ObjectiveTo observe the dynamic changes of neuroglobin (NGB) expression in hippocampus after status epilepticus(SE) in rats, and to explore the role of NGB in epileptic seizures.Methods40 healthy male Sprague Dawley rats were randomly divided into two group according to random number table method:control group (n=5) and epilepsy model group(n=35).Epilepsy model group according to observation time was divided into:0h, 1h, 3h, 12h, 24h, 10d and 30d.Intraperitoneal injection Lithium-pilocarpine (20 mg/kg~127 mg/kg, Li-PC) to establish the rat model of SE.Observe the behavioral changes in rats with epilepsy.Nissl staining was used to detect the neuronal damage in hippocampus. Streptavidin-biotin-peroxidase complex immunohistochemical method was used to detect the expression level of NGB in hippocampus;ResultsAfter SE, the neurons in hippocampus were severely damaged with the progress of epileptic seizures, the number of surviving neurons in CA1, CA3 regions showed a near linear decline.Among them, the number of surviving neurons in (12h, 24h, 10d, 30d)CA1, (0h, 12h, 24h, 10d, 30d)CA3 and(12h, 24h, 10d, 30d) DG area were significantly lower than that of the control group (P < 0.05).The expression level of NGB in CA1, CA3 and DG region of hippocampus were increased after SE, and both of CA1 and DG were reached peak in 24h after SE, but was still higher than the control group.And the CA3 area showed a continue rising trend.Among them, CA1(24h, 10d, 30d), CA3(24h, 10d, 30d) and DG(12h, 24h, 10d, 30d) were higher than that of control group significantly (P < 0.05).In addition, it was found that there was a positive correlation between the number of surviving neurons in CA3 area and the expression level of NGB (R=0.306, P=0.011).ConclusionUp-regulation of NGB expression in hippocampus after status epilepticus, and was positively correlated with the number of neurons in the CA3 area, suggesting that up regulation of NGB expression may be a compensatory protective mechanism of ischemic injury induced by seizures, and participate in the protection of epilepsy related neuronal damage.
Objective To observe the release pattern of the microcysts and the effect of ectopic osteogenesis of combined micromorselized bone by optimized preparation of microcysts. Methods Optimized poly-DLlactide-co-glycolide (PLGA) microcysts manufacturing method was performed with the orthogonal design, and the accumulated release amount of microcysts was calculated at 2 h, 4 h, 8 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, 96 h, 120 h, 144 h, 168 h, 192 h, 216 h, 240 h and 264 h. Twentyfour Wistar rats were divided into 4 groups (n=6) and 1 cm length incision was cut in their bilateral thighs skin, forming 48 gluteus maximus muscle sackmodels. In group A,collagen was implanted to bilateral muscle sacks respectively. In group B, collagen and autologous morselized bone were implanted to bilateral muscle sacks. Ingroup C, collagen and rhBMP-2/PLGA delayed release microcysts were implanted to bilateralmuscle sacks respectively. In group D, collagen and morselized bone/rhBMP-2/PLGA delayed release microcysts were implanted to bilateral muscle sacks. Gross and histologic observations were made at 3, 4 and 5 weeks postoperatively.Results Every optimized variance had an effect on particle diameter of microcyst and its encapsulating rate. The microcyst’s surface was smooth and had a fine spheroplast, which released slowly within 11 days in vitro. In thethird week postoperatively, the graft in group A could not be touched, while the graft in all other 3 groups was still found. After 3 weeks, collagen was absorbed completely in group A, the residual collagen could be seen in groups B, C andD. After 4 weeks, collagen could be seen in group A; micromorselized bone continued to be absorbed and became smaller in group B; microsphere became smaller, osteoblasts increased in group C; micromorselized bone and microsphere continuedto be absorbed, oteoblasts and chondroblasts increased. After 5 weeks, implantsbecame small, microsphere was absorbed, osteoblasts and chondroblasts became more in groups B, C and D. Microcysts presented with white granuloshape and were packaged in tissue pieces. Histologic observation showed that the PLGA microcysts in 3 weeks and 4 weeks could be absorbed gradually as the time in vivo, if combining with morselzed bone they could produce abundant induced osteoblasts and chondroblasts. Conclusion Optimizing the preparation technology of microcysts has delayed their release during a long period in vitro. Autologous micromorselized bone can be ectopicly induced to produce large amount of osteoblasts in gluteus maximus muscle sack, where PLGA microcysts can combine organically and bring about the bone formation with less amount of growth factors.
Objective Bone marrow mesenchymal stem cells (BMSCs) play an important role in repairing nerve injury, meanwhile external temperature has significant effect on BMSCs transplantation, prol iferation, and differentiation. To investigate the effect of BMSCs transplantation and mild hypothermia on repair of rat spinal cord injury (SCI). Methods Forty-five female adult SD rats (weighing 200-250 g) were made the models of hemitransection SCI and divided randomly into 3 groups according to different treatments: group A (SCI group), group B (BMSCs transplantation group), and group C [BMSCs transplantation combined with mild hypothermia (33-35 ) group]. At 1, 2, 4, 6, and 8 weeks after injury, the fuction of hind l imb was evaluated with Basso Beattie and Bresnahan (BBB) score and incl ined plane test. At 4 weeks after injury, histopathology and BrdU immunohistochemistry staining were performed. At 8 weeks after injury, horseradishperoxidase (HRP) retrograde nerve trace and transmission electron microscope (TEM) testing were performed to observe the regeneration of axon. Results After 4 weeks, the function of hind l imb obviously recovered in groups B and C, there were significant differences in BBB score between groups B, C and group A (P lt; 0.05), between group B and group C (P lt; 0.05). There was no significant difference (P gt; 0.05) in tilt angle among 3 groups after 1 and 2 weeks, and there were significant differences (P lt; 0.05) among 3 groups after 4 weeks. HE staining showed that significant cavity could be seen in group A, l ittle in group B, and no cavity in group C. BrdU immunohistochemistry staining showed that the number of positive cells was 0, 90.54 ± 6.23, and 121.22 ± 7.54 in groups A, B, and C, respectively; showing significant differences (P lt; 0.01) among 3 groups. HRP retrograde neural tracing observation showed that the number of HRP positive nerve fibers was 10.35 ± 1.72, 43.25 ± 2.65, and 84.37 ± 4.59 in groups A, B, and C, respectively, showing significant differences (P lt; 0.01) among 3 groups. TEM observation showed that a great amount of unmyel inated nerve fibers and myel inated nerve fibers were found in central transverse plane in group C. Conclusion The BMSCs transplantation play an impontant role in promotion of recovering the function of hind l imb after SCI, and mild hypothermia has synergism effects.
Objective To study the relationship between the expression ratio of induced nitric oxide synthase (iNOS) over glial fibrillary acidic protein (GFAP) and the time of injury after brain concussion in rat, in order to acquire a new visual angle for determining injury time of cerebral concussion. Methods Eighty-five healthy Sprague-Dawley rats were divided into three groups randomly: model group (n=25), experimental group (n=55), and control group (n=5). The rats in the model group were used to confirm the attack hight to make the model of brain concussion; according to the time of execution, rats in the experimental group were then subdivided into 11 groups with 5 rats in each subgroup, and their execution time was respectively hour 0.5, 1, 3, 6, 12, 24, 48, 96, 168, 240, and 336; the rats in the control group were executed after fed for 24 hours. After the model of cerebral concussion was established through freefalling dart method, hematoxylin-eosin staining and immunohistochemistry staining of iNOS and GFAP were conducted for the brain of the rats. All related experimental results were studied by using microscope with image analytical system and homologous statistics. Results The ratio of positive expression of iNOS over that of GFAP increased gradually during hour 0.5- 3 after injury in brain (from 5.03 to 10.47). At the same time, the positive expression of iNOS increased significantly (from 14.61% to 37.45%). However, the increase of the positive expression of GFAP was not obvious. Between hour 3 and 12, the ratio began to decline to 4.98, which was still at a high level, and during the same time period, the positive expressions of iNOS and GFAP also experienced the same change pattern. Later, the ratio began to decline between hour 12 and 336 after injury (from 4.98 to 0.95). All ratios at this time were lower than those between hour 0.5 and 12. The positive expression of iNOS and GFAP both increased to a climax before declining. Conclusions The ratio of positive expression of iNOS over GFAP and the respective change pattern of iNOS and GFAP can be used as the evidence of estimating the injury time of cerebral concussion. We can use the ratio of two or more markers to provide a new visual angle for concluding the concussion injury time.
【Abstract】ObjectiveTo investigate the effects of liver transplantation on splenic function in rats with hepatic cirrhosis. MethodsHepatic cirrhosis model was established in rats by subcutaneous injections of carbon tetrachloride. Liver transplantation model was established with twocuff technique. Spleen index, morphological changes of spleen were observed before and after liver transplantation in hepatic cirrhosis rats. Spleen T lymphocyte subgroups before and after liver transplantation were also assayed by immunofluorescence staining and flow cytometry. ResultsBefore liver transplantation, spleen index was increased from (2.42±0.11) mg/g to (3.62±0.14) mg/g， P＜0.01; pathological examination of spleen samples showed that the areas of white pulp were decreased from (23.47±2.30)% to (7.70±2.01)%， P＜0.01， and the areas of spleen trabecula were increased from (1.75±0.61)% to (4.46±0.71)%， P＜0.01. Meanwhile, the ratio of CD4/CD8 of spleen T lymphocyte subgroups was decreased from 2.67±0.15 to 1.18±0.15, P＜0.01. After liver transplantation, spleen index was decreased from (3.62±0.14) mg/g to (2.62±0.11) mg/g， P＜0.01; pathological examination of spleen showed that the areas of white pulp were increased from (7.70±2.01)% to (15.07±1.97)%， P＜0.01, and those of spleen trabecula were decreased from (4.46±0.71)% to (3.11±0.51)%， P＜0.05. Meanwhile, the ratio of CD4/CD8 of spleen T lymphocyte subgroups was increased from 1.18±0.15 to 2.32±0.11, P＜0.01. ConclusionImpaired function of spleen resulting from liver function damage can be improved in rats with hepatic cirrhosis after liver transplantation.
Objective To explore the effects of several immunosuppressants on the cell numbers of cultured rat macrophages and Schwann’s cells. Methods The macrophages and Schwann’s cells were cultured from the newborn Wistar rats. Different concentrations of methylprednisolone(10-3, 10－4,10－6 and 10－8 mol/L), CsA(10－5, 10－6, 10－7 and 10－8 mol/L) and FK506(10-6, 10－7, 10－8 and 10－9 mol/L) were administrated to the cells, while control group was given no drugs. Twentyfour, 48 and 72 hours after administration, the cells from different concentrations were measured with MTT methods respectively. Theresults were compared and analyzed statistically. Results Only high concentration methylprednisolone (10-4 mol/L) and a certain range of concentrations of CsA (10-6，10-7 and 10-8 mol/L) and FK506 (10-7,10-8 and 10-9 mol/L) can provide protection to culturedrat macrophages. Under most concentrations, CsA and FK506 had no effects onthe cell number of cultured rat Schwann’s cell. Only with high concentration CsA (10-5 mol/L) and methylprednisolone (10-3 mol/L) could significantly decreased the cell number of Schwann’s cell. Long time (72 hours) and low dosage (10-8 mol/L) administration of methylprednisolone could significantlyprotect Schwann’s cell. Conclusion High concentration methylprednisolone and some certain concentration CsA and FK506 can protect cultured rat macrophages. But high concentration CsA and methylprednisolone prohibit the proliferation of Schwann’s cells. Only long time and low dosage methylprednisolonecan protect cultured rat Schwann’s cells.
Objective To investigate the effects of ectomesenchymalstem cells on hematopoiesis after total body irradiation in rats. Methods The primary ectomesenchymal stem cells were isolated from E11.5 SD fetal mandibular processes by 25g/L trypsin and cultured with DMEM/F12. The morphology and growthrate were observed by inverted microscope. Eighty SD male rats randomly dividedinto ectomesenchymal stem cells group (n=20), fibroblast group(n=20), saline group(n=20) and control group(n=20), the first three groups were irradiated with 60Co γ rays at 6.0 Gy. The number of their bone marrow nucleated cells was counted after 4 weeks; the forming ability of colony-forming unit-granulocyte macrophage(CFU-GM) and histopathology of bone marrow were also observed. Results The cultured cells displayed monolayer growth and fibroblast-like with 2-4 processes. The ectomesenchymal stem cells could increase the number of bone marrow nucleated cells and peripheral blood white cell count, and improve the forming ability of CFU-GM. After 4 weeks of transplantation, the number of the peripheral blood white cells in group A was more than that in groups B and C(Plt;0.05), the contents of Hb in groups A and D was significantly higher than those in groups B and C(Plt;0.0). After 4 weeks, the bone morrow nucleated cells in group A were significant more than those in groups B and C(Plt;001); CFU-GM in groups A and D was higher than that in groups B and C(Plt;0.01). Conclusion Ectomesenchymal stem cells have characteristics of stem cells. It may improve hematopoiesis recovery of irradiated rats.
ObjectiveTo investigate the changes of inflammatory cytokines in bronchoalveolar lavage fluid (BALF) in rats exposed to paraquat (PQ). MethodsAdult healthy SD rats were randomly divided into a control group (n=8) and three experimental groups (PQ in low dosage of 15 mg/kg,medium dosage of 30 mg/kg,and high dosage of 60 mg/kg,n=24 in each group). The rats in three experimental groups were intragastrically administered with PQ,and the rats in the control group were treated with saline by gavage. Two rats in the control group and six rats in three experimental groups were sacrificed on 1st,7th,14th,and 21st day after exposure respectively. BALF was collected for measurement of interleukin-1(IL-1),IL-6,macrophage inflammatory protein-2(MIP-2),monocyte chemoattractant protein-1(MCP-1),and biopterin by ELISA. ResultsThe levels of cytokines in all experimental groups were higher than those in the control group at any time point. In the exposure day 1 to day 14, IL-1 and biopterin levels in BALF increased significantly with the increase in PQ dose. On 14th and 21st day,IL-6 level in BALF increased significantly with the increase in PQ dosage. The levels of IL-1,IL-6,and biopterin in the experimental groups reached the peak on 14th day. On 14th day,the MIP-2 level in BALF of high-dosage group was significantly higher than that of low-dosage and medium-dosage groups (all P<0.05). The level of MCP-1 in the low-dosage group was lower than that in the medium-dosage and high-dosage groups at any time point (P<0.05). ConclusionIL-1,IL-6,MIP-2,MCP-1,and biopterin may play important roles in the development and progression of PQ-induce lung inflammation.
ObjectiveTo characterize the dynamic expression of Robo3 in the rat model of temporal lobe epilepsy(TLE), and assess the potential contribution of Robo3 to epileptogenesis. MethodsMale Sprague-Dawley (SD) rats were randomly divided into the control group (n=6) and the experimental groups (n=30, 6 per group). The experimental groups were injected intraperitoneally (i.p.) with an aqueous solution of lithium-pilocarpine, and sacrificed at different time points (1, 7, 14, 30 and 60 days) following the seizure. The control group was i.p. with 0.9% sodium chloride instead of pilocarpine. Quantitative real-time PCR were used to detected the mRNA expression of Robo3 and Western bolt were used to detected the protein expression of Robo3. ResultsQuantitative real-time PCR showed that the expression of Robo3 were significantly lower in the rat temporal lobe tissues of the latent and the chronic period group as compared with the controls(P < 0.05), but no significant differences were identified between the acute period group and the controls(P > 0.05). Western blot showed that the protein expression of Robo3 were significantly lower in the rat temporal lobe tissues of the latent and the chronic period group as compared with the controls(P < 0.05), no significant differences were identified between the acute period group and the controls(P > 0.05). ConclusionsRobo3 may be involved in the pathogenesis of temporal lobe epilepsy.
ObjectiveTo investigate the effect of emodin on the expression of hypoxia inducible factor (HIF)-1α protein in rats with severe acute pancreatitis-associated renal injury and explore the possible mechanisms. MethodsA total of 72 rats were randomly divided into sham-operated group (n=24), severe acute pancreatitis with renal injury group (injury group, n=24), and treatment group (n=24). The sham-operated and injury groups were given 1.5 mL saline through intragastric administration before operation while the treatment group was fed with the same amount of 50 mg/kg emodin diluent. The pancreas and pancreatic tail-segment was dissociated and the head of pancreas was occluded in rats to form the model, and blood vessel forceps were loosed after three hours. All the rats were sacrificed 12, 24 and 36 hours after modeling. The level of ascites, serum amylase, creatinine, blood urea nitrogen were detected. Hematoxylin-eosin staining was used to observe the pancreatic and renal pathological changes, and immunohistochemical method was used to detect the expression of HIF-1α protein level in the kidney. ResultsCompared with the sham-operated group, the level of ascites, serum amylase, creatinine, blood urea nitrogen and the expression of HIF-1α protein level increased significantly. The tissue damage of pancreas and the kidney became more serious. Compared with the injury group, the kidney and pancreas function of the treatment group had a better performance. HIF-1α protein level significantly increased in the treatment group, and the difference had a statistical significance (P<0.05). ConclusionEmodin has a good protective effect on severe acute pancreatitis-associated renal injury. It may function through up-regulation expression of HIF-1α protein level to improve the ability of the kidney to tolerate hypoxia, and then reduce the cell apoptosis and necrosis of the kidney.