Local Immunosuppression With Long Term Intra-arterial Cyclosporine A In Rat Hindlimb Allograft

Part1: Local immunosuppression model in rats With long term intra-arterial cyclosporine ABackgroundToxicity caused by high blood concentration is a complication of large dose immunosuppression in transplantation. On the other hand, drug density in tissue is crucial to the survival of grafts. Former researches in tumor chemotherapy have confirmed that a local drug delivery, especially directly into the nourish artery, is helpful in reducing whole blood level and enhancing the drug availability in target tissue. Kinds of chemicals have been proved can be worked for local immunosuppression with intra-arterial infusion. However, none of them is efficient enough and can not last for a long time untill the cyclosporine come out. As new infusion apparatus widely spread, a long term local immunosuppression therapy become available.ObjectiveTo analyze the pharmacokinetics advantages of intra-arterial immunosuppression, an autograft model was established on rats’ hindlimb to imitate the blood supply status of allotransplant. Cyclosporine A (CSA), an immunosuppressant widely used in clinic, was constantly infused into the nourishing artery of replanted limb to determine the feasibility of long term local immunosuppression.Methods1. Experimental designSixty adult male SD rats received right hindlimb amputation and then replanted with standard microsurgical techniques. After autotransplantation, all animals were randomly divided into four equal groups for diverse dosing approaches: GroupI.G.(intragastric irrigation), GroupI.P.(intraperitoneal injection), Group I.V.(intravenous infusion) and Group I.A.(intra-arterial infusion). In each group, three subgroups were established separately according to the dosage of CSA administration.2. Limb autograft model Rats receivedright hindlimb replantation. Aamputation was made at the level of thigh,including femur, muscles,nervi and all ood vessels. Then a18G needle was used as a intramedullary rod to fix the femur. Femoral vessel and sciatic nerve were respectively anastomosed end-to-end to the recipient’s, using standard microsurgical techniques. Thigh muscles were approximated using silk sutures.3. Drug administrationCSA was administrated to each subgroups respectively at three dose grade of2.5,5.0and10mg·kg-1·day-1and then last for60days. To rats for oral gavage and intraperitoneal study, CSA was intermittently administrated once a day. In groups for intravenous or intra-arterial infusion, Smith infusion ports were implanted under rats’ dorsal skin. Using a micro-tubing inserted into right femoral blood vessels, CSA was constantly infused into rats’ replanted limbs for long term observation.4. Whole blood drug concentration analysisAfter60-days continuous administration, whole blood sample was collected from rats’ thoracic aorta. CSA concentration inblood was determined with Enzyme amplification immunoassay test (EMIT).5. Tissue CSA density analysisAt the end of research, replanted right hindlimb was collected. Mass of skin and muscle were homogenized and then test with EMIT to determine the drug density tissue.6. Statistical analysisData was recorded as MEAN±SD and then analyzed with Statistical Product and Service Solutions (SPSS) software in variance analysis procedures. Differences were compared between each groups including on bioavailabilities and tissue distribution rate for diverse administration approaches.Results1. General observationForty percent (2in5) rats received intraperitoneal cyclosporine underwenttemporary peritonitis. Another two rats in Group I.V. died for toxicity of high dose cyclosporine. None visible side reaction was noticed in rats receiving oral dose or intra-arterial CSA.2. Whole blood drug concentrationIn Groupl.A, blood CSA level was reduced by28%～40%than in rats received intravenous cyclosporine at equal dose. Difference was significant at0.05level. When treated with low dose CSA at2.5mg·kg-1·day-1level, blood drug level was slightly higher in GroupI.A. than in GroupI.G. and GroupI.P.. As CSA dose elevated to5～10mg·kg-1·day-1, no significant difference was found among these three groups. Biological conversion rate of CSA in each administration approach is about23%～52%for oral dose,43%～69%for intraperitoneal injection, and60%～72%for intra-arterial infusion. Absorption rate increased with dose grade when CSA administrated orally which indicated a saturation of gastrointestinalelimination. Absorption slightly decreased as treated with high dose cyclosporine intraperitoneally while keep constant in Group I.A.3. Tissue drug concentration comparisonSkin CSA level in GroupI.A.was4.6-25.9fold to GroupI.G.,4.1-14.5fold to Groupl.P., and2.4-5.1.fold toGroupI.V.. Similar elevation was found in muscle from rats received intra-arterial CSA:3.2-12fold to Groupl.G,4.1-14.5times to Group I.P. and1.7-3.3fold to GroupI.V..ConclusionsIntra-arterial delivery of CSA has advantages than systemic administration including reducing whole blood drug level and enhancing drug absorption in target tissue. A long term intra-arterial immunosuppression is safe and feasible.Part2: Prolonged limb allograft survival with Continuous intra-arterial cyclosporineBackgroundKinds of chemicals have been proved useful in prolonging allograft survival by local administration. However, none of them are effective enough to create a robust immunotolerance. Pharmacokinetics study indicates that intra-arterial administration of cyclosporine A (CSA) can multiple the drug density in target tissue which may be helpful to graft survival. However there is no report about long term local immunosuppression in composite tissue allograft scope.objectiveTo discuss the feasible of long term administration of intravascular cyclosporine, allogeneic limb transplantation model was established between two species rats. CSA was constant infused into the femoral artery of transplanted limbs. Graft survival days was recorded to evaluate the efficiency of long term local immunosuppression. Drug deposition was examined in solid organs for toxicity evaluation after long term CSA infusion.Methods1. Hindlimb allotransplant Male, inbred Brown Norway (BN; RT1n) rats were used as donors, and Lewis (LEW; RT11) rats as recipients. To induce immunotolerance anti-lymphocyte serum (ALS) 1.0ml and cyclopsporine10mg·kg-1·day-1was administeredintraperitoneally the day before operation. Donor’s right hindlimb, including bone, muscle, femoral vessels, sciatic nerve, and skin, were amputated at midfemoral level. A similar amputation was made in the recipient rat. Osteosynthesis was performed using an18-gauge needle as an intramedullary rod. The donor femoral vessel and sciatic nerve were respectively anastomosed end-to-end to the recipient’s, using standard microsurgical techniques. Thigh muscles were approximated using silk sutures.2. Experimental designFive limbs amputated on Lewis rats and then replanted as GroupISO for isogeneic controls. Another five rats served a frank allotransplant contrast (GroupALLO) experienced allograft without maintained immunosuppression. Fifty-four rats involved in immunosuppression study. Animals were randomly divided into three groups:GroupI.P.(intraperitoneal injection), GroupI.V.(intravenous infusion) and GroupI.A.(intra-arterial infusion). Three subgroups were established to each dose approach in accord with diverse CSA dose at2.5,5.0and10mg·kg-1·day-1.3. Long term infusion protocolTo rat for infusion study, a two-phrase infusion system was established. The vivo part contained a Smith infusion port implanted under dorsal skin. Polyurethane catheter was inserted into the femoral artery or vein for intravascular infusion. The vivo part of infusion system was totally sealed under skin to prevent infection. The external part linked with a micropump and connected with the subcutaneous port using special adaptor. The vitro segment was replaced and sterilized weekly. A dual-channel infusion design was adopted for cyclosporine infusion and storage. Original CSA solution was stored in syringe as one channel. Heparinized Dextran was sealed in the other channel as a diluting liquid. Drug was pre-diluted when infused into rats blood vessel. Such a pre-diluting system was helpful to minish CSA decade when exposed in room temperature and ease to against blood clots.4. Limb survival observationAny sign of rejection on grafts was recorded. An irreversible rejection occurred or graft survive beyond100days was regarded as end point of research. 5. Histological analysisAt the end of research, grafts were collected for hematoxylin and eosin (HE) stain. Histological feature was compared between each group.6. Mean rejection grading (MRG) evaluationFor grafts achieved full term survival, Banff2007rejection score classification was adopted. Four location of skin from grafts, including anterior, posterior, medial and lateral, was sectioned for rejection grading. By average the four results derived from four skin part aMRG score was obtained to evaluate the difference caused by administration methods.7. Drug accumulation in solid organTo animals achieved full term limb survival, tissue mass was collected from both liver and right kidney. Sample was homogenized with mechine and then for CSA concentration analysis with Enzyme amplification immunoassay test (EMIT).8. Statistical analysisData was recorded as MEAN±SD and then analyzed with one-way ANOVA test in SPSS13.Over software. Grading data of rejection score was analyzed with nonparametric statistical approach.Results1. Clinical observationNo macroscopic rejection was observed in ratsreceived self replantation. All grafts without maintained immunosuppression underwent typical rejection course. Limbs showed progressive skin erythema, swelling, skin ulcers, hair loss and mummificationand then fell off in a week. Two rats in GroupI.P. experiencedtemporary abdominal infection since excessive injection. Another two died in GroupI.V. for toxicity of long term high dose immunosuppression. Intra-arterial CSA showed no adverse reactions except mean wound healing delay which concerning with inhibition of cell metabolism.2. Graft survival timeA mono therapy with low dose intra-arterial CSA (2.5mg·kg-1·day-1) did not achieved full time survival, however prolonged the mean survival time to39.4±9.1 days. Elevation was significant at P<0.05level when compared with GroupI.P.(13.6·1.9days)and GroupI.V.(21.0±3.9days). As CSA dose doubled to5.0mg kg-1·day-1only rats in GroupI.A. got their limbs survived above100days, far beyond the46.4±7.0days in GroupI.P. and the56.4±7.0days in GroupI.V.. When a large dose CSA at10mg·kg-1·day-1level was used, all rats got full term immunotolerance no matter dosing approach. No difference on mean survival days was found in three groups at this dose grade.3. HistologicalHistological results showed typical sign of rejection in frank allografted group. Dense monocyte infiltration was found in skin and subcutaneous tissue. Rats treated with low dose CSA showed a similar histological result. Lymphocyte infiltration was alleviated in GourpI.A.which mainly presented an inflammation surround the small vessel. When CSA dosage was raised to over5.0mg·g-1·day-1rejection sign could still be observed in GroupI.P. And GroupI.V.,ranged from moderate to slight. At the same time rare infiltration except mean periangiitis was found in grafts treated with intra-arterial CSA. When CSA dose doubled to10mg·kg-1·day-1rejection sign was totally free from GroupI.A.4. Mean rejection grade evaluationA lower rejection score was found in GroupI.A. than the other two groups. Difference was significant at0.01level.5. Toxicity accumulationAfter intra-arterial infusion, liver CSA density was reduced by19%and35%respectively than in intraperitoneally or intravenously treated rats. Similar, drug concentration in kidney was reduced by31%than in that treated with intravenous CSA. All differences were significant at0.01levels. No statistical difference was found on kidney drug density between intra arterial or intraperitoneal administration.ConclusionsCyclosporine interference is helpful to induce immunotolerance. However, mono therapy of CSA is insufficient to prevent rejection thoroughly when administrated systemically. Intra-arterial CSA strongly prolongs graft survival than systemicadministration and highly reduces rejection level. Grafts can get a robust immuno tolerance with intra-arterial CSA over median dose. Though it can not be achieved under a low CSA dose even it used in the artery. Result indicates that a certain level of CSA in peripheral blood was essential for graft survival. Local immunosuppression with intra-arterial CSA was safe and available.