A acute partial obstructive hepatocholangitis model by selective ligation and injection of E coli into left hepatic bile duct was successfully founded in rat. Using parameters including mortality, mitochondrial glutamic oxalacetic transaminase and ornithine carbamoytransferase activity, pathological observation and blood culture of bacteria, we evaluated the model. The authors emphasize that this models is superior to the wole-bile-duct-challenged cholangitis model, which is characterized by liver injury.
ObjectiveTo investigate the expression of caspase-3 and Toll-like receptor 4 (TLR4) in the incised rat skin healing process and its relationship with the wound time and to provide an experimental evidence for the prediction of injury time. MethodsAfter the rat incised wound model was established, hematoxylin-eosin dyeing technology and immunohistochemical staining technique were used to observe the expression of caspase-3 and TLR4. Then Image Pro Plus Image analysis software and SPSS statistical analysis software were used to deal with the experimental results. ResultsCaspase-3- and TLR4-positive cells were detected in epidermis, hair follicle and sebaceous gland cells in the control skin. The expression of caspase-3 and TLR4 of the ante mortem groups were significantly different compared with the control group except the 0 h group (P<0.05). Caspase-3- and TLR4-positive cells were detected in neutrophils around the hair follicle half an hour later. Caspase-3- and TLR4-positive cell rate increased with the infiltration of inflammatory cells. Caspase-3- and TLR4-positive cell rate reached the maximum on the 3 rd day, and then it began to decrease, and they were mainly expressed in fibroblasts and mononuclear macrophages. Caspase-3- and TLR4-positive cells were mainly expressed in fibroblasts on the 10th day. There was no significant differences between the postmortem injury groups and the normal control groups (P>0.05). ConclusionCaspase-3- and TLR4-positive cell rate is time dependent and stable in 25℃ temperature environment which makes it possible to determine the time of injury.
ObjectiveTo evaluate the effect of pre-infusion of allogeneic lymphoyctes treated with 5-FU on the rat liver graft. MethodsRat liver transplant models from Wistar to SD were established. Four groups were designed as following: control group: only liver transplantation without any other intervention; lymphocytes group: 1 ml of untreated lymphocytes (5×106/ml) from Wistar rats were preinfused into SD rats on day 7 and 4 separately before transplantation; lymphocytes with low concentration of 5-FU group: low concentration 5-FU (7.5 μg) treated lymphocytes were preinfused as above; lymphocytes with high concentration of 5-FU group: high concentration 5-FU (15 μg) treated lymphocytes were preinfused as above. Fas-L and CD8 expression were detected by immunohistochemistry method on day 7 after transplantation. ResultsThe integral opticaldensity (IOD) of Fas-L positive lymphocytes in the lobules of liver and portal areas were higher in lymphocytes with low concentration of 5-FU group than in the other groups （Plt;0.05）. There was no difference between lymphocyte group and lymphocytes with high concentration of 5-FU group （Pgt;0.05）. The IOD of CD8+ expression in lobules of liver was not different among all the three lymphocytes treated groups （Pgt;0.05）. But in portal areas, CD8+ expression was lower in the lymphocytes with low concentration of 5-FU group than in the other groups （Plt;0.05）. ConclusionPreinfusion of lymphocytes treated with low concentration 5-FU can induce graft immune tolerance, the probable mecanism of which is the increasing Fas-L expression in graft.
Objective To explore the liver regeneration following partial liver transplantation. MethodsPartial liver transplantation in the rats were established, three experimental groups were: Ⅰ=control, partial liver resection; Ⅱ=orthotopic liver transplantation (OLT); Ⅲ=partial orthotopic liver transplantation (POLT). Liver function test, morphological investigations and liver regeneration were performed in different time after transplantation. The regenerative response of transplanted partial liver graft in rats were evaluated by Flow Cytometry and compared it to liver regeneration following resection.Results The serum concentrations of ALT, BILI increased in one week, but returned gradually to normal level within one month after transplantation. Large numbers of mononuclear cells infiltrating into the portal areas. Hepatocyte necrosis was observed on day 14 after transplantation. On day 30, the parenchyma cell showed a nearly normal appearance, bile duct proliferation was seen in portal areas. In addition, after liver resection and POLT some diploid hepatocytes were found. Dilation of the central veins, adjoining sinusoids and interlobar veins were seen in group Ⅲ. The partial liver graft is capable of regeneration similar to the situation following partial hepatectomy. The peak of liver regeneration was seen on day 1,2,4 following a hepatectomy and POLT and OLT, respectively.Conclusion The transplanted liver shows the same and/or enhanced regeneration compared to controls. There are several possible explanations for the slight delay in achieving the maximal regenerative response in rats undergoing the POLT and OLT. These may include damage that is induced by the operation itself, preservation, and reperfusion injuries. These suggest that this be caused by activation of the immune system and it might be related to the regulation of cytokines and hormone.
【Abstract】Objective To investigate the surgical technique of orthotopic liver transplantation using two-cuff technique and prevention of operational complications in rats. Methods The model was established with modified cuff technique.Before donor livers were harvested,the portal vein and hepatic artery were interrupted for 10 min,and reflow was initiated for another 10 min.The donor liver was perfused through abdominal aorta and portal vein respectively.The infrahepatic vena vein and portal vein were anastomosed by means of cuff method； the anastomosis of the suprahepatic vena vein was performed with suture method. The anastomosis of the common bile duct was performed with an internal stent. Results One hundred and twenty rats underwent orthotopic liver transplantation using twocuff technique and the successful rate was 90.8%. The average nonhepatic time of recipients was (21.0±3.5) min and the total surgical time was (46.0±4.5) min. The oneweek survival rate of recipients was 87.2%. Conclusion Good exposure of operative field, sophisticated microsurgical technique and delicate surgical manipulation can benefit decreasing nonhepatic and total surgical time of recipients and increasing the survival rate of recipients.
OBJECTIVE To determine the role of endogenous carbon monoxide(CO) in oxidant-mediated organ injury following limb ischemia-reperfusion (I/R) in rats. METHODS: Sixty-four SD rats were divided into 4 groups: Sham group, Sham + zinc protoporphyrin (ZnPP, an inhibitor of heme oxygenase activity), 2-hour ischemia followed by 4-hour reperfusion (I/R) group and I/R + ZnPP group. Carboxyhemoglobin (COHb) level in the artery blood, malondialdehyde (MDA) content and superoxide dismutase (SOD) activity in the lung, heart, liver and kidney were detected. The 24-hour survival rate of rats was studied. RESULTS: Compared with the sham group, the COHb level and MDA content significantly increased, while the SOD activity and the survival rate significantly decreased in I/R group (P lt; 0.05). Compared with the I/R group, MDA content significantly increased, while the SOD activity, the 24-hour survival rate and COHb level significantly decreased in I/R + ZnPP group (P lt; 0.05, respectively). CONCLUSION: Limb I/R could lead to the oxidant-mediated multiple organ injury accompanied by the increase of CO level which play an important role in the defense against I/R-induced remote multiple organ injury in rats.
Objective To investigate the effect of heme oxygenase-1 (HO-1) activity on rat liver transplantation model. Methods One hundred and thirty-seven rats were divided into 4 groups. Study control group (n＝44)： 24 h before operation, saline 5 ml/kg was infused into peritoneal cavity of donor rats; Hemin group (n＝44)： hemin 100 mg/kg was infused into peritoneal cavity of donor rats 24 h before operation, and hemin 100 mg/kg was infused into portal vein during the preserve time in 4 ℃ saline； ZnPP group (n＝44)： ZnPP 5 mg/kg was infused into peritoneal cavity of donor rats 24 h before operation, and ZnPP 5 mg/kg was infused into portal vein during the preserve time in 4 ℃ saline； Normal control group (n＝5)： normal rats as normal control group. Expressions of HO-1 protein and mRNA were detected with immunohistochemistry and RT-PCR technique respectively. The apoptosis rate of hepatocytes was analyzed by flow cytometry. Results Expression of HO-1 mRNA in the liver of hemin group after transplantation was higher than that in study control group obviously, serum ALT and AST levels were lower than those in study control group (P＜0.05); HO-1 mRNA expression in ZnPP group liver was lower than that in study control group, serum ALT and AST levels were higher than those in study control group (P＜0.05). About liver cell apoptosis rate 48 h after liver transplantation, ZnPP group was the highest, hemin group was the minimum, and there had a significant difference between two groups (P＜0.05). Seven days after transplantation, the survival ratios of control study group, hemin group and ZnPP group were 7/12, 9/12 and 4/12 in turn, the inter-group differences had statistical significance (P＜0.05). Conclusion Activity of HO-1 could be induced by the transplant operation. HO-1 increases the survival rate after liver transplantation which was related with reducing apoptotic ratio of hepatocyte and improve hepatic function.
【Abstract】Objective To investigate the effect of donor blood transfusion on inducing pancreatic allograft tolerance in outbred rat model. Methods Wistar male rats were used as blood and pancreas donor, and diabetic recipients. One ml of donor blood injected into abdomen of diabetic recipients on the day of transplantation and azathioprine given 2 days pretransplant and continued for three days. Results Pancreas allograft survival was significantly prolonged (28 to 112 days, media survival time 64.2 days). One ml of donor blood alone injected into the abdomen and azathioprine given alone 2 days pretransplant did not improve allograft survival (media survival time 9.8 vs 10.2 days). Conclusion Donor blood injected on the day of transplantation and a 3 days course of azathioprine started 2 days pretransplant have b synergism in inducing long term graft survival in this rat model.
【Abstract】 Objective To investigate the expression of connexin 40 (Cx40) and hyperpolarization-activated cycl icnucleotide-gated cation channel 4 (HCN4) in rat bone marrow mesenchymal stem cells (BMSCs) cocultured with the sinoatrialnode (SAN) tissues in vitro, so as to evaluate the possibil ity of BMSCs differentiation into SAN cells. Methods BMSCs wereisolated from Sprague Dawley rats (aged 4-6 weeks, male or female) by the adhesive method and cultured; BMSCs at the 3rdpassage were marked with carboxyfluorescein succinimidyl ester, and then were incubated on 6-well culture plate; cell climingsl ices were prepared at the same time. SAN tissue was taken and cut into 0.3 cm × 0.3 cm mass, and then placed into 4℃ PBSsolution. The SAN tissue mass was cocultured with marked BMSCs at the 3rd passage for 3 weeks as the experimental group, andBMSCs at 3rd passage were cultured alone for 1 week as the control group. At 1, 2, and 3 weeks after coculture, the mean integratedabsorbance (MIA) values of Cx40 and HCN4 were measured by Image pro plus 5.0 through the method of immunohistochemistry,and the mRNA expressions of Cx40 and HCN4 were identified by real-time fluorescent quantitative PCR. Results TheMIA values of Cx40 and HCN4 in the experimental group were higher than that in the control group, showing significantdifferences (P lt; 0.01). In the experimental group, the expressions of Cx40 and HCN4 increased gradually with time. The longerthe culture time was, the higher the expressions of Cx40 and HCN4 were, showing significant differences (P lt; 0.05). The mRNAexpressions of Cx40 and HCN4 in the experimental group were significantly higher than those in the control group (P lt; 0.01); inthe experimental group, the mRNA expressions of Cx40 and HCN4 increased gradually with time, showing significant differencesbetween different time points (P lt; 0.05). Conclusion The expressions of Cx40 and HCN4 increase obviously after coculturingBMSCs with SAN tissue, indicating that BMSCs could differentiate into SAN cells by coculturing with SAN tissue in vitro.
Objective To investigate effect of the removal of epithelium and mixed glands from the tracheal allografts on the graftimmunosuppression. Methods Fresh untreated tracheal allografts， cryopreserved tracheal allografts, and 10 off-epithelium tracheal allografts were obtained from 25 male SD rats. Fresh untreated tracheal allografts(40) were divided into 4 groups and dipped respectively in the solution of protease ⅩⅣ in 0, 0.1, 0.3 and 0.5 mg/ml at 4℃ for 12 hours. Thirty recipient male SD rats were randomly and equally divided into group A (fresh untreated tracheal allografts), group B(cryopreserved tracheal allografts), and group C(offepithelium tracheal allografts). The transplanted allografts were implanted into the abdominal cavity of other rats by being embedded in the greater omentum. Twenty-one days after transplantation, the tracheal graft segments were surgically removed, and then were initially fixed in cold 10% neutral buffered formalin solution for hematoxylineosin staining. Histological observation and lymphocyte infiltration were performed on the grafts to evaluate rejection. Results The 0.3 mg/ml protease ⅩⅣ could remove the epithelium and mixed glands of the grafts completely, but did no damage to cartilage. The cartilages of each group all survived and were revascularized. The lumens of group A were filled with granulation and necrosis tissue. In contrast, group B was filled with a few granulation tissues and group C was not at all. The number of lymphocyte infiltration in group A, B, and C was 29.16±2.69/HP, 15.17±2.19/HP, and 11.56±0.87/HP respectively. There was significant difference between group A and both group B and group C (Plt;0.05), and there was significant difference between group B and group C (Plt;0.05). Therefore, the grade of graftrejectionwas group Agt;group Bgt;group C. Conclusion The 0.3 mg/ml protease ⅩⅣ can completely remove the epithelium and mixed glands of grafts at 4℃ for 12 hours, and it preserves the normal structure of cartilage. The antigenicity of tracheal grafts can be greatly reduced by removing the epithelium and by the cryopreservation. The prior tracheal allograft in the omentum is feasible for the revascularization of the grafts.