30-day-old TgN(SOD1-G93A)G1H mice was twice as high as the level found in age-matched nontransgenic mice. However, at 60 and 100 days of age, the levels were the same. Then, between 100 and 120 days of age, the levels in the TgN(SOD1-G93A)G1H mice increased dramatically (557%) compared with either the nontransgenic mice or transgenic animals that overexpress the wild-type human Cu,Zn-SOD [TgN(SOD1)N29]. The 100–120-day increase in spinal cord protein carbonyl levels was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoretic separation and western blot immunoassay, which enabled the identification of heavily oxidized individual proteins using a monoclonal antibody against DNPH-derivatized proteins. One of the more heavily oxidized protein bands (14 kDa) was identified by immunoprecipitation as largely Cu,Zn-SOD. Western blot comparison of the extent of Cu,Zn-SOD protein carbonylation revealed that the level in spinal cord samples from 120-day-old TgN(SOD1-G93A)G1H mice was significantly higher than that found in age-matched nontransgenic or TgN(SOD1)N29 mice. These results suggest that the increased hydroxyl radical production associated with the G93A SOD1 mutation and/or lipid peroxidation-derived radical species (peroxyl or alkoxyl) causes extensive protein oxidative injury and that the Cu,Zn-SOD itself is a key target, which may compromise its antioxidant function.     

Functional recovery after transplantation of bone marrow-derived human mesenchymal stromal cells in a rat model of spinal cord injury

Background aimsSpinal cord injury (SCI) is a medically untreatable condition for which stem cells have created hope. Pre-clinical and clinical studies have established that these cells are safe for transplantation. The dose dependency, survivability, route of administration, cell migration to injury site and effect on sensory and motor behavior in an SCI-induced paraplegic model were studied.MethodsA spinal cord contusion injury model was established in rats. Bone marrow (BM) mesenchymal stromal cells (MSC) were tagged to facilitate tracing in vivo. Two different doses (2 and 5 million cells/kg body weight) and two different routes of infusion (site of injury and lumbar puncture) were tested during and after the spinal shock period. The animals were tested post-transplantation for locomotor capacity, motor control, sensory reflex, posture and body position. Stem cell migration was observed 1 month post-transplantation in spinal cord sections.ResultsThe overall results demonstrated that transplantation of BM MSC significantly improved the locomotor and sensory behavior score in the experimental group compared with the sham control group, and these results were dose dependent. All the infused stem cells could be visualized at the site of injury and none was visualized at the injected site. This indicated that the cells had survived in vivo, were probably chemoattracted and had migrated to the lesion site.ConclusionsMSC transplanted with a lumbar puncture method migrate to the site of injury and are the most suitable for SCI healing. These cells demonstrate a dose-dependent effect and promote functional recovery when injected during or after the spinal shock period.     

大鼠脊髓损伤后感觉、运动及电生理变化

在应用磁控机械夹断法复制的大鼠脊髓损伤模型上,动态地观察了脊髓损伤后的感觉及运动机能变化,并进行了电生理学研究。结果表明,0.3A电流未能导致永久性瘫痪。术后2周,后肢的感觉及运动功能逐渐恢复;可记录到体感诱发电位(SEP)。0.4,0.5和0.8A电流均能导致大鼠永久性瘫痪;倾斜板及开阔场地行走分数均显著低于0.3A组;术后4周这些大鼠可产生行走样动作,于损伤部位再次切断脊髓后仍能出现这些动作;0.4A组可记录到早期SEP,再次切断脊髓后SEP消失。结果提示:(1)脊髓不全横断后,由于残留纤维活动,可在相当程度上导致大鼠感觉和运动机能的恢复;(2)脊髓完全横断后,后肢的上行冲动可能经再生的神经纤维向中枢端传导至脑;(3)大鼠脊髓内可能存在行走中枢模式发生器(CPG),适当刺激可激发其活动,并产生行走样运动。     

Human neural stem cell replacement therapy for amyotrophic lateral sclerosis by spinal transplantation

Background

Mutation in the ubiquitously expressed cytoplasmic superoxide dismutase (SOD1) causes an inherited form of Amyotrophic Lateral Sclerosis (ALS). Mutant synthesis in motor neurons drives disease onset and early disease progression. Previous experimental studies have shown that spinal grafting of human fetal spinal neural stem cells (hNSCs) into the lumbar spinal cord of SOD1^G93A rats leads to a moderate therapeutical effect as evidenced by local α-motoneuron sparing and extension of lifespan. The aim of the present study was to analyze the degree of therapeutical effect of hNSCs once grafted into the lumbar spinal ventral horn in presymptomatic immunosuppressed SOD1^G93A rats and to assess the presence and functional integrity of the descending motor system in symptomatic SOD1^G93A animals.

Methods/Principal Findings

Presymptomatic SOD1^G93A rats (60–65 days old) received spinal lumbar injections of hNSCs. After cell grafting, disease onset, disease progression and lifespan were analyzed. In separate symptomatic SOD1^G93A rats, the presence and functional conductivity of descending motor tracts (corticospinal and rubrospinal) was analyzed by spinal surface recording electrodes after electrical stimulation of the motor cortex. Silver impregnation of lumbar spinal cord sections and descending motor axon counting in plastic spinal cord sections were used to validate morphologically the integrity of descending motor tracts. Grafting of hNSCs into the lumbar spinal cord of SOD1^G93A rats protected α-motoneurons in the vicinity of grafted cells, provided transient functional improvement, but offered no protection to α-motoneuron pools distant from grafted lumbar segments. Analysis of motor-evoked potentials recorded from the thoracic spinal cord of symptomatic SOD1^G93A rats showed a near complete loss of descending motor tract conduction, corresponding to a significant (50–65%) loss of large caliber descending motor axons.

Conclusions/Significance

These data demonstrate that in order to achieve a more clinically-adequate treatment, cell-replacement/gene therapy strategies will likely require both spinal and supraspinal targets.     

Local Delivery of High-Dose Chondroitinase ABC in the Sub-Acute Stage Promotes Axonal Outgrowth and Functional Recovery after Complete Spinal Cord Transection

Chondroitin sulfate proteoglycans (CSPGs) are glial scar-associated molecules considered axonal regeneration inhibitors and can be digested by chondroitinase ABC (ChABC) to promote axonal regeneration after spinal cord injury (SCI). We previously demonstrated that intrathecal delivery of low-dose ChABC (1 U) in the acute stage of SCI promoted axonal regrowth and functional recovery. In this study, high-dose ChABC (50 U) introduced via intrathecal delivery induced subarachnoid hemorrhage and death within 48 h. However, most SCI patients are treated in the sub-acute or chronic stages, when the dense glial scar has formed and is minimally digested by intrathecal delivery of ChABC at the injury site. The present study investigated whether intraparenchymal delivery of ChABC in the sub-acute stage of complete spinal cord transection would promote axonal outgrowth and improve functional recovery. We observed no functional recovery following the low-dose ChABC (1 U or 5 U) treatments. Furthermore, animals treated with high-dose ChABC (50 U or 100 U) showed decreased CSPGs levels. The extent and area of the lesion were also dramatically decreased after ChABC treatment. The outgrowth of the regenerating axons was significantly increased, and some partially crossed the lesion site in the ChABC-treated groups. In addition, retrograde Fluoro-Gold (FG) labeling showed that the outgrowing axons could cross the lesion site and reach several brain stem nuclei involved in sensory and motor functions. The Basso, Beattie and Bresnahan (BBB) open field locomotor scores revealed that the ChABC treatment significantly improved functional recovery compared to the control group at eight weeks after treatment. Our study demonstrates that high-dose ChABC treatment in the sub-acute stage of SCI effectively improves glial scar digestion by reducing the lesion size and increasing axonal regrowth to the related functional nuclei, which promotes locomotor recovery. Thus, our results will aid in the treatment of spinal cord injury.     

Neurochemical plasticity of nitric oxide synthase isoforms in neurogenic detrusor overactivity after spinal cord injury

Nitric oxide (NO) participates in the neural pathways controlling the lower urinary tract (LUT). Expression of NO synthase (NOS) can be upregulated after spinal cord injury (SCI), and altered NOS activity may participate in resulting LUT dysfunction. To investigate distribution of NOS-immunoreactivity (NOS-IR) in neurons of rats following SCI and the possible effects of NOS inhibitors. Expression of neuronal and inducible NOS-IR in lumbosacral spinal cord was assessed in rats. Cystometry was performed to examine effects of intrathecal injection of NOS inhibitor. There was increased expression of neuronal NOS-IR after trauma. Maximum bladder capacity was increased by neuronal NOS (nNOS) inhibitors. Upregulation of nNOS may facilitate emergence of the spinal micturition reflex following SCI; nNOS inhibitor suppressed SCI-induced urinary incontinence by increasing bladder capacity. Our results indicate manipulation of NO production could help treat LUT dysfunction after SCI.