[Influence of electroacupuncture of "Dazhui"（GV14） and "Mingmen" （GV4） on expressions of NL1 and NF-200 in spinal cord injury rats].

OBJECTIVE: To observe the effect of electroacupuncture(EA) on activities of A2 type astrocytes(A2s)and A1 type astrocytes (A1s) , expressions of neurofilament protein 200 (NF-200, a marker of axon regeneration), nexin 1(NL1, a marker of synaptic regeneration), and regeneration of Nissl bodies in rats with spinal cord injury (SCI), so as to explore its mechanisms underlying improvement of SCI. METHODS: A total of 75 male SD rats were rando-mized into sham operation, model, antibody neutralizing (AN), EA and EA+AN groups, with 15 rats in each group. The SCI model was established by using an infinite field impactor to deliver an about 200 k dyne weight onto the exposed spinal cord after making a dorsal laminectomy at vertebral level T10. EA (2 Hz, 1 mA) was applied to"Dazhui"(GV14) and "Mingmen"(GV4) for 20 min, once daily for 28 days. After modeling, intraspinal injection of neutralizing antibodies IL-1α, TNF-α and complement 1q (C1q, 2 µL) to the injured spinal locus for inhibition of A1 type astrocytes (A1s) was conducted on the 1st, 7th , 14th and 21st day for rats of AN and EA+AN groups. BBB rating scale was used to evaluate hindlimb locomotor function on day 1, 7, 14, 21 and 28 after modeling. The activation of A2s (its specific marker S100a10), astrocyte (its specific marker glial fibrillary acidic protein, GFAP), and A1s (its specific marker C3) in the spinal cord was detected by immunofluorescence, and the protein expressions of NF-200 and NL1 in the spinal cord detected by Western blot and immunohistochemistry, separately, and the neuronal regeneration was observed after Nissl staining. RESULTS: After SCI, the BBB scores at 1 , 7, 14, 21 and 28 day, and the immunoactivity of NL1 and NF-200 were significantly decreased (P<0.01), and the fluorescence intensity of double labelled S100a10 (A2s)/GFAP and C3, and the expression of NF-200 were considerably increased in the model group (P<0.05, P<0.01). In contrast to the model group, the BBB scores at 7, 14, 21 and 28 day, and the immunoactivity of NL1 and NF-200, and the fluorescence intensity of A2s/GFAP in the AN, EA and AN+EA groups, and the expressions of NL1 in the EA and AN+EA groups, and expression of NF-200 protein in the AN+EA group were evidently increased (P<0.05, P<0.01), and the fluorescence intensity of C3 was strikingly decreased in the EA group (P<0.01). The effect of AN+EA was significantly superior to that of single AN and EA in increasing BBB scores at 14, 21 and 28 day, and in up-regulating the immunoactivity of NF-200(P<0.01, P<0.05). Nissl staining showed damaged structure of the gray matter of the spinal cord, atrophy of the Nissl body, and pyknosis of neurons, which was milder in the AN and EA groups, particularly in the AN+EA group. CONCLUSION: EA at GV14 and GV4 may promote activation of A2s and promote regeneration of axons and synapses in SCI model rats.

Neuronal differentiation of rat bone marrow mesenchymal stem cells vialentivirus-mediated bone morphogenetic protein 7 transfection / 中国组织工程研究

BACKGROUND: Bone marrow mesenchymal stem cells have the potential to differentiate into neuron-like cells, which have been listed as the preferred stem cells for the treatment of spinal cord injury. However, due to their low differentiation efficiency, it is particularly important to find a factor with high induction ability. Based on literature review and our previous studies, it is speculated that bone morphogenetic protein 7 (BMP-7) gene may play a vital role in promoting the differentiation of bone marrow mesenchymal stem cells into neuron-like cells. OBJECTIVE: To investigate the differentiation of rat bone marrow mesenchymal stem cells into neurons induced by BMP-7 lentivirus vector transfection. METHODS: Bone marrow mesenchymal stem cells of Sprague-Dawley rats were cultured by whole bone marrow adherence method, and then were transfected with LV-GFP when multiplicities of infection were 50, 25, 10, and 1. Green fluorescent protein expression was observed using fluorescence inversion microscope in each group at 3 days after transfection, to confirm the best multiplicity of infection. Passage 3 bone marrow mesenchymal stem cells were divided into blank control group (routine culture), LV-GFP group, and LV-BMP-7-GFP group, followed by transfection at the best multiplicity of infection. After 24, 48, 72, 96, and 120 hours of transfection, MTT assay was used to detect cell survival rate in each group. Immunocytochemical assay was used to detect the expression of nerve cell markers (neurofilament protein 200, synaptophysin-1) after 3 days of transfection. RESULTS AND CONCLUSION: (1) After 3 days of LV-GFP transfection, GFP-positive cells were observed under fluorescence microscopy when multiplicities of infection were 10, 25, and 50, whereas no GFP-positive cells were found when the multiplicity of infection was 1. The average fluorescence intensity was the highest when the multiplicity of infection was 10 (P < 0.05), indicating that multiplicity of infection=10 had the best infection effect. (2) Immunocytochemical results showed that the expression of neurofilament-200 and synaptophysin-1 was negative in the blank control group and LV-GFP group, but positive in the LV-BMP-7-GFP group. The cell body and axon in the LV-BMP-7-GFP group were dyed bright brown. In summary, lentivirus-mediated BMP-7 transfection can promote the differentiation of rat bone marrow mesenchymal stem cells into neuron-like cells.

[Repair effect of bFGF combined with bone marrow mesenchymal stem cells on spinal cord injury in rats].

OBJECTIVE: To observe the repair effect of bone marrow mesenchymal stem cells (BMSCs) combined with basic fibroblast growth factor (bFGF) on spinal cord injury in rats and explore its mechanism. METHODS: SD rat BMSCs were obtained by serum culture technique. Eighty healthy 6-week-old male SD rats(weight about 240 g) were randomly divided into 4 groups with 20 each. The sham operation group underwent simple laminectomy without damaging spinal cord and was kept in the same condition as the other 3 groups. The other 3 groups underwent left T9 spinal cord hemisection to establish spinal cord injury model. After 9 days of modeling the local transplantation was performed. The Control group was implanted with gelatin sponge containing normal saline. The BMSCs transplantation group was implanted with gelatin sponge containing BMSCs. The bFGF+BMSCs transplantation group was implanted with gelatin sponge containing bFGF+BMSCs. After 4 and 8 weeks, the expression of NF-200 and GFAP in injured spinal cord tissue was analyzed by Western blotting and the recovery of hind limb function was evaluated by Basso Beattie Bresnahan(BBB) motor function score scale. RESULTS: The BBB scores of BMSCs transplantation group and bFGF+BMSCs transplantation group were better than control group at 4 and 8 weeks after operation (P<0.05) and there was significant difference between bFGF+BMSCs transplantation group and BMSCs transplantation group (P<0.05). After 4 and 8 weeks postoperatively, NF-200 expression was minimal in control group and only a small amount was expressed in BMSCs transplantation group while in bFGF+BMSCs transplantation group NF-200 was highly expressed(P<0.05). GFAP expression was high in control group, middle in BMSCs transplantation group and low in bFGF BMSCs transplantation group(P<0.05). There was significant difference between bFGF+BMSCs transplantation group, BMSCs transplantation group and control group(P<0.05). CONCLUSIONS: The combined transplantation of BMSCs and bFGF can repair the spinal cord injury in rats. The mechanism may be related to the decrease of GFAP expression and the increase of NF-200 expression.

Repair effect of bFGF combined with bone marrow mesenchymal stem cells on spinal cord injury in rats / 中国骨伤

OBJECTIVE@#To observe the repair effect of bone marrow mesenchymal stem cells (BMSCs) combined with basic fibroblast growth factor (bFGF) on spinal cord injury in rats and explore its mechanism.@*METHODS@#SD rat BMSCs were obtained by serum culture technique. Eighty healthy 6-week-old male SD rats(weight about 240 g) were randomly divided into 4 groups with 20 each. The sham operation group underwent simple laminectomy without damaging spinal cord and was kept in the same condition as the other 3 groups. The other 3 groups underwent left T9 spinal cord hemisection to establish spinal cord injury model. After 9 days of modeling the local transplantation was performed. The Control group was implanted with gelatin sponge containing normal saline. The BMSCs transplantation group was implanted with gelatin sponge containing BMSCs. The bFGF+BMSCs transplantation group was implanted with gelatin sponge containing bFGF+BMSCs. After 4 and 8 weeks, the expression of NF-200 and GFAP in injured spinal cord tissue was analyzed by Western blotting and the recovery of hind limb function was evaluated by Basso Beattie Bresnahan(BBB) motor function score scale.@*RESULTS@#The BBB scores of BMSCs transplantation group and bFGF+BMSCs transplantation group were better than control group at 4 and 8 weeks after operation (<0.05) and there was significant difference between bFGF+BMSCs transplantation group and BMSCs transplantation group (<0.05). After 4 and 8 weeks postoperatively, NF-200 expression was minimal in control group and only a small amount was expressed in BMSCs transplantation group while in bFGF+BMSCs transplantation group NF-200 was highly expressed(<0.05). GFAP expression was high in control group, middle in BMSCs transplantation group and low in bFGF BMSCs transplantation group(<0.05). There was significant difference between bFGF+BMSCs transplantation group, BMSCs transplantation group and control group(<0.05).@*CONCLUSIONS@#The combined transplantation of BMSCs and bFGF can repair the spinal cord injury in rats. The mechanism may be related to the decrease of GFAP expression and the increase of NF-200 expression.

Local injection of bone morphogenetic protein 7 promotes neuronal regeneration and motor function recovery after acute spinal cord injury.

After spinal cord injury, the number of glial cells and motor neurons expressing bone morphogenetic protein 7 (BMP7) increases, indicating that upregulation of BMP7 can promote nerve repair. We, therefore, tested whether direct injection of BMP7 into acutely injured rat spinal cord can affect neurological recovery. Allen's impactor was used to create spinal cord injury at T10. The injury site was then injected with 50 ng BMP7 (BMP7 group) or physiological saline (control group) for 7 consecutive days. Electrophysiological examination showed that the amplitude of N1 in motor evoked potentials (MEP) decreased after spinal cord injury. At 8 weeks post-operation, the amplitude of N1 in the BMP7 group was remarkably higher than that at 1 week post-operation and was higher than that of the control group. Basso, Beattie, Bresnahan scale (BBB) scores, hematoxylin-eosin staining, and western blot assay showed that at 1, 2, 4 and 8 weeks post-operation, BBB scores were increased; Nissl body staining was stronger; the number of Nissl-stained bodies was increased; the number of vacuoles gradually decreased; the number of synapses was increased; and the expression of neuronal marker, neurofilament protein 200, was increased in the hind limbs of the BMP7 group compared with the control group. Western blot assay showed that the expression of GFAP protein in BMP7 group and control group did not change significantly and there was no significant difference between the BMP7 and control groups. These data confirmed that local injection of BMP7 can promote neuronal regeneration after spinal cord injury and promote recovery of motor function in rats.

Effects of quercetin on glial scar formation and axonal regeneration after acute spinal cord injury in rats and its association with p38 mitogen activated protein kinase signal pathway / 中华创伤骨科杂志

Objective To investigate the effects of quercetin on glial scar formation and axonal regeneration after spinal cord injury (SCI) and its association with the p38 mitogen activated protein kinase (MAPK) signal pathway.Methods 128 female Sprague-Dawley (SD) rats were randomly divided into a control group (SCI + saline),an intervention group (SCI + quercetin + anisomycin),a treatment group (SCI + quercetin) and a sham-operation group (n =32).Basso Beattie Bresnahan (BBB) assessment and footprint analysis of the hind limb were performed on days 1,3,7,14,21 and 28 postoperation in each group.The expression levels of p38MAPK,phosphorylation p38MAPK,glial fibrillary acidic protein (GFAP) and neurofilament protein-200 (NF-200) were detected by Western blot.The numbers of GFAP and NF-200 positive staining cells in the injured spinal cord in each group were detected by immunohistochemistry.Results The BBB scores in the treatment group were significantly higher than in the intervention and control groups at each time point after SCI except on day 3 postoperation (P ＜ 0.05).The expression levels of phosphorylation p38MAPK protein in each SCI group were significantly higher than in the sham-operation group on days 3 and 7 postoperation (P ＜ 0.05).The expression levels of phosphorylation p38MAPK protein in the treatment group were significantly lower than in the control and intervention groups on days 3,7 and 14 postoperation (P ＜ 0.05),but there was no significant difference on day 28 postoperation among all the groups (P ＞ 0.05).The numbers of NF-200 and GFAP positive staining cells were significantly greater than in the sham-operation group at each time point postoperation (P ＜ 0.05);the NF-200 positive staining cells in the treatment group were significantly increased in comparison with the control and intervention groups (P ＜ 0.05);the GFAP positive staining cells in the treatment group were significandy fewer than in the control and intervention groups on days 7,14 and 28 postoperation (P ＜ 0.05).Conclusions Quercetin may have protective effects against acute SCI by decreasing glial scar formation,increasing axonal regeneration and promoting recovery of locomotor and nerve function in rats.The effects may be correlated with inhibition of the p38MAPK signal pathway.

Functional recovery after acute intravenous administration of human umbilical cord mesenchymal stem cells in rats with cerebral ischemia-reperfusion injury.

Cell therapy is a potential approach for treatment of strokes. Mesenchymal stem cells (MSCs) are a potential cell source for clinical use because they are safe and easy to obtain. A peptide solution can promote neural regeneration. Previously, such a solution was stereotactically injected into the brain of rats with cerebral infarction, resulting in improvement in the animal's neurological function and reduction in the infarction volume, but the injury was relatively severe. The current study established a rat model of cerebral ischemia-reperfusion (I/R) injury. MSCs isolated from Wharton's jelly of human umbilical cords (HUMSCs) were injected intravenously immediately after cerebral I/R injury(3 × 10(6) cells per rat). Twenty-four h and 14 d after surgery, animal behavior was evaluated using the Rogers test and infarct lesion volume was evaluated by 2,3,5-triphenyltetrazolium chloride staining. Fourteen d after surgery, brain tissues were collected at 14 d to study migration/implantation of HUMSCs, cellular proliferation, neural regeneration and astrocyte activation. Compared to cerebral I/R injury alone, HUMSC treatment improved function at 14 d after surgery, with no reduction in infarct volume or migration or implantation of cells into the damaged brain areas. Nevertheless, 14 d after surgery, HUMSC administration increased cellular proliferation and the level of neurofilament 200 level and decreased the level of glial fibrillary acidic protein. After cerebral I/R injury, acute intravenous administration of HUMSCs could promote recovery by activating endogenous neural regeneration and inhibiting astrocyte activation, without migration and implantation directly into lesions.