Effects of continuous peripheral nerve block by tetrodotoxin on growth associated protein-43 expression during neuropathic pain development

BACKGROUND： Peripheral nerve injury may lead to neuropathic pain and cause a markedly increase expression of growth associated protein-43 （GAP-43） in the spinal cord and dorsal root ganglion, local anesthetics blocking electrical impulse propagation of nerve fibers may also affect the expression of GAP-43 in the spinal cord and dorsal root ganglion. OBJECTIVE： To determine the effects of continuous peripheral nerve block by tetrodotoxin before and after nerve injury on GAP-43 expression in the dorsal root ganglion during the development of neuropathic pain. DESIGN： A randomized controlled animal experiment. SETTINGS： Department of Anesthesiology, the Second Hospital of Xiamen City; Department of Anesthesiology, the Second Affiliated Hospital of Shantou University Medical College. MATERIALS： Thirty-five Spragne Dawley （SD） rats, weighing 200 - 250 g, were randomly divided into four groups： control group （n =5）, simple sciatic nerve transection group （n =10）, peripheral nerve block before and after sciatic nerve transection groups （n =10）. All the sciatic nerve transection groups were divided into two subgroups according to the different postoperative survival periods： 3 and 7 days （n =5） respectively. Mouse anti-GAP-43 monoclonal antibody （Sigma Co., Ltd.）, supervision TM anti-mouse reagent （HRP, Changdao antibody diagnosis reagent Co., Ltd., Shanghai）, and HMIAS-100 image analysis system （Qianping Image Engineering Company, Tongji Medical University） were employed in this study. METHODS： This experiment was carried out in the Department of Surgery and Pathological Laboratory, the Second Affiliated Hospital of Shantou University Medical College from April 2005 to April 2006. ①The animals were anesthetized and the right sciatic nerve was exposed and transected at 1 cm distal to sciatic notch. ② Tetrodotoxin 10 μg/kg was injected percutaneously between the greater trochanter and the posterior superior iliac spine of fight hind limb to block the sciatic nerve proximally at 1 hour before or 4 hours after nerve injury respectively, the injection was repeated in all the rats every 12 hours.③ At 3 or 7 days after nerve injury, immunohistochemistry and image analysis were used to evaluate the expression of GAP-43 in the dorsal root ganglions of L5 to the transected sciatic nerve, and quantitative analysis was also performed. ④ Statistical analysis was performed using one way analysis of variance followed by t test. MAIN OUTCOME MEASURE： Expression of GAP-43 in the fight dorsal root ganglions of L5. RESULTS： All the 35 SD rats were involved in the final analysis of results. In normal rats, there were very low expressions of GAP-43 in the dorsal root ganglions. In simple sciatic nerve transection rats 3 and 7 days after sciatic nerve transection, the average absorbance value of GAP-43 immunopositive neurons were significantly different from that in normal rats （t =8.806, 6.771, P 〈 0.01）. Whereas 3 and 7 days after sciatic nerve transection in rats with peripheral nerve block before and after nerve injury, the average absorbance value of GAP-43 immunopositive neurons were not significantly different from that in normal rats （P 〉 0.05）. CONCLUSION： Local anesthetic continuous peripheral nerve block before or after nerve injury can suppress nerve injury induced high expression of GAP-43 during the development of neuropathic pain.

Pathological changes in the retina and growth associated protein-43 expression following treatment of intracanalicular optic nerve injury via optic canal decompression,dexamethasone or their combination

BACKGROUND： The main clinical treatments for optic nerve injury are optic canal decompression and systemic administration of hormones, but both treatments have disadvantages. OBJECTIVE： To observe the pathological changes in the retina and growth associated protein-43 （GAP-43） expression, to compare the treatment of optic canal decompression, hormones, and their combination with the intracanalicular optic nerve injury.DESIGN, TIME AND SETTING： A randomized, controlled animal study was performed at the Department of Anatomy, Weifang Medical University, China, from September 2007 to November 2008.MATERIALS： Dexamethasone （Shandong Huaxin Pharmaceutical, China） and rabbit anti-GAP-43 polyclonal antibody （Boster, China） were used.METHODS： All 36 healthy adult rabbits were randomly assigned to control group （n = 4）, simple injury group （n = 20）, and treatment group （n = 12）. Intracanalicular optic nerve injury models were established using the metal cylinder free-fall impact method. The control group was left intact. The treatment group （four rabbits in each subgroup） was treated by optic nerve decompression, dexamethasone treatment （1 mg/kg daily via two intravenous infusions, 1/5 total dose reduction every 3 days, for 14 days）, and simultaneously giving surgery and hormone treatment.MAIN OUTCOME MEASURES： Pathological changes in the retina were determined using hematoxylin-eosin staining. GAP-43 expression was detected using immunohistochemistry in the retina.RESULTS： Retina injury induced obvious pathological changes in the retina. With prolonged time after optic nerve injury, the number of retinal ganglion cells was gradually decreased, and reached the minimum on day 14 （P〈0.01）. All three treatments increased the number of retinal ganglion cells （P〈0.01）, but surgery ＋ hormone treatment was most effective. No GAP-43 cells were present in the normal retinal, but they appeared 3 days after injury, peaked 7 days after injury, and then began to decline.CONCLUSION： Intracanalicular optic nerve injury induced obvious pathological changes in the retina, including increased GAP-43 expression. Optic canal decompression and hormones improved nerve repair after injury, and their combination produced better outcomes.

Enriched environment elevates expression of growth associated protein-43 in the substantia nigra of SAMP8 mice

An enriched environment protects dopaminergic neurons from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine（MPTP）-induced neuronal injury, but the underlying mechanism for this is not clear. Growth associated protein-43（GAP-43） is closely associated with neurite outgrowth and axon regeneration during neural development. We speculate that an enriched environment can reduce damage to dopaminergic neurons by affecting the expression of GAP-43. This study is designed to test this hypothesis. Three-month-old female senescence-accelerated mouse prone 8（SAMP8） mice were housed for 3 months in an enriched environment or a standard environment. These mice were then subcutaneously injected in the abdomen with 14 mg/kg MPTP four times at 2-hour intervals. Morris water maze testing demonstrated that learning and memory abilities were better in the enriched environment group than in the standard environment group. Reverse-transcription polymerase chain reaction, immunohistochemistry and western blot assays showed that m RNA and protein levels of GAP-43 in the substantia nigra were higher after MPTP application in the enriched environment group compared with the standard environment group. These findings indicate that an enriched environment can increase GAP-43 expression in SAMP8 mice. The upregulation of GAP-43 may be a mechanism by which an enriched environment protects against MPTP-induced neuronal damage.

Neurofilament 200 expression in a rat model of complete spinal cord injury following growth-associated protein-43 treatment

BACKGROUND： Growth-associated protein-43 （GAP-43） expression in the nervous system has been demonstrated to promote neural regeneration, neuronal growth and development, as well as synaptic reconstruction. Neurofilament 200 （NF200） expression could reflect degree of injury and repair in injured spinal axons. OBJECTIVE： To observe NF200 expression changes in a rat model of complete spinal cord injury following GAP-43 treatment and to explore the effects of GAP-43 following spinal cord injury. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment was performed at the Laboratory of Histology and Embryology of Kunming Medical University between March 2007 and October 2008. MATERIALS： GAP-43 and GAP-43 antibody were provided by Beijing Boao Biology, China; mouse anti-rat NF200 antibody was purchased from Chemicon, USA. METHODS： Female, 8-week-old, Sprague Dawley rats were randomly assigned into three groups following complete spinal cord injury, with 20 animals in each group： GAP-43 antibody, GAP-43, and model groups. In addition, each group was subdivided into four subgroups according to sampling time after modeling, Le., 3-, 5-, 9-, and 15-day groups, with 5 rats in each group. GAP-43 antibody or GAP-43 was injected into injury sites of the spinal cord, 5 μg/0.2 mL, respectively, twice daily for three consecutive days, followed by three additional days of injection, once daily. The model group did not receive any treatment following injury. MAIN OUTCOME MEASURES： NF200 expression in the damaged spinal area at different stages was detected by immunohistochemistry; lower limb motion function following injury was evaluated using the Basso, Beattie and Bresnahan （BBB） locomotor rating scale. RESULTS： NF200 expression was significantly reduced in the GAP-43 antibody group, compared with GAP-43 and model groups, at 3 and 5 days after spinal cord injury （P 〈 0.05）. In addition, the model group expressed significantly less NF200 than the GAP-43 group （P 〈 0.05）. BBB scores from the GAP-43 antibody and model groups were remarkably less than the GAP-43 group （P 〈 0.05）. At 9 and 15 days of injury after drug withdrawal, NF200 expression was increased in the GAP-43 antibody group, and NF200 expression and BBB scores in the GAP-43 antibody and GAP-43 groups were significantly greater than in the model group （P 〈 0.05）. In particular, the GAP-43 group exhibited greater BBB scores than the GAP-43 antibody group at day 9 （P 〈 0.05）. CONCLUSION： GAP-43 promoted NF200 expression and recovery of lower limb function. Early administration of GAP-43 antibody produced reversible nerve inhibition, which was rapidly restored following withdrawal.

益气通脉胶囊对脑缺血大鼠学习记忆能力的影响

目的观察益气通脉胶囊对脑缺血损伤后大鼠学习记忆能力的影响及相关机理研究。方法选择SD大鼠90只,按随机数字表法选择10只作为对照组,其余大鼠采用反复夹闭双侧颈总动脉结合硝普钠降压法复制SD大鼠拟血管性痴呆模型。造模后选存活大鼠50只随机分为模型组、脑心通组和益气通脉胶囊高、中、低剂量组,每组各10只,连续给药15 d后通过Morris水迷宫测试大鼠的学习记忆能力,之后取大鼠脑的海马组织,提取蛋白测定生长相关蛋白43(growth associated protein 43,GAP43)和神经丝蛋白-200(Neurofilaments Protein-200,NF200)的表达,以及测定大鼠海马组织NOS活性和NO含量。结果 (1)大鼠的学习记忆能力:益气通脉胶囊高、中剂量组大鼠的学习记忆能力均高于模型组。(2)GAP43和NF200的表达:益气通脉胶囊高、中、低剂量组的蛋白表达均明显大于模型组。(3)益气通脉胶囊高、中剂量组大鼠的NOS活性和NO含量均显著低于模型组。结论益气通脉胶囊对脑缺血损伤后大鼠学习记忆能力的恢复有明显的促进作用,能使神经可塑性相关蛋白表达上调,降低大鼠海马组织NOS活性和NO含量。

The mechanism of astragaloside Ⅳ promoting sciatic nerve regeneration

3-O-beta-D-xylopyranosyl-6-O-beta-D-glucopyranosyl-cycloastragenol （astragaloside IV）, the main active component of the traditional Chinese medicine astragalus membranaceus, has been shown to be neuroprotective. This study investigated whether astragaloside IV could promote the repair of injured sciatic nerve. Denervated sciatic nerve of mice was subjected to anastomosis. The mice were intraperitoneally injected with 10, 5, 2.5 mg/kg astragaloside IV per day for 8 consecutive days Western blot assay and real-time PCR results demonstrated that growth-associated protein-43 ex- pression was upregulated in mouse spinal cord segments L4-6 after intervention with 10, 5, 2.5 mg/kg astragaloside IV per day in a dose-dependent manner. Luxol fast blue staining and elec- trophysiological detection suggested that astragaloside IV elevated the number and diameter of myelinated nerve fibers, and simultaneously increased motor nerve conduction velocity and action potential amplitude in the sciatic nerve of mice. These results indicated that astragaloside IV con- tributed to sciatic nerve regeneration and functional recovery in mice. The mechanism underlying this effect may be associated with the upregulation of growth-associated protein-43 expression.

Buyang Huanwu Decoction regulates neural stem cell behavior in ischemic brain

The traditional Chinese medicine Buyang Huanwu Decoction has been shown to improve the neu- rological function of patients with stroke. However, the precise mechanisms underlying its effect remain poorly understood. In this study, we established a rat model of cerebral ischemia by middle cerebral artery occlusion and intragastrically administered 5 g/kg Buyang Huanwu Decoction, once per day, for 1, 7, 14 and 28 days after cerebral ischemia. Immunohistochemical staining revealed a number of cells positive for the neural stem cell marker nestin in the cerebral cortex, the subven- tricular zone and the ipsilateral hippocampal dentate gyrus in rat models of cerebral ischemia. Buyang Huanwu Decoction significantly increased the number of cells positive for 5-bromodeoxyuridine （BrdU）, a cell proliferation-related marker, microtubule-associated protein-2, a marker of neuronal differentiation, and growth-associated protein 43, a marker of synaptic plasticity in the ischemic rat cerebral regions. The number of positive cells peaked at 14 and 28 days after intragastric administration of Buyang Huanwu Decoction. These findings suggest that Buyang Huanwu Decoction can promote the proliferation and differentiation of neural stem cells and en- hance synaptic plasticity in ischemic rat brain tissue.

Rho kinase:A new target for treatment of cerebral ischemia/reperfusion injury

Rho kinase inhibitor fasudil hydrochloride has been shown to reduce cerebral vasospasm, to inhibit inflammation and apoptosis and to promote the recovery of neurological function. However, the effect of fasudil hydrochloride on claudin-5 protein expression has not been reported after cerebral ischemia/reperfusion. Therefore, this study sought to explore the effects of fasudil hydrochloride on blood-brain barrier permeability, growth-associated protein-43 and claudin-5 protein expression, and to further understand the neuroprotective effect of fasudil hydrochloride. A focal cerebral ischemia/reperfusion model was established using the intraluminal suture technique. Fasudil hydrochloride （15 mg/kg） was intraperitoneally injected once a day. Neurological deficit was evaluated using Longa＇s method. Changes in permeability of blood-brain barrier were measured using Evans blue. Changes in RhoA, growth-associated protein-43 and claudin-5 protein expression were detected using immunohistochemistry and western blotting. Results revealed that fasudil hydrochloride noticeably contributed to the recovery of neurological function, improved the function of blood-brain barrier, inhibited RhoA protein expression, and upregulated growth-associated protein-43 and claudin-5 protein expression following cerebral ischemia/reperfusion. Results indicated that Rho kinase exhibits a certain effect on neurovascular damage following cerebral ischemia/reperfusion. Intervention targeted Rho kinase might be a new therapeutic target in the treatment of cerebral ischemia/reperfusion.

Treadmill step training promotes spinal cord neural plasticity after incomplete spinal cord injury

A large body of evidence shows that spinal circuits are significantly affected by training, and that intrinsic circuits that drive locomotor tasks are located in lumbosacral spinal segments in rats with complete spinal cord transection. However, after incomplete lesions, the effect of treadmil training has been debated, which is likely because of the difficulty of separating spontaneous stepping from specific training-induced effects. In this study, rats with moderate spinal cord contusion were sub-jected to either step training on a treadmil or used in the model （control） group. The treadmil training began at day 7 post-injury and lasted 20 ± 10 minutes per day, 5 days per week for 10 weeks. The speed of the treadmil was set to 3 m/min and was increased on a daily basis according to the tolerance of each rat. After 3 weeks of step training, the step training group exhibited a sig-nificantly greater improvement in the Basso, Beattie and Bresnahan score than the model group. The expression of growth-associated protein-43 in the spinal cord lesion site and the number of tyrosine hydroxylase-positive ventral neurons in the second lumbar spinal segment were greater in the step training group than in the model group at 11 weeks post-injury, while the levels of brain-derived neurotrophic factor protein in the spinal cord lesion site showed no difference between the two groups. These results suggest that treadmil training significantly improves functional re-covery and neural plasticity after incomplete spinal cord injury.

Extract of Ginkgo biloba promotes neuronal regeneration in the hippocampus after exposure to acrylamide

Previous studies have demonstrated a neuroprotective effect of extract of Ginkgo biloba against neuronal damage, but have mainly focused on antioxidation of extract of Ginkgo biloba. To date, limited studies have determined whether extrasct of Ginkgo biloba has a protective effect on neuronal damage. In the present study, acrylamide and 30, 60, and 120 mg/kg extract of Ginkgo biloba were administered for 4 weeks by gavage to establish mouse models. Our results showed that 30, 60, and 120 mg/kg extract of Ginkgo biloba effectively alleviated the abnormal gait of poisoned mice, and up-regulated protein expression levels of doublecortin（DCX）, brain-derived neurotrophic factor, and growth associated protein-43（GAP-43） in the hippocampus. Simultaneously, DCX-and GAP-43-immunoreactive cells increased. These findings suggest that extract of Ginkgo biloba can mitigate neurotoxicity induced by acrylamide, and thereby promote neuronal regeneration in the hippocampus of acrylamide-treated mice.

Neuroprotective effects of ginsenoside Rb1 on hippocampal neuronal injury and neurite outgrowth

Ginsenoside Rb1 has been reported to exert anti-aging and anti-neurodegenerative effects. In the present study, we investigate whether ginsenoside Rb1 is involved in neurite outgrowth and neuroprotection against damage induced by amyloid beta（25–35） in cultured hippocampal neurons, and explore the underlying mechanisms. Ginsenoside Rb1 significantly increased neurite outgrowth in hippocampal neurons, and increased the expression of phosphorylated-Akt and phosphorylated extracellular signal-regulated kinase 1/2. These effects were abrogated by API-2 and PD98059, inhibitors of the signaling proteins Akt and MEK. Additionally, cultured hippocampal neurons were exposed to amyloid beta（25–35） for 30 minutes; ginsenoside Rb1 prevented apoptosis induced by amyloid beta（25–35）, and this effect was blocked by API-2 and PD98059. Furthermore, ginsenoside Rb1 significantly reversed the reduction in phosphorylated-Akt and phosphorylated extracellular signal-regulated kinase 1/2 levels induced by amyloid beta（25–35）, and API-2 neutralized the effect of ginsenoside Rb1. The present results indicate that ginsenoside Rb1 enhances neurite outgrowth and protects against neurotoxicity induced by amyloid beta（25–35） via a mechanism involving Akt and extracellular signal-regulated kinase 1/2 signaling.

Transfection of the glial cell line-derived neurotrophic factor gene promotes neuronal differentiation

Glial cell line-derived neurotrophic factor recombinant adenovirus vector-transfected bone marrow mesenchymal stem cells were induced to differentiate into neuron-like cells using inductive medium containing retinoic acid and epidermal growth factor. Cell viability, micro- tubule-associated protein 2-positive cell ratio, and the expression levels of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43 protein in the su- pernatant were significantly higher in glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells compared with empty virus plasmid-transfected bone marrow mes- enchymal stem cells. Furthermore, microtubule-associated protein 2, glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein743 mRNA levels in cell pellets were statistically higher in glial cell line-derived neurotrophic factor/bone marrow mesen- chymal stem cells compared with empty virus plasmid-transfected bone marrow mesenchymal stem cells. These results suggest that glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells have a higher rate of induction into neuron-like cells, and this enhanced differentiation into neuron-like cells may be associated with up-regulated expression of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43.

Preemptive analgesic effects of low-dose ketamine on growth-associated protein expression in dorsal root ganglion of chronic constriction injury model rats

BACKGROUND： Ketamine is a noncompetitive N-methyl-D-aspartate （NMDA） receptor antagonists and plays an important role in the treatment of pain. OBJECTIVE： To analyze the preemptive analgesic effects of different doses of ketamine on growth-associated protein-43 （GAP43） expression in dorsal root ganglion in a rat model of chronic sciatic nerve constricted injury, and to study the differences between high-dose and low-dose ketamine DESIGN： Randomized controlled animal study. SETTING： Medical College of Shantou University. MATERIALS： Thirty-five adult male Sprague Dawley rats were provided by the Experimental Animal Center of Guangzhou University of Traditional Chinese Medicine. Ketamine hydrochloride injection was provided by Hengrui Pharmaceutical Co., Ltd., Jiangsu. METHODS： This study was performed at the Immunological Laboratory, Medical College of Shantou University from September to December 2006. Model of chronic sciatic nerve constricted injury： after anesthesia, the right sciatic nerve was exposed and ligated l-cm distal to the ischiadic tuberosity with a No. 3-0 cat gut suture. Grouping and intervention： 35 rats were randomly divided into 4 groups： normal control group （n = 5）, chronic constriction injury （CCI） group （n = 10）, low-dose ketamine group （n = 10）, and high-dose ketamine group （n = 10）. Rats in the normal control group did not undergo any surgery or drug intervention. Rats in the CCI group received intraperitoneal injection of saline （1 mL）, and their sciatic nerves were ligated after 10 minutes. Rats in the low-dose ketamine group underwent intraperitoneal injection of ketamine （25 mg/kg） 10 minutes prior to ligation of sciatic nerve; while, rats in the high-dose ketamine group were given intraperitoneal injection of ketamine （50 mg/kg） 10 minutes prior to ligation of sciatic nerve. On the third and the seventh days after surgery, dorsal root ganglion were resected from the sciatic nerve and cut into sections. MAIN OUTCOME MEASURES： GAP-43 expression in dorsal root ganglion was detected by immunohistochemistry and image analysis system, as well as semi-quantitative analysis. RESULTS： Thirty-five Sprague Dawley rats were included in the final analysis. Qualitative analysis： GAP-43 expression in the CCI group was higher than in the normal control group. Quantitative analysis： after three post-operative days, GAP-43 expression in the CCI group was significantly higher than in the normal control group （t = 22.919, 7.319, P 〈 0.05）. GAP-43 expression in the low-dose and high-dose ketamine group was significantly lower than in the CCI group （t = 11.166, 26.474, P 〈 0.05）. After seven postoperative days, GAP-43 expression in the low-dose and high-dose ketamine groups was significantly lower than in the CCI group （t = 2.382, 5.016, P 〈 0.05）. CONCLUSION： Preoperative administration of ketamine inhibited the increased GAP-43 expression in dorsal root ganglion during neuropathic pain.

Optimal compatible doses and effects of ephedrine and naloxone on neural plasticity in cerebral ischemia/reperfusion rats

BACKGROUND： Ephedrine promotes neural plasticity in rats following cerebral ischemia/reperfusion injury. Ephedrine has been combined with naloxone in some studies, and it has been confirmed that their combination has synergistic effects on increasing neural plasticity following cerebral ischemia/reperfusion injury. OBJECTIVE： To investigate the effects of ephedrine combined with various doses of naloxone on neural plasticity and to find an optimal dose of naloxone in rats after cerebral ischemia/reperfusion injury by analyzing growth associated protein-43 （GAP-43）, synaptophysin and β-endorphin expression in the hippocampal CA3 area. DESIGN, TIME AND SETTING： This immunohistochemical, randomized, controlled, animal experiment was performed at the Chongqing Research Institute of Pediatrics, China from September 2007 to June 2008. MATERIALS： Ephedrine hydrochloride injection and naloxone hydrochloride injection were respectively purchased from Shandong Lvliang Pharmaceutical Factory, China and Sichuan Jingwei Pharmaceutical Co., Ltd., China. A total of 192 healthy adult Sprague Dawley rats were used to establish models of left middle cerebral artery occlusion using the suture occlusion method. METHODS： At 2 hours following cerebral ischemia, the rats were intraperitoneally injected with 1.5 mg/kg/d ephedrine （ephedrine group）, with 0.1, 0.2, or 0.3 mg/kg/d naloxone （low, moderate and high doses of naloxone groups）, with 1.5 mg/kg/d ephedrine ＋ 0.1, 0.2, or 0.3 mg/kg/d naloxone （ephedrine ＋ low, moderate and high doses of naloxone groups）, and with 0.5 mL saline （model group）, respectively. MAIN OUTCOME MEASURES： GAP-43, synaptophysin and β -endorphin expression were detected in the hippocampal CA3 area using immunohistochemistry 1-4 weeks after surgery. Sensorimotor integration in rats was assessed using the beam walking test. RESULTS： GAP-43 and synaptophysin expression was greater in the ephedrine group, and in the ephedrine ＋ moderate and high doses of naloxone groups compared with the model group. GAP-43 and synaptophysin expression was greatest in the ephedrine ＋ high dose of naloxone group at 2 and 3 weeks alter surgery. β -endorphin expression was significantly lower in the ephedrine group, and in the ephedrine ＋ moderate and high doses of naloxone groups compared with the model group （P 〈 0.05）. β -endorphin expression was persistently low in the ephedrine ＋ high dose of naloxone group. At 1-3 weeks after surgery, the beam walking test score was significantly higher in the ephedrine group and ephedrine ＋ various doses of naloxone groups than in the model group （P 〈 0.05）. The score was higher in the ephedrine ＋ moderate and high doses of naloxone groups than in the ephedrine group （P 〈 0.05）. Moreover, the score was increased as the dose of naloxone increased in the ephedrine ＋ various doses of naloxone groups. CONCLUSION： Ephedrine promotes GAP-43 and synaptophysin expression, inhibits /3 -endorphin expression in the hippocampal CA3 area, and improves motor function in rats following cerebral ischemia/reperfusion injury. Naloxone does not have the above-mentioned effects. During combined treatment with ephedrine and naloxone, however, the above-described effects are enhanced with an increased dose of naloxone. The combination of ephedrine （1.5 mg/kg/d） and naloxone （0.3 mg/kg/d） can produce optimal therapeutic efficacy in treatment of cerebral ischemic injury.

Nischarin-siRNA delivered by polyethyleniminealginate nanoparticles accelerates motor function recovery after spinal cord injury

A previous study by our group found that inhibition of nischarin promotes neurite outgrowth and neuronal regeneration in Neuro-2 a cells and primary cortical neurons.In recent years,more and more studies have shown that nanomaterials have good prospects in treatment of spinal cord injury.We proposed that small interfering RNA targeting nischarin（Nis-si RNA） delivered by polyethyleneimine-alginate（PEIALG） nanoparticles promoted motor function recovery in rats with spinal cord injury.Direct microinjection of 5 μL PEI-ALG/Nis-si RNA into the spinal cord lesion area of spinal cord injury rats was performed.From day 7 after surgery,Basso,Beattie and Bresnahan score was significantly higher in rats from the PEI-ALG/Nis-si RNA group compared with the spinal cord injury group and PEI-ALG/Control-si RNA group.On day 21 after injection,hematoxylin-eosin staining showed that the necrotic area was reduced in the PEI-ALG/Nis-si RNA group.Immunohistochemistry and western blot assay results confirmed successful inhibition of nischarin expression and increased protein expression of growth-associated protein-43 in the PEI-ALG/Nis-si RNA group.These findings suggest that a complex of PEI-ALG nanoparticles and Nis-si RNA effectively suppresses nischarin expression,induces expression of growth-associated protein-43,and accelerates motor function recovery after spinal cord injury.

Effects of exogenous ganglioside-1 on learning and memory in a neonatal rat model of hypoxia-ischemia brain injury

BACKGROUND： Exogenous ganglioside-1 （GM1） can cross the blood-brain barrier and play a protective role against hypoxia-ischemia-induced brain damage. OBJECTIVE： To examine the possible mechanisms of exogenous GM1 protection in hypoxia-ischemia-induced brain damage in a neonatal rat model by measuring changes in brain mass, pathological morphology, growth-associated protein-43 expression, and neurobehavioral manifestations. DESIGN, TIME AND SETTING： A randomized block-design study was performed at the Immunohistochemistry Laboratory of the Pediatric Research Institute, Children＇s Hospital of Chongqing Medical University from August 2005 to August 2006. MATERIALS： A total of 36 neonatal, 7-day-old, Sprague Dawley rats were used in this experiment. The hypoxia-ischemia-induced brain damage model was established by permanently occluding the right carotid artery, followed by oxygen inhalation at a low concentration （8% O2, 92% N2） for 2 hours, METHODS： All rats were randomly divided into the following groups： GMI, model, and sham operation, with 12 rats each group. Rats in the GM 1 and model groups received hypoxic/ischemic-induced brain damage. Rats in the GM1 group received injections of GM1 （i.p., 20 mg/kg） at 0, 24, 48, 72, 96, 120, and 144 hours following models established, and rats in the model group were administered （i.p.） the same amount of saline. The right carotid artery was separated, but not ligated, in the sham operation group rats. MAIN OUTCOME MEASURES： At 1 week after surgery, expression of growth-associated protein-43, a marker of neural development and plasticity, was detected in the hippocampal CA3 region by immunohistochemistry. Brain mass was measured, and the pathological morphology was observed. At 4 weeks after surgery, behavioral changes in the remaining rats were tested by Morris water maze, and growth-associated protein-43 expression was measured. RESULTS： （1） In the GMI and sham operation groups, growth-associated protein-43 expression was greater in the hippocampal CA3 region compared to the model group 1 week after surgery （P 〈 0.05）. In all three groups, brain weight of the right hemisphere was significantly less than the left hemisphere, in particular in the model group （P 〈 0.05）. In the GMI group, the weight difference between two hemispheres, as well as the extent of damage in the right hemisphere, was less than the model group （P 〈 0.01 ）. In the sham operation Uoup, brain tissue consisted of integrated structures and ordered cells. In the model group, the cerebral cortex layers of the right hemisphere were not defined, neurons were damaged, and neurons were disarranged in the hippocampal area. In the GM1 group, neurons were dense in the right cerebral cortex and hippocampal area, with no significant change in glial proliferation. （2） The average time of escape latency in the GM1 group was shortened 4 weeks alter surgery, and significantly less than the model group （P 〈 0.05）. In addition, the frequency platform passing in the GMI group was significantly greater than the model group （P 〈 0.01）. CONCLUSION： Exogenous GM1 may reduce brain injury and improve learning and memory in hypoxia-ischemia-induced brain damage rats. This protection may be associated with increased growth-associated protein-43 expression, which is involved in neuronal remodeling processes.

SYNGR4 and PLEKHB1 deregulation in motor neurons of amyotrophic lateral sclerosis models: potential contributions to pathobiology

Amyotrophic lateral sclerosis is the most common adult-onset neurodegenerative disease affecting motor neurons. Its defining feature is progressive loss of motor neuron function in the cortex, brainstem, and spinal cord, leading to paralysis and death. Despite major advances in identifying genes that can cause disease when mutated and model the disease in animals and cellular models, it still remains unclear why motor symptoms suddenly appear after a long pre-symptomatic phase of apparently normal function. One hypothesis is that age-related deregulation of specific proteins within key cell types, especially motor neurons themselves, initiates disease symptom appearance and may also drive progressive degeneration. Genome-wide in vivo cell-type-specific screening tools are enabling identification of candidates for such proteins. In this minireview, we first briefly discuss the methodology used in a recent study that applied a motor neuron-specific RNASeq screening approach to a standard model of TAR DNA-binding protein-43(TDP-43)-driven amyotrophic lateral sclerosis. A key finding of this study is that synaptogyrin-4 and pleckstrin homology domain-containing family B member 1 are also deregulated at the protein level within motor neurons of two unrelated mouse models of mutant TDP-43 driven amyotrophic lateral sclerosis. Guided by what is known about molecular and cellular functions of these proteins and their orthologs, we outline here specific hypotheses for how changes in their levels might potentially alter cellular physiology of motor neurons and detrimentally affect motor neuron function. Where possible, we also discuss how this information could potentially be used in a translational context to develop new therapeutic strategies for this currently incurable, devastating disease.

Lipopolysaccharide-induced dysregulation of connexin 43 via nuclear factor κB /JNK-dependent signaling

Connexin 43 （Cx43） is the major structural protein of gap junctions in the ventricular myocardium and a major determinant of myocardial electrical properties. Cx43 expression is decreased in the wild type mice after myocardial infarction and this effect is attenuated in MMP-7-/- mice. Matrix metalloproteinase expression is regulated at the transcription level by the modulation of the activation of transcription factors such as activator protein （AP）-I and nuclear factor kappa-light-chain enhancer of activated B cells （NF-KB）. Methods Rat myocardial cells （H9c2） were cultured and maintained at 37 ~C and 5% CO2. H9c2 cells in 6-well plates were treated with lipopolysaccharides （LPS）, NF-kB inhibitor （JSH 23, 30 μ, Santa Cruz） ＋ LPS and c-Jun N-terminal kinase （JNK） inhibitor （SP600125, 10μM, Sigma） ＋ LPS for 6, 12 and 24 h. Apoptosis rate of cells was determined by flow cytometry. Cx43 expression was assessed by Western blotting. Results LPS induced a time-dependent apoptosis in all cell line. We have found that the treatment with LPS induced increase of apoptosis in H9c2 cells at 6 h, 12 h and 24 h, but the effect was decreased by the addition of JSH-23 and SP600125 to LPS respectively （P 〈 0.05） . LPS resulted in decreased expression of Cx43 expression at 6 h, 12 h and 24 h. However, JSH-23 and SP600125 attenuated the loss of Cx43 respectively （P 〈 0.05）. Conclusion Transcription factors NF-kB and JNK/AP-1 signaling pathway participates in the regulation of LPS-induced Cx43 expression in the H9c2 cells, and maybe play an important role in regulation of Cx43 expression.