Search

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 1 st , 7 th , 14 th and 21 st 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.

Objective: To study the expressions of microRNA-221 (miR-221) and the protein of phosphatase and tension protein homologue (PTEN) in the proximal and distal stumps after sciatic nerve injury in rats and their correlation with the repair of peripheral nerve injury, so as to provide a new target for clinical diagnosis of peripheral nerve injury., Methods: Ninety-six male Sprague-Dawley rats of SPF grade were selected to establish sciatic nerve injury models. Twenty-four rats were sacrificed at 0 (immediately after operation), 1, 4, and 7 days after operation. The proximal and distal sciatic nerve fragments were taken under aseptic conditions. The expression of miR-221 was detected by real-time fluorescent quantitative PCR, and the expression of PTEN protein was detected by Western blot and immunofluorescent staining. The relationship between miR-221 and PTEN was verified by dual-luciferase reporter gene. At the same time, the ultrastructure of nerve stump was observed by transmission electron microscopy., Results: The results of real-time fluorescent quantitative PCR, Western blot, and immunofluorescence staining showed that the relative expression of miR-221 in the proximal and distal stumps increased gradually with time, and the relative expression of PTEN protein decreased gradually, and the differences between different time points after operation were significant ( P <0.05). At 1, 4, and 7 days after operation, the relative expression of miR-221 in proximal stump was significantly higher than that in distal stump, and the relative expression of PTEN protein in proximal stump was significantly lower than that in distal stump ( P <0.05). Dual-luciferase reporter gene suggested that PTEN was the target for miR-221. Transmission electron microscopy observation showed that the normal morphological structure was observed at 0 day after operation, and the proliferation of Schwann cells and degeneration of axons and myelin sheaths gradually increased with time. There was no significant difference between proximal and distal stumps at 1 day after operation. At 4 and 7 days, Schwann cells proliferated more in proximal stump than in distal stump, and the degeneration of axons and myelin sheaths was less., Conclusion: After sciatic nerve injury in rats, the up-regulation of the miR-221 expression targets the down-regulation of PTEN expression, which results in the difference of expression levels of miR-221 and PTEN in proximal and distal stumps. This phenomenon may play a role in promoting nerve repair after peripheral nerve injury.

MicroRNA is a class of endogenous non-coding RNA, which regulates the expression of target genes at the post-transcriptional level. In recent years, researches have shown that microRNA expresses specifically and plays an important role in the nervous system under physiological condition. Furthermore, the distribution and expression level of microRNA were changed after the injury of nervous system, which presents important effect on nerve regeneration. This effect of microRNA is significant to the clinical diagnosis and treatment on the injury of nervous system.

Objective: To explore the changes in the expressions of glial fibrillary acidic protein (GFAP) and growth- associated protein-43 (GAP-43) in retinal ganglial cells after neural transplantation., Methods: Thirty-nine rats were randomized into normal control group, nerve amputation group and nerve amputation with peripheral nerve transplantation group. Immunohistochemistry was used to detect the changes in the expressions of GFAP and GAP-43 at different time points after the operations, and real-time PCR was employed to detect the mRNA expressions of 13 genes in the retinal ganglial cells of the rats., Results: Immunohistochemistry showed obviously increased GFAP expressions in the retina following the nerve amputation. GFAP expression was down-regulated while GAP-43 expression upregulated in the retinal ganglial cells after peripheral nerve transplantation. Real-time PCR results showed that 5 days after the operations, retinal GFAP and GAP-43 expressions increased significantly in the nerve amputation group and peripheral nerve transplantation groups as compared with those in the control group, but GAP-43 expression decreased significantly in the former two groups afterwards., Conclusion: The regenerated retina may adjust the production of GFAP. The retinal ganglial cells express GAP-43 during retinal regeneration. Up-regulation of the expression of GAP-43 provides the evidence for nerve regeneration following the nerve transplantation.

Objective: To enhance the neurotrophic effect of Schwann cells, promote optic nerve regeneration, and simplify the means of observation, the CNTF-GFP fusion plasmid was constructed and transferred into Schwann cells by electroporation., Methods: It was an experimental study. Plasmid pcDNA3. 1/CNTF-GFP was constructed and verified by auto-sequence. Cultured Schwann cells were transfected by electroporation. The transfection efficiency was counted by flow cytometry, the transcription of the gene was evaluated by RT-PCR, and the expression of protein was observed by fluorescence microsphere and cell immunofluorescence., Results: CNTF-GFP plasmid was verified correctly. After electroporation, the green fluorescence of gene-transfected Schwann cells can be seen at 3 hours later, increased at 12 hours later, reached the peak during 24 h to 72 h later, and still could be seen at 7 days later. The transfection efficiency was evaluated at 44.93% +/- 12.92% by flow cytometry. The transcription of CNTF gene and the expression of CNTF protein have been proven by RT-PCR and cell immunofluorescence., Conclusions: CNTF-GFP plasmid was constructed correctly and Schwann cells were transfected by electroporation successfully and CNTF-GFP fusion protein can be expressed well, which became a good foundation for our future's study on the transplantation of gene-modified Schwann cells to promote optic nerve regeneration.

Objective: To determine whether overexpression of Bcl-2 is sufficient to support optic nerve regeneration in mouse in the early neonatal stage, when the brain environment remains permissive for axonal growth, and whether regenerating axons follow the guidance cues to reach the appropriate targets in the brain., Methods: Optic nerve crush was performed in wild-type (C57BL/6J) and Bcl-2 transgenic mouse pups at 3 days after birth (P3). To assess optic nerve regeneration, mouse pups were allowed to survive for 1-4 days post-surgery. An anterograde tracer-cholera toxin B subunit (CTB) and anti-GAP-43 immunofluorescence were applied to reveal regenerating axons., Results: In wild-type mice, severed optic nerves failed to regenerate, and most severed axons retracted within 24 hour post-surgery. In contrast, in all of the Bcl-2 transgenic mice examined, optic nerves regenerated robustly over long distances and reached their brain targets in 4 days after optic nerve crush. However, the majority of regenerating axons, appearing to follow the existing optic tract and visual pathways, entered the visual targets that were ipsilateral to the nerve injury. Much less number of regenerating axons innervated their contralateral visual targets., Conclusion: Overexpression of Bcl-2 is sufficient to support the intrinsic growth mechanisms of retinal ganglion cell axons and enables robust optic nerve regeneration over long distances in vivo if the injury is incurred at 3 days after birth. However, the regenerating axons appear to follow the existing CNS pathways and innervate uninjured brain areas.

Objective: To determine the age-related expression features of slit2 mRNA at various intervals at the proximal and distal parts of sciatic nerve during re-innervation after severance injury, and to investigate the molecular biological reason why co-contraction occurs more commonly in the young rats than in the adults after peripheral nerve injury., Methods: The right sciatic nerve was transected sharply at the midthigh in the young and adult rats, 28 in each group. The proximal and distal stumps were inserted into a medical silicone tube with a gap of 3 mm. Before the injury, and 1, 3 and 7 days (6 rats at each interval) after sciatic nerve severance injury, both stumps of the sciatic nerve were harvested. Reverse transcription polymerase chain reaction (RT-PCR) was use to examine the expression of slit2 mRNA in the young and adult group rats. In situ hybridization was done to detect the cell location of slit2 mRNA expression in young and adult rats, 4 rats in each group, 2 rats used before the injury and 2 rats used on the postoperative third day., Results: RT-PCR results suggested that slit2 mRNA at both stumps was significantly lower in the young group of rats than in the adult except that at the proximal part on the postoperative 7th day. There was statistically significant difference (P < 0.01). In situ hybridization studies indicated that the slit2 mRNA was specifically expressed in the cytoplasm of Schwann cells both in the young and adult rats before injury and 3 days after injury., Conclusion: The expression of slit2 mRNA by Schwann cells in the young rats is lower than that in the adult rats after peripheral nerve injury. These findings suggests the lower expression of chemotactic factor (slit2) by Schwann cells in the young group is likely to be the molecular biological reason for the higher incidence of co-contraction between agonists and antagonists in the younger rats.

After crushing the right sciatic nerve of rat, the contents of both tubulin and beta tubulin mRNA increased significantly in the ventral horns of spinal cord. Analysis of tubulin in bilateral ventral horns by SDS-PAGE showed that tubulin in the injured side increased by 17%-121% of the control side. The results of in situ hybridization indicated with the signals of hybridization with beta tubulin cDNA probes were 27%-70% higher in motoneurons of the ventral horns in the injury side than the control. Northern blotting analysis of tubulin mRNA in ventral horns also revealed an increase of tubulin gene expression.