Key Clinical and Histopathological Features of a Pterygium-Like Induced Lesion in a Rabbit Model

Abstract

Purpose: Surgery is the definitive treatment for pterygium; therefore, reliable animal models are required for translational research. The goal of this investigation was to establish a standardized preclinical model of pterygium-like lesion.

Methods: A subconjunctival injection of fibroblasts (NIH3T3) and extracellular matrix was administered to 22 New Zealand rabbits. Clinical evaluation was assessed at different points, the severity of the lesion was scored according to four grades and correlated with the area of hyperemia and the histopathological findings on day 23.

Results: Thirteen of 22 eyes (60%) developed pterygium-like lesions after 7 days and progressed through different grades. Initially, grade 3, characterized by an elevated and fleshiness conjunctiva with tortuous hyperemia, was evident on day 7. By day 15, lesion decreased to grade 2, with less elevation and hyperemia. Subsequent improvement was noted, with grade 1 on day 18. Finally, day 23 was marked by a white‒yellow lesion, classified as grade 4. The area of hyperemia increased from grade 2 to grade 3 (P < 0.05) and decreased from grade 3 to grade 4 (P ≤ 0.05). Histopathological analysis revealed a tendency toward increasing inflammation at grades 2, 3, and 4. There was a correlation between clinical features and the degree of inflammation.

Conclusions: Subconjunctival injection of NIH3T3 and extracellular matrix induces a pterygium-like lesion that progresses across four grades, beginning with an acute inflammatory process that evolve a chronic form. This study provides a replicable model for simulating pterygium.

Translational Relevance: The development of a standardized preclinical model of pterygium to evaluate new pharmacological or surgical treatments.

Introduction

Pterygium is a common ocular surface disease that represents a challenge for ophthalmologists. This abnormal growth of conjunctival tissue is associated with discomfort, cosmetic concerns, dry eye syndrome, and severe corneal vision problems caused by astigmatism.¹ Various approaches have been developed for pterygium treatment, ranging from the use of artificial drops to surgical intervention involving conjunctival grafting with or without limbal tissue.^2,3 

Despite ongoing efforts to address pterygium, there have been limited advancements in the definitive surgical treatment of this disease. The use of tissue glue in treatment is probably the most recent development, eliminating the need for traditional suture stitches to attach the conjunctival graft.⁴ After excision, the recurrence of pterygium is the most common complication, with variable rates across different ophthalmology centers.⁵ Although it is a common disorder, the etiopathogenesis of pterygium is not understood completely. It is thought that limbal damage owing to chronic ultraviolet (UV) exposure is an important factor in pterygium development.⁶ Additionally, an increase in the number of proinflammatory molecules related to UV irradiation may contribute to pterygium formation.⁷ 

To further our understanding of this challenging ocular condition, it is essential to establish a reliable animal model that can mimic the inflammatory environment for the development of pterygium-like lesion to evaluate new pharmacological or surgical treatments. A limited number of animal models for pterygium have been described. 

Although the role of UV irradiation in the development of pterygium has been recognized, no appropriate in vivo model has been established. A closer approximation is a computer simulation using data from rabbit cornea demonstrating that chronic UV irradiation of the limbus results in a wing-shaped mass similar to pterygium.⁸ Additionally, a mouse model injected with human pterygium fibroblasts reproduces the histological features of human pterygium⁹; however, the size of the eye in this animal model may challenge the evaluation of surgical procedures. 

In a previous study, we demonstrated the development of a pterygium-like lesion 7 days after the injection of fibroblasts and extracellular matrix in a rabbit animal model.¹⁰ In this study, we conducted a follow-up analysis and correlated clinical characteristics with the histopathological progression of pterygium-like lesions. Our study aimed to contribute to the standardization of a pterygium-like lesion useful in preclinical models. 

Materials and Methods

Animals

Twenty-two 3-month-old male white New Zealand rabbits with 2 to 3 kg of body weight were used in the experiment. They were kept at constant temperature (22 ± 1°C) and humidity (60 ± 10%) with a 12:12 hour light–dark cycle, and unrestricted access to standard diet and water. All animals were treated in accordance with the ARVO statement for the use of animals in ophthalmic and vision research. The protocols were approved by the Committee of Ethics for the Use of Experimental Animals for the School of Medicine of Tecnologico de Monterrey (2023-002-R2). 

Induction of a Pterygium-like Lesion

The experiment was carried out in accordance with our previous model.¹⁰ All animals were anesthetized via intravenous injection of a mixture of xylazine (5 mg/kg, Xilapet 10 Vets Pharma, Mexico City, Mexico) and zolazepam + tiletamine (2.5/2.5 mg/kg, Zelazol 100 Lab Zoetis, Mexico City, Mexico). The procedure was carried out on the right eye of each animal under an ophthalmic microscope (World Precision Instruments, Sarasota, FL) and aseptic conditions. The eyes were topically anesthetized with a drop of tetracaine (5 mg/mL, Sophia, Mexico City, Mexico), and a wire lid speculum was inserted to retract the lid for conjunctival exposure. 

A subconjunctival injection of 20,000 murine fibroblasts (NIH3T3 cell line, CRL 1658, ATCC) in 10 µL of Matrigel extracellular matrix (Corning, Corning, NY) was performed in the superotemporal quadrant of each eye, 5 mm outside the limbus, using a 1-mL 30G needle. After the procedure, postoperative antibiotic-corticosteroid eye drops (dexamethasone 1 mg/mL + tobramycin 3 mg/mL, Sophia) were administered three times daily for 1 day. 

Clinical Evaluation

The macroscopic aspect of the eyes was registered with a digital camera (EOS rebel T7, Canon, Taichung, Taiwan) at 7, 10, 15, 18, and 23 days after injection. On a scale of 1 to 4, the clinical aspect of the conjunctiva at the site of injection was classified as follows: grade 1, slight changes: flat appearance of conjunctiva, hyperemia composed of fine and thin vessels in a nondefined area of the quadrant; grade 2, moderate changes: elevated appearance of conjunctiva, hyperemia composed of tortuous and enlarged vessels with involvement of 25% to 50% of the quadrant; grade 3, severe changes: elevated and fleshiness appearance of conjunctiva, hyperemia composed of tortuous and enlarged vessels with involvement of >50% of the quadrant; and grade 4, chronic lesion: elevated and white‒yellow lesion, no defined hyperemia with involvement of <25% of the quadrant (Table 1). One masked examiner (ocular surface and cornea specialist) classified the severity of the lesion based on this classification. 

Table 1.

 

The Clinical Features of Each Pterygium-Like Lesion Grade

 

Conjunctival Hyperemia Evaluation

The area of conjunctival hyperemia was measured by analyzing images of the different grades of pterygium-like lesion using the ImageJ program (version 1.54f, National Institutes of Health, Bethesda, MD) and reported in square millimeters. 

Histopathological Analysis

On day 23, all animals were euthanized under anesthesia and an intracardiac overdose of embutramide (200 mg/kg) + mebezonium iodide (50 mg/kg) + tetracaine hydrochloride (5 mg/kg), (T61, MSD, Salud Animal, Mexico City, Mexico). Subconjunctival tissue samples from the site of injection and from the normal conjunctiva of the contralateral eye were excised, rinsed with phosphate-buffered saline, and fixed in 10% buffered formalin. Conjunctival tissues were embedded in paraffin, and 5-µm-thick sections were obtained with a microtome and stained with hematoxylin and eosin. The histological analysis included classification of the level (1, 2, 3, or 4), localization and cell composition of inflammation, cell organization, presence of edema, and epithelial changes. The analysis was performed by one masked examiner (a pathologist specialist). 

Statistical Analysis

Statistical analysis was performed using analysis of variance for mean comparisons, and a P value of < 0.05 indicated statistical significance. 

Results

Clinical Evaluation

Thirteen of 22 eyes (60%) developed pterygium-like lesions after subconjunctival injection (Fig. 1). On day 1 (Fig. 1A) after subconjunctival injection, the eyes showed a normal conjunctiva, characterized by a flat, smooth appearance and the absence of hyperemia. After 7 days, changes in the conjunctiva became evident (Figs. 1B–F). Between postoperative days 7 and 10 (Figs. 1B, 1C), severe changes characterized by an elevated lesion, with a fleshiness aspect close to the limbus, and hyperemia composed of tortuous and enlarged vessels in >50% of the quadrant were observed (grade 3). On day 15 (Fig. 1D), an improvement in the clinical aspect was noted with a decrease in the size of the affected area of the quadrant, although the appearance of the conjunctiva was still elevated, and with hyperemia of tortuous and enlarged vessels (grade 2). On day 18 (Fig. 1E), slight changes characterized by a flat conjunctiva with hyperemia composed of fine and thin vessels (grade 1) were observed. On day 23 (Fig. 1F), a white‒yellow elevated lesion was observed at the site of injury (grade 4). The lesion did not extend to the cornea. 

Figure 1.

 

Progression of pterygium-like lesion of different grades during the study period. (A) Normal conjunctiva. (B and C) Grade 3. (D) Grade 2. (E) Grade 1. (F) Grade 4.

Conjunctival Hyperemia Evaluation

The mean areas of conjunctival hyperemia in grades 2, 3 and 4 were 8.56 ± 2.04 mm², 17.32 ± 5.01 mm², and 2.14 ± 0.68 mm², respectively (Fig. 2). There was a significant difference (P < 0.05) in the areas of hyperemic lesions among the different grades. The hyperemic areas of grade 1 were not measured owing to the absence of lesion development. A significant increase in conjunctival hyperemia was observed between grade 2 and grade 3 (P = 0.0023). In contrast, a significant decrease in hyperemia was observed between grades 3 and 4 (P ≤ 0.0001). Additionally, a significant difference was observed between grades 2 and 4 (P = 0.0185) (Fig. 2). 

Figure 2.

 

Analysis of the difference between areas of conjunctival hyperemia (mm²) observed at grades 2, 3 and 4 (n = 5). *Statistically significant.

Histopathological Analysis

On day 23 after subconjunctival injection, the results showed different levels of inflammation (Fig. 3), with different characteristics (Table 2). Histopathological analysis revealed a trend toward greater inflammation in grade 2 (Fig. 3E), grade 3 (Fig. 3F), and grade 4 (Figs. 3G–I) eyes than in normal conjunctiva (Fig. 3A) and grade 1 (Figs. 3B–D) eyes. It was observed that the level of inflammation was correlated with the grade of the lesion according to clinical observation. In the normal conjunctiva (Fig. 3A), no inflammation was observed, whereas grade 1, inflammation (Figs. 3B–D) included lymphocytes and plasmocytes at level 1. Grade 2 showed an increase in inflammation to level 2, with presence of lymphocytes, plasmocytes and eosinophils. An increase in the level of inflammation (level 4) was more evident in grade 3 (Fig. 3F). 

Figure 3.

 

Histopathological analysis throughout the study period and its correlation with the different grades of the lesion: (A) normal conjunctiva; (B–D) grade 1; (E) grade 2; (F) grade 3; and (G–I) grade 4.

Table 2.

 

Histopathological Characteristics of the Different Grades of Pterygium-Like Lesions

 

At this advanced grade, similar to grade 2, lymphocytes, plasmocytes, and eosinophils were still observed in the characteristic cell infiltration. In contrast, grade 4 showed a decrease in the level of inflammation (level 3) but with a particular organization of the cell infiltrate: subepithelial multifocal organization (Fig. 3G), lymphoid mass (Fig. 3H) and granuloma (Fig. 3I). This cell organization contrasts with the unspecific organization of the infiltrate reported in the early stages of lesion development. The histopathological characteristics of the different grades of pterygium-like lesions are summarized in Table 2. 

Discussion

Pterygium is considered a chronic degenerative process associated with UV light exposure in which acanthosis and elastosis are common findings,¹¹ along with chronic inflammation, which plays an important role in the pathogenesis of this disease.^12,13 In the past, mouse models have been used to induce lesions similar to those in human pterygium.^9,14 Yang et al.⁹ reproduced the characteristics of human pterygium in mice after the injection of human pterygial cells and proposed a model for studies of therapeutic agents. However, the small size of the eye in the mouse model may limit clinical evaluation or the possibility of studying surgical innovations. Previous studies by our group demonstrated that it is possible to reproduce human pterygium-like changes in a rabbit model.¹⁰ In the present study, we correlated the progression of the clinical aspect of pterygium-like lesions with the histopathological analysis for a sustained period. 

Between days 7 and 15, after a subconjunctival injection of NIH3T3 cells and extracellular matrix, we were able to produce an elevated, fleshiness and hyperemic lesion with a high level of inflammation, suggesting an acute inflammatory process. Some of the described characteristics of our lesion in the first 15 days were observed in inflamed pterygia reported by Mansour.¹⁵ Additionally, the microscopic characteristic of lymphocytic infiltration, with the presence of plasma cells reported by Hill and Maske,¹³ is present in our pterygium-like lesions. 

A prospective study by Mushtaq et al.,¹² in which pterygium samples were collected after resection, revealed a correlation between lesion size and inflammation grade. It seems that lesions ˃4 mm are associated with increased inflammation and vascularization. The management of pterygium with these characteristics involves surgical resection of the lesion. In our study, the lesions between 7 and 15 days after injection were the largest and most hyperemic during the follow-up period (Figs. 1B, 1C). According to Zhao et al.,¹⁶ the presence of conjunctival hyperemia is associated with a high rate of recurrence. Vascular alterations in the pterygium have been proposed as a therapeutic target; for this reason, different studies have used anti-vascular endothelial growth factor therapy for pterygium, which has shown good results, especially in decreasing recurrence after surgery.¹⁷ 

It is possible that the presence of intense hyperemia permits the arrival of inflammatory cells and enhances and perpetuates an immune reaction at the site of damage independent of the cause. Seven to 15 days after subconjunctival injection, it is possible to reproduce the characteristics of inflamed pterygium with a high rate of recurrence described by Mushtaq et al.¹² in our animal model. This point is important because recurrence is one of the greatest concerns in the treatment of pterygium. 

Our results provide evidence to support the idea that, in our model, the pterygium-like lesion between days 7 and 15 allows the evaluation of surgical treatments and therapeutic agents against inflammatory and vascular components. To our knowledge, there are no animal models supporting this possibility. 

In contrast, on day 18, a decrease in inflammation composed of lymphocytes and plasmocytes was observed after conjunctival injection. This attenuation suggests that conjunctival tissue is an active mechanism for resolving acute insult. However, on day 23, a notable clinical change characterized by a decrease in the area of conjunctival hyperemia, but with an elevated lesion was observed. Histopathological analysis revealed high levels of inflammation organized as multifocal infiltrates, granulomas, and lymphoid masses. This is in accordance with the findings of Safi et al.,¹⁸ who demonstrated that leukocytic infiltration can be detected without evidence of redness in pterygium samples collected after surgical excision. The possibility of having a model of pterygium with a chronic phase might be useful in the evaluation of pharmaceutical treatments focused on preventing the establishment of the precursor environment of the chronic changes related to a pterygium-like lesion. 

In our study, histopathological analysis revealed the presence of lymphocytes and plasmocytes in the different stages of lesions; this identified cellular profile is similar to that of other studies that evaluated the immunologic basis involved in the pathogenesis of pterygium.^19,20 According to Džunić et al.,²¹ lymphocytic infiltration and the presence of plasma cells are histopathological hallmarks of chronic inflammation; these findings were also observed in our pterygium-like lesions at the end of the study. Another finding in our study was the presence of histiocytes (macrophages) in the infiltrate; these cells are present in the chronic inflammatory response to promote phagocytosis, fibrosis, and angiogenesis.²² 

Although we were unable to replicate the elastosis or acanthosis alterations and the typical triangular shape of the pterygium that invades the cornea in our model, we were able to reproduce the inflammatory environment precursor of these changes. Because the inflammatory process is a dynamic phenomenon, an animal model that assesses different phases of the inflammatory process involved in the development of pterygium-like lesions would be very useful for evaluating different therapeutic strategies during specific periods. 

Conclusions

Subconjunctival injection of fibroblasts (NIH3T3) combined with extracellular matrix can induce different grades of pterygium-like lesions throughout the follow-up period in a rabbit model. The clinical examination of the lesion between 7 and 15 days revealed an elevated lesion with fleshiness aspect associated with an acute inflammatory process and a white‒yellow elevated lesion on day 23 associated with a chronic process. The model can be used to replicate the inflammatory and vascular components of pterygium, which in turn enables the evaluation of drug preventive, therapeutic, and surgical approaches at different stages of pterygium-like lesion. 

Acknowledgments

The authors thank the team of the CORELAB Preclinical Research Unit of Tecnologico de Monterrey for their skillful technical assistance. 

Supported by grant Challenge Based Research Program Funding 2022 E015-EMCS-GI02-D-T1, an Institutional funding grant of Tecnologico de Monterrey. 

Disclosure: C.A. Rodríguez-Barrientos, None; G.R. Ayala-Villegas, None; J.E. Valdez-García, None; J. Zavala, None