Electroacupuncture in the repair of spinal cord injury:inhibiting the Notch signaling pathway and promoting neural stem cell proliferation

Electroacupuncture for the treatment of spinal cord injury has a good clinical curative effect, but the underlying mechanism is unclear. In our experiments, the spinal cord of adult Sprague-Dawley rats was clamped for 60 seconds. Dazhui(GV14) and Mingmen(GV4) acupoints of rats were subjected to electroacupuncture. Enzyme-linked immunosorbent assay revealed that the expression of serum inflammatory factors was apparently downregulated in rat models of spinal cord injury after electroacupuncture. Hematoxylin-eosin staining and immunohistochemistry results demonstrated that electroacupuncture contributed to the proliferation of neural stem cells in rat injured spinal cord, and suppressed their differentiation into astrocytes. Real-time quantitative PCR and western blot assays showed that electroacupuncture inhibited activation of the Notch signaling pathway induced by spinal cord injury. These findings indicate that electroacupuncture repaired the injured spinal cord by suppressing the Notch signaling pathway and promoting the proliferation of endogenous neural stem cells.     

Optimal time for subarachnoid transplantation of neural progenitor cells in the treatment of contusive spinal cord injury

This study aimed to identify the optimal neural progenitor cell transplantation time for spinal cord injury in rats via the subarachnoid space. Cultured neural progenitor cells from 14-day embryonic rats, constitutively expressing enhanced green fluorescence protein, or media alone, were injected into the subarachnoid space of adult rats at 1 hour (acute stage), 7 days (subacute stage) and 28 days (chronic stage) after contusive spinal cord injury. Results showed that grafted neural progenitor cells migrated and aggregated around the blood vessels of the injured region, and infiltrated the spinal cord parenchyma along the tissue spaces in the acute stage transplantation group. However, this was not observed in subacute and chronic stage transplantation groups. O4- and glial fibrillary acidic protein-positive cells, representing oligodendrocytes and astrocytes respectively, were detected in the core of the grafted cluster attached to the cauda equina pia surface in the chronic stage transplantation group 8 weeks after transplantation. Both acute and subacute stage transplantation groups were negative for O4 and glial fibrillary acidic protein cells. Basso, Beattie and Bresnahan scale score comparisons indicated that rat hind limb locomotor activity showed better recovery after acute stage transplantation than after subacute and chronic transplantation. Our experimental findings suggest that the subarachnoid route could be useful for transplantation of neural progenitor cells at the acute stage of spinal cord injury. Although grafted cells survived only for a short time and did not differentiate into astrocytes or neurons, they were able to reach the parenchyma of the injured spinal cord and improve neurological function in rats. Transplantation efficacy was enhanced at the acute stage in comparison with subacute and chronic stages.     

Jisuikang,a Chinese herbal formula,increases neurotrophic factor expression and promotes the recovery of neurological function after spinal cord injur y

The Chinese medicine compound, Jisuikang, can promote recovery of neurological function by inhibiting lipid peroxidation, scavenging oxygen free radicals, and effectively improving the local microenvironment after spinal cord injury. However, the mechanism remains unclear. Thus, we established a rat model of acute spinal cord injury using a modified version of Allen's method. Jisuikang(50, 25, and 12.5 g/kg/d) and prednisolone were administered 30 minutes after anesthesia. Basso, Beattie, and Bresnahan locomotor scale scores and the oblique board test showed improved motor function recovery in the prednisone group and moderate-dose Jisuikang group compared with the other groups at 3–7 days post-injury. The rats in the moderate-dose Jisuikang group recovered best at 14 days post-injury. Hematoxylin-eosin staining and transmission electron microscopy showed that the survival rate of neurons in treatment groups increased after 3–7 days of administration. Further, the structure of neurons and glial cells was more distinct, especially in prednisolone and moderate-dose Jisuikang groups. Western blot assay and immunohistochemistry showed that expression of brain-derived neurotrophic factor(BDNF) in injured segments was maintained at a high level after 7–14 days of treatment. In contrast, expression of nerve growth factor(NGF) was down-regulated at 7 days after spinal cord injury. Real-time fluorescence quantitative polymerase chain reaction showed that expression of BDNF and NGF m RNA was induced in injured segments by prednisolone and Jisuikang. At 3–7 days after injury, the effect of prednisolone was greater, while 14 days after injury, the effect of moderate-dose Jisuikang was greater. These results confirm that Jisuikang can upregulate BDNF and NGF expression for a prolonged period after spinal cord injury and promote repair of acute spinal cord injury, with its effect being similar to prednisolone.     

Electroacupuncture promotes the recovery of motor neuron function in the anterior horn of the injured spinal cord

Acupuncture has been shown to lessen the inflammatory reaction after acute spinal cord injury and reduce secondary injury.However,the mechanism of action remains unclear.In this study,a rat model of spinal cord injury was established by compressing the T8–9 segments using a modified Nystrom method.Twenty-four hours after injury,Zusanli(ST36),Xuanzhong(GB39),Futu(ST32)and Sanyinjiao(SP6)were stimulated with electroacupuncture.Rats with spinal cord injury alone were used as controls.At 2,4 and 6 weeks after injury,acetylcholinesterase(ACh E)activity at the site of injury,the number of medium and large neurons in the spinal cord anterior horn,glial cell line-derived neurotrophic factor(GDNF)m RNA expression,and Basso,Beattie and Bresnahan locomotor rating scale scores were greater in the electroacupuncture group compared with the control group.These results demonstrate that electroacupuncture increases ACh E activity,up-regulates GDNF m RNA expression,and promotes the recovery of motor neuron function in the anterior horn after spinal cord injury.     

Electroacupuncture promotes the proliferation of endogenous neural stem cells and oligodendrocytes in the injured spinal cord of adult rats

A contusive model of spinal cord injury at spinal segment T8-9 was established in rats. Huantiao (GB30) and Huatuojiaji (Ex-B05) were punctured with needles, and endogenous neural stem cells were labeled with 5-bromo-2’-deoxyuridine (BrdU) and NG2. Double immunofluorescence staining showed that electroacupuncture markedly increased the numbers of BrdU+/NG2+ cells at spinal cord tissue 15 mm away from the injury center in the rostral and caudal directions. The results suggest that electroacupuncture promotes the proliferation of endogenous neural stem cells and oligodendrocytes in rats with spinal cord injury.     

Synaptosomal-associated protein 25 may be an intervention target for improving sensory and locomotor functions after spinal cord contusion

Synaptosomal-associated protein 25 kDa (SNAP-25) is localized on the synapse and participates in exocytosis and neurotransmitter release. Decreased expression of SNAP-25 is associated with Alzheimer's disease and attention deficit/hyperactivity disorder. However, the expres-sion of SNAP-25 in spinal cord contusion injury is still unclear. We hypothesized that SNAP-25 is associated with sensory and locomotor functions after spinal cord injury. We established rat models of spinal cord contusion injury to detect gene changes with a gene array. A de-creased level of SNAP-25 was detected by quantitative real time-polymerase chain reaction and western blot assay at 1, 3, 7, 14 and 28 days post injury. SNAP-25 was localized in the cytoplasm of neurons of the anterior and posterior horns, which are involved in locomotor and sensory functions. Our data suggest that reduced levels of SNAP-25 are associated with sensory and locomotor functions in rats with spinal cord contusion injury.     

Rat hair follicle stem cells differentiate and promote recovery following spinal cord injury

Emerging studies of treating spinal cord injury （SCI） with adult stem cells led us to evaluate the effects of transplantation of hair follicle stem cells in rats with a compression-induced spinal cord lesion. Here, we proposed a hypothesis that rat hair follicle stem cell transplantation can promote the recovery of injured spinal cord. Compression-induced spinal cord injury was induced in Wistar rats in this study. The bulge area of the rat vibdssa follicles was isolated, cultivated and characterized with nestin as a stem cell marker. 5-Bromo-2＇-deoxyuridine （BrdU） labeled bulge stem cells were transplanted into rats with spinal cord injury. Immunohistochemical staining results showed that some of the grafted cells could survive and differentiate into oligodendrocytes （receptor-interacting protein positive cells） and neuronal-like cells （~lll-tubulin positive cells） at 3 weeks after transplantation. In addition, recovery of hind limb locomotor function in spinal cord injury rats at 8 weeks following cell transplantation was assessed using the Basso, Beattie and Bresnahan （BBB） locomotor rating scale. The results demon- strate that the grafted hair follicle stem cells can survive for a long time period in vivo and differentiate into neuronal- and glial-like cells. These results suggest that hair follicle stem cells can promote the recovery of spinal cord injury.     

Inhibition of inflammatory cytokines after early decompression may mediate recovery of neurological function in rats with spinal cord injury

A variety of inflammatory cytokines are involved in spinal cord injury and influence the recovery of neuronal function. In the present study, we established a rat model of acute spinal cord injury by cerclage. The cerclage suture was released 8 or 72 hours later, to simulate decompression surgery. Neurological function was evaluated behaviorally for 3 weeks after surgery, and tumor necrosis factor α immunoreactivity and apoptosis were quantified in the region of injury. Rats that underwent decompression surgery had significantly weaker immunoreactivity of tumor necrosis factor α and significantly fewer apoptotic cells, and showed faster improvement of locomotor function than animals in which decompression surgery was not performed. Decompression at 8 hours resulted in significantly faster recovery than that at 72 hours. These data indicate that early decompression may improve neurological function after spinal cord injury by inhibiting the expression of tumor necrosis factor α.     

Large-conductance Ca2+-activated K+ channel involvement in suppression of cerebral ischemia/reperfusion injury after electroacupuncture atShuigou (GV26) acupoint in rats

Excess activation and expression of large-conductance Ca2+-activated K+ channels (BKCa channels) may be an important mechanism for delayed neuronal death after cerebral ischemia/reperfusion injury. Electroacupuncture can regulate BKCa channels after cerebral ischemia/reperfusion injury, but the precise mechanism remains unclear. In this study, we established a rat model of cerebral ischemia/reperfusion injury. Model rats received electroacupuncture of 1 mA and 2 Hz atShuigou (GV26) for 10 minutes, once every 12 hours for a total of six times in 72 hours. We found that in cerebral ischemia/reperfusion injury rats, ischemic changes in the cerebral cortex were mitigated after electroacupuncture. Moreover, BKCa channel protein and mRNA expression were reduced in the cerebral cortex and neurological function noticeably improved. These changes did not occur after electroacupuncture at a non-acupoint (5 mm lateral to the left side of Shuigou). Thus, our ifndings indicate that electroacupuncture atShuigou improves neurological function in rats following cerebral ischemia/reperfu-sion injury, and may be associated with down-regulation of BKCa channel protein and mRNA expression. Additionally, our results suggest that theShuigou acupoint has functional speciifcity.     

Effect of electroacupuncture on the mRNA and protein expression of Rho-A and Rho-associated kinase Ⅱ in spinal cord injury rats

Electroacupuncture is beneficial for the recovery of spinal cord injury, but the underlying mechanism is unclear. The Rho/Rho-associated kinase(ROCK) signaling pathway regulates the actin cytoskeleton by controlling the adhesive and migratory behaviors of cells that could inhibit neurite regrowth after neural injury and consequently hinder the recovery from spinal cord injury. Therefore, we hypothesized electroacupuncture could affect the Rho/ROCK signaling pathway to promote the recovery of spinal cord injury. In our experiments, the spinal cord injury in adult Sprague-Dawley rats was caused by an impact device. Those rats were subjected to electroacupuncture at Yaoyangguan(GV3), Dazhui(GV14), Zusanli(ST36) and Ciliao(BL32) and/or monosialoganglioside treatment. Behavioral scores revealed that the hindlimb motor functions improved with those treatments. Real-time quantitative polymerase chain reaction, fluorescence in situ hybridization and western blot assay showed that electroacupuncture suppressed the m RNA and protein expression of Rho-A and Rho-associated kinase Ⅱ(ROCKⅡ) of injured spinal cord. Although monosialoganglioside promoted the recovery of hindlimb motor function, monosialoganglioside did not affect the expression of Rho-A and ROCKⅡ. However, electroacupuncture combined with monosialoganglioside did not further improve the motor function or suppress the expression of Rho-A and ROCKⅡ. Our data suggested that the electroacupuncture could specifically inhibit the activation of the Rho/ROCK signaling pathway thus partially contributing to the repair of injured spinal cord. Monosialoganglioside could promote the motor function but did not suppress expression of Rho A and ROCKⅡ. There was no synergistic effect of electroacupuncture combined with monosialoganglioside.     

Electrophysiological functional recovery in a rat model of spinal cord hemisection injury following bone marrow-derived mesenchymal stem cell transplantation under hypothermia

Following successful establishment of a rat model of spinal cord hemisection injury by resecting right spinal cord tissues, bone marrow stem cells were transplanted into the spinal cord lesions via the caudal vein while maintaining rectal temperature at 34 ± 0.5°C for 6 hours (mild hypothermia). Hematoxylin-eosin staining showed that astrocytes gathered around the injury site and formed scars at 4 weeks post-transplantation. Compared with rats transplanted with bone marrow stem cells under normal temperature, rats transplanted with bone marrow stem cells under hypothermia showed increased numbers of proliferating cells (bromodeoxyuridine-positive cells), better recovery of somatosensory-evoked and motor-evoked potentials, greater Basso, Beattie, and Bresnahan locomotor rating scores, and an increased degree of angle in the incline plate test. These findings suggested that hypothermia combined with bone marrow mesenchymal stem cells transplantation effectively promoted electrical conduction and nerve functional repair in a rat model of spinal cord hemisection injury.     

A progressive compression model of thoracic spinal cord injury in mice: function assessment and pathological changes in spinal cord

Non-traumatic injury accounts for approximately half of clinical spinal cord injury, including chronic spinal cord compression. However, previous rodent spinal cord compression models are mainly designed for rats, few are available for mice. Our aim is to develop a thoracic progressive compression mice model of spinal cord injury. In this study, adult wild-type C57BL/6 mice were divided into two groups: in the surgery group, a screw was inserted at T9 lamina to compress the spinal cord, and the compression was increased by turning it further into the canal (0.2 mm) post-surgery every 2 weeks up to 8 weeks. In the control group, a hole was drilled into the lamina without inserting a screw. The results showed that Basso Mouse Scale scores were lower and gait worsened. In addition, the degree of hindlimb dysfunction in mice was consistent with the degree of spinal cord compression. The number of motor neurons in the anterior horn of the spinal cord was reduced in all groups of mice, whereas astrocytes and microglia were gradually activated and proliferated. In conclusion, this progressive compression of thoracic spinal cord injury in mice is a preferable model for chronic progressive spinal cord compression injury.     

Basic fibroblast growth factor attenuates the degeneration of injured spinal cord motor endplates**

The distal end of the spinal cord and neuromuscular junction may develop secondary degeneration and damage following spinal cord injury because of the loss of neural connections. In this study, a rat model of spinal cord injury, established using a modified Allen’s method, was injected with basic fibroblast growth factor solution via subarachnoid catheter. After injection, rats with spinal cord injury displayed higher scores on the Basso, Beattie and Bresnahan locomotor scale. Motor function was also well recovered and hematoxylin-eosin staining showed that spinal glial scar hyperplasia was not apparent. Additionally, anterior tibial muscle fibers slowly, but progressively, atrophied. Immunohistochemical staining showed that the absorbance values of calcitonin gene related peptide and acetylcholinesterase in anterior tibial muscle and spinal cord were similar, and injection of basic fibroblast growth factor increased this absorbance. Results showed that after spinal cord injury, the distal motor neurons and motor endplate degenerated. Changes in calcitonin gene related peptide and acetylcholinesterase in the spinal cord anterior horn motor neurons and motor endplate then occurred that were consistent with this regeneration. Our findings indicate that basic fibroblast growth factor can protect the endplate through attenuating the decreased expression of calcitonin gene related peptide and acetylcholinesterase in anterior horn motor neurons of the injured spinal cord.     

Injection of bone marrow mesenchymal stem cells by intravenous or intraperitoneal routes is a viable alternative to spinal cord injury treatment in mice

In spite of advances in surgical care and rehabilitation,the consequences of spinal cord injury(SCI)ar still challenging.Several experimental therapeutic strategies have been studied in the SCI field,and recen advances have led to the development of therapies that may act on the inhibitory microenvironment.As sorted lineages of stem cells are considered a good treatment for SCI.This study investigated the effect o systemic transplantation of mesenchymal stem cells(MSCs)in a compressive SCI model.Here we presen results of the intraperitoneal route,which has not been used previously for MSC administration after comveI.WeafC57BL/6nt lmy atb spinal cord compression for 1 minute with a 30-g vascular clip.The animals were divided into five groups DMEM at 7 days after SCI),DMEM i.v.(intravenous injection of 500μL of DMEM at 7 days after SCI)The effects of MSCs transplantation in white matter sparing were analyzed by luxol fast blue staining.Th number of preserved fibers was counted in semithin sections stained with toluidine blue and the presenc of trophic factors was analyzed by immunohistochemistry.In addition,we analyzed the locomotor perfor mance with Basso Mouse Scale and Global Mobility Test.Our results showed white matter preservation and a larger number of preserved fibers in the MSC groups than in the DMEM groups.Furthermore,th MSC groups had higher levels of trophic factors(brain-derived neurotrophic factor,nerve growth factor neurotrophin-3 and neurotrophin-4)in the spinal cord and improved locomotor performance.Our result indicate that injection of MSCs by either intraperitoneal or intravenous routes results in beneficial out comes and can be elected as a choice for SCI treatment.     

Senegenin inhibits neuronal apoptosis after spinal cord contusion injury

Senegenin has been shown to inhibit neuronal apoptosis, thereby exerting a neuroprotective effect. In the present study, we established a rat model of spinal cord contusion injury using the modiifed Allen’s method. Three hours after injury, senegenin (30 mg/g) was injected into the tail vein for 3 consecutive days. Senegenin reduced the size of syringomyelic cavities, and it substantially reduced the number of apop-totic cells in the spinal cord. At the site of injury, Bax and Caspase-3 mRNA and protein levels were decreased by senegenin, while Bcl-2 mRNA and protein levels were increased. Nerve ifber density was increased in the spinal cord proximal to the brain, and hindlimb motor function and electrophysiological properties of rat hindlimb were improved. Taken together, our results suggest that senegenin exerts a neuroprotective effect by suppressing neuronal apoptosis at the site of spinal cord injury.     

Cell transplantation for the treatment of spinal cord injury–bone marrow stromal cells and choroid plexus epithelial cells

Transplantation of bone marrow stromal cells(BMSCs) enhanced the outgrowth of regenerating axons and promoted locomotor improvements of rats with spinal cord injury(SCI).BMSCs did not survive long-term,disappearing from the spinal cord within 2–3 weeks after transplantation.Astrocyte-devoid areas,in which no astrocytes or oligodendrocytes were found,formed at the epicenter of the lesion.It was remarkable that numerous regenerating axons extended through such astrocyte-devoid areas.Regenerating axons were associated with Schwann cells embedded in extracellular matrices.Transplantation of choroid plexus epithelial cells(CPECs) also enhanced axonal regeneration and locomotor improvements in rats with SCI.Although CPECs disappeared from the spinal cord shortly after transplantation,an extensive outgrowth of regenerating axons occurred through astrocyte-devoid areas,as in the case of BMSC transplantation.These findings suggest that BMSCs and CPECs secret neurotrophic factors that promote tissue repair of the spinal cord,including axonal regeneration and reduced cavity formation.This means that transplantation of BMSCs and CPECs promotes "intrinsic" ability of the spinal cord to regenerate.The treatment to stimulate the intrinsic regeneration ability of the spinal cord is the safest method of clinical application for SCI.It should be emphasized that the generally anticipated long-term survival,proliferation and differentiation of transplanted cells are not necessarily desirable from the clinical point of view of safety.     

BMS-345541对脊髓损伤后大鼠NF-κB表达及运动功能的影响

This study sought to elucidate the changes of nuclear factor kappa B (NF-κB) expression and locomotor function of hind limb after subdural injection of BMS-345541 was applied in rats with acute spinal cord injury.The results indicated that BMS-345541 treatment reduced the expression of NF-kB at 24 hours after injury,compared with normal saline-tre ated rats.This treatment also led to a significant improvement in locomotor functional recovery at 14 days after injury.Overall,the findings demonstrated that BMS-345541 significantly ameliorated spinal cord injury-induced hind limb dysfunction by inhibiting the expression of NF-kB after spinal cord injury.     

Methylprednisolone promotes recovery of neurological function after spinal cord injury:association with Wnt/β-catenin signaling pathway activation

Some studies have indicated that the Wnt/β-catenin signaling pathway is activated following spinal cord injury,and expression levels of specific proteins,including low-density lipoprotein receptor related protein-6 phosphorylation,β-catenin,and glycogen synthase kinase-3β,are significantly altered.We hypothesized that methylprednisolone treatment contributes to functional recovery after spinal cord injury by inhibiting apoptosis and activating the Wnt/β-catenin signaling pathway.In the current study,30 mg/kg methylprednisolone was injected into rats with spinal cord injury immediately post-injury and at 1 and 2 days post-injury.Basso,Beattie,and Bresnahan scores showed that methylprednisolone treatment significantly promoted locomotor functional recovery between 2 and 6 weeks post-injury.The number of surviving motor neurons increased,whereas the lesion size significantly decreased following methylprednisolone treatment at 7 days post-injury.Additionally,caspase-3,caspase-9,and Bax protein expression levels and the number of apoptotic cells were reduced at 3 and 7days post-injury,while Bcl-2 levels at 7 days post-injury were higher in methylprednisolone-treated rats compared with saline-treated rats.At 3 and 7 days post-injury,methylprednisolone up-regulated expression and activation of the Wnt/β-catenin signaling pathway,including low-density lipoprotein receptor related protein-6 phosphorylation,β-catenin,and glycogen synthase kinase-3β phosphorylation.These results indicate that methylprednisolone-induced neuroprotection may correlate with activation of the Wnt/β-catenin signaling pathway.