Simultaneous Blockade Of Inducible Co-stimulator And CD28 Pathway On Prevention Of Rabbit Corneal Allograft Rejection

Simultaneous Blockade of Inducible Co-stimulator and CD28 Pathway on Prevention of Rabbit Corneal Allograft RejectionIntroductionAllograft rejection is the most frequent cause of corneal graft failure, above 60% in high risk corneal transplantation. The newest study show that T cells activation is a most complex process and co-stimulatory signal pathway is multiple during the process of transplant rejection. Although a sole blocking pathway can prevent acute rejection in some degree, the stable and long immune tolerance can not be induced. In this study, we research the effection of CTLA-4Ig and ICOSmAb on corneal allografts rejection through co-simultaneous blocking CD28 and ICOS based on high-risk rabbit corneal transplantion model, which reduce the allograft rejection and prolong survival days.Material and MethodsNew zealand White(NZW) rabbits of both sexes weighing between 1.5 and 2.0kg were used, 8-12w, from china medical university. Throughout this study, all animals were handled according to the ARVO Resolution on the Use of Animals in Research. Rabbit vascularization of the recipient corneas before transplantationThe NZW recipients were anesthetized with an intramuscular injection of ketamine/xylazine, two drops of phenylephrine hydrochloride and cyclopentolate hydrochlofide were placed on the ocular surface. Three loops of 5-0 silk suture were placed in the anterior cornea of right eyes. The distance is 5ram and the depth is in 2/3 cornea. 2w later, when blood vessels had grown into the recipient cornea for 7mm, the sutures were removed, and rabbits with blood vessels cover 3/4 cornea were selected as recipient. 1. Penetrating KeratoplastyThe surgery of PKP was conducted after removing sutures for 2-4days. First, the same anesthetize as the above reported. Vascularized rabbits were as recipients and healthy rabbita were as domors. A 7mm Hessburg-Barron trephine was used to make a circular incision in the recipient cornea. Subsequently a sterile blade was used to enter the anterior chamber beginning at one pole and continuing circularly for 360 degree and deepened with a viscoelastic substance. Final removal of the recipient cornea was completed with right- and left- handed curved corneal-scleral scissors. A 7.5mm donor cornea was sutured into position with four 10-0 nylon cardinal sutures.Next, 12 10-0 interrupted sutures were placed in a symmetrically radial fashion around the wound, achieving watertight closure. All suture knots were rotated into the stroma with tying forceps. The viscoelasticsubstance was injected into the anterior chamber to ensure that a well-formed anterior chamber resulted at the completion of surgery. Finally neomycin-polymyxin and antibiotic ointment was applied to the ocular. The animals were retured to their cages for recovery from the anesthesia.2.Experiment oneNZW recipients were used in two parts One part had normal, clear, nonvascularized graft sites, and the other part had vascularized graft sites, 20 rabbits for each group. Another 20 rabbits were used as donors. Then, each group was divided into control and experimental group. Transplantant corneas were incubated in preserving solutions for control group, whereas contained CTLA4Ig (10ug/ml) for experimental group (4℃, 18h) All recipients of control and experimental corneas were examined and the appearance of the graft was recorded with a slit-lamp photomicroscope. Each group corneal grafts were examined on the 4 week and rejection happened with histology method and in situ hybridization method. Tumor necrosis factor-α(TNF-α) on corneal graft was determined. Compare survial days between two parts.3.Experiment two rabbits were divided into five groups in random(n=10) according to the dosage of ICOS, named as: the control group(grafts were in preserving solution); the A experiment group(grafts were in preserving solution with ICOS-Ig at the dosage of 1ug/kg), the B experiment group(with ICOS-Ig at the dosage of 10ug/kg), the C experiment group(with ICOS-Ig at the dosage of 25ug/kg), the D experiment group (with ICOS-Ig at the dosage of 250ug/kg). All recipients of control and experimental corneas were examined and the appearance of the graft was recorded with a slit-lamp photomicroscope. The test of T lymphocyte increment were performed to detect the ability of T lymphocyte increment after operation on the 4 week. Each group corneal grafts were examined on the 4 week and rejection happened with histology method(HE srain). IL-2 and IL-10 were detected by the way of RT-PCR from the aqueous humor. The T cells were detected by flow cytometry through 1/2 comeas.4.Experiment three40 rabbits from vascularized model were divided into four groups in random(n=10), named as: the co-blockade group(with CTLA4-Ig at the dosage of 1ug/ml and ICOSmAb at the dosage of 10ug/kg), the ICOSmAb group(with ICOSmAb at the dosage of 10ug/kg), the CTLA4 group(with CTLA4-Ig 10ug/ml), the control group(preserving solution) All recipients of control and experimental corneas were examined and the appearance of the graft was recorded with a slit-lamp photomicroscope. The test of T lymphocyte increment were performed to detect the ability of T lymphocyte increment after operation on the 4 week. Each group comeal grafts were examined on the 4 week and rejection happened with histology method and in situ hybridization method. Tumor necrosis factor-α(TNF-α) on comeal graft was determined.Data are expressed as mean and SD or SEM. The significant of difference was evaluated by q test of ANOVA(Newrnan-keuls). A p value＜0.05 was considered statistical significant. Results1 .Experiment one(1) 25 of 65 rabbits were used as recipient of comea vascularized models, 20 rabbits were used as the recipients successfully for PKP surgery. Other 5 rabbits were gived up because of infection and unstandarded condition. (2) In nonvascularized graft sites, control allografts and all experimental allografts exhibited similar frequencies of rejection. More than half of the grafts(16/30, 53%) survived the 100-day observation period. In vascularized graft sites, the grafts incubated in preserved solution containing CTLA4Ig at concentration of 10ug/ml had significantly longer mean survival times, compared with the control group(p＜0.05). (3) In vascularized graft sites, the result of histology(HE stain ): on the 4 week or rejection happened corneas grafts in contral group became edema and vascularized, which were heavily infiltrated with inflammatory cells, there were no inflammatory cells in experimental group. (4) in situ hybridization method: on the 4 week or rejection happened, the expression of Tumor necrosis factor-α(TNF-α) was determined on corneal graft, whereas no expression in experimental group.2. Experiment two(1) 58 of 78 rabbits were used as recipient of cornea vascularized models, 55 rabbits were used as the recipients successfully for PKP surgery. Other 3 rabbits were gived up because of infection and unstandarded condition. (2) the grafts incubated in preserved solution containing ICOSmAb at concentration of 1ug/ml or ICOSmAb at concentration of 10ug/ml had significantly longer mean survival times, compared with the control group and other two experimental group(p＜0.05), the grafts incubated in preserved solution containing ICOSmAb at concentration of 250ug/ml were rejected soon after operation, which the mean survival time was 10±3d. there was significantly difference between experimental A and B(p＜0.05), which no difference between experimental A and B(p＞0.05). Compared with control group experimental group A and B, there was significantly difference(p＜0.05). (3) the result of histology(HE stain ): on the 4 week or rejection happened corneas grafts in contral group and ICOSmAb 250ug/ml became edema and vascularized, which were heavily infiltrated with inflammatory cells, there were few inflammatory cells in experimental group 25ug/ml, which no inflammatory cells in other experimental groups. (4) on the 4 week or rejection happened, the expression IL-2 and IL-10 from the aqueous humor were detected by the way of RT-PCR in control and ICOSmAb 250ug/ml, whereas no expression in experimental group 10ug/ml. (5) On the 4 week, there didn't have significant different compared with each others by the blood test of T lymphocyte increment (P＞0.05). (6)The expression of CD4~+ and CD8~+ molecule in control and experimental D was up-regulation on the 4 week or rejection happened after transplantation, but both the A experiment and the B experiment group didn't have the same change. That of the C experiment group was down-regulation to appropriate extent, there did have a significant difference compared the control group. From the result of the mean positive percent ICOS expression on CD4~+, control and the D experimental group had a great up-regulation, which the A and B experimental groups had significant down-regulation, the above had difference (p＜0.05), there was no difference between the A and B group, the C experiment group was down-regulation to appropriate extent. From the result of the mean positive percent ICOS expression on CD8~+, there was no difference between control and experimental group, which there was significant difference compared with other experimental groups. The expression of ICOS was up-regulation in control and the D group, whereas there were no significant up-regulation in the A and B group. The expression of ICOS on CD8~+ was not great change compared that of on CD4~+3. Experiment three(1)45 of 65 rabbits were used as recipient of cornea vascularized models, 40 rabbits were used as the recipients successfully for PKP surgery. Other 5 rabbits were gived up because of infection and unstandarded condition. (2) the grafts in the associated blockade group had significantly longer mean survival times, compared with the control and the other experimental group(p＜0.05). There did have a significant difference between ICOSmAb and CTLA4-Ig group. (3) the result of histology(HE stain): on the 4 week or rejection happened corneas grafts in contral group became edema and vascularized, which were heavily infiltrated with inflammatory cells, there were few inflammatory cells in the B and C experimental group, there were no inflammatory cells in the A experimental group. (4) in situ hybridization method: on the 4 week or rejection happened, the expression of Tumor necrosis factor-α(TNF-α) was determined on corneal graft, whereas no expression in the A experimental group, which there were few expression of TNF-αin the B and C groups. (5) on 4 week or rejection happened, there didn't have significant different compared with each others by the blood test of T lymphocyte increment (P＞0.05).Conclusion1. The rejection of conreal grafts in high risk rabbit corneal transplantation model could be inhibited significant by blockade with the way of CD28/B7.2. The rejection of conreal grafts in non-vascularized rabbit comeal transplantation model could not be affected significant by blockade with the way of CD28/B7.3. The expression of the molecule of ICOS is up-regulation on T lymphocytes when rejection happens.4. The rejection of conreal grafts could be inhibited significant with ICOSmAb by blockade with the way of ICOS/ICOSL.5. The best dosage of ICOSmAb is 10μg/ml.6. The rejection of conreal grafts could be significant inhibited by co-stimulatory blockade with the way of CD28/B7 and ICOS/ICOSL, which is the better way than that of single blockade.