ABSTRACT
Background Cyclosporine A (CsA) is an effective drug to prevent rejection response after penetrating keratoplasty (PKP).Appropriate dosage forms and right administrating route is very important for improving the bioavailability of CsA.Objective This study was to investigate the preventing effect of CsA microspheres via subconjunctive and anterior chamber injection and CsA eye drops on immune rejection after PKP.Methods Sixty eyes of 60 clean adult New Zealand white rabbits served as receipts,and 60 eyes of 30 clean adult pigmented rabbits were used as the donors.The receipts were randomized into the blank control group (only PKP group),subconjunctival CsA injection group,subconjunctival vector injection group,anterior chamber CsA injection group,anterior chamber vector injection group,and 1％ CsA eye drops group.PKP was performed on the all rabbits,and then the CsA microsphere (0.1 ml,12 g/L) or blank microsphere (0.1 ml) were respectively administered in the corresponding groups.The corneal grafts were examined by slit lamp microscope regularly,and rejection index (RI)was calculated based on the corneal opacity,edema and neovascularization.The intraocular pressure (IOP) of operative eyes was measured by Tono-pen tonometer before operation,3 days,1 week,2 weeks,3 weeks,1 month,2 months and 3 months after operation,respectively.Histopathological examination on corneal grafts was performed 1month and 3 months after operation.Results The IOP of all the rabbits lowed after operation,but there was no statistical difference in different time points and various groups (Ftime =29.210,P =0.000; Fgroup =0.254,P =0.938).The grafts of the blank control group,subconjunctival vector injection group and the anterior chamber vector injection group showed varied degrees of corneal opacity and neovascularization 2-3 weeks after operation,and the degree of graft opacity was aggravated obviously in the fourth week,with the RI 8.60±1.52,8.60±0.55 and 8.80±0.84 individually.Neovascularization of the grafts in the subconjunctival CsA injection group,anterior chamber CsA injection group,and 1％ CsA eye drops group was found 3 weeks after operation,and the RI were 4.40±0.89,3.20±0.84 and 3.00±0.71,showing a significantly lower than that of the control groups (P＜0.05).Mild inflammatory response of the grafts was seen in the anterior chamber CsA injection group,but it was lightened over time.The histopathological examination revealed obvious thickening of corneal grafts,neovascularization and infiltration of lots of inflammatory cells in the blank control group,subconjunctival vector injection group and the anterior chamber vector injection group.However,only slight new blood vessels and inflammatory response were seen on the subconjunctival CsA injection group,anterior chamber CsA injection group,and 1％ CsA eye drops group.Conclusions Administration of CsA in different methods can prevent the immune rejection after PKP in rabbit eyes,the effect of giving drugs via anterior chamber injection is better than that via subconjunctive pathyway.
ABSTRACT
Background How to harvest the purified corneal endothelial seed cells is very important for the corneal tissue engineering technology. Herein,to establish a good culture method and effective identification method of corneal endothelial cells ( CECs) is the key. Objective Present study wag to establish the cultivating and identifying approach of the rabbit CECs and detect the biological characteristics of passaged cells. Methods Rabbits CECs were isolated from Descemet's membrane peeled off completely in 30 New Zealand white rabbits and then digested with 0. 25% trypsin-0. 02% EDTA and primarily cultured in CECs medium containing 15% fetal bovine serum. The growth situate and cellular morphology of rabbit CECs were observed under the inverted phase-contrast microscope following the alizarin red staining. Rabbit CECs were identified by cellular morphology as well as in gene and protein level, including the detection of expressions of collagen type IV α2(COL4A2) ,vascular endothelial growth factor receptor 2 ( FLK1 ) , Na+-K+ ATPase alpha 1subunit ( ATP1 A1 ) , aquaporin 1( AQP1 ) , voltage-dependent anion channels ( VDACs) by reverse transcription-polymerase chain reaction ( RT-PCR ). The expression and distribution of neurone specific enolase ( NSE) , Na+-K+ ATPase, zonula occludens-1 ( ZO-1 ) were also detected by immunocytochemistry under the fluorescence microscope. The proliferation activity of the passage cells was dynamically observed by MTT assay,and Na+-K+ ATPase activity of different generations cells was detected by ATPase kit. Results Majority of the primarily cultured cells adhered in 24 hours, infused in 2-3 days with the hexagon shape in appearance. The morphology of the cells was very varied with passage. Alizarin red staining showed a well- defined and well-lined cellular morphology similar to the corneal cells in vivo. Target genes of C0L4A2, FLK1, ATPIAI ,AQPI and VDACs were positively expressed in the cells. However,the expression of CK12 was absent in the cells. NSE, Na+-K+ ATPase, ZO-1 positive cells were respectively observed under the laser confocal scanning microscopy. MTT results showed a gradually low growth curve with the passage. Quantitative results also revealed that the Na+-K+ ATPase activity was gradually declined in different generations of cells ( F = 77. 174, P = 0. 000 ). Conclusion The enzyme digestion is a better approach of isolating and culturing comeal endothelial cells. Cultured cells can be identified by cells staining, gene expression and protein level. Earlier generation of CECs are ideal seed cells for cornea tissue engineering.