This study aims to investigate the neuroprotective effect of tetramethylpyrazine on mice after spinal cord injury and its mechanism. Seventy-five female C57BL/6 mice were randomly divided into 5 groups, namely, a sham operation group, a model group, a tetramethylpyrazine low-dose group(25 mg·kg~(-1)), a tetramethylpyrazine medium-dose group(50 mg·kg~(-1)), and a tetramethylpyrazine high-dose group(100 mg·kg~(-1)), with 15 mice in each group. Modified Rivlin method was used to establish the mouse model of acute spinal cord injury. After 14 d of tetramethylpyrazine intervention, the motor function of hind limbs of mice was evaluated by basso mouse scale(BMS) and inclined plate test. The levels of inflammatory cytokines tumor necrosis factor-α(TNF-α), interleukin-6(IL-6), and interleukin-1β(IL-1β) in the spinal cord homogenate were determined by enzyme-linked immunosorbent assay(ELISA). Hematoxylin-eosin(HE) staining was used to observe the histology of the spinal cord, and Nissl's staining was used to observe the changes in the number of neurons. Western blot and immunofluorescence method were used to detect the expression of glial fibrillary acidic protein(GFAP) and C3 protein. Tetramethylpyrazine significantly improved the motor function of the hind limbs of mice after spinal cord injury, and the BMS score and inclined plate test score of the tetramethylpyrazine high-dose group were significantly higher than those of the model group(P<0.01). The levels of TNF-α, IL-6, and IL-1β in spinal cord homogenate of the tetramethylpyrazine high-dose group were significantly decreased(P<0.01). After tetramethylpyrazine treatment, the spinal cord morphology recovered, the number of Nissl bodies increased obviously with regular shape, and the loss of neurons decreased. As compared with the model group, the expression of GFAP and C3 protein was significantly decreased(P<0.05,P<0.01) in tetramethylpyrazine high-dose group. In conclusion, tetramethylpyrazine can promote the improvement of motor function and play a neuroprotective role in mice after spinal cord injury, and its mechanism may be related to inhibiting inflammatory response and improving the hyperplasia of glial scar.
Rats ; Mice ; Female ; Animals ; Rats, Sprague-Dawley ; Neuroprotective Agents/pharmacology* ; Tumor Necrosis Factor-alpha/metabolism* ; Interleukin-6 ; Mice, Inbred C57BL ; Spinal Cord Injuries/genetics* ; Spinal Cord/metabolism*

OBJECTIVE: To observe the effect of moxibustion at oppositely-located points "Mingmen" (GV 4) and "Shenque" (CV 8) on the motor function of the hind limbs and bladder function in rats with neurogenic bladder after suprasacral spinal cord injury (SCI), so as to explore the effect of this therapy on bladder tissue apoptosis mediated by endoplasmic reticulum stress pathway. METHODS: Twenty-eight female Wistar rats were randomly divided into a sham-operation group (8 rats) and a model establishment group (20 rats). Using the modified Allen's method, the spinal cord of T₁₀ segment was injured to establish a neurogenic bladder model in the model establishment group. Sixteen rats were modeled successfully and then divided into a model group (8 rats) and a moxibustion group (8 rats). In the moxibustion group, 2 h after consciousness regaining from modeling anesthesia, moxibustion was exerted at "Shenque" (CV 8) and "Mingmen" (GV 4), 2 cones at each acupoint in one intervention. The intervention was administered once every two days and 5-time intervention was required totally. After intervention, Basso, Beattie and Bresnahan locomotor rating scale (BBB) score for the motor function of the hind limbs, and the urodynamics indexes (maximum bladder capacity, urine leakage pressure and bladder compliance) were compared among groups. HE staining method was adopted to observe the morphological changes of bladder tissue. With Western blot method and real-time PCR assay, the protein and mRNA expressions of the endoplasmic reticulum stress-related genes (glucose- regulated protein 78 [GRP78], activating transcription factor 4 [ATF4] and cysteinyl aspartate specific proteinase-12 [Caspase-12]) were determined. RESULTS: The transitional epithelial cells were arranged irregularly, the bladder wall was getting thinner, and the cellular vacuolar degeneration and neutrophil infiltration were found in the model group. Whereas, compared with the model group, in the moxibustion group, the arrangement of transitional epithelial cells was clear and continuous in layers, the cellular vacuolar degeneration was mild and the infiltration presented in a small amount of neutrophil granulocytes. Compared with the sham-operation group, in the model group, the BBB score was reduced (P<0.01), the maximum bladder capacity and bladder compliance were increased (P<0.01), and the protein expression levels of GRP78, ATF4 and Caspase-12, as well as mRNA expressions were all increased (P<0.01). In comparison with the model group, in the moxibustion group, BBB score was increased (P<0.01), the maximum bladder capacity and bladder compliance were decreased (P<0.01), and the protein and mRNA expression levels of GRP78, ATF4 and Caspase-12 were all decreased (P<0.01). CONCLUSION Moxibustion at the "oppositely-located points" improves the urination function, alleviate urine retention in neurogenic bladder rats after spinal cord injury. The underlying mechanism may be related to the down-regulation of the expressions of GRP78, ATF4 and Caspase-12 in the endoplasmic reticulum stress pathway of the bladder tissues, and thus to alleviate the apoptosis of bladder tissue.
Animals ; Caspase 12/genetics* ; Electroacupuncture ; Endoplasmic Reticulum Stress ; Female ; Moxibustion ; RNA, Messenger ; Rats ; Rats, Sprague-Dawley ; Rats, Wistar ; Spinal Cord ; Spinal Cord Injuries/therapy* ; Urinary Bladder, Neurogenic/therapy*

Spinal cord injury is a severe central nervous system disease, which will cause a series of complex pathophysiological changes and activate a variety of signaling pathways including Notch signaling. Studies have evidenced that activation of the Notch signaling pathway is not conducive to nerve repair and symptom improvement after spinal cord injury. Its mechanisms include inhibiting neuronal differentiation and axon regeneration, promoting reactive astrocyte proliferation, promoting M1 macrophage polarization and the release of proinflammatory factors, and inhibiting angiogenesis. Therefore, it has become a promising therapeutic strategy to inhibit Notch signal as a target in the treatment of spinal cord injury. In recent years, some researchers have used drugs, cell transplantation or genetic modification to regulate Notch signaling, which can promote the recovery of nerve function after spinal cord injury, thereby providing new treatment strategies for the treatment of spinal cord injury. This article will summarize the mechanism of Notch signaling pathway in spinal cord injury, and at the same time review the research progress in the treatment of spinal cord injury by modulating Notch signaling pathway in recent years, so as to provide new research ideas for further exploring new strategies for spinal cord injury.
Axons/metabolism* ; Cell Transplantation ; Humans ; Nerve Regeneration ; Signal Transduction/genetics* ; Spinal Cord/metabolism* ; Spinal Cord Injuries/metabolism*

OBJECTIVE: To explore the influence of electroacupuncture (EA) on the expression of AMPA receptor subunit GluR1 in the rats with acute spinal cord injury (SCI) and explore the potential effect mechanism of EA in treatment of acute SCI. METHODS: A total of 80 SD rats were randomly divided into five groups, i.e. a sham-operation group, a model group, an AMPA antagonist (DNQX) group, an EA group and a DNQX+EA group, 16 rats in each group. The modified Allen's impacting method was adopted to prepare the rat model of acute SCI at T RESULTS: Compared to the sham-operation group in 6 h, 24 h and 48 h after modeling, the BBB scores were all significantly decreased in the model group ( CONCLUSION The intervention with EA at "Dazhui" and "Mingmen" promotes the repair of the injured nerve in the spinal anterior horn probably through inhibiting GluR1 expression in the spinal injured area in the rats with acute SCI.
Animals ; Electroacupuncture ; Rats ; Rats, Sprague-Dawley ; Receptors, AMPA/genetics* ; Spinal Cord ; Spinal Cord Injuries/therapy*

OBJECTIVE: To investigate expression of Semaphorin 3A in rats after spinal cord injury and explore possible mechanism of inhibiting of axonal regeneration after SCI. METHODS: Forty healthy female SD rats, 8 weeks old, weighing (210.00±9.88) g, were randomly divided into control group(20 rats in group A) and model group(20 rats in group B). In control group, removal of T RESULTS: After a simple spinal cord transection injury, hemorrhagic necrosis, localized edema, neurodegeneration, necrosis, and cyst formation occurred in the injured area, and glial scar formation occurred in glial cells. Semaphorin 3A expression levels in control group was low in the gray matter area. There was no expression of Semaphorin 3A in the injured area of spinal cord injury in model group 3 days after operation. On the 14th day, the expression of Semaphorin 3A in the injured area of spinal cord injury increased significantly and was at a high level. On the 28th day, the expression of Semaphorin 3A was moderate. On the 42th day, the positive expression of Semaphorin 3A returned to normal level. CONCLUSION The increased expression of Semaphorin 3A after spinal cord injury may be one of the mechanisms that inhibit axonal regeneration.
Animals ; Female ; Rats ; Rats, Sprague-Dawley ; Semaphorin-3A/genetics* ; Spinal Cord ; Spinal Cord Injuries/genetics*

Evidence suggested that glycogen synthase kinase-3β (GSK-3β) is involved in Nogo-66 inhibiting axonal regeneration in vitro, but its effect in vivo was poorly understood. We showed that stereotactic injection of shRNA GSK-3β-adeno associated virus (GSK-3β-AAV) diminished syringomyelia and promoted axonal regeneration after spinal cord injury (SCI), using stereotactic injection of shRNA GSK-3β-AAV (tested with Western blotting and RT-PCR) into the sensorimotor cortex of rats with SCI and by the detection of biotin dextran amine (BDA)-labeled axonal regeneration. We also determined the right position to inject into the sensorimotor cortex. Our findings consolidate the hypothesis that downregulation of GSK-3β promotes axonal regeneration after SCI.
Animals ; Axons ; drug effects ; metabolism ; Dependovirus ; genetics ; Glycogen Synthase Kinase 3 beta ; genetics ; metabolism ; Humans ; Nerve Regeneration ; genetics ; RNA, Small Interfering ; administration & dosage ; genetics ; Rats ; Sensorimotor Cortex ; drug effects ; pathology ; Spinal Cord Injuries ; genetics ; pathology ; therapy ; Syringomyelia ; genetics ; pathology ; therapy

We induced percutaneous spinal cord injuries (SCI) using a balloon catheter in 45 rats and transplanted human umbilical cord blood derived mesenchymal stem cells (hUCB-MSCs) at the injury site. Locomotor function was significantly improved in hUCB-MSCs transplanted groups. Quantitative ELISA of extract from entire injured spinal cord showed increased expression of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and neurotrophin-3 (NT-3). Our results show that treatment of SCI with hUCB-MSCs can improve locomotor functions, and suggest that increased levels of BDNF, NGF and NT-3 in the injured spinal cord were the main therapeutic effect.
Animals ; Brain-Derived Neurotrophic Factor/*genetics ; *Cord Blood Stem Cell Transplantation ; Enzyme-Linked Immunosorbent Assay ; Gene Expression Profiling ; *Gene Expression Regulation ; Humans ; Locomotion ; Nerve Growth Factor/genetics ; Rats ; Spinal Cord Injuries/*therapy

PURPOSE: Spinal cord injury (SCI) is associated with permanent neurological damage, and treatment thereof with a single modality often does not provide sufficient therapeutic outcomes. Therefore, a strategy that combines two or more techniques might show better therapeutic effects. MATERIALS AND METHODS: In this study, we designed a combined treatment strategy based on neural stem cells (NSCs) introduced via a neuronal cell type-inducible transgene expression system (NSE::) controlled by a neuron-specific enolase (NSE) promoter to maximize therapeutic efficiency and neuronal differentiation. The luciferase gene was chosen to confirm whether this combined system was working properly prior to using a therapeutic gene. The luciferase expression levels of NSCs introduced via the neuronal cell type-inducible luciferase expression system (NSE::Luci) or via a general luciferase expressing system (SV::Luci) were measured and compared in vitro and in vivo. RESULTS: NSCs introduced via the neuronal cell type-inducible luciferase expressing system (NSE::Luci-NSCs) showed a high level of luciferase expression, compared to NSCs introduced via a general luciferase expressing system (SV::Luci-NSCs). Interestingly, the luciferase expression level of NSE::Luci-NSCs increased greatly after differentiation into neurons. CONCLUSION: We demonstrated that a neuronal cell type-inducible gene expression system is suitable for introducing NSCs in combined treatment strategies. We suggest that the proposed strategy may be a promising tool for the treatment of neurodegenerative disorders, including SCI.
Cell Differentiation/genetics/physiology ; *Gene Expression ; Gene Regulatory Networks ; *Genetic Therapy ; Humans ; Luciferases/genetics/*metabolism ; *Neural Stem Cells ; Neurons/metabolism ; Phosphopyruvate Hydratase/metabolism ; Promoter Regions, Genetic ; Spinal Cord Injuries/*therapy ; Stem Cells/*metabolism

OBJECTIVETo study the effects of Jisuikang (Chinese characters) on Nogo-NgR gene expression, and to explore the protective effects and mechanism of Jisuikang (Chinese characters) on spinal cord injury in rats.

METHODSOne hundred eighty female rats were randomly assigned to 6 groups(30 rats per group). Sham group: T10 lamina was resected only and spinal cord was untreated. Model group: spine cord injury (SCI) was created with a modified impinger of Allen's by impacting on the T10 spinal cord. Prednisolone group: Prednisolone (0.06 g/kg) was given by intragastric administration at a time interval of 24 hours after operation. The Jisuikang (Chinese characters) high, moderate and low dose groups: Jisuikang (Chinese characters) was supplied with different dose (50 g/kg, 25 g/kg, 12.5 g/kg) by intragastric administration in rats after operation,for the first time at 30 min after surgery. Animals were killed 3, 7, 14 days after surgery. The expression levels of Nogo-A and NgR were observed by Western Blot and Real-time PCR.

RESULTSThe expression of Nogo-A and NgR was at the basic level at all time points in sham group. Compared with model group, the protein expression levels of Nogo-A and NgR in sham, prednisolone, Jisuikang (Chinese characters) moderate dose groups were statistically significant at all time points (P < 0.05). No difference was found in Jisuikang (Chinese characters) high and low dose groups (P > 0.05). Three days after surgery, the mRNA levels of Nogo-A and NgR in treatment group were significantly lower than that in model group (P < 0.01); 7 days after surgery,Nogo-A and NgR mRNA expression were dramatically upregulated and peaked; 14 days after operation, the expression was decreased, but still significantly higher than that in other treatment groups (P < 0.01). Prednisolone and Jisuikang (Chinese characters) moderate dose groups showed the most significant effects among all groups,but there was no statistically significant difference between two groups (P > 0.05).

CONCLUSIONThe decoction Jisuikang (Chinese characters) can promote the nerve cell regeneration by regulating Nogo-A and NgR gene expression, activating Nogo- NgR signaling pathways after acute spinal cord injury.

Animals ; Female ; GPI-Linked Proteins ; analysis ; genetics ; physiology ; Medicine, Chinese Traditional ; Myelin Proteins ; analysis ; genetics ; physiology ; Nerve Regeneration ; drug effects ; Nogo Proteins ; Nogo Receptor 1 ; Rats ; Rats, Sprague-Dawley ; Receptors, Cell Surface ; analysis ; genetics ; physiology ; Signal Transduction ; drug effects ; Spinal Cord Injuries ; drug therapy ; metabolism

OBJECTIVETo observe the neurological protection effects of "paraplegia-triple-needling method" on rats with incomplete spinal cord injury (SCI), so as to make a preliminary exploration on its mechanism.

METHODSA total of 45 SD rats were randomly divided into a paraplegia-triple-needling method group (group A), a regular acupuncture group (group B) and a model group (group C), 15 rats in each one. The rats model of incomplete spinal cord injury was established by modified Allen's method. The acupoints of governor vessel and back-shu points next to the vertebras of upper end and lower end of injured segment as well as motor points in key muscle of lower extremities were treated with acupuncture in the group A; the acupoints of governor vessel and back-shu points next to the vertebras of upper end and lower end of injured segment as well as "Huantiao" (GB 30), "Housanli" (ST 36), "Yanglingquan" (GB 34) and "Genduan"(Extra) were treated with acupuncture in the group B; rats in the group C received no treatment after model establishment but grabbing and immobilization. The needles were retained for 15 min in the group A and group B, once a day for 14 times. 1 d, 7 d and 14 d after model establishment, Basso Beattie Bresnahan (BBB) scores were observed in each group; the morphologic change of injured spinal cord and expression of positive cells of calcitonin gene-related peptide (CGRP) were observed. Results (1) One day after SCI, there was no significant difference of BBB scores among three groups (P> 0. 05); 7 days and 14 days after SCI, BBB scores in the group A and group B were significantly superior to those in the group C (all P<0. 05), and the BBB scores in the group A were superior to those in the group B ( both P<0. 05). (2) There was expression of CGRP positive cells in all three groups, and that in the group A and group B was significantly higher than that in group C (both P<0. 05); 14 days after treatment, the expression in the group A was higher than that in the group B (P<0. 05).

CONCLUSIONThe "paraplegia-triple-needling method" could obviously! improve the motor function of rats with SCI, especially the expression of neuroprotective factor CGRP, which is likely to be one of the mechanisms of neurological protection effect.

Acupuncture Points ; Acupuncture Therapy ; instrumentation ; methods ; Animals ; Calcitonin Gene-Related Peptide ; genetics ; metabolism ; Disease Models, Animal ; Humans ; Male ; Rats ; Rats, Sprague-Dawley ; Spinal Cord Injuries ; genetics ; metabolism ; therapy