ABSTRACT
This paper was aimed to investigate the effects of emodin on oxidative stress and inflammatory response in rats with acute spinal cord injury（SCI）, and to explore the protective mechanism of emodin on neurons after SCI. Rat SCI models were established using a modified Allen's method. One hundred and ninety five Sprague-Dawley rats were randomly divded into sham (group A), model (group B), emodin group of 20 mg·kg⁻¹(group C), emodin group of 40 mg·kg⁻¹(group D), emodin group of 80 mg·kg⁻¹(group E). Functional recovery was evaluated using the Basso-Beattie-Bresnahan (BBB) scale and inclined plate test on the 3rd, 7th, 14th and 28th day. On the 7th day after SCI, neuron nylon body was observed with toluidine blue staining. The changes of myelinated nerve fibers were observed by transmission electron microscope. Nrf2, HO-1, NQO1, GFAP, NF-κB protein were detected by Western blot. The content of TNF-α, IL-1β, IL-6 were detected by ELISA. Immunofluorescence staining was performed to observe the expression of the GFAP, NG2 and ED-1. The BBB and inclined plate scores of group C, D and E were higher than group B on the 7th, 14th, 28th day,the difference was statistically significant (<0.05). On the 7th day, Nylon Body of group B and C started to fuse,the fusion of group D and E were significantly alleviated than group B and C. Transmission electron microscope showed that the changes of demyelination were obvious in group B and C, group D and E were significantly improved than group B and C. Western blot showed that Nrf2, HO-1, GFAP, NF-κB protein expression,group C, D, E compared with group B, NQO1 protein expression, group D, E compared with group B, the difference was statistically significant (<0.05). ELISA showed that the content of TNF-α, IL-6,group C, D, E compared with group B,the content of IL-1β,group D, E compared with group B, the difference was statistically significant (<0.05)；Immunofluorescence showed that the expressions of GFAP and NG2 in group C, D and E were higher than that in group B, and the group D and E were more obvious. The expression of ED-1 in group C, D, E were decreased significantly compared with group B. Emodin has protective effect on neurons after SCI. The mechanism may connect with activting Nrf2-ARE pathway, reducing the expression of NF-κB, ED-1, TNF-α, IL-1β, IL-6, and promoting the expression of GFAP and NG2.
ABSTRACT
In order to investigate the protective effect of tanshinone ⅡA sulfonate on the sciatic nerve activty in rats after cryopreservation as well as the nerve regeneration and functional recovery after allograft and its possible mechanism, Sprague-Dawley (SD) rats were divded into four groups at different doses of tanshinone ⅡA sulfonate (A 0 mg·L⁻¹, B 80 mg·L⁻¹, C 160 mg·L⁻¹, D 480 mg·L⁻¹) cryopreserved at -80 °C for 24 weeks. Fresh control group nerve segments were harvested without cryopreservation. The ultrastructure and the viable cells of the nerve segments after cryopreservation were observed by electron microscopy, calcein-AM/propidium iodide staining, respectively. The expression of Bax and Bcl-2 was detected by Western blot. After cryopreservation, the nerve segments were cultured in vitro for one week, the mRNA and protein level of NGF and GDNF were detected by PCR and Western blot respectively. In addition, the above four cryopreserved groups transplanted to the Wistar rats by allografting (A', B', C', D'). At 16-week postoperation, muscle compound action potential latency and nerve conduction velocity were examined by electrophysiological. The number and the thickness of myelinated nerve fibers were analyzed by toluidine blue staining. The ultrastructure of the sciatic nerve by electron microscopy was observed. According to the results, after the cryopreserved for 24 weeks, compared with groups A and B, the nerve demyelination and vacuolation were weak, and the more viable cells, the decreased Bax and increased Bcl-2, the increased NGF and GDNF in group C and D. At 16-week poseoperation, the results demonstrated that the more larger and thickly regenerated myelinated axons, the shorter latency of muscle compound action potentials and higher nerve conduction velocity in groups C' and D' compared with groups A' and B'. According to these results, tanshinone ⅡA sulfonate exerted a significant protective effect on the viability of the nerves during cryopreservation at -80 °C and promoted nerve regeneration and functional recovery after transplantation especially in middle- and high-dose of tanshinone ⅡA sulfonate.
ABSTRACT
@#Objective To investigate the effect of cold-inducible RNA binding protein(CIRP)on the viability of cryopreserved sciat-ic nerve and nerve regeneration after allograft. Methods Sciatic nerve segments of 15 mm from male Sprague-Dawley rats were placed in DMEM solution and pretreat-ed with 4 ℃, 15 ℃ and 32 ℃ for 24 hours (group A, group B and group C, respectively). Fresh nerve group (group D)without pretreatment was set up.The mRNA and protein level of CIRP was detected by RT-PCR and Western blotting,respectively.The above nerves were cryopreserved in liquid nitrogen for four weeks.The via-ble cells of the nerve segments after cryopreservation were observed by calcein-AM/propidium iodide staining. The expression of Bax and Bcl-2 was detected by Western blotting.After cryopreservation,the nerve segments were cultured in vitro for one week, the protein level of nerve growth factor (NGF) and glial cell line-derived neurotrophic factor(GDNF)was detected by Western blotting.In addition,the above four cryopreserved groups were transplanted to the Wistar rats by allografting(groups A',B',C'and D').Fresh nerve allograft group(E')and isograft group(F')were set up.At four-week post operation,the expression of CD4of the nerve and plasma level of interleukin(IL)-6 and interferon(IFN)-γ were detected by immunohistochemistry and ELISA,respectively.At 20-week postoperation, the muscle compound action potential (CMAP) and motor nerve conduction velocity (MNCV) were examined by electrophysiological examination. The number and the thickness of myelinated nerve fibers were analyzed by toluidine blue staining. The ultrastructure of the sciatic nerve was observed by electron microscopy. Results The mRNA and protein of CIRP were significantly higher in group C than in groups A and B(P<0.05).After 4 weeks of cryopreservation,compared with groups A,B and D,the viable cells increased,the expression of Bax decreased and the expression of Bcl-2 increased in group C (P<0.05). The expression of NGF and GDNF in-creased in group C than in groups A and B(P<0.05).At four-week postoperation,the expression of CD4and plas-ma concentration of IL-6 and IFN-γ significantly decreased in group C'than in group E'(P<0.05),however,no significant difference was found in group C'compared with groups D'and F'(P>0.05).At 20-week postopera-tion, CMAP, MNCV, the number of axon, and thickness of myelin sheath were significantly better in group C' than in groups A',B',D'and E'(P<0.05).Compared with groups A',B',D'and E',the myelinated nerve fibers were more, the fiber thickness was more uniform, the fiber distribution was wider, and the myelin sheath was thicker in groups C'and F'. Conclusion CIRP was induced at 32℃in the sciatic nerve,which exerted a significant protective effect on the viability of the nerves during cryopreservation,and promoted nerve regeneration and functional recovery after transplanta-tion.
ABSTRACT
Objective To investigate the effect of emodin on spinal cord edema induced by acute spinal cord injury (SCI) and its mechanism. Methods A total of 180 healthy female Sprague-Dawley rats were randomly divided into sham group(group A),model group(group B),and low-dose group(group C),middle-dose group(group D)and high-dose group(group E)of emodin,with 36 cases in each group.The SCI model was established with the modified Allen's method.Function-al recovery was evaluated with Basso-Beattie-Bresnahan(BBB)score and inclined plate test three days,seven days,14 days and 28 days after modeling.Three days after modeling,the pathological changes of the spinal cord were observed by HE staining; the water content of spinal cord was detected by dry-wet weight method, the blood-spinal cord barrier(BSCB)permeability was detected by Evans blue(EB)staining,and the expression of aquaporin-4 (AQP-4) and matrix metalloproteinase-2 (MMP-2) mRNA and protein were detected by RT-PCR and Western blotting respectively.Results The BBB score and inclined plate scores were better in groups C,D and E than in group B(P<0.05)seven days, 14 days and 28 days after modeling, especially in group E (P<0.05). Three days after modeling, HE staining showed that there was a large hemorrhage in the section of group B,the nerve cells were swollen and damaged, and a large number of inflammatory cells were infiltrated,the tissue gap was widened and the edema was severe. The above pathological changes were better in groups C,D and E than in group B,especially in group E.The spi-nal cord water content was higher in group B than in group A(P<0.05),and was lower in groups D and E than in groups B and C(P<0.05).EB content was higher in group B than in group A(P<0.05),and was lower in groups C, D and E than in group B (P<0.05). The expression of AQP-4, MMP-2 mRNA and protein were lower in groups C,D and E than in group B(P<0.05),especially in group E(P<0.05). Conclusion Emodin can alleviate spinal cord edema,and improve hind limb movement function after SCI,which could be related with the down-regulation of AQP-4 and MMP-2 expression,and the reduction of the permeability of BSCB.