Chronic morphine drinking establishes morphine tolerance, but not addiction in Wistar rats

Objective： Some animal models apply morphine in the drinking water to generate addiction, but related reports are not free of conflicting results. Accordingly, this study aimed to figure out if chronic consumption of morphine in the drinking water can induce morphine addiction in Wistar rats. Methods： For 3 weeks, the animals received a daily morphine dose of 35 mg/kg by offering a calculated volume of sugar water （5% sucrose） with morphine （0. l mg/ml） to each rat; animals receiving just sugar water served as controls. Immediately after the treatment phase, the tail immersion test was used to check for morphine tolerance, and all animals were then kept on tap water for one week （withdrawal phase）. Afterwards, all rats were allowed to choose their drinking source by offering two bottles, containing sugar water without and with morphine, simultaneously for two days （preference phase）. Results： While the chronic consumption of morphine led to a reduction in body weight and to morphine tolerance, the morphine-treated Wistar rats did not show any preference for the opiate-containing sugar water. Conclusion： Body weight loss and tolerance do not reveal a condition of drug craving, and current animal models should be re-evaluated regarding their potential to establish morphine addicted animals.

Expression of hypoxia inducible factor-1 alpha and ischemic erythropoietin tolerance in the brain of cerebral ischemic tolerance model rats

BACKGROUND： Hypoxia inducible factor-1 alpha （HIF-1 （x） and erythropoietin（EPO）, possessing neuroprotective effect in the cerebral ischemia, might play an important role in the formation of cerebral ischemic tolerance （IT）. OBJECTIVE：To observe the neuroprotective effect of cerebral ischemic preconditioning（IPC） of rats, and the expression and mechanism of HIF-1α and target gene erythropoietin in the brain tissue following the formation of cerebral IT. DESIGN ： A randomized and controlled observation SETTING： Department of Neurology, the Affiliated Hospital of Medical College, Qingdao University MATERIALS： Totally 84 enrolled adult healthy male Wistar rats of clean grade, weighing 250 to 300 g, were provided by the Animal Experimental Department, Tongji Medical College of Huazhong University of Science and Technology. Ready-to-use SABC reagent kit and rabbit anti-rat HIF-1α monoclonal antibody were purchased from Boshide Bioengineering Co.Ltd （Wuhan）; Rabbit anti-rat EPO monoclonal antibody was purchased from Santa Cruz Company （USA）. METHODS： This experiment was carried out in the Department of Anatomy, Medical College, Qingdao University during March 2005 to March 2006. ① The 84 rats were divided into 3 groups by a lot： IPC group （n=40）, sham-operation group （n=40） and control group （n=4）. In the IPC group, middle cerebral artery was occluded for 2 hours respectively on the 1^st, 3^rd, 7^th, 14^th and 21^st days of the reperfusion following 10-minute preischemia was made using a modified middle cerebral artery second suture method from Zea-Longa. The rats were sacrificed 22 hours after reperfusion in the end of middle cerebral artery occlusion （MCAO）. That was to say, after 10-minute preischemia, suture was exited to the extemal carotid artery and embedded subcutaneously. Middle cerebral artery was occluded again to form the second reperfusion at the set time point after reperfusion. Twenty-two hours later, rats were sacrificed; In the sham-operation group,the preischemia was substituted by sham-operation（only common carotid artery and crotch were exposed, and MCAO by suture was omitted）, and the other procedures were the same as those in the IPC group. In the control group, rats were given sham-operation twice at an interval of one day, and they were sacrificed 24 hours after the second sham-operation. ② Brain tissue was taken from the rats in each group. Cerebral infarction area of each layer was measured with TTC staining, and total cerebral infarction volume （The total cerebral infarction area of each layerxinterspace ） was calculated. After brain tissue was stained by haematoxylin-esoin （HE）, the form of nerve cells was observed under an optical microscope, and the expressions of HIF-1α（and EPO protein in the brain tissue were detected with immunohistochemical method. MAIN OUTCOME MEASURES： ①Cerebral infarction volume;②form of nerve cell; ③ the expression of HIF-1α and EPO protein in the brain tissue. RESULTS：Totally 84 rats were enrolled in the experiment. The dead rats were randomly supplied during the experiment, and finally 84 rats entered the stage of result analysis. ① Detection of cerebral infarction volume of rats in each group： Cerebral infarction volume in the IPC group was significantly smaller than that in the sham-operation group on the 1^st, 3^rd and 7^th days after reperfusion respectively [（161.2±6.9） mm^3 vs （219.9±11.2） mm^3, （134.9±9.0） mm^3 vs （218.6±13.0） mm^3, （142.9±13.7） mm^3 vs （221.3±14.2） mm^3, t=-8.924, 10.587,7.947, P〈 0.01]. ② Observation of nerve cell form of brain tissue： HE staining showed that the ischemic degree, range and cerebral edema degree of IPC group were significantly milder than those of sham-operation group. ③ The expressions of HIF-1α and EPO protein in cerebral cortex and hippocampus ： The expression of HIF-1αof IPC group was significantly higher than that of sham-operation group on the 1^st, 3^rd and 7^th days after reperfusion respectively （125.93±3.79 vs 117.65±5.60, 140.63±4.64 vs 119.33±4.26, 131.15±2.74 vs 107.60±3.89, t=2.449, 6.763,9.899,P 〈 0.05-0.01）. The expression of EPO of IPC group was significantly higher than that of sham-operation group on the 3^rd and 7^th days after perfusion respectively （141.68±3.29 vs 126.33±4.51, 138.88±2.59 vs 125.58±6.18,t=5.499,3.970, P〈 0.05）. CONCLUSION ： ①IPC can protect the never cells in rat brain and the best time to onset of cerebral IT induced by IPC is 1 to 7 days after reperfusion. ② Neuroprotective effect of cerebral IT might be related to the expression of HIF-1α and its target gene EPO.

Inhibitory effect of cyclosporin A on the immunological rejection of rats with cerebral hemorrhage following transplantation of nerve growth factors transfected glial cells

BACKGROUND： At present, it has been confirmed that immunological rejection exists in the cell transplantation in brain tissue, the effects of immunosuppressant on the immunological rejection and the survival of grafts in brain cell transplantation are worthy being investigated further. OBJECTIVE： To observe the immunological rejection after transgeneic cell transplantation in treating cerebra hemorrhage in rats, and investigate the interventional effect of cyclosprin. DESIGN ： A randomized controlled study SETTINGS： Second Affiliated Hospital of Xuzhou Medical College; First Affiliated Hospital of Nanjing Medica University. MATERIALS： Thirty-five healthy clean-degree SD rats of 6-8 weeks old were used, weighing 200-250 g, either male or female; The FACSort flow cytometer （American BD Company） and NYD-1000 image analytical system were used, The rat-anti-rat CD4 monoclonal antibody, rat-anti-rat CD8 monoclonal antibody, and rat-anti-rat MHC Ⅱ antigen monoclonal antibody were purchased from Santa Cruz Company; SP and DAB kits were purchased from Beijing Zhongshan Bio-engineering Company. XSP-8C2 light microscope was the product of Shanghai Zousun Optical Instrument, Co.,Ltd, and KYKY-3800B electron microscope was the product of China KYKY Technology Development Co.,Ltd. METHODS ： The experiments were carried out in the animal experimental center of Nanjing Medical University from April to July in 2003. ① Model establishment： The rats were anesthetized, and then the coordinates of left internal capsule were identified, and the needle was withdrawn after 120 μL blood was injected into the internal capsule. Adenoviruses were taken as the carriers, after the astrocytes were successfully transfected by nerve growth factor（NGF） gene, 0.2 mL cell suspension was injected into the sites of cerebral hemorrhage. Thirty successfully established rat models were randomly divided into cyclosporin A group （n=18） and control group, the rats were treated with intraperitoneal injection of cyclosporin A （10 mg/kg per day） intraperitoneal injection of saline of the same dosage from the 1^st day after transplantation, once a day for 7 days continuously.② CD4^＋ and CD4^＋ detection： The CD4^＋ and CD4^＋ T lymphocytes in caudal vein were counted with flow cytometer at 15 days after treatment. ③ Morphological observation in the transplanted sites： The rats were killed and then brain tissues were taken out, the transplanted sites and the structure of the normal brain tissue around the transplanted sites were observed with light and electron microscopes. ④Detections of the infiltration of T lymphocyte subsets and expression of major histocompatibility complex （MHC） Ⅱ antigen in the transplanted sites： The image analysis of immunohistochemical sections was performed with the image analytical system, and the integral optical density （IOD） was taken as the statistical value to observe the infiltration of T lymphocyte subsets and expression of MHC Ⅱ antigen in the transplanted sites, and the normal brain tissue around the transplanted sites were taken as controls. MATN OUTCOME MEASURES： ① Countings of CD4^＋ and CD4^＋ in peripheral blood; ②Results of the morphological observation in the transplanted sites; ③ Infiltration of T lymphocyte subsets and expression of MHC Ⅱ antigen in the transplanted sites RESULTS ： Totally 35 rats were used, and 30 were successfully made into models, 5 died during the treatment, the other 25 were involved in the analysis of results. ① Results of CD4^＋ and CD4^＋ T lymphocytes in pedpherel blood： The percentages of CD4^＋ and CD4^＋ T lymphocytes in the cyclosporin A group were （29.20±3.97）% and （20.65±2,02）%, respectively, which were obviously lower than those in the control group [（47,39±3,01）%, （28.30±2.36）%, t=-4.983, 4.012, P 〈 0.05], and the CDC/CD4^＋ ratio was obviously lower than that in the control group （1,41±0.86, 1,64^＋0.69, t=-3. 871, P〈 0.05）.② Morphological results in the transplanted sites： Under optical and electron microscopes, the survival region of the transplant was round, and it had an unobvious migration region with the normal brain tissues, the grafts had normal cellular form. Infiltrations of lymphocytes and monocytes were observed in both groups, and mainly located in the transplanted sites, and the expression of lymphocytes in the cyclosporin A group was markedly lower than that in the control group, and no above-mentioned changes were observed in the normal brain tissue around the transplanted sites. ③ Results of CD. and CD4^＋ T lymphocytes and expression of MHC Ⅱ antigen in the transplanted sites： The CD4^＋ and CD4^＋ T lymphocytes and expression of MHC Ⅱ antigen in the transplanted sites were observed in both groups. The IOD of CD4^＋ and CD4^＋ antigen positive cells in the cyclosporin A group were obviously lower than those in the control group （1.85±0.38, 1.44^＋0.33; 3.33±0.37, 2.648±0.56, /=-4.122, 4.434, P〈 0.05）, and the IOD of MHC Ⅱantigen positive cells was markedly lower than that in the control group （0.76±0.22, 0.94±0.24, t=3.885, P 〈 0.05）. CONCLUSION： There is immunological rejection in brain tissue after the transplantation of NSC transgeneic glial cells. ② The immunosuppressant of cyclosporin A can reduce the immunological rejection after the cell transplantation.

The diagnostic role of heat shock protein 70 in acute rejection after pancreatioduodenal transplantation in rats

Objective To explore the diagnositc role of heat shock protein ( HSP) 70 in acute rejection after pancreatioduodenal transplantation in rats. Methods Groups of Wistar rats underwent total pancreatioduodenal transplantation from allogeneic SD or syngeneic Wistar rats. The grafts were harvested on the posttransplantation day 3,5 and 7 and were used to detect the expression of HSP70 by immunohistochemistry and Western Blotting quantitative methods. The correlation between HSP70 expression and pathological findings was observed. Results The level of HSP70 in the isografts did not change greatly( P 】 0.05); the level of HSP70 in the allografts was increased progressively on the posttransplantation day3, 5 and 7 ( P 【 0.01). There was a significant correlation between HSP70 and pathological score in the allograft group (P 【 0.01, r = 0.934). Conclusion HSP70 involved in pancreas allograft rejection and could be useful for early diagnosis of acute rejection following pancreas transplantation. 8 refs,2 tabs.

Gene transfer of huCTLA4-Ig to inhibit the acute rejection of liver allograft in rats

To observe the effect of gene transfer of huCTLA4-Ig to inhibit the acute rejection of liver allograft in rats.Methods With AdEasy vector system,the recombinant adenovirus containing huCTLA4-Ig gene was constructed.Using ex vivo gene transfer technique,exogenous gene was introduced to the liver graft during cold preservation and expressed locally in the graft.The effect of inhibition of acute rejection and inducing liver graft tolerance was observed.Results No recipients in group A (without any treatment,n=5) or group B (treated with Ad-GFP,n=4) died within 3 weeks after transplantation and severe acute rejection (massive periportal infiltration,endothelilitis,damage to biliary epithelium and severe tissue destruction) was confirmed pathologically in the graft.In contrast,all recipients in group C (treated with Ad-huCTLA4-Ig,n=5) achieved long-term liver allograft survival (>150 days).Histological examination of Ad-huCTLA4-Ig transduced allografts demonstrated a mild to moderate periportal inflammation and mild injury to liver graft on day 8 posttransplant.A mild mononuclear infiltration was observed;however,there was complete preservation of the bild ducts and no evidence of vascular injury on day 150 posttransplant.The mean IL-2 concentration in serum was (362.09±45.84) ng/L at day 1 pretransplant.In control animals (groups A and B),serum IL-2 concentration was elevated to a high level within 7 days posttransplant,which was about 1.5 to 2.5 times as much as that before transplant.In contrast,in huCTLA4-Ig-treated animals (groups C),IL-2 concentration in serum was maintained at a relative low level,which was near or less than that before transplant (P<0.01).Conclusion Using ex vivo gene transfer technique,huCTLA4-Ig gene can be introduced to the liver graft during cold preservation.The modified graft can express and excrete immunoregulatory protein locally,which can suppress acute alloimmune response and is responsible for prolongation of graft survival without using routine immunosuppressive drugs.These findings provide some experimental evidence that gene delivery of sequences encoding immunoregulatory proteins can be applied to clinical liver transplantation for inhibiting the acute alloimmune response and achieving graft tolerance.7 refs,2 tabs.

Induction of immune tolerance with heart-thymus composite allotransplantation in rats

Objective To study on the role of thymus transplantation for heart allograft in rats. Methods Vascularized heart-thymus combined transplantation was performed with microsurgical technique. Graft survival, histopathology, level of IL-2, IL-4 and its mRNA expression in serum and cardiac grafts were investigated. Results Heart-thymus combined transplantation achieved effect in the prolongation of cardiac graft survival with short-term administration of cyclosporine. Conclusions Vascularized thymus transplantation induced immune tolerance in thymectomized rats.

Tolerance and dependence of edomorphin-1 in rats and possible mechanisms

Objective：To observe the tolerance and the dependence of endomorphin-1 （EM-1） in rats and the possible mechanisms. Methods：Sixty Sprague-Dawley rats were randomly allocated into saline, acute EM-1-treated and chronic EM-1-treated groups. The rats were intracerebroventricularly injected with saline, acute EM-1 10 μg/kg 30 rain prior to sacrifice,and chronic EM-1 by daily administration at 8：00 A.M. and 15：00 P.M. from 10 μg/kg on the 1^st day to 50 μg/kg on the 94 day, respectively. In chronic EM-1-treated group, the median antinociceptive dose （AD50） and the catatonic median effective dose （ED50） were determined by the improved Dixon＇s method. Natural withdrawl test was used to assess the dependence of EM-1. Maximal binding capacity （Bmax） and dissociation constant （Kd） of 3H-DAMGO, binding to mu-opioid receptor （MOR） in brain tissue, was measured by Scatchard analysis. Gene expression of MOR was measured by reverse transcription-polymerase chain reaction（RT-PCR）. Results ：Tolerance of the antinociceptic and catatonic effects on the 3rd day （3.1-fold and 1.9-fold ） and the 9th day （28.4-fold and 8.5-fold） were observed in chronic EM-1-treated group （P 〈 0.05）. Jumping times and withdrawal scores of rats were significantly higher in the chronic EM-1-treated group than those in saline group on the 94 day （P 〈 0.05）. Bmax and mRNA expression of MOR in cortex, midbrain and striatum were lower in chronic EM-1-treated group on the 94 day than the other two groups（P 〈 0.05）, but Kd had no significant difference （P 〉 0.05）. AD50,ED50,Bmax ,Kd and gene expression of MOR were recorded. Conclusion： EM-1 possesses the tolerance and the dependence. After a long-term treatment, EM-1 down regulates the binding capacity and mRNA of MOR, which somewhat accounts for the dependence.

Studies of CTLA4Ig in acute rejection of pancreas transplantation in rats

Objective： To investigate the protective effect of CTLA4Ig in rejection of pancreaticoduodenal transplantation model of rat. Methods ： Pancreaticoduodenal transplantion models were established from the donor F344 rats to the Lewis recipients. The models were divided into 2 groups： Group A and B with 12 rats in each group.2 days after transplantation, reciepients in group A were treated with i.p. injection of sailine, and those in group B CTLA41 were injected（200μg）. On day 1,4,7, 10 after transplantation, the grafts were harvested for histopathological examination. On day 4 after transplantation, the CD4^＋CD25^＋ T cells in the grafts were detected by Flow Cytometry. Results： Compared with group A： the degree of the rejection of grafts in group B was lower. The number of CD4^CD25^＋ T cells of graft was （7.91±1.26）% in group A and （13.81±1.71）% in group B, which had significant difference （P〈0.01）. Conclusion： CTLA4Ig could inhibit T cell costimulatory pathway, prevent acute rejection, which might be mediated bv increasing the number of CD4^＋CD25^＋ regulatory T cells.

DIFFERENCE OF REJECTION IN SINGLE VERSUS COMBINED PANCREAS AND KIDNEY TRANSPLANTATION IN RATS

Objective.To investigate the difference of rejection in single versus combined pancreas and kidney transplantation in rats. Methods.Allograft models including simultaneous pancreas and kidney(SPK)transplant and pancreas or kidney transplant alone were established in SD-Wistar rats, rejections of pancreas and kidney in different models were compared morphologically and functionally. Results.Mean survival time(MST)of pancreas was significantly prolonged in SPK than in pancreas transplant alone(PTA)(115 days vs. 92 days, P<005). Incidence of interstitial pancreatic rejection at grade Ⅱ and grade Ⅲ was much obvious in PTA than in SPK(429% vs. 125% at grade Ⅱ and 286% vs 63% at grade Ⅲ , P<005). No significant difference was found in MST between SPK and kidney transplant alone(KTA). Administration of cyclosporine A prolonged the MST of pancreas and kidney, without altering the tendency stated above. Conclusions.In SPK, the function of pancreas is protected by kidney hence the severity of rejection is reduced, whereas the function of kidney is not protected by pancreas. It suggests that different organs differ in immunoallergization and immunoregulation, and immune response tend to attack organs with greater immunoactivity, those organs with minor one could be protected. Cyclosporine A is effective on prolonging the MST of pancreas and kidney.

Effect of Sugar on the Process of Cold-Acclimation-Induced Freezing Tolerance of Populus tomentosa

Populus tomentosa seedlings for cold-acclimating were pretreated wit h or without 20% saccharose. Changes in the concentrations of total soluble sugar , the survival rates, and freezing tolerance of seedlings during cold acclimatio n were investigated. The results showed that cold acclimation increased the conc entrations of total soluble sugar, the survival rates and freezing tolerance. Co ld acclimation, combined with the saccharose-pretreatment, enhanced the above- ment ioned effect of cold acclimation, and obviously increased the concentrations of total soluble sugar, the survival rates and freezing tolerance of seedlings. Fur ther analysis found that the concentrations of total soluble sugar in branches i ncreased greater than that in leaves during both cold acclimation with or withou t the pretreatment of saccharose. Moreover, an increase of the concentrations of total soluble sugar in branches and leaves was closely related to the freezing tolerance of seedlings. The results indicate the accumulation of soluble sugar i n seedlings induced by cold acclimation may be involved in the induction of free zing tolerance .

Immunological tolerance of human hepatocyte xenograft induced by adenovirus vector-mediated CTLA4Ig gene transfer

BACKGROUND:Systemic administration of CTLA4Ig has been applied in inducing immunological tolerance of hepatocyte implants,but has potential for systemic immune inhibition.This study was designed to induce hepatocyte immunological tolerance by locally expressing CTLA4Ig in an attempt to improve the effectiveness of cell transplantation.METHODS:A normal human liver cell line(L02) was transfected with adenovirus vector containing the CTLA4Ig gene(Ad-CTLA4Ig-EGFP) in vitro,and the expression of CTLA4Ig by transfected cells was assessed by fluorescent imaging and immunocytochemical staining.Transfected cells then were injected into the spleen of Sprague-Dawley rats,the survival of cells was determined by immunohistochemistry,and the immune status was examined through CD4 + and CD69 + T cellcounts and ELISA detection of IL-2 in peripheral blood.RESULTS:L02 cells expressed CTLA4Ig in the cytoplasm for >4 weeks.Surviving L02 cells were observed in the experimental group at 3 and 4 weeks post-transplantation,while none was detected in the control group.Furthermore,the percentages of CD4 + and CD4 + CD69 + T cells in the CTLA4-transfected group were 24.5% and 45.1%,markedly lower than those in the control group at 4 weeks post-transplantation(P<0.01).Furthermore,the IL-2 level was also lower in the CTLA4transfected group than in the control group.CONCLUSION:Adenovirus-mediated CTLA4Ig gene transfer into human hepatocytes has the potential to become an effective method of inducing immunological tolerance in hepatocyte transplantation.

A proteomic analysis of allograft rejection in rats after liver transplantation

In order to understand the allograft rejection in orthotopic liver transplantation (OLT), an allograft re- jection rat model was established and studied by proteomic approach. The protein expression profiles of liver tissues were acquired by fluorescence two-dimensional difference gel electrophoresis (2D DIGE) that incorporated a pooled internal standard and reverse fluorescent labeling method. The expression levels of 27 protein spots showed significant changes in acute rejection rats. Among these spots, 19 were identified with peptide mass fingerprinting using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) after tryptic in-gel digestion. The results of the present paper could be helpful for our better understanding of allograft rejection in organ transplantation.

Transplantation of human hepatocytes into tolerized genetically immunocompetent rats

AIM: To determine whether normal genetically immunocompetent rodent hosts could be manipulated to accept human hepatocyte transplants with long term survival without immunosuppression. METHODS: Tolerance towards human hepatocytes was established by injection of primary human hepatocytes or Huh7 human hepatoma cells into the peritoneal cavities of fetal rats. Corresponding cells were subsequently transplanted into newborn rats via intrasplenic injection within 24h after birth. RESULTS: Mixed lymphocyte assays showed that spleen cells from non-tolerized rats were stimulated to proliferate when exposed to human hepatocytes, while cells from tolerized rats were not. Injections made between 15 d and 17 d of gestation produced optimal tolerization. Transplanted human hepatocytes in rat livers were visualized by immunohistochemical staining of human albumin. By dot blotting of genomic DNA in livers of tolerized rats 16 weeks after hepatocyte transplantation, it was found that approximately 2.5 X 10(5) human hepatocytes survived per rat liver. Human albumin mRNA was detected in rat livers by RT-PCR for 15 wk, and human albumin protein was also detectable in rat serum. CONCLUSION: Tolerization of an immuno-competent rat can permit transplantation, and survival of functional human hepatocytes.

The Dual Regulatory Roles of Macrophages in Acute Allogeneic Organ Graft Rejection

Innate immune cells are critical for transplant response.As an important cellular component of innate immune cells,macrophages are the predominate infiltrated cells in allografts,and macrophage accumulation in allografts is negatively associated with the short-and long-term outcomes of organ transplantation.Macrophages are functionally heterogeneous and plastic.They participate in organ graft rejection through multiple pathways,including antigen presentation,the expression of costimulatory molecules and cytokines,and direct cytotoxicity and injury ability to allografts.However,some macrophage subpopulations,such as regulatory macrophages,can protect allografts from immune rejection and promote transplant immune tolerance with their immune regulatory properties.Although researchers recognize the potential roles macrophages play in allograft injury,they pay insufficient attention to the diverse roles of macrophages in allograft rejection.We herein briefly summarize the distinctive roles of macrophages in acute transplant immune response and the effect of immunosuppressive drugs on macrophages.Greater attention should be paid to the complex and critical function of macrophages in allograft rejection,and more effort should be put into developing immunosuppressive drugs that specifically target macrophages,which would ultimately improve the long-term survival of organ grafts in patients.

Effect of FTY720 and ICAM-1 mAb mono and combination therapy in cardiac allo-transplantation in rats

Objective To observe the effect of FTY720 and ICAM-1 mAb mono and combination therapy in cardiac silo-transplantation in rats.Methods Rats were randomly assigned to 9 groups,heart allo-transplantation were performed in abdominal site with micro-surgical technique.Recipients with allografts were treated with different doses of FTY720 and(or)ICAM-1 mAb.Graft survival,histopathology andlevel of serum IL-2,IFN-γ,IL-4,IL-10were investigated.Results Low doses of FTY720(lmg/kg)combined with ICAM-1 mAb achieved synergistic effect in the prolongation of cardiac graft survival,combination index CD=0.67.Conclusion Concomitant therapy of FTY720 and ICAM-1 mAb achieved a synergistic effect in the prolongation of heart allograft survival in rats.

Low-dose morphine elicits ventilatory excitant and depressant responses in conscious rats: Role of peripheral <i>µ</i>-opioid receptors

The systemic administration of morphine affects ventilation via a mixture of central and peripheral actions. The aims of this study were to characterize the ventilatory responses elicited by a low dose of morphine in conscious rats;to determine whether tolerance develops to these responses;and to determine the potential roles of peripheral μ-opioid receptors (μ-ORs) in these responses. Ventilatory parameters were monitored via unrestrained whole-body plethysmography. Conscious male Sprague-Dawley rats received an intravenous injection of vehicle or the peripherally-restricted μ-OR antagonist, naloxone methiodide (NLXmi), and then three successive injections of morphine (1 mg/kg) given 30 min apart. The first injection of morphine in vehicle-treated rats elicited an array of ventilatory excitant (i.e., increases in frequency of breathing, minute volume, respiratory drive, peak inspiratory and expiratory flows, accompanied by decreases in inspiratory time and end inspiratory pause) and inhibitory (i.e., a decrease in tidal volume and an increase in expiratory time) responses. Subsequent injections of morphine elicited progressively and substantially smaller responses. The pattern of ventilatory responses elicited by the first injection of morphine was substantially affected by pretreatment with NLXmi whereas NLXmi minimally affected the development of tolerance to these responses. Low-dose morphine elicits an array of ventilatory excitant and depressant effects in conscious rats that are subject to the development of tolerance. Many of these initial actions of morphine appear to involve activation of peripheral μ-ORs whereas the development of tolerance to these responses does not.

Correlation of hypoxia-inducible factor-1 alpha and erythropoietin protein and mRNA to cerebral ischemic tolerance in a focal ischemia/reperfusion model using the twice suture method

BACKGROUND： Numerous studies have shown that transient ischemic preconditioning induces cerebral ischemic tolerance. However, the underlying mechanisms of endogenous protection following ischemic preconditioning remain unclear. OBJECTIVE： To dynamically measure erythropoietin and hypoxia-inducible factor-1α （HIF-1α） mRNA and protein expression at various times following preconditioning, and to investigate effects of erythropoietin and HIF-1α on cerebral ischemic tolerance in a model of focal ischemia/reperfusion established using the twice suture method. DESIGN, TIME AND SETTING： The randomized, controlled study was performed at the Institute of Anatomy, Medical College, Qingdao University, China from March 2006 to March 2007. MATERIALS： Rabbit anti-rat HIF-1α monoclonal antibody and biotinylated goat anti-rabbit IgG （Boster, China）, rabbit anti-rat erythropoietin monoclonal antibody （Santa Cruz Biotechnology, USA）, and one-step RT-PCR kit （Qiagen, Germany） were used in this study. METHODS： A total of 99 healthy, male, Wistar rats were randomly assigned to three groups： sham surgery （n = 9）, non-ischemic preconditioning （n = 45）, and ischemic preconditioning （n = 45）. In the ischemic preconditioning group, rat models of pre-ischemia-reperfusion-ischemia-reperfusion were established by occluding the left middle cerebral artery using the twice suture method. In the non-ischemic preconditioning group, pre-ischemia was replaced by sham surgery. Subsequently, the ischemic preconditioning and non-ischemic preconditioning groups were equally divided into five subgroups according to time of first reperfusion, including 1-, 3-, 7-, 14-, and 21-day subgroups. The sham surgery group received the sham surgery twice. MAIN OUTCOME MEASURES： HIF-la and erythropoietin protein expression was measured in the cerebral cortex, corpus striatum, and hippocampus of the ischemic hemisphere. HIF-1α and erythropoietin mRNA expression were determined in the frontal and parietal cortex of the ischemic hemisphere. RESULTS： （1） Intergroup comparison： compared with the non-ischemic preconditioning group, HIF-1α protein expression significantly increased in the rat cerebral cortex, corpus striatum, and hippocampus in the ischemic hemisphere at 1,3, and 7 days following reperfusion in the ischemic preconditioning group （P 〈 0.05 or P 〈 0.01）. Erythropoietin protein expression significantly increased in the cerebral cortex, corpus striatum, and hippocampus, as well as HIF-1α and erythropoietin mRNA expression in the frontal and parietal cortex in the ischemic hemisphere, at 3 and 7 days following reperfusion in the ischemic preconditioning group （P 〈 0.05）. （2） Temporal expression： HIF-1α protein expression in the rat cerebral cortex, corpus striatum, and hippocampus, as well as HIF-la mRNA expression in the frontal and parietal cortex, in the ischemic hemisphere increased at 3 days, and gradually decreased from 7 days following reperfusion in the ischemic preconditioning group. Temporal erythropoietin protein and mRNA expression was consistent with HIF-1α protein expression. （3） Correlation： erythropoietin mRNA expression positively correlated with HIF-1α mRNA expression （r= 0.737, P 〈 0.01）. CONCLUSION： Ischemic preconditioning induced cerebral ischemic tolerance. Pre-ischemiainduced increase in endogenous HIF-1αexpression, as well as its target gene erythropoietin, participated in the formation of cerebral ischemic tolerance.

A New Carotid Artery Transplantation Model of Rats

To establish a murine carotid artery transplantation model for the study of the chronic rejection, 80 rats were divided into two groups, an allotransplant （ACI-Lewis） group and an isotransplant （Lewis-Lewis） group （control group）. The donor carotid artery and the recipient carotid artery were anastomosed by using a polyethylene cuff （internal diameter： 0.7 mm, length： 3 mm）.The pathological changes of carotid artery transplant were observed 14, 28 and 56 days after the transplantation. The results showed that the model was successfully established in 95% of the animals. The chronic rejection-associated arteriosclerosis was induced 28 days after the transplantation. The new chronic rejection model of carotid artery by using cuff technique caused fewer traumas and was easy to make. The pathological changes of the transplant mimicked the chronic rejection-associated arteriosclerosis found in human transplant.

Length of warm ischemic tolerance for epithelial regeneration in heterotopic rat tracheal isografts

Objective To determine the length of warm ischemic ( WI) tolerance in bronchial graft from non - heart - beating donors. Methods Forty - eight rats were randomly divided into 4 groups ( each group having 12 rats) according to different WI durations including WI - 0 min ( group A) ,WI - 30 min ( group B) ,WI - 45