Study On Correlation Of Immunological Rejection And Changes Of TNF-α After High-risk Penetrativity Penetrating Keratoplasty In Canine

Objective:Local dogs of Chongqing Province were taken as experimental animals in this rsearch. High risk cornea pathologic model were established and PKP were conducted subsequently. Correlection of rejection reaction and TNF-a in peripheral blood and corneal tissue was analysed after PKP.Methods:(1)Thirty-four healthy local dogs were chosen as paticipants whose left eyes were treated by1mol/L NaOH to establish high risk pathologic model. Subsequently, the situation of the operative eyes was observed by slip lamp and vessels were measured everyday.(2)Seventeen healthy dogs were selected as donors for PKP. Complications both intraoperative and postoperative were monitored closely. The operated dogs were divided into two groups:the half was treated by normal saline, and the rest by1%RAPA, corneal condition was observed and recorded everyday.(3)Eight dogs of each group were blooded at anterior limb before establishing high risk pathologic model, on operating day,7th、14th、21th、28th、35th、42th、49th and56th after operation and the blood were used to test the concentration of TNF-a by enzyme-linked immunosorbent assay(ELISA); The rest of the eighteen dogs were randomly divided into two groups and made the same treatment as normal saline and1%RAPA. The corneas of three dogs were taken randomly each group on the day of7th、28th and56th respectively, corneas from another three healthy dogs were selected as control.The corneas were divided into three groups equally, one was used to detcet the concentration of TNF-a, another was used to observe the histological pathological changes of the corneas after dyeing by HE.(4)The last group was assayed by fluorescence quantitative PCR and the relative expression level of the corneal TNF-a gene was analysed by△△Ct.Results:(1)Whole layers of cornea were actually show silvery fog on burning day, different degrees of edema appeared three days later, turbidity aggravated significantly, neonatal vascular began infiltrating at limbus and grew to the center of cornea. Inflammation aggravated after seven days, the vascular diffused towards corneal center, Some corneal matrix layer dissolved aggravatively and the neonatal vascular became coarse after nine days. All of these appearance demonstrated that the pathological model was successively constructed.(2)All the surgeried dogs didn’t occur suture bursting crack, but two cases occurred acute rejection reaction; There was lots of secrection aroud the surgeried eyes after operation in the saline group, the the inserted slice was slightly edema on3th day after operation, Inflammation ocurred on the juncture of the the inserted slice and recipient bed on6th day, part of vascular began inserting into corneal, all of the operated dogs showed rejection until15th day, the inserted slice began to turn hard on28th day and the corneal tissue turn carnification on56th day; The secretion was letter in1%RAPA treating group, there was no edema on3th day after operation, the neonatal vascular inserted into the transplant slice on6th day, parts of dogs’corneas became slightly edema, the neonatal vascular increased to a certain extaint but still didn’t reach the level to induce rejection, edema and opacity of the the inserted slice was improved obviously and the neonatal vascular began regressive on15th day, the slice turn transparent on28th day and all of the corneal transplant slice kept transparent except for three cases, there was no rejection.(3)The content of TNF-a in peripheral blood increased in all dogs after alkali burning and operation. The content reach the highest level on the28th day(94.10±1.66pg·ml-1) and kept high in subsquently monitoring period, but the therapy group reached highest level on14th day(68.22±0.90pg·ml-1) and descended soon later, reached the normal level before operation on56th day(37.76±1.07pg·ml-1), the difference had statistical significance between the saline group and control group(P<0.01); The content of TNF-a in corneal tissues of each group dogs increased except the control group after PKP. The therapy group reached highest level(31.99±0.84pg·ml-1) on28th day after PKP and reduced subseqently, but the saline group kept increasing after28th day, reached37.53±0.94pg·ml-1and stilled had trend of growth; Histopathology results showed that7th day after PKP:cells in the corneal tissue of saline group appeared serious edema, some disorders including dissolving and loss ocurred in epithelial layer, matrix layer turned thick and some neonatal vascular and erythrocyte infiltrated, parts of endothelial cells appeared faulture and loss, but layers of cells ofthe therapy group occured slightly edema, endothelial cells thicken to a certain extaint, a little erythrocyte infiltrated, the structure of cllagen fiber and endodermis were integrity.28th day post PKP. corneal tissue of saline group became increasingly edema, epithelial cells became crack, lots of neonatal vascular and erythrocyte inliltrated in stroma layer, the original arrangement of the collagen fibers were completely disrupted, posterior elastic layer and endothelial cells turn detachment and loss in large quantities. Cells in all layers of cornea of the therapy group arrange in order, edema fade obviously, epithelial cells kept integrity, fiber in stroma layer was compact and uniform, the extant of neonatal vascular and erythrocyte inliltration was improved, posterior elastic layer and endothelial layer still kept integrity.56th day after PKP:all the epithelial cells dissolved completely, the remaining cells were also necrosis, the erythrocyte invased the stormal layer completely, almost no endothelial cells left. Histological structure of each cell layer was identical in treatment group, there was no obvious edema, the stormal fiber arrange evenly, only a small amout of endodermal cells loss.(4) Corneal content of TNF-a gene expression and either group or between groups in different time periods corneal TNF-a gene expression were showing some differences. Consistent with the change in the amount of tissue in the cornea TNF-a gene expression in different time periods.Conclusion:(1)RAPA played effective role of coping immune rejection in dogs with high-risk penetrating keratoplasty, inhibitting the growth of neonatal vascular and played strong immunosuppressive effect.(2)Changes of TNF-a concentration with the slit lamp microscope observation and histopathological biopsy results. Peripheral blood changes of TNF-a content may be associated with the the rejection.(3)Correlation in the peripheral blood of TNF-a content changes and local surgery corneal tissue TNF-a content changes, and correlation in the corneal tissue TNF-a content changes and the expression of TNF-amRNA.