Objective To investigate the effect of chondroitinase ABC (ChABC) on the expression of growth associated protein 43 (GAP-43) and gl ial fibrillary acidic protein (GFAP) after spinal cord injury (SCI) in rats. Methods A total of 150 adult female SD rats, weighing 250-300 g, were randomly divided into ChABC treatment group (group A), sal ine treatment group (group B), and sham operation group (group C) with 50 rats in each group. In groups A and B, the rats were made the SCI models and were treated by subarachnoid injection of ChABC and sal ine; in group C, the rats were not treated as a control. At 1, 3, 7, 14, and 21 days after operation, the Basso, Beattie, and Bresnahan (BBB) score system was used toevaluate the motion function, and immunofluorescent histochemical staining was used to observe the expressions of GAP-43 and GFAP. Results At different time points, the BBB scores of groups A and B were significantly lower than those of group C (P lt; 0.05); there was no significant difference in BBB score between groups A and B after 1, 3, and 7 days of operation (P gt; 0.05), but the BBB score of group A was significantly higher than that of group B after 14 and 21 days of operation (P lt; 0.01). At different time points, the GAP-43 and GFAP positive neurons of groups A and B were significantly higher than those of group C (P lt; 0.05). After 14 and 21 days of operation, the GAP-43 positive neurons of group A were more than those of group B (P lt; 0.01). After 7, 14, and 21 days of operation, the GFAP positive neurons of group A were significantly less than those of group B (P lt; 0.01). Conclusion ChABC can degrade gl ial scar, improve the microenvironment of the injured region and enhance the expression of GAP-43, which promotes axonal growth and extension.
ObjectiveTo observe the effect of transplantation of neural stem cells (NSCs) induced by all-trans-retinoic acid (ATRA) combined with glial cell line derived neurotrophic factor (GDNF) and chondroitinase ABC (ChABC) on the neurological functional recovery of injured spinal cord in Sprague Dawley (SD) rats.
MethodsSixty adult SD female rats, weighing 200-250 g, were randomly divided into 5 groups (n=12): sham operation group (group A), SCI model group (group B), NSCs+GDNF treatment group (group C), NSCs+ChABC treatment group (group D), and NSCs+GDNF+ChABC treatment group (group E). T10 segmental transversal injury model of the spinal cord was established except group A. NSCs induced by ATRA and marked with BrdU were injected into the site of injury at 8 days after operation in groups C-E. Groups C-E were treated with GDNF, ChABC, and GDNF+ChABC respectively at 8-14 days after operation;and group A and B were treated with the same amount of saline solution. Basso Beattie Bresnahan (BBB) score and somatosensory evoked potentials (SEP) test were used to study the functional improvement at 1 day before remodeling, 7 days after remodeling, and at 1, 2, 5, and 8 weeks after transplantation. Immunofluorescence staining and HE staining were performed to observe the cells survival and differentiation in the spinal cord.
ResultsFive mouse died but another rats were added. At each time point after modeling, BBB score of groups B, C, D, and E was significantly lower than that of group A, and SEP latent period was significantly longer than that of group A (P<0.05), but no difference was found among groups B, C, D, and E at 7 days after remodeling and 1 week after transplantation (P>0.05). BBB score of groups C, D, and E was significantly higher than that of group B, and SEP latent period was significantly shorter than that of group B at 2, 5, and 8 weeks after transplantation (P<0.05);group E had higher BBB score and shorter SEP latent period than groups C and D at 5 and 8 weeks, showing significant difference (P<0.05). HE staining showed that there was a clear boundary between gray and white matter of spinal cord and regular arrangement of cells in group A;there were incomplete vascular morphology, irregular arrangement of cells, scar, and cysts in group B;there were obvious cell hyperplasia and smaller cysts in groups C, D, and E. BrdU positive cells were not observed in groups A and B, but could be found in groups C, D and E. Group E had more positive cells than groups C and D, and difference was significant (P<0.05). The number of glial fibrillary acidic protein positive cells of groups C, D, and E was significantly less than that of groups A and B, and it was significantly less in group E than groups C and D (P<0.05). The number of microtubule-associated protein 2 positive cells of groups C, D, and E was significantly more than that of groups A and B, and it was significantly more in group E than groups C and D (P<0.05).
ConclusionThe NSCs transplantation combined with GDNF and ChABC could significantly promote the functional recovery of spinal cord injury, suggesting that GDNF and ChABC have a synergistic effect in the treatment of spinal cord injury.
Objective The purity and activity of islets will greatly affect the outcome of xenotransplantation therapy of type 1 diabetes mell itus. To set up an improved method of the isolation and purification of rat islets, which can obtain highpurity,high-yield, and high-viabil ity islets. Methods Ten healthy and adult male SD rats, weighing 250-300 g were used asorgan donors. Collagenase V was perfused into pancreas via pancreatic duct. Pancreas was digested with collagenase in water bath at 38℃ about 15 minutes, islet purification was performed using two techniques: with Ficoll 400 density gradient (group A), and Ficoll-Paque? PLUS (group B). Dithizone (DTZ) was util ized for identifying islets, counting islets equivalent quantity (IEQ) and islets’ purity. Trypan blue staining was used to detect the viabil ity of islets. Islets of group B was encapsulated with alginate/poly-L-lysine/alginate (APA). Islets function of microencapsulated and nonmicroencapsulated was evaluated by the insul in release test. Results DTZ staining showed that islets shape were round, ell ipse and irregular with a clear edge and a diameter range of 50-300 μm. The IEQ values were 338.04 ± 76.61 and 834.80 ± 54.00 in groups A and B, respectively, showing significant difference (P lt; 0.05). The purities were 88.31% ± 2.67% and 95.63% ± 1.96% in groups A and B, respectively, showing no significant difference (P gt; 0.05). The activities of islets were 67.40% ± 5.15% and 86.05% ± 2.52% in groups A and B, showing significant difference (P lt; 0.05). Islet APA microcapsules had round shape, unified size, and its diameter was between 1.5 and 2.0 mm. Each microcapsule was encapsulated of 1 to 3 islets. The result of insul in release assay was that the concentrations of insul in secretion with islets of microencapsulated and nonmicroencapsulated were (5.53 ± 1.64) ng/ mL and (4.76 ± 0.26) ng/mL in low glucose, and its concentrations of insul in secretion in high glucose were (11.95 ± 2.07) ng/ mL and (14.34 ± 3.18) ng/mL. Stimulated insul in secretion in high glucose was 2 times more than that in low glucose (P lt; 0.05), but there was no significant difference (P gt; 0.05) in the stimulation index between group A (2.16 ± 0.30) and group B (3.01 ± 0.59). Conclusion The method of islets isolation and purification using Ficoll-Paque? PLUS own the virtues of more convenient, high islet yield, and high islet purity. Both microencapsulated and nonmicroencapsulated islets show high-viabil ity while culture in vitro.
Objective
To investigate the expressions of heat shock protein 27 (HSP27), Bcl-2, and Bax proteins of the nerve cells after spinal cord ischemia/reperfusion injury (SCII) in rats and their relationship.
Methods
Seventy adult male Sprague Dawley rats (weighing, 200-220 g) were randomly divided into the sham operated group (sham group, n=35) and the SCII group (n=35). Only the left renal artery was exposed with no occlusion of the abdominal aorta in the rats of sham group. The left renal artery was exposed with occlusion of the abdominal aorta for 20 minutes in the rats of SCII group. At 4, 8, and 12 hours and at 1, 2, 3, and 5 days, reperfusion treatment was performed in 5 rats respectively, and then the spinal cord tissue was harvested to detect the expressions of HSP27, Bcl-2, and Bax protein of the nerve cells by using immunohistochemistry staining.
Results
The HSP27 began to express at 4 hours, reached the peak at 3 days, and decreased at 5 days in SCII group; significant differences were found between at 3 and 5 days and at the other time points (P lt; 0.05). The Bcl-2 expression increased at 4 hours, reached the peak at 1 day and maintained a high level at 2 days, and then gradually decreased; significant differences were found between at 1 and 2 days and at the other time points (P lt; 0.05). The Bax expression reached the peak at 12 hours and 3 days, and decreased at 5 days; significant differences were found between at 12 hours and 3 days and at the other time points (P lt; 0.05). A little expression of each protein was observed in sham group at different time points; the expressions of HSP27, Bcl-2, and Bax proteins in SCII group were significantly higher than those in sham group at different time points (P lt; 0.05).
Conclusion
There may be the time window of self repair after SCII. High expression of HSP27 has an obvious protective effect on the SCII in rat, by promoting the expression of the anti-apoptotic protein Bcl-2 and reducing the expression of the pro-apoptotic protein Bax so as to inhibit spinal cord cell apoptosis.
Objective To investigate the delay of the denervated skeletal muscle atrophy with the method of restraining the increment of the connective tissues by tetrandrine and hormone. Methods The left hind limbs of 42 male adult SD rats were made into models of the denervated gastrocnemius, and then the rats were randomly divided into 3 groups, with 14 rats in each. In Group A, tetrandrine (8 mg/L）was injected into the denervated gastrocnemius; in Group B, triamcinolone acetonide（1.6 g/L） was injected; in Group C (the control group),normal saline was injected. Enough samples were obtained according to the different observation indexes at 30 days after operation. Electromyography, muscle wet weight measurement, light microscopy,electron microscopy,and microimage analysis were performed. ResultsThe fibrillation potential amplitude was 0.195 8±0.041 9 μV in Group A and 0.185 2±0.050 3 μV in Group B, and there was no significant difference betweenthe two groups (Pgt;0.05). However,in Group C the fibrillation potential amplitude was 0.137 7±0.058 9μV. The fibrillation potential amplitude was significantly greater in Group A than in Group C（Plt;0.05）. The muscle wet weight was 1.740 0±0.415 9 g in Group A and 1.940 1±0.389 4 gin Group B, and there was no significant difference between the two groups(Pgt;0.05).However, in Group C the muscle wet weight was 0.800 0±0.100 0 g. The muscle wet weight was significantly greater in Group A than in Group C（Plt;0.05）.The microscopy showed that more remarkable atrophy occurred in the control group. The muscle fibers were more complete, thicker and larger, with more nuclei and clearer cross-lines. More connective tissue and flat cells could be observed in Groups A and B. The myogenic protein amount was 440.124 2±46.135 6 in Group A and 476.211 4±41.668 8in Group B, and there was no significant difference between the two groups(Pgt;0.05).However, in Group C the amount was 380.040 0±86.315 9.The myogenic protein amount was significantly greater in Group A thanin Group C（Plt;0.05）. The muscle fiber number, diameter, cross section, and connective tissue increment were all significantly greater in Group A than in Group C（Plt;0.05）; however, there wasno significant difference between Groups A and B (Pgt;0.05). The electron microscopy showed that there were more degeneration changes, such as muscle silk disorder, chondriosome disappearance, and hepatin reduction, could be observed inGroup C than in Groups A and B. Conclusion Tetrandrine and hormone can delay the denervated skeletal muscle atrophy by restraining the increment of the connective tissues.
Objective To improve arterial anastomosis method for rat renal transplantation. Methods Renal transplantations were performed on 72 wistar rats. The donor superior mesenteric artery was end-to-end anastomosed to the recipient left renal artery by using of sleeve anastomosis technique. The external diameters of the vessels anastomosed were 0.60±0.05 mm (left renal artery) or 0.80±0.07 mm (superior mesenteric artery). The procedure consisted of a guidingsuture and two fixing sutures. The guiding suture was used to “telescope” therecipient left renal artery into the donor superior mesenteric artery about 2 millimetre. Two fixing sutures were applied 180°apart from each other and tied. Three sutures passed through all layers of the donor superior mesenteric artery andconstricted the vessel lumen, but only penetrated the adventitia of the recipient left renal artery. Results The time for arterial anastomoses was approximately 6 to 8 minutes. The renal grafts perfused very well after the recipient left renal artery clamp was removed. Complications included anastomotic hemorrhage(1 case) and thrombosis (1 case). Histologic examination of 34 grafts at different postoperative time ranging from 6 to 30 days revealed that renal artery was fully patent, with no evidence of ischemic injury. Conclusion The modified arterial sleeve anastomosis technique is simple and feasible regardless of experimentalcondition and can be easily performed.
ObjectiveTo evaluate the dynamic changes of blood flow and blood pressure of acute hindlimb ischemia of rats by laser Doppler flowmetry (LDF) and laser Doppler perfusion imaging (LDPI). MethodsThe acute hindlimb ischemia model of rats was established by resection of rats femoral arteries of left hindlimb. The blood flow and blood pressure between operated and nonoperated hindlimbs were examined by LDF on 2, 7, 14, 28, and 49 d after operation. And the blood flow was evaluated by LDPI on 7 d after operation. ResultsAll rats survived after operation and no hindlimb necrosis occurred. The mean score was 2 on 14 d after operation and 1 on 49 d after operation. The ratio of blood flow between operated and nonoperated hindlimbs on 2 d after operation significantly increased from 1 to 1.31±0.439 (P=0.021). The ratio of blood flow on 7 d (0.82±0.538) and 14 d (0.93±0.294) after operation was significantly lower than that on 2 d after operation (P=0.032 and P=0.019), although the difference between the two former was not significant (P=0.502). Furthermore, the ratio of blood flow on 28 d after operation reached the bottom (0.41±1.970), which was obviously lower than that on 2, 7, and 14 d after operation (P=0.004, P=0.007, and P=0.006). The blood flow of operated hindlimbs recovered approximately the value before operation (0.98±0.093), which was significantly lower than that on 2 d (P=0.010), higher than that on 28 d (P=0.005), but not different from that on 7 d and 14 d after operation (P=0.126 and P=0.382). The ratio of blood pressure between operated and nonoperated hindlimbs on 2 d after operation significantly increased from 1 to 0.47±0.375 (P=0.031). The ratio of blood pressure decreased on 7 d after operation (0.44±0.118), which was not different from that on 2 d after operation (P=0.203). Furthermore, the ratio of blood pressure on 14 d after operation reached the bottom (0.35±0.115), which was obviously lower than that on 2 d and 7 d after operation (P=0.001 and P=0.036). On 28 d after operation, the ratio of blood pressure increased (0.54±0.146), which was significantly higher than that on 14 d after operation (P=0.008), while not different from that on 2 d (P=0.493) and 7 d after operation (P=0.551). The ratio of blood pressure recovered approximately the value before operation (0.97±0.094), which was significantly higher than that on 2, 7, 14, and 28 d (P=0.013, P=0.021, P=0.002, and P=0.031). ConclusionAcute hindlimb ischemia model of rats can be established by resection of rats femoral arteries of left hindlimb and the most serious stage of hindlimb ischemia is on 14-28 d after operation. LDF and LDPI are of importance for monitoring the dynamic changes of rats hindlimb ischemia after operation.
Objective To establish a reliable rats model of orthotopic liver transplantation with non-heart beating donors. Methods The model was established with modified double-cuff method. According to obtain pre-liver warm ischemia time experiencing non-heart-beat the rats were divided into 3 groups: 10 min (R10 group), 20 min (R20 group) and 30 min (R30 group), then one week survival after operation was compared in rats. Results The operative time of donor was 30 min approximately except warm ischemia time and the cold preservation time of donor liver was 1 h. The anastomotic time for suprahepatic vena cava was 12-22 min (mean 15 min). The anastomotic time for portal vein and infrahepatic vena cava was about 2 min and 1 min, respectively. The anhepatic phase sustained 14-24 min (mean 19 min). The operative time of receptor was 50-65 min (mean 60 min). Twelve rats died at 24 h after operation, which was considered as operative failure. The success rates of operation in R10 group, R20 group, and R30 group were 95% (19/20), 80% (16/20), and 65% (13/20), respectively. After one week the survival rate was 95% (18/19), 81% (13/16), and 54% (7/13), respectively. Conclusions Improved non-heart donor liver transplantation model of rat on the basis of Kamada’s “twocuff technique” acts as a good simulation in clinical non-heart-donor liver transplantation. This study showes that rat liver can tolerate warm ischemia time less than 30 min, the short-term survival after transplantation can reach satisfactory results. However, long-term survival requires further study.
ObjectiveTo investigate the effect of electrospun chitosan/polylactic acid (ch/PLA) nerve conduit for repairing peripheral nerve defect in rats.
MethodsNerve conducts loaded with ch/PLA was made by the way of electrospun. The mechanical property, hydrophility, biocompatibility were tested, and the scanning electron microscope was used to observe the ultrastructure. The same experiments were also performed on pure PLA nerve conducts as a comparison. Then, 54 Sprague Dawley rats were divided into 3 groups randomly, 18 rats in each group. Firstly, the 10 mm defects in the right sciatic nerves were made in the rats and were respectively repaired with ch/PLA (group A), autografts (group B), and no implant (group C). At 4, 8, and 12 weeks after operation, general observations, sciatic functional index (SFI), electrophysiological evaluation, wet weight of gastrocnemius and soleus muscles, histological examination, immunohistological analysis, and transmission electron microscopy were performed to evaluate the effects.
ResultsCompared with pure PLA nerve conducts, the addition of chitosan could improve the mechanical property, hydrophility, biocompatibility, and ultrastructure of the nerve conducts. At 4 weeks postoperatively, the regenerated nerve bridged the nerve defect in group A. The SFI improved gradually in both group A and group B, showing no significant difference (P>0.05). Compound muscle action potentials and nerve conduction velocity could be detected in both group A and group B at 8 and 12 weeks after operation, and significant improvements were shown in both groups (P<0.05). The wet weight and myocyte cross section of gastrocnemius and soleus muscles showed no significant difference between group A and group B (P>0.05), but there was significant difference when compared with group C (P<0.05) at 12 weeks postoperatively. Immunohistological analysis revealed that S-100 positive Schwann cells migrated in both group A and group B, and axon also regenerated by immunohistological staining for growth associated protein 43 and neurofilaments 160. Transmission electron microscopy showed no significant difference in the diameter of nerve fiber between group A and group B (P>0.05), but the thickness of myelin sheath in group A was significantly larger than that in group B (P<0.05).
ConclusionThe electrospun ch/PLA nerve conduits can effectively promote the peripheral nerve regeneration, and may promise an alternative to nerve autograft for repairing peripheral nerve defect.
