Investigating the effect of intracameral cefuroxime on oxidative stress and apoptosis in the rat cornea / Investigando o efeito de cefuroxima intracameral sobre o estresse oxidativo e a apoptose na córnea de ratos

ABSTRACT PURPOSE: We examined the effect of intracameral administration of cefuroxime on oxidative stress and endothelial apoptosis in rat corneal tissue. METHODS: In total, 30 rats were divided into 3 groups of 10 rats each (intracameral administration of cefuroxime 0.1 mg/0.01 mL (cefuroxime group); intracameral administration of balanced salt solution 0.01 mL (control group); or absence of intracameral injection (sham group). Corneal endothelial apoptosis was assessed by immunohistochemical analysis using caspase-3 and caspase-8. Total oxidant status, total antioxidant status, oxidative stress index, and paraoxonase and arylesterase levels were examined in corneal endothelial tissue and serum. RESULTS: Paraoxonase levels in the serum were significantly different between the sham and cefuroxime groups (p=0.027). A significant difference was also observed in total oxidant status levels between the cefuroxime and balanced salt solution groups (p=0.023). In addition, there were significant differences in total antioxidant status levels in corneal tissue between the cefuroxime and sham groups (p<0.001) and between the cefuroxime and balanced salt solution groups (p<0.001). Furthermore, significant differences were also observed in oxidative stress index levels between the cefuroxime and balanced salt solution groups (p=0.001) and between the cefuroxime and sham groups (p=0.026). According to the immunohistochemical staining results, a significant association with caspase-3 activity existed between the cefuroxime and balanced salt solution groups (p=0.007), while no significant difference was found with caspase-8 activity (p=0.541). Caspase-3 activity exhibited a significant relationship between the sham and balanced salt solution groups (p=0.018), but no relationship was found with caspase-8 activity (p=0.623). CONCLUSION: Immunohistochemical examination revealed that intracameral cefuroxime increased apoptosis when compared to the sham and balanced salt solution groups. Moreover, intracameral cefuroxime increased oxidative stress in the cornea and simultaneously induced apoptosis.

RESUMO OBJETIVO: Examinamos o efeito da administração intracameral da cefuroxima sobre o estresse oxidativo e a apoptose endotelial no tecido corneano de ratos. MÉTODOS: No total, 30 ratos foram divididos em 3 grupos de 10 ratos cada (administração intracameral de cefuroxima 0,1 mg/0,01 mL (grupo cefuroxima), administração intracameral de solução salina balanceada 0,01 mL (grupo controle) ou ausência de injeção intracameral (grupo sham)). A apoptose endotelial da córnea foi avaliada por análise imuno-histoquimica usando caspase-3 e -8. O status oxidante total, o status antioxidante total, o índice de estresse oxidativo e os níveis de a paraoxonase e arilesterase foram investigados no tecido endotelial da córnea e no soro. RESULTADOS: Os níveis de paraoxonase no soro foram significativamente diferentes entre os grupos sham e cefuroxima (p=0,027). Foi também observada uma diferença significativa nos níveis de estado oxidante total entre os grupos cefuroxima e solução salina balanceada (p=0,023). Além disso, houve diferenças significativas nos níveis de status antioxidante total no tecido da córnea entre os grupos cefuroxima e sham (p<0,001) e entre os grupos cefuroxima e solução salina balanceada (p<0,001). Diferenças significativas também foram observadas nos níveis do índice de estresse oxidativo entre os grupos cefuroxima e solução salina balanceada (p=0,001) e entre os grupos cefuroxima e sham (p=0,026). De acordo com os resultados de coloração imuno-histoquimica, houve associação significativa com a atividade da caspase-3 entre os grupos cefuroxima e solução salina balanceada (p=0,007), enquanto não houve diferença significativa com a atividade da caspase-8 (p=0,541). A atividade da caspase-3 exibiu uma relação significativa entre os grupos sham e solução salina balanceada (p=0,018), mas nenhuma relação foi encontrada com a atividade da caspase-8 (p=0,623). CONCLUSÃO: O exame imuno-histoquímico revelou que a cefuroxima intracameral aumentou a apoptose quando comparada com os grupos sham e solução salina balanceada. Além disso, a cefuroxima intracameral aumentou o estresse oxidativo na córnea e induziu simultaneamente a apoptose.

Comparison of Cytotoxic Effects on Rabbit Corneal Endothelium between Preservative-free and Preservative-containing Dorzolamide/timolol

PURPOSE: To evaluate and compare the toxic effects of eyedrops containing a fixed combination of 2.0% dorzolamide and 0.5% maleate timolol with or without preservatives on rabbit corneal endothelium. METHODS: This study was performed with 22 eyes of New Zealand white rabbits. Dorzolamide/timolol eyedrops with preservative (Cosopt group) or without preservative (Cosopt-S group) were diluted with a balanced salt solution at a 1 : 1 ratio. We injected 0.1 mL of diluted Cosopt into the anterior chamber of left eyes and an equal volume of diluted Cosopt-S into the anterior chamber of right eyes. Corneal thickness, corneal haze, and conjunctival injection were measured before and 24 hours after treatment. Endothelial damage was compared between both eyes by vital staining (alizarin red/trypan blue staining), live/dead cell assay, TUNEL assay, and scanning electron microscopy. RESULTS: Corneal endothelial damage was severe in the Cosopt group. Cosopt-treated eyes exhibited remarkable corneal edema and prominent apoptosis of endothelial cells. In addition, the live/dead cell assay revealed many dead cells in the endothelium, and scanning electron microscopy analysis showed that corneal endothelial cells exhibited a partial loss of microvilli on the surface as well as extensive destruction of intercellular junctions. However, in the Cosopt-S group, corneal edema was mild and the damage to the corneal endothelium was minimal. CONCLUSIONS: The main cause of corneal endothelial toxicity was due to the preservative in the dorzolamide/timolol fixed combination eyedrops, and not the active ingredient. Thus, it appears to be safer to use preservative-free eyedrops during the early postoperative period.

Effect of intracameral use of dexamethasone on corneal endothelial cells

To evaluate the effect of intracameral dexamethasone on corneal endothelium. Quasi experimental study. Layton Rehmatulla Benevolent Trust Eye Hospital, Lahore, from May 2011 to January 2012. Study subjects were adults of either gender with senile cataract who underwent phacoemulsification. They were divided in two groups, each had 110 patients. Group-A received subconjunctival injection of dexamethasone [2 mg/0.5 ml] at the end of surgery while group-B received intracameral injection of dexamethasone [0.4 mg/0.1 ml] at the end of surgery. Endothelial cell count was performed by specular microscopy pre-operatively and postoperatively at first week, first month and three months. Outcome measures included changes in endothelial cell count. Results were compared using t-test for means. There were 55 [50%] males and 55 [50%] females in group-A and 44 [40%] males and 66 [60%] females in group-B. In group-A, there were 66 [60%] right and 44 [40%] left eyes while group-B had 62 [56.36%] right and 48 [43.63%] left eyes. Mean age in group-A was 55.17 +/- 5.93 years and 54.87 +/- 5.55 years in group-B. Mean phacoemulsification time in group-A was 1.92 +/- 0.63 minutes and 1.82 +/- 0.54 minutes in group-B. After 3 months, in group-A, there was 7.55 +/- 1.19% endothelial cell loss while in group-B, there was 7.63 +/- 1.10% endothelial cell loss. The difference between the two groups was not statistically significant [p=0.614]. Use of intracameral dexamethasone at the end of cataract surgery is safe for corneal endothelium.

The effect of subconjunctival bevacizumab on corneal neovascularization, inflammation and re-epithelization in a rabbit model

PURPOSE: To evaluate the use of subconjunctival bevacizumab on corneal neovascularization in an experimental rabbit model for its effect on vessel extension, inflammation, and corneal epithelialization. METHODS: In this prospective, randomized, blinded, experimental study, 20 rabbits were submitted to a chemical trauma with sodium hydroxide and subsequently divided into two groups. The experimental group received a subconjunctival injection of bevacizumab (0.15 m; 3.75 mg), and the control group received an injection of 0.15 ml saline solution. After 14 days, two blinded digital photograph analyses were conducted to evaluate the inflammation/diameter of the vessels according to pre-established criteria. A histopathological analysis of the cornea evaluated the state of the epithelium and the number of polymorphonuclear cells. RESULTS: A concordance analysis using Kappa's statistic showed a satisfactory level of agreement between the two blinded digital photography analyses. The neovascular vessel length was greater in the control group (p<0.01) than in the study group. However, the histopathological examination revealed no statistically significant differences between the groups in terms of the state of the epithelium and the number of polymorphonuclear cells. CONCLUSIONS: Subconjunctival bevacizumab inhibited neovascularization in the rabbit cornea. However, this drug was not effective at reducing inflammation. The drug did not induce persistent corneal epithelial defects.

Toxicidade da mitomicina C ao endotélio da córnea de coelhos / Mitomycin C toxicity in rabbit corneal endothelium

OBJETIVO: Avaliar alterações do endotélio corneano após aplicação de mitomicina C na esclera por meio de microscopia eletrônica de transmissão e de varredura e correlacionar as alterações com tempo, concentração e entre os dois métodos de avaliação. MÉTODOS: Foi avaliado o endotélio corneano dos olhos de 32 coelhos albinos distribuídos em 4 grupos experimentais com 8 coelhos cada um. A mitomicina C foi aplicada sob retalho escleral no olho direito por 5 minutos. Nos grupos G1 e G2 a concentração da mitomicina C foi de 0,5 mg/ml e nos grupos G3 e G4 a concentração foi de 0,2 mg/ml. O exame foi realizado com 15 dias após nos grupos G1 e G3 e com 30 dias nos grupos G2 e G4. Dos 8 animais 4 foram preparados para microscopia eletrônica de transmissão e 4 para microscopia eletrônica de varredura. Os olhos esquerdos de todos animais serviram como controle. RESULTADOS: À microscopia eletrônica de transmissão foram observadas alterações do endotélio corneano em todos os grupos experimentais: rarefação do citoplasma, dilatação e fragmentação das cisternas do retículo endoplasmático rugoso, aparelhos de Golgi com dilatação das cisternas, redução de vacúolos e irregularidades da membrana celular interna sendo mais intensas em G1 e G2. À microscopia eletrônica de varredura foram observadas alterações em todos grupos experimentais, exceto G1: alteração de forma e tamanho das células e projeções filopoidais mais longas. CONCLUSÕES: 1 - A mitomicina C causou alteração no endotélio da córnea tanto na concentração de 0,5 mg/ml como de 0,2 mg/ml observadas 15 e 30 dias após a aplicação; 2 - As alterações foram mais intensas com a maior concentração de mitomicina C (0,5 mg/ml) na microscopia eletrônica de transmissão e não na microscopia eletrônica de varredura; 3 - As alterações tiveram correlação com o tempo na microscopia eletrônica de varredura e não na microscopia eletrônica de transmissão.

PURPOSE: To evaluate corneal endothelium alterations after applying mitomycin C to the sclera using transmission and scanning electron microscopy, correlating alterations with time, concentration, and evaluation methods. METHODS: The corneal endothelium of both eyes of 32 albino rabbits was evaluated and distributed into four groups of 8. Mitomycin C was applied under a scleral flap in the right eye for 5 minutes. Mitomycin C concentrations were 0.5 mg/ml for G1 and G2 and 0.2 mg/ml for G3 and G4. Examinations were performed 15 days after application to G1 and G3, and 30 days after application to G2 and G4. Four cornea in each group were prepared for transmission electron microscopy and four for scanning electron microscopy. Left eyes of all animals were used as controls. RESULTS: Transmission electron microscopy showed corneal endothelium alterations in all groups: rarefied cytoplasm, dilation and fragmentation of rough endoplasmic reticulum cisternae, Golgi apparatus with cisternal dilation, reduced vacuoles, and irregularities of internal membrane more noticeable in G1 and G2. Scanning electron microscopy revealed alterations in all groups except G1: changes in the shape and size of cells and longer filopodial projections. CONCLUSIONS: 1 - Corneal endothelium alterations were seen at both 0.5 and 0.2 mg/ml concentrations and at 15 and 30 days after mytomicin C application; 2 - Alterations were more intense with higher mytomicin C concentration by transmission electron but not by scanning electron microscopy; 3 - The alterations correlated with time by scanning electron microscopy but not by transmission electron microscopy.

Corneal endothelial safety of intracameral preservative-free 1% xylocaine.

PURPOSE: To evaluate the effect of intracameral preservative-free 1% xylocaine on the corneal endothelium as an adjuvant to topical anaesthesia during phacoemulsification and Acrysof foldable IOL implantation. MATERIAL & METHODS: This is a prospective, controlled, randomised, double-masked study. 106 patients with soft to moderately dense (Grade 1-3) senile cataract and corneal endothelial cell density of >1500/mm2 were randomised to the xylocaine group (n=53) and control group(n=53). Central endothelial specular microscopy and ultrasound corneal pachymetry were performed preoperatively. On the first postoperative day the eyes were evaluated for corneal oedema and Descemet's folds. Ultrasound corneal pachymetry was performed at 1, 3 and 12 months. Specular microscopy was performed at 3 and 12 months. Cell loss was expressed as a percentage of preoperative cell density. Six patients could not complete one year follow-up. Chi-square and paired t test (2 tail) statistical tests were applied for analysis. RESULTS: Four (7.54%) patients in the xylocaine group and 5 (9.43%) in the control group had a few Descemet's folds associated with mild central stromal oedema. Corneal thickness increased from 549.3micro +/- 37.2micro to 555.5micro +/- 36.5micro in the xylocaine group and from 553.1micro +/- 36.2micro to 559.3micro +/- 40.5micro in the control group at the one-month postoperative visit. Thickness returned to the preoperative level in xylocaine group 549.6micro +/- 34.5micro and control group 554.7micro +/- 41.1micro at three months. (P=0.484) The percentage of cell loss was 4.47 +/- 2.53% in the xylocaine group and 4.49 +/- 3.09% in the control group at one year. (P=0.97) CONCLUSION: Intracameral preservative-free 1% xylocaine does not appear to affect corneal endothelium adversely during phacoemulsification.

The expression of the plasmid DNA encoding TGF-beta 1 in endothelium after injection into the anterior chamber / 华中科技大学学报（医学）（英德文版）

The method of gene transfer into corneal endothelium was investigated to provide a foundation for the study of TGF-beta 1 gene transfer to inhibit corneal graft rejection. Two days after direct injection of pMAM TGF-beta 1 mediated by liposome into the anterior chamber of rabbits, one half of corneas were made into paraffin slides and the endothelial layer was carefully torn from the other half to make a single layer slide of endothelia. By means of immunohistochemical technique, the plasmid pMAM TGF-beta 1 expression product TGF-beta 1 in the endothelia was detected. Specific TGF-beta 1 expression was positive in the endothelia on both the paraffin slide and the single layer slide. The results showed that by direct injection into the anterior chamber, foreign plasmid DNA could be transferred into the endothelia and its expression was obtained. This may provide a foundation for further study on TGF-beta 1 participating in local induction of corneal immune tolerance.

Effect of topically applied Na-hyaluronan on experimental corneal alkali wound healing

The effect of topically applied 1% sodium hyaluronate (Na-HA) on the healing of a standardized corneal alkali wound was studied. The healing of the epithelium, stroma, and endothelium was evaluated separately, using quantitative methods. Central corneal alkali wound was produced in one eye of the rabbits by applying a 5.5-mm round filter paper, soaked in 1 N NaOH, for 60 seconds. 1% Na-HA in the treatment group and phosphate buffered saline (PBS) in the control group were given topically 4 times per day for 2 days, 1- and 3-weeks. Epithelial and endothelial healing was assessed morphometrically from standardized photographs and micrographs, respectively. Stromal healing was determined by counting polymorphonuclear leukocytes (PMN) and keratocytes in the central and marginal wound areas. A positive healing influence was observed in the epithelium. In stromal healing, 1% Na-HA treated corneas showed less PMNs and more keratocytes than the control group, suggesting that topically applied 1% Na-HA may suppress the stromal PMN infiltration and enhance the keratocyte repopulation during corneal alkali wound healing. However, no significant difference was found in morphometric evaluation of endothelial healing between the two groups.

Silicone oil keratopathy

Light microscopy and electron microscopic examination were carried out on the corneal buttons of two patients who required penetrating keratoplasty for treatment of corneal complication following the intraocular injection of silicone oil to repair recurrent retinal detachments in aphakic eyes. Light microscopic examination demonstrated increased cellularity and irregularity of collagen fibers of stromal layer, defect of endothelial cell layer and endothelial degeneration. Electron microscopy examination demonstrated marked decrease in endothelial cell population density, accompanied by flattening and thinning of the remaining cells and attenuation of cell borders. There were silicone droplets in the endothelial cell layer and collagenous layer posterior to endothelial layer. These findings are well correlated to clinical manifestation and are thought to be rather due to barrier effect of silicone oil than direct toxicity.

The effect of steroids on the viability of endothelial cells of stored cornea

The change of endothelial cell viability due to corticosteroid treatment in stored rabbit corneas was investigated. Hydrocortisone was injected into the anterior chamber of enucleated eyeballs which were stored in a moist chamber. After 24,48, or 72 hours of storage, the cornea was removed and stained with trypan blue. The unstained endothelial cells were counted under the light microscope in order to determine the density of viable endothelial cells. The same procedures were done on the contralateral eye with normal saline injected into the anterior chamber instead of hydrocortisone as a control. The density of viable endothelial cells in the corticosteroid-treated group was higher than that of the control group by 1.75%,14.39%, and 27.40% in 24,45, and 72 hour-stored corneas, respectively.