Objective: To evaluate our modifed corneal alkali rat model and compare with the Ma et al. corneal alkali model. Methods: Corneal alkali injury by the method of Ma et al. was applied to 10 rats in Group 1 and corneal alkali injury with our modifed method was applied to 10 rats in Group 2. Hypopyon, hyphema, corneal fstula, phthisis bulbi occurrence and expected epithelial damage success rate and ultrastructural changes were evaluated in both groups. Results: Phthisis bulbi incidence was higher in Group 1 and expected epithelial damage success rate was higher in Group 2. Histological analysis revealed that stability of basal layers of epithelium of cornea was much more disturbed in Group 1. Conclusion: Our modifed corneal alkali injury animal model is an efective animal model with an increased epithelial success rate and decreased incidence of phthisis bulbi. Key words: Corneal alkali burn, Rat model Niche, 2012; 1: 31-3 doi : 10.5152/niche.2012.07 A Modifed Rat Model for Corneal Alkali Burn Emrullah Beyazyldz 1 , Ferda Alpaslan Pnarl 2 , zlem Beyazyldz 2 , mit Beden 3 , Tuncay Deliba 3 1 Gazi Mustafa Kemal State Hospital, Department of Ophthalmology, Ankara, Turkey 2 Dskap Yldrm Beyazt Research and Training Hospital, Clinic of Cell Research and Genetic Diagnosis Center, Ankara, Turkey 3 Ulucanlar Eye Research and Training Hospital, Department of Ophthalmology, Ankara, Turkey 4 Memorial Ataehir Hospital, Department of Ophthalmology, stanbul, Turkey 5 Dskap Yldrm Beyazt Research and Training Hospital, Department of Endocrinology and Metabolism, Ankara, Turkey Submitted : 17.10.2012 Accepted : 26.01.2012 Correspondence: Dr. Emrullah Beyazyldz Clinic of Ophthalmology, Gazi Mustafa Kemal Government Hospital, Ankara, Turkey Phone: +90 312 271 83 58 E-mail: dremrullah@hotmail.com Copyright 2012 by Cellular Therapy and Regenerative Medicine Society Available online at www.nichejournal.org Introduction Chemical exposure to the external eye can result in trauma, ranging from mild irritation to severe damage to both the ocular surface and anterior segment, leading to permanent vision loss. Severe damage occurs when alkali chemicals penetrate the cornea within seconds, leaving a white non- transparent corneal opacity (1). There is a wide range of therapeutic approaches used to treat alkali eye injuries. However, more ef- fective strategies are still required to treat severe cases, and new treatment modalities such as stem cell-based therapeutics are being studied in cor- neal alkali burn animal models. The Ormerod et al. (2) animal model is widely ac- cepted and commonly used for corneal alkali burn research. However, although this model is consid- ered the standard for corneal alkali burn, severe corneal ulcers with hypopyon, hyphema, corneal fstula and phthisis bulbi have been reported in several studies using this model (3). Ma et al. (3) modifed the Ormerod et al. (2) method, but still observed high levels of excessive corneal burn rates. Therefore, for these studies, we sought to modify the corneal alkali injury model in an efort to decrease excessive corneal burn rates and other complications associated with other models , spe- cifcally comparing the results from our modifed corneal alkali rat model with the model employed by Ma et al. (3). Methods Animals This study was approved by the Animal Research Ethics Committee of the Dkap Yldrm Beyazt Training and Research Hospital, and the study was undertaken at the Experimental Animal Re- search Center of Dkap Yldrm Beyazt Training and Research Hospital. Animal experiments were performed in a manner consistent with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Twenty Spraque-Dawley rats (200-250 g) were used in this study. Rats were anes- thetized by ether inhalation. The experiment was carried out on the right eye of each animal. At the end of the experiment, all rats were sacrifced with an overdose of 10% chloral hydrate and corneas of the rats were harvested for histological analysis. Grouping and Corneal Injury Methods For these studies, rats were randomly divided into two groups: 10 rats were subjected to the Ma et al. (3) model of corneal alkali injury (Group 1) and 10 rats received our modifed model of corneal alkali 32 Beyazyldz et al. A Modifed Rat Model for Corneal Alkali Burn Niche 2012; 1: 31-3 injury (Group 2). Briefy, for Group 1, a flter paper ring (ID 4 mm and OD 8 mm) was saturated with 1 N NaOH and applied to the center of the cornea for 30 s. The cornea was then rinsed with saline (60 mL) for 20 s. In animals from Group 2, a flter paper ring (ID 4 mm and OD 8 mm) saturated with 1 N NaOH was applied to the center of the cornea for 20 s. The cornea was then rinsed with saline (60 mL) for 10 s. Following the corneal alkali burn, animals from both groups were treated with gentamicin and dexametha- sone eye drops twice a day for one week (Genta oft 0,35% -Bilim drug, Dexa-sine 5 mL-Liba medical). Clinical Outcome and Analysis Following corneal injury, the eye of each animal was monitored daily for hypopyon, hyphema, corneal fstula and phthisis bulbi development. Corneas were also examined to see whether there was any corneal epithelial damage throughout the entire thick- ness of the tissue. After excision, corneas were examined under a direct light microscope to assess corneal clarity and neovascular- ization. Corneal transparency was evaluated as: completely trans- parent, iris not clear, or pupil could not be seen. Corneal neovas- cularization was evaluated as: no neovascularization, detected within 2 mm from the limbus, or detected over 2 mm from the limbus. Corneal epithelial defects were assessed in tissue stained with fuorescein using a microscope. Corneal epithelial defects were evaluated as: staining more than 50% of whole cornea or less than 50% of whole cornea (Figure 1). Histological Analysis Excised corneas were collected, fxed in 10% paraformaldehyde, and embedded in parafn. Preparations with 5 m sections were stained with hematoxylin and eosin (H&E). Preparations were an- alyzed using a Leica HMLB45 light microscope (Leica, Germany). Statistical Analysis The SPSS 13.0 program was used for statistical analysis. The Pear- sons Chi-square test, Fischer exact test and continuity-adjusted Chi-square tests were used. A probability level of p<0.05 was con- sidered signifcant. Results Following the surgical procedure, infection, hyphema, and cor- neal fstula incidence were assessed. No signifcant diferences were observed between the two groups (p>0.05). Infection and corneal fstula were observed in 1 rat from Group 1 but hyphema did not occur in animals from either group. Phthisis bulbi inci- dence and expected epithelial damage success rate were com- pared between the two models of injury. Phthisis bulbi incidence was signifcantly greater in Group 1 (50%) when compared with Group 2 (10%) (p<0.05). In contrast, expected epithelial success rate was signifcantly greater in Group 2 (70%) compared with Group 1 (30%) (p<0.05). A summary of all measurements can be found in Table 1. Corneal epithelium of the rats were histologically analyzed. The corneal epithelial basal layer was separated from the stroma and stability of the cornea was disrupted in Group 1 and basal layers of the corneal epithelium remain attached to stroma of cornea and stability in corneal epithelial basal layers was preserved in Group 1. Histological analysis showed greater disruption to the stabil- ity of the basal layers of cornea epithelium in rats from Group 1 (Figure 2) when compared with those in Group 2 (Figure 3). Discussion Alkali injuries constitute one of the most urgent ocular emer- gencies and are notoriously destructive due to their rapid pen- etration of ocular tissues. Alkali injuries can change the corneal transparency, resulting in a dramatic decrease in visual acuity or Figure 1. Evaluation of rat cornea stained with fuorescein using a co- balt blue-light under the microscope. Cornea epithelial damage oc- curred in the absence of phthisis bulbi Figure 2. Histological analysis (H&E stain, x200) of rat cornea alkali burn generated with Ma. et. al. (3) model. Disruption in corneal epi- thelial basal layers is clearly observed. Corneal epithelial basal layer is separated from the stroma and stability of cornea is disrupted Figure 3. Histological analysis (H&E stain, x200) of rat cornea alkali burn generated with our modifed model. Basal layers of cornea epithelium remain attached to stroma of cornea and stability in corneal epithelial basal layers is preserved 33 Beyazyldz et al. A Modifed Rat Model for Corneal Alkali Burn Niche 2012; 1: 31-3 complete loss of sight. Corneal ulceration, perforation and neo- vascularizations are the most common acute complications of corneal chemical burns. Thus acute management of corneal al- kali burns is crucial for successful clinical treatment of the injury (4). Although there are a number of therapeutic modalities for the treatment of alkali burn, to date, no ideal method is available, especially for severe burns. Recent studies have investigated the efects of mesenchymal stem cells in animal models of corneal alkali burn, but further tests are needed to elucidate the poten- tial therapeutic efcacy of mesenchymal stem cells. For the cur- rent study, we aimed to fnd a more efective corneal alkali burn rat model to decrease the number of animal loss. This should be particularly useful for future studies testing new potential thera- peutic strategies. A standardized corneal alkali injury animal model was frst devel- oped by Ormerod et al. (2). Before this, corneal burn was only pro- duced using an alkali-flled well applied to the corneal surface (5). After a variable period of time, the well was drained and removed and the eye irrigated. However, it was difcult to produce uniform lesions with this method. Ormerod et al. (2) achieved a central dis- coid corneal burn in rabbits which permitted clinical evaluation of the corneal neovascular response. They used 7 mm flter paper discs which were soaked for 10-20 s in 1 N NaOH and applied to the cornea for 2 min. Ye et al. (6) studied a corneal alkali injury model using 3 mm diameter flter paper soaked with 1 N NaOH and applied to the center of the cornea for 40 s; followed imme- diately by 1 min of corneal rinsing. They did not report any inci- dence of corneal ulcer, hypopyon or hyphema. However, when Ma et. al. (3) used identical methods, they reported corneal ulcer, hypopyon, hypema, corneal fstula and severely damaged corne- al stroma . Even with a modifed method using a flter paper ring (ID 4 mm and OD 8 mm), applied to the cornea for 30 s, and then immediately rinsed with saline 20 s, Ma et al. (3) still observed corneal ulcers and other surgical complications. For the current study, we used the method of Ma et al. (3) for Group 1, and phthi- sis bulbi occurred in 50% of rats, along with a lower epithelial success rate. For Group 2, we utilized a modifed method which decreased both the NaOH application time (20 s) and the rins- ing time (10 s). With these modifcations, phthisis bulbi incidence was decreased to 10% and epithelial success rate was increased to 70%. Conclusion These results suggest that our modifed corneal alkali injury is an efective animal model for inducing corneal injury with an in- creased epithelial success rate and decreased incidence of phthi- sis bulbi, and could be useful for future therapeutic studies. Conflict of interest: No conflict of interest was declared by the authors. References 1. Wagoner MD. Chemical injuries of the eye: current concepts in pathophysiology and therapy. Surv Ophthalmol 1997; 41: 275-313. [CrossRef] 2. Ormerod LD, Abelson MB, Kenyon KR. Standard models of corneal injury using alkali-immersed filter discs. Invest Ophthalmol Vis Sci 1989; 30: 2148-53. 3. Ma Y, Xu Y, Xiao Z, Yang W, Zhang C, Song E, et al. Reconstruction of chemically burned rat corneal surface by bone marrow-derived human mesenchymal stem cells. Stem Cells 2006; 24: 315-21. [CrossRef] 4. William Tasman Duanes Ophthalmology. 2006 edition Philadelphia: Lippincott Williams & Wilkins, 2006. 5. Pfister RR, Burstein N. The alkali burned cornea I. Epithelial and stromal repair. Exp Eye Res 1976; 23: 519-35. [CrossRef] 6. Ye J, Yao K, Kim JC. Mesenchymal stem cell transplantation in a rabbit corneal alkali burn model: engraftment and involvement in wound healing. Eye 2006; 20: 482-90. [CrossRef] Hyphema Corneal Phytisis Expected epithelial (n-%) Fistula Bulbi damage (n-%) (n-%) (n-%) Group 1 0 (0) 1 (10) 5 (50) 3 (30) Group 2 0 (0) 1 (10) 1 (10) 7 (70) Table 1. Comparison of groups according to infection, hyphema, corneal fstula, phytisis bulbi and expected epithelial damage rates