T in the control groups, as judged by the degree of neovascularisation and inflammatory cell infiltration (Figure 3).Graft expression of Title Loaded From File TGF-bDuring the acute Title Loaded From File corneal rejection, there was extensive TGF-b1 expression in the corneal grafts from rats in the negative control group. In addition, TGF-b1 was also expressed in the corneal stroma, endothelial cells, and some inflammatory cells, which showed dark brown staining (+++). Specifically, in the corneal grafts of groups II, III, and IV, the basal layer of corneal epithelial cells and fibroblasts and the cytoplasm of corneal endothelial cells showed light yellowish-brown staining (+). The quantity of positive inflammatory cells was lower than that of the rats in the control group (Fig 4).Figure 1. The appearance of the corneal graft 14 days after the operation. A, In group I, the graft showed oedema and new blood vessel growth into the centre of the graft. B, in the group II, the graft showed mild oedema, and fewer new blood vessels were observed than in controls. C-D, in the groups III and group IV, the graft was transparent, and no neovascularisation was found in the centre of the graft. doi:10.1371/journal.pone.0060714.gFigure 2. The survival curve of the grafts for the four groups. The recipients in group I exhibited accelerated rejection. The median survival was significantly different among the four groups according to a log-rank test (p,0.01). doi:10.1371/journal.pone.0060714.gCorneal Graft Rejection with the IL-1ra GeneTable 2. Scores on corneal transplant indices 14 days after surgery.*Group Group I Group II Group III Group IV F PTransparency 2.8860.64 2.0060.43 2.0860.29 2.0060.54 7.097 0.Stromal Edema 1.8860.35 1.2560.45 1.3360.49 1.2560.46 3.799 0.Neovascularization 3.0060.54 2.0060.95 2.0860.52 2.0060.54 4.298 0.Rejection Index 7.7560.45 5.2561.14 5.5061.00 5.3860.74 14.292 0.*Mean 6 standard deviation. F = Fisher T-test values. P = probability value. doi:10.1371/journal.pone.0060714.tGraft expression of RANTESDuring acute corneal rejection, RANTES expression was observed in the cell membrane and cytoplasm. The average colour intensities of the corneal epithelium, neovascular basement membrane and the few inflammatory cells in the control group were increased compared to groups II, III and IV (Fig 5).0.394). Two weeks after rejection, the IL-1a and IL-1b levels in groups II, III, and IV were lower than those in group I (P,0.05). The IL-1a and IL-1b levels in groups II and III were significantly different from those in group IV; however, there was no significant difference between groups II and III (P = 0.066, 0.166) (Fig. 7).Detection of IL-1ra protein and mRNA in corneal grafts CD4 and CD8 T cell graft infiltrationBefore acute corneal rejection, there were only a few CD4+ cells in the control group. During acute corneal rejection, there were many CD4+ and CD8+ cells in all of the groups. Furthermore, the numbers of CD4+ and CD8+ cells in the control group were higher than those in groups II, III and IV. There was no significant difference in the experimental groups (Figures 6, Table 3). Corneal grafts injected with the IL-1ra gene in the anterior chamber (group III) showed IL-1ra protein expression at postoperative day 3. After acute rejection, IL-1ra protein expression was weak in the corneas of the group that underwent anterior chamber injection; IL-1ra expression was also low in the group that received a PEI/DNA injection in the corneal stroma 1 hour before donor graft c.T in the control groups, as judged by the degree of neovascularisation and inflammatory cell infiltration (Figure 3).Graft expression of TGF-bDuring the acute corneal rejection, there was extensive TGF-b1 expression in the corneal grafts from rats in the negative control group. In addition, TGF-b1 was also expressed in the corneal stroma, endothelial cells, and some inflammatory cells, which showed dark brown staining (+++). Specifically, in the corneal grafts of groups II, III, and IV, the basal layer of corneal epithelial cells and fibroblasts and the cytoplasm of corneal endothelial cells showed light yellowish-brown staining (+). The quantity of positive inflammatory cells was lower than that of the rats in the control group (Fig 4).Figure 1. The appearance of the corneal graft 14 days after the operation. A, In group I, the graft showed oedema and new blood vessel growth into the centre of the graft. B, in the group II, the graft showed mild oedema, and fewer new blood vessels were observed than in controls. C-D, in the groups III and group IV, the graft was transparent, and no neovascularisation was found in the centre of the graft. doi:10.1371/journal.pone.0060714.gFigure 2. The survival curve of the grafts for the four groups. The recipients in group I exhibited accelerated rejection. The median survival was significantly different among the four groups according to a log-rank test (p,0.01). doi:10.1371/journal.pone.0060714.gCorneal Graft Rejection with the IL-1ra GeneTable 2. Scores on corneal transplant indices 14 days after surgery.*Group Group I Group II Group III Group IV F PTransparency 2.8860.64 2.0060.43 2.0860.29 2.0060.54 7.097 0.Stromal Edema 1.8860.35 1.2560.45 1.3360.49 1.2560.46 3.799 0.Neovascularization 3.0060.54 2.0060.95 2.0860.52 2.0060.54 4.298 0.Rejection Index 7.7560.45 5.2561.14 5.5061.00 5.3860.74 14.292 0.*Mean 6 standard deviation. F = Fisher T-test values. P = probability value. doi:10.1371/journal.pone.0060714.tGraft expression of RANTESDuring acute corneal rejection, RANTES expression was observed in the cell membrane and cytoplasm. The average colour intensities of the corneal epithelium, neovascular basement membrane and the few inflammatory cells in the control group were increased compared to groups II, III and IV (Fig 5).0.394). Two weeks after rejection, the IL-1a and IL-1b levels in groups II, III, and IV were lower than those in group I (P,0.05). The IL-1a and IL-1b levels in groups II and III were significantly different from those in group IV; however, there was no significant difference between groups II and III (P = 0.066, 0.166) (Fig. 7).Detection of IL-1ra protein and mRNA in corneal grafts CD4 and CD8 T cell graft infiltrationBefore acute corneal rejection, there were only a few CD4+ cells in the control group. During acute corneal rejection, there were many CD4+ and CD8+ cells in all of the groups. Furthermore, the numbers of CD4+ and CD8+ cells in the control group were higher than those in groups II, III and IV. There was no significant difference in the experimental groups (Figures 6, Table 3). Corneal grafts injected with the IL-1ra gene in the anterior chamber (group III) showed IL-1ra protein expression at postoperative day 3. After acute rejection, IL-1ra protein expression was weak in the corneas of the group that underwent anterior chamber injection; IL-1ra expression was also low in the group that received a PEI/DNA injection in the corneal stroma 1 hour before donor graft c.
