Revisiting rabbit models for keratoconus: A long-term study on collagenase-induced disease progression.

Keratoconus (KC) is a degenerative disorder resulting from the degradation of the stromal collagen fibril network in the cornea, leading to its thinning and conical deformation. Various studies have established animal models of KC by using the collagenase type II enzyme to gain a better understanding of the pathogenesis, however, long-term monitoring or follow-up of the models have not been reported so far. This study evaluates the long-term stability of collagenase type II-induced KC in a rabbit model. Six New Zealand rabbits were divided into 4 study groups with 3 eyes per group. The groups were control (group 1), 0.5% proparacaine + 5 min collagenase treatment on day 0 and day 30 (group 2), 0.5% proparacaine + 10 min collagenase treatment on day 0 (group 3) and, mechanical debridement + 2 min collagenase treatment on day 0 (group 4). Inflammation was observed in group 4 till week 10. Significant decrease in the central corneal thickness was observed in group 3 by week 4 (p < 0.001) however, the thickness was regained in the subsequent follow-ups in all the groups. Keratography results showed no changes in Km values but an increased astigmatic power in all groups. Scanning electron microscopy images revealed thinner collagen fibrils arranged in a mesh-like pattern above the uniform layer of the collagen lamellae in the central part of the treated corneas. Similarly, histological staining revealed loosely packed stromal fibrils in the anterior portion of the cornea which corroborates with the immunofluorescent staining results. This study revealed the remodeling of the corneal structure by eight weeks of collagenase treatment. Consequently, the possibility of creating a rabbit keratoconus model induced by collagenase may warrant further consideration.

Modelo animal de ectasia corneal en conejo mediante inyección intraestromal de colagenasa tipo ii / Animal model of corneal ectasia in rabbits by intrastromal injection of type II collagenase

Introducción La colagenasa ii ha sido utilizada para inducir queratocono experimental en modelos animales. Sin embargo, no ha sido estudiado su efecto cuando se administra por inyección intraestromal, por lo que el propósito de este estudio fue estudiar los efectos de la inyección intraestromal de colagenasa ii sobre la superficie corneal y la morfología de la córnea. Método Se trabajó con 6 conejos Nueva Zelanda, se administró colagenasa ii por inyección intraestromal (5μL de 2,5mg/mL) en los ojos derechos y solución salina balanceada en los ojos izquierdos. Se realizaron queratometrías para evaluar la alteración de la curvatura, también al séptimo día se obtuvieron las córneas y se realizó tinción hematoxilina-eosina para examinar los cambios morfológicos. Asimismo, se investigaron los cambios en la expresión de colágeno tipo i por tinción rojo sirio y PCR semicuantitativa. Resultados K1, K2 y Km presentaron diferencias en los promedios con cambios estadísticamente significativos. Los cambios morfológicos que se demostraron fueron degradación y disposición irregular del estroma corneal, incremento en la densidad celular de queratocitos y ligera infiltración celular. Finalmente se demostró que hay mayor expresión de fibras de colágeno tipo i en el grupo experimental a diferencia de los controles y el grosor de las fibras también aumentó por acción de la colagenasa ii; sin embargo, en cuestión génica no hubo cambios en la expresión de colágeno tipo i a nivel molecular entre el grupo control y experimental. Conclusiones La colagenasa ii administrada por inyección intraestromal es capaz de inducir cambios en la superficie corneal y el estroma, pudiendo simular un modelo de queratocono (AU)

Introduction Collagenase II has been used to induce experimental keratoconus in animal models. However, its effect when administered by intrastromal injection has not been studied, so the purpose of this study was to study the effects of intrastromal injection of collagenase II on corneal surface and corneal morphology. Method Six New Zealand rabbits were used, collagenase II was administered by intrastromal injection (5μL of 2.5mg/mL) in the right eyes and balanced salt solution in the left eyes. Keratometry was performed to evaluate curvature alteration, also at day 7 corneas were obtained and hematoxylin–eosin staining was performed to examine morphologic changes. Likewise, changes in type I collagen expression were investigated by Sirius Red staining and semi-quantitative PCR. Results K1, K2, and Km presented differences in the means with statistically significant changes. The morphological changes that were demonstrated were degradation and irregular arrangement of the corneal stroma, increase in the cellular density of keratocytes and slight cellular infiltration. Finally, it was demonstrated that there is greater expression of type I collagen fibers in the experimental group as opposed to the controls and the thickness of the fibers also increased due to the action of collagenase II, however, in terms of genetics there were no changes in the expression of type I collagen at molecular level between the control and experimental groups. Conclusions Collagenase II administered by intrastromal injection is able to induce changes in the corneal surface and stroma, being able to simulate a model of keratoconus (AU)

Induction of the integrated stress response in the rat cornea.

Keratoconus (KC), a progressive, degenerative corneal disease, represents the second leading indication for corneal transplantation globally. We have previously demonstrated that components of the Integrated Stress Response (ISR) are upregulated in human keratoconic donor tissue, and treatment of normal tissue with ISR agonists attenuates collagen production. With no consistently accepted animal models available for translational KC research, we sought to establish an in vivo model based on ISR activation to elucidate its role in the development of the KC phenotype. Four-week-old female SD rats were treated with topical SAL003 formulated as a nanosuspension or vehicle every 48 h for four doses. Animals were subject to monitoring for ocular inflammation and discomfort before being euthanized at 1, 14, or 28 days after treatment was withdrawn. Schirmer's tear test, intraocular pressure, and body weight measurements were obtained at baseline and prior to euthanasia. Globes were subject to routine histopathology, immunohistochemistry for ATF4, and qPCR for Col1a1 expression. ANOVAs and Student's t tests were used to assess statistical significance (α = 0.05). SAL003 treatment did not produce any adverse ocular or systemic phenotype but did result in decreased keratocyte density. Col1a1 transcripts were reduced, corresponding to nuclear ATF4 expression within the axial cornea. In vivo topical treatment with a gel-formulated ISR agonist recapitulates key features of the activated ISR including nuclear ATF4 expression and decreased extracellular matrix (ECM) production. Exogenous ISR agonists may present one approach to establishing a rodent model for keratoconus, a charge essential for future evaluations of pathogenesis and therapeutic interventions.

Case report: acute hydrops and spontaneous corneal perforation in a patient with keratoconus treated with colchicine for familial Mediterranean fever.

PURPOSE: To report a rare case of spontaneous corneal perforation after hydrops in keratoconus patients who suffer from familial Mediterranean fever and was treated systemically with Colchicine. METHODS: Case report. RESULTS: We report a case of a 30-year-old male with keratoconus and familial Mediterranean fever treated with colchicine, presented with acute hydrops in his left eye, which progressed to a spontaneous corneal perforation. Attempts to treat the perforation with cyanoacrylate glue failed, and he underwent penetrating keratoplasty with an excellent visual outcome. CONCLUSION: Colchicine treatment may have had a role in the pathogenesis of this rare case.

Late-onset progression of keratoconus after therapy with selective tissue estrogenic activity regulator.

We describe the association of rapid progression of keratoconus in a 49-year-old woman on selective tissue estrogenic activity regulator (STEAR) therapy for endometriosis. Approximately 4 months after initiation of therapy with STEAR therapy and 3 months after ovariectomy, Scheimpflug images showed a massive increase in the previously stable ectasia. During this period, the maximum increase in the keratometry values was 2.7 diopters (D) in the right eye and 3.8 D in the left eye. Corneal crosslinking (CXL) was performed in both eyes. This resulted in excessive flattening of 5.5 D in the right eye and 6.1 D in the left eye at 9 months postoperatively. Patients having STEAR therapy must be monitored closely for corneal changes.

A rabbit model of corneal Ectasia generated by treatment with collagenase type II.

BACKGROUND: To investigate use of collagenase type II for generating a rabbit model of corneal ectasia. METHODS: Ten New Zealand white rabbits were used with right eyes treated as the experimental group and left eyes treated as the control group. After epithelial debridement, a collagenase type II solution (200 µL of 5 mg/mL) was applied in the experimental group at room temperature (24 °C) for 30 min, and a 200 µL solution without collagenase was applied in the control group. Slit-lamp microscopy, the mean keratometry (Km), and central cornea thickness (CCT) were examined before and after the procedure. Corneas were obtained on day 14 for biomechanical evaluation. RESULTS: No obvious inflammatory reaction was observed in all eyes after the procedure. A statistically significant increase in Km (1.54 ± 1.29D vs - 0.82 ± 0.44D at day7 and 0.89 ± 0.89D vs - 2.11 ± 1.02D at day14) and a statistically significant decrease in CCT (- 23.10 ± 12.17 µm vs 6.20 ± 16.51 µm at day7 and - 16.10 ± 10.46 µm vs 11.60 ± 0.88 µm at day14) were observed in the experimental group compared with the control group. The mean stresses and elastic modulus at 5%, 10%, 15%, and 20% deformities in the experimental group decreased and the differences in elastic modulus between the two groups were statistically significant at 10% and 15% deformities. CONCLUSIONS: Collagenase type II treatment results in mimic KC with increased corneal keratometry and corneal thinning and a lower elastic modulus. An animal model for corneal ectasia can be generated by treatment with collagenase type II.

Chronic pesticide exposure and consequential keratectasia & corneal neovascularisation.

Ocular toxicity as a consequence of chronic pesticide exposure is one of the health hazards caused due to extended exposure to pesticides. The cornea, due to its position as the outer ocular layer and its role in protecting the internal layers of the eye; is gravely affected by this xenobiotic insult to the eye, leading to ocular irritation and damage to normal vision. The deleterious effects of chronic pesticide exposure on the various corneal layers and the ocular risks involved therein, were explored by mimicking the on-field scenario. Cytological, histological and flowcytometric parameters were taken into consideration to determine the enhanced risk of corneal neovascularisation and keratectasia, specifically, keratoconus. Chronic exposure to pesticides leads to heightened ocular morbidity wherein there were visible pathophysiological changes to the ocular surface. The cornea was found to be adversely affected with visible protuberance in a cone-like shape, characteristic of keratoconus in a majority of the experimental animals. Further analyses revealed a detrimental impact on all the corneal layers and an amplified expression of inflammation markers such as TNF-α, VCAM-1 and ICAM-1. Additionally, it was found that post pesticide exposure, the corneal surface developed hypoxia, leading to a significant increase of angiogenesis promoting factors and consequential neovascularisation. Apart from ocular toxicity, chronic exposure to pesticides significantly increases the risks of keratectasia and corneal neovascularisation; disorders which lead to diminished vision and if untreated, blindness.