Nea was cut, and the graft was placed on the host

Nea was cut, and the graft was placed on the host bed. The wound was sutured with 10? nylon stitches using an interrupted suture technique, the anterior chamber was reconstructed by BSS, and the knot was exposed. After surgery, the pupil was dilated, 2000 U of gentamicin was injected subconjunctivally, and the palpebral margins were sutured. The eyelid suture was removed after 24 hours for drug administration.Corneal graft observation and evaluation after surgeryBased on the scoring criteria of Larkin [12], the corneal grafts were scored using three indices: opacity, oedema and neovascularisation. The sum of the scores for these 3 indicators was the rejection index (RI). An RI 5 or a corneal oedema score of 3 was defined as the occurrence of immune rejection.Graft histopathologyCorneal grafts were placed in 10 formalin, dehydrated with routine methods and embedded in paraffin. Samples were serially sectioned into 5-mm-thick slices. After haematoxylin-eosin staining, the sections were mounted with neutral balsam. The pathological changes in each layer of the cornea were observed under optical microscopy.ImmunohistochemistryAfter deparaffinisation for 30 min, the samples were Title Loaded From File cleared using a graded ethanol series of 95 , 90 and 85 . The specimen was then placed in sodium citrate solution and subjected to microwave antigen 16985061 retrieval for 10 min, followed by three 5min Title Loaded From File irrigations in PBS. Specimens were examined by immunohistochemistry in accordance with the instructions of the SP reagent kits. Antibodies against transforming growth factor b1 (TGF-b1), RANTES and CD4/CD8 T cells were used as the primary antibodies (1:100 dilution) and were allowed to bind for 5 min, followed by irrigation with distilled water. The specimen was then mounted onto a slide using neutral balsam and observed under the microscope. The negative control sample was prepared in the same manner, except that PBS was used in place of the primary antibody solution. The results were interpreted as follows: clear cell membrane boundary with no specific staining (2); light brown, mild specific staining in the cell membrane or cytoplasm (+); brown, moderate specific staining in the cell membrane or 23148522 cytoplasm (++); significant, specific staining in the cell membrane or cytoplasm with brown or dark brown colouring (+++). The average number of positive T cells was counted in the central areas of the corneal grafts at low magnification.Animal grouping and gene transfer protocolThe PEI/DNA transfection mixture was formulated as 20 mg of plasmid in 10 mL of mixture, and the mixture was incubated at room temperature for 30 minutes before use. The concentration of hIL-1ra purified protein solution was 1.5 mg/mL, which was diluted to 500 mg/mL with normal saline and stored at 4uC. The animals were divided into 4 groups. Group I (n = 20) was the negative control, which received a subconjunctival injection of saline after surgery. Group II (n = 34) was the IL-1ra gene corneal injection group, which received a 20 mg injection of PEI/DNA mixture into the corneal stroma before donor graft collection [10]. Group III (n = 34) was the IL-1ra gene anterior chamber injection group, which received an injection of 20 mg of the PEI/DNADetection of IL-1a and IL-1b in corneal graftsSamples were ground in liquid nitrogen, and the tissue debris of each cornea was resuspended in 1 mL PBS and centrifuged at 1500 gram at 4uC for 10 minutes. The supernatant was collected into EP tubes and preserved at 2.Nea was cut, and the graft was placed on the host bed. The wound was sutured with 10? nylon stitches using an interrupted suture technique, the anterior chamber was reconstructed by BSS, and the knot was exposed. After surgery, the pupil was dilated, 2000 U of gentamicin was injected subconjunctivally, and the palpebral margins were sutured. The eyelid suture was removed after 24 hours for drug administration.Corneal graft observation and evaluation after surgeryBased on the scoring criteria of Larkin [12], the corneal grafts were scored using three indices: opacity, oedema and neovascularisation. The sum of the scores for these 3 indicators was the rejection index (RI). An RI 5 or a corneal oedema score of 3 was defined as the occurrence of immune rejection.Graft histopathologyCorneal grafts were placed in 10 formalin, dehydrated with routine methods and embedded in paraffin. Samples were serially sectioned into 5-mm-thick slices. After haematoxylin-eosin staining, the sections were mounted with neutral balsam. The pathological changes in each layer of the cornea were observed under optical microscopy.ImmunohistochemistryAfter deparaffinisation for 30 min, the samples were cleared using a graded ethanol series of 95 , 90 and 85 . The specimen was then placed in sodium citrate solution and subjected to microwave antigen 16985061 retrieval for 10 min, followed by three 5min irrigations in PBS. Specimens were examined by immunohistochemistry in accordance with the instructions of the SP reagent kits. Antibodies against transforming growth factor b1 (TGF-b1), RANTES and CD4/CD8 T cells were used as the primary antibodies (1:100 dilution) and were allowed to bind for 5 min, followed by irrigation with distilled water. The specimen was then mounted onto a slide using neutral balsam and observed under the microscope. The negative control sample was prepared in the same manner, except that PBS was used in place of the primary antibody solution. The results were interpreted as follows: clear cell membrane boundary with no specific staining (2); light brown, mild specific staining in the cell membrane or cytoplasm (+); brown, moderate specific staining in the cell membrane or 23148522 cytoplasm (++); significant, specific staining in the cell membrane or cytoplasm with brown or dark brown colouring (+++). The average number of positive T cells was counted in the central areas of the corneal grafts at low magnification.Animal grouping and gene transfer protocolThe PEI/DNA transfection mixture was formulated as 20 mg of plasmid in 10 mL of mixture, and the mixture was incubated at room temperature for 30 minutes before use. The concentration of hIL-1ra purified protein solution was 1.5 mg/mL, which was diluted to 500 mg/mL with normal saline and stored at 4uC. The animals were divided into 4 groups. Group I (n = 20) was the negative control, which received a subconjunctival injection of saline after surgery. Group II (n = 34) was the IL-1ra gene corneal injection group, which received a 20 mg injection of PEI/DNA mixture into the corneal stroma before donor graft collection [10]. Group III (n = 34) was the IL-1ra gene anterior chamber injection group, which received an injection of 20 mg of the PEI/DNADetection of IL-1a and IL-1b in corneal graftsSamples were ground in liquid nitrogen, and the tissue debris of each cornea was resuspended in 1 mL PBS and centrifuged at 1500 gram at 4uC for 10 minutes. The supernatant was collected into EP tubes and preserved at 2.