Microbiological contamination in donor corneas preserved for medium-term.

To evaluate the characteristics of microbiological contamination in donor corneas preserved for medium-term. A total of 82 donated corneas from June 1, 2014 to November 30, 2014 were retrospectively analyzed. The corneas were preserved in cornea chambers medium-term solution at 4-8 °C for keratoplasty. After removal of the central corneas for transplantation, the corneoscleral rims were put back into the medium for 1 month at room temperature (20-25 °C). The suspicious contaminated storage solutions indicated with transparency or color change were examined with bacteria and fungi cultivation for strain identification. The data collected included gender, age, procurement site and causes of death of donors, and follow-up of recipients. Statistical analysis was performed using Microsoft Excel and SPSS 24.0. Significance level was set at a P value < 0.05. The overall pathogen positive rate was 9.8% (n = 8), including 7 (87.5%) fungi and 1 (12.5%) bacteria. They were 2 (2.44%) Fusarium, 2 (2.44%) Chromomycosis, 1 (1.22%) Candida albicans, 1 (1.22%) Aspergillus versicolor, 1 (1.22%) Acremonium species, and 1 (1.22%) Enterococcus. 5 contaminated corneas were used for penetrating keratoplasty; although four out of five (80%) had not been given antifungal drugs during more than 6 months following-up period, none of the recipients was infected with a graft. Donor age (P = 0.839), gender (P = 0.062), procurement sites (P = 0.713) and cause of death (P = 0.711) had no statistically significant influence on the contamination rate. All donor corneas have a possibility of microbiological contamination. Strict tissue preservation protocol but not antifungal drugs following keratoplasty seems necessary to prevent graft infection.

Effects of preservation time on proliferative potential of human limbal stem/progenitor cells.

AIM: To determine the proliferative potential and the maintenance of stem cell activity in stored human limbal tissues, and correlate this with the preservation time, cell viability and the expression of stem cell markers. METHODS: Thirty limbal rims were split into 4 parts and stored in corneal preservation medium at 4°C for 0, 1, 4, or 7 days. The limbal stem cell and mitotic markers P63, CK19, proliferating cell nuclear antigen (PCNA), and Ki67 were determined by immunohistochemical staining. The proliferative potential of limbal epithelial cells was assessed by cell viability, the ability of generating stratified epithelium, and colony forming assay. RESULTS: The stored tissues maintained limbal stratified structure to 7 days and exhibited comparable expression level of stem cell and mitotic markers. The proportion of viable cells decreased with the prolonged preservation time, while colony forming efficiency decreased from the 1(st) day and disappeared at the 4(th) day. When inoculated on amniotic membrane, the cells preserved for 1 day formed a stratified epithelium, while the cells from 4 days' preservation formed a discontinuous layer. CONCLUSION: The colony forming efficiency of limbal epithelial stem/progenitor cells decreased rapidly with the increasing preservation time, while the expression level of markers and capacity of forming epithelial monolayer on amniotic membrane decreased gradually. The limbal epithelial stem cells lost their function earlier than the lost expression level of stem cell markers. This may help us to better choose the appropriate preservation grafts for future limbal stem cell transplantation.

Lymphocyte infiltration and activation in iris-ciliary body and anterior chamber of mice in corneal allograft rejection.

AIM: To investigate the infiltration and activation of lymphocyte in iris-ciliary body and anterior chamber after allogenic penetrating keratoplasty (PK), for further revealing the role of iris-ciliary body in corneal allograft immune rejection. METHODS: In the mice models of PK, BALB/C mice received orthotopic isografts (n =35) or C57BL/6 donor allografts (n =25). Grafts were examined daily for 3 weeks by slit-lamp microscopy and scored for opacity. The infiltration of CD4(+) T lymphocyte in iris-ciliary body and anterior chamber was examined by immunohistology and the mRNA of CD80 and CD86 in both cornea graft and iris-ciliary body by RT-PCR was analyzed in allograft recipient at days 3, 6, 10 and the day when graft rejection occurred. Isograft recipients were examined as control at the corresponding time points. Transmission electron microscope was used to study the ultrastructure, especially cell infiltration, of iris-cilary body and corneal graft at day 3, 7 and the day when rejection occurred after allogenic PK. RESULTS: Rejection was observed in all the allograft recipients followed more than 10 days, at a median time of 15 days (range 12-18 days), but not in any of isografts. CD4(+) T cells were first detected at day 6 after transplantation in limbus and Ciliary body, and then in the stroma of recipient, iris, anterior chamber and corneal allograft with an increased number until graft rejection occurred. CD80 and CD86 mRNA were detected under RT-PCR examination in both graft and iris-ciliary body of allograft recipient, but not in any of isograft recipient. Three days after operation, lymphocytes and monocytes macrophages were visible in iris blood vessels and the anterior chamber, and vascular endothelial cell proliferation and activation were significant under transmission electron microscopy examination. At day 7, corneal endothelial cells became thinner. Lymphocytes and mononuclear macrophages were found with great number in the anterior chamber and adhered to the corneal endothelium. Blood vessels in iris increased and were filled with lymphocytes. And lymphocytes were detected to migrate through endothelial cell gap out of vessels. When allograft rejection occurred, macrophages attached to endothelial cells with large number of lymphocytes and macrophages infiltrating in iris. CONCLUSION: Lymphocyte infiltration and activation occurred in iris-ciliary body after allogenic PK, and the lymphocytes could migrate from iris blood vessel to the anterior chamber, which might play an important role in corneal allograft immune rejection.

[Correlations of telomere length changing and pathogeny of keratoconus].

OBJECTIVE: To study telomere length, senescence-associated-beta-galactosidase (SA-beta-galactosidase) and senescence marker protein-30 (SMP-30) in the stromal cells of keratoconus or normal corneas respectively, aiming finding the association of these indexes with the phenotype of keratoconus. METHODS: Experiment research. 37 keratoconus lesions corneas were removed from 32 keratoconus patients who were operated in Shangdong Eye Institute between January 2006 and December 2006, and 20 normal corneas were collected from eye bank. The keratoconus corneas ages were from 13 to 34 years [mean ages (19 + or - 5) years] and the control group consists of 20 normal corneas donor ages from 9 to 30 years [mean ages (19 + or - 4) years]. And there was no statistical difference of ages between keratoconus and normal corneas. Southern blot method was utilized to detect telomere length of genomic DNA. SA-beta-galactosidase was detected by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) staining method respectively in keratoconus and normal corneas. Isolated mRNA from keratoconus and normal corneas were reverse-transcribed to cDNA and SMP-30 was detected using PCR with specific primers (sense: 5' ccg tgg atg cct ttg act at 3'; anti-sense: 5' caa ctt cat gc tgct ttg ga 3'). To compare normal corneas and keratoconus corneas by histopathological study. Statistical analysis by t test. RESULTS: The telomere length in stromal cells in keratoconus corneas were from 10.29 to 14.12 kb, mean (11.54 + or - 1.41) kb, while that of normal corneas were from 12.64 to 15.32 kb, mean (13.45 + or - 0.99) kb. The difference of telomere length in stromal cells of keratoconus and normal corneas reached a statistical significant level (t = 4.753, P < 0.05). That means the telomere length of keratoconus stroma was shorter than that of normal corneal stroma. Light microscopy revealed that collagen fibers in keratoconus corneal stroma were arranged in an irregular manner. Cells density in keratoconus stroma appeared lower than in normal ones but the decrease was not significant. The staining of SA-beta-galactosidase in the keratoconus section was evident, but there was no staining in the normal corneas. SMP-30 was not detectable with RT-PCR method in either keratoconus or normal corneas. CONCLUSION: Telomeres in the keratoconus stromas manifest higher SA-beta-galactosidase than control, implying that improper senescence might be involved in pathogenesis of keratoconus.

[Transfer of endostatin gene for inhibition of retinal angiogenesis in mice].

OBJECTIVE: To evaluate the effect of liposome mediated plasmids encoding endostatin (ES) injected into the vitreous to inhibit experimental retinal neovascularization. METHODS: Cationic liposome mediated ES expression plasmid PCDNA(3)-ES was constructed. One-week-old C57Bl/6N mice were exposed to (75 +/- 2)% oxygen for 5 days, then returned to the room air to induce retinal neovascularization. Cationic liposome mediated ES complex (2 microl) was injected into the vitreous in the treatment group. PBS 2 microl or liposome with carrier DNA complex were injected in the control group. The ES protein expression in the retina was tested with immunohistological methods at 1, 3, 7 and 14 days after injection. Retinal neovascularization was evaluated by angiography with injection of fluorescein dextran and quantification of neovascular proliferative retinopathy after 5 days in room air. To examine the toxicity of the liposome and plasmid PCDNA(3)-ES complex, the histological changes in the retina were examined by light and electron microscopy. RESULTS: ES protein was expressed in the retina 24 hours after injection. Most of them presented in the retinal ganglion layer. This could last for 2 weeks at least. Retina of the PBS-injected eyes of retinal neovascular animal model showed prominent neovascular tuft and fluorescein leakage. Fewer neovascular tufts could be seen after ES injection. Retinal neovascularization in the eyes injected with ES plasmids complex was reduced as compared with the control group. No side effect on the retina was observed by light and electron microscopy. CONCLUSIONS: Liposome mediated plasmids encoding ES can be transferred to retinal cells by vitreous injection and can suppress retinal neovascularization, no side or toxic effects are presented in the retina. Further studies should be done to improve the treatment results.

[Organ culture for preservation of the cornea: human umbilical cord serum versus fetal bovine serum].

OBJECTIVE: To evaluate the changes of porcine corneal endothelium, the morphology, histology, ultrastructure, enzymes activity and metabolism of the cornea induced by organ culture with two different media containing fetal bovine serum (FBS) or human umbilical cord serum (HCS). METHODS: Fifty pairs of porcine corneas were preserved at 31 degrees C for 7, 14, 21, 28 days. One cornea of each pair was cultivated in medium I containing 10% FBS (group 1); the other one was stored in medium II containing 10% HCS (group 2). Thirteen fresh porcine corneas served as controls. All stored corneas were dehydrated for 24 hours. Twelve corneas from each group were evaluated each week, including the morphology, histology and enzyme histochemical staining of the cornea. Scanning electron microscopy was performed on one cornea from each group at 14 and 28 days and compared with the fresh cornea. pH value, glucose and lactate concentration of the culture media before and after culture were examined. Microbiological evaluation was also performed. RESULTS: Endothelium evaluation did not differ statistically between the two groups of porcine corneas. The morphological endothelium study showed some alterations such as pleomorphism. After 28 days of cultivation, the mean cell losses of endothelium were 10.98% and 10.85% in medium I and medium II stored corneas, respectively. There were no statistical differences of the histology, ultrastructure and enzymes activity of corneas between the two groups. The histological study showed corneal swelling and epithelial sloughing after preservation. Scanning electron microscopy showed an intact endothelial layer in all corneas. Enzyme histochemical staining showed vigorous enzyme activity in the corneal epithelium and endothelium. Enzyme activity in stroma decreased with preservation time. Corneas showed good glucose metabolism. Incidence of contamination was 6% for storage medium. CONCLUSIONS: The corneal endothelium can maintain a good viability for 4 weeks in these two organ culture media. HCS can replace FBS in the organ culture medium.