Background: Translational research to develop pharmaceutical and surgical treatments for pterygium requires a reli‑ able and easy to produce animal model. Extracellular matrix and fibroblast are important components of pterygium. The aim of this study was to analyze the effect of the subconjunctival injection of fibroblast cells (NIH3T3 cell line) and exogenous extracellular matrix in rabbits in producing a pterygium‑ like lesion. Methods: Six 3‑ month‑ old white New Zealand rabbits were injected with 20,000 NIH3T3 cells and 5 µL of Matrigel in the right conjunctiva, and with only 5 µL of Matrigel in the left conjunctiva. The eyes were photographed under a magnification of 16 × using a 12‑ megapixel digital camera attached to the microscope on day 1, 3 and 7. Conjunctival vascularization was measured by analyzing images to measure red pixel saturation. Area of corneal and conjunctival fibrovascular tissue formation on the site of injection was assessed by analyzing the images on day 3 and 7 using area measurement software. Histopathologic characteristics were determined in the rabbit tissues and compared with a human primary pterygium. Results: The two treatments promoted growth of conjunctival fibrovascular tissue at day 7. The red pixel saturation and area of fibrovascular tissue developed was significantly higher in right eyes (p < 0.05). Tissues from both treat‑ ments showed neovascularization in lesser extent to that observed in human pterygium. Acanthosis, stromal inflam‑ mation, and edema were found in tissues of both treatments. No elastosis was found in either treatment. Conclusions: Matrigel alone or in combination with NIH3T3 cells injected into the rabbits’ conjunctiva can promote tissue growth with characteristics of human pterygium, including neovascularization, acanthosis, stromal inflamma‑ tion, and edema. The combination of Matrigel with NIH3T3 cells seems to have an additive effect on the size and redness of the pterygium‑ like tissue developed. Keywords: Pterygium, Fibroblast, Rabbit, Extracellular matrix, Animal model Background of pro-inflammatory cytokines induced by fibrogenic Pterygium is a benign fibrovascular growth of the ocu - growth factors, oxidative stress and DNA methylation lar surface commonly associated to discomfort and have been implicated in the pathogenesis of pterygium. red eye, and as disease progresses is often related to Since some of these factors are affected by exposure to decreased vision (topographic astigmatism) and ocular ultraviolet (UV) light, current evidence from multiple motility restriction in severe cases . A wide variety sources suggests that individuals with high exposure to sunlight are at increased risk of pterygium . Despite the extensive research, there is no clear understanding of the pathogenesis of pterygium, but one of the most *Correspondence: firstname.lastname@example.org Tecnologico de Monterrey, Escuela de Medicina y Ciencias de la Salud, important factors contributing to the pathogenesis are Ave. Morones Prieto 3000, 64710 Monterrey, NL, Mexico the neoplastic changes of limbal stem cells associated Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zavala et al. Biol Res (2018) 51:15 Page 2 of 7 to UV light exposure and the possible role of oncogenic and 7. Conjunctival vascularization was measured on day virus (Human papillomavirus) . 1, 3 and 7 by analyzing images using Adobe Photoshop Epidemiological studies report that the prevalence of CS5 (Version 12.0, Adobe, San Jose, CA) color histo- pterygium in Latino population is elevated (16% overall), grams to measure red pixel saturation on a 6 × 6 mm area especially in people with low income and low educational on the site of injection. Area of corneal and conjunctival status, increased age and with men having a higher preva- fibrovascular tissue formation on the site of injection and lence compared to female (2:1) . The definite treatment surrounding area was assessed by analyzing the images is often surgical excision with the use of adjuvant anti- on day 3 and 7 using area measurement software (Adobe proliferative drugs (mitomycin C, 5-fluorouracil, doxycy - Photoshop CS5). cline or bevacizumab), which still need standardization After 7 days, surgical specimens were obtained under and, in the best scenario, still report variable recurrence anesthesia and were immediately fixed in 10% buffered rates (0.1–33.3%) [5–7]. In vitro models focused on the formalin (pH = 7.3) for at least 8 h, routine histopatho- pathogenesis of pterygium to explore the intercellular logic process was followed and tissue was embedded in signaling pathways in epithelial cells and fibroblasts show paraffin. Immediately after obtaining conjunctival speci - promising results [8, 9], but animal models to support the mens, all rabbits were euthanized (day 7) using a lethal evidence are needed. To date, three animal models for dose (200 mg/kg) of intravenously-administered pento- pterygium have been described, using injection of human barbital (Penta-Hypnol , Agrovet MArket, Lima, Peru). epithelial pterygium cells, exogenous extracellular matrix Each sample was serially cut into at least 10 four micron- or UV scattered radiation in rabbit and mice [3, 10, 11]. thick sections, mounted and stained with hematoxylin/ The results of the rabbit model using UV scattered light eosin staining method. All specimens were evaluated by are focused on the computational prediction of the size the same pathologist to limit subjective variation during and shape of the tissue growth, with no histological the observation and counting process. Histopathologic analysis. The mouse model showed histological charac - characteristics including neovascularization, epithelial teristics of human pterygium, however, manipulation of inflammation, stromal inflammation, acanthosis, elasto - rabbits for ophthalmological procedures is easier given sis and edema were determined in the rabbit tissues and that the ocular structure and size resembles more to that compared with a human primary pterygium. of human. Histological changes were semi-quantitatively clas- Hence, the purpose of this study was to develop an ani- sified into six groups. Vascular density was defined as mal model for pterygium using subconjunctival injection the average vessel count in six high power fields (HPF, of murine fibroblast cells (NIH 3T3 cell line) and exog - 400×) in the areas appearing as the most vascularized enous extracellular matrix (Matrigel) in white New Zea- foci. The whole pterygium specimen was examined and 6 land rabbits suitable for its implementation on both basic HPF which seemed to have the greatest vascular density pharmacologic/molecular research on pterygium patho- were selected; all vessels lined by endothelium were con- genesis and on novel surgical techniques. sidered, given that the presence of endothelium makes it possible to differentiate a vessel from the pseudo-vas - Methods cular canal. Epithelial and stromal inflammation were All procedures were conducted according to the Guide graded with the following scale: grade 0, absent or sparse for the Care and Use of Laboratory Animals and were inflammatory cells in the tissue; and grade 1, significant approved by Institutional Animal Care and Use Com- patchy or diffuse inflammatory cells infiltration. Acan - mittee. A total of six 3-month-old white New Zealand thosis was graded based on the number of epithelial cells rabbits were used. Animals were anesthetized with found (N) in the unaltered epithelium, using the follow- intramuscular injection of ketamine HCl (30 mg/kg) ing scale: grade 0, absent or any number smaller than N; and xylazine (5 mg/kg) and topical anesthesia with tet- grade 1, N – 1½N; and grade 2, 1½N – 2 N. The percent - racaine hydrochloride (2 eye drops). Rabbits received age of elastosis and edema were described as the propor- subconjunctival injection of 20,000 murine fibroblast tion of elastotic changes and edema, respectively, to the cells (NIH 3T3 cell line) and 5 µL of Matrigel (Corning whole fibro connective stroma and graded with the fol - Inc., New York, US) on the perilimbal temporal bulbar lowing scale: grade 0, absent; grade 1, element found on conjunctiva of the right eye using a 1 ml 30G needle. Left less than 50% of the tissue; and grade 2, element found eyes were injected with 5 µL of Matrigel under the per- on more than 50% of the tissue. The sections were ana - ilimbal temporal bulbar conjunctiva (control). The eyes lyzed using standard light microscopy (Carl Zeiss, Axi- were photographed under a magnification of 16× using a ostar plus, Germany) under 400× and 40× magnification 12-megapixel digital camera attached to the microscope for vascular density, epithelial and stromal inflammation, (World Precision Instruments, Inc. FL, US) on day 1, 3 and acanthosis; and elastosis and edema, respectively. Zavala et al. Biol Res (2018) 51:15 Page 3 of 7 Statistical analysis was performed using paired t-test and analysis of variance (ANOVA) for mean compari- sons, statistical significance was considered with a p value < 0.05. Results All eyes (12 eyes) of the 6 rabbits developed conjuncti- val fibrovascular tissue after injection of Matrigel and NIH3T3 cells (n = 6, right eyes) or only Matrigel (n = 6, left eyes) at day 7 (Fig. 1). The mean area of fibrovascular tissue developed in the right eyes at day 1, 3 and 7 was 2.92 ± 1.21, 14.32 ± 3.47 and 33.97 ± 4.40 mm respec- tively. Left eyes had a mean fibrovascular tissue area of 2.91 ± 1.36, 8.41 ± 2.08 and 15.36 ± 3.89 mm at days 1, 3 and 7 respectively. The difference in fibrovascular growth areas observed between right and left eyes at day 3 and 7 was significant (p < 0.05) (Fig. 2). A significant increase in fibrovascular growth was also observed between days 1 and 3 (p = 0.001), days 1 and 7 (p < 0.001) and days 3 and Fig. 2 Analysis of the difference between the fibrovascular growth 7 (p < 0.001) in right eyes. Likewise, a significant increase areas (mm ) observed at day 1, 3 and 7, post injection site in the in fibrovascular growth was also observed between days 1 animal model right (n = 6) and left eyes (n = 6) (6 rabbits, 12 eyes) and 3 (p = 0.006), days 1 and 7 (p = 0.001) and days 3 and 7 (p = 0.001) in left eyes. No difference was seen between red pixel saturation of the fibrovascular tissue in right and left eyes at day 1 and 3. At day 7, right eyes exhib- left eyes was 27; which is far from the 134 found in the ited significant (p < 0.05) higher red pixel saturation of human tissue. Five eyes of each treatment (right and left the developed fibrovascular tissue, related to conjunctival eyes) registered non- epithelial inflammation, resem - vascularization, when compared with left eyes (Fig. 3). bling the changes observed in human pterygium. Stromal Significant differences in the neovascularization pat - inflammation, absent in human pterygium, was observed tern between rabbit induced pterygium tissue and human in 1 of 6 right eyes and in 4 of the 6 left eyes. Acantho- pterygium was observed in the histopathological analysis sis was observed in 4 right eyes and in 6 left eyes (Posi- (Table 1). The average number of vessels found in the 6 tive, grade 2 in human pterygium sample). Only one eye analyzed lamellas of the right eyes was 29, while in the in each group (Matrigel + NIH 3T3 and Matrigel alone) Fig. 1 Right eye of rabbit 4 at 1, 3 and 7 days (a, b and c respectively) post injection of Matrigel and NIH3T3; left eye of rabbit 4 at 1, 3 and 7 days (d, e and f respectively) post injection of Matrigel only Zavala et al. Biol Res (2018) 51:15 Page 4 of 7 2 edema. Figure 4 shows a representative image of the fibrovascular tissue developed in one rabbit (right eye, Matrigel + NIH 3T3) and the human pterygium. Discussion Pterygium is a frequent ophthalmological disease whose pathogenesis is not fully understood. The only defini - tive treatment available is surgical excision, with the dis- advantage of a variable but persistent recurrence rate. Surgical alternatives and novel approaches involving autologous conjunctival grafts, extensive Tenon’s resec- tion and amniotic membrane transplant in the excision area combined with the use of postsurgical antimetabo- Fig. 3 Red pixel saturation comparison analysis at day 1, 3 and 7, lites to decrease the recurrence rate have shown promis- animal model right (n = 6) and left eyes (n = 6) (6 rabbits, 12 eyes) ing results [5, 7]. However, the lack of knowledge about the subjacent molecular and cellular mechanisms have hindered the development of novel pharmacological showed elastosis in the same grade as human pterygium, treatments and therapeutic approaches. Consequently, while edema was observed in a similar grade between the development of a pterygium animal model is an all right eyes specimens and human pterygium. Three essential tool in the research for pathogenic events and left eyes showed grade 1 edema and three showed grade novel surgical and pharmacological approaches. Table 1 Comparison of the tissue characteristics of 13 lamellae, corresponding to the 6 right and 6 left eyes after 7 days of injection and a human pterygium Eye Rabbit 1 Rabbit 2 Rabbit 3 Rabbit 4 Rabbit 5 Rabbit 6 Human RE LE RE LE RE LE RE LE RE LE RE LE Neovascularization 21 30 30 18 17 54 30 20 46 19 31 23 134 Epithelial inflammation 0 1 0 0 0 0 1 0 0 0 0 0 0 Stromal inflammation 1 1 1 0 0 0 1 0 1 0 1 1 0 Acanthosis 2 2 0 2 0 2 2 2 2 2 2 2 2 Elastosis 0 0 0 1 1 0 0 0 0 0 0 0 1 Edema 2 2 2 2 2 1 2 1 2 1 2 2 2 RE right eye, LE left eye Fig. 4 Comparison of the histopathological criteria evaluated in human pterygium a and the rabbit conjunctival fibrovascular tissue in a right eye b. Fibroconnective tissue (asterisk) is observed with a great amount of blood vessel (open triangle) coated by acanthotic squamous epithelium (filled triangle). In the rabbit tissue, edema interstitial is observed but not in human pterygium (H&E ×100) Zavala et al. Biol Res (2018) 51:15 Page 5 of 7 Previous attempts in developing a rabbit models for and left eyes (Matrigel). Hence, we decided to per- pterygium were carried out by Kowk and Coroneo , form the histologic examination as early as day 7 post who reported the use of incident ultraviolet (UV) scat- injection. However, relevant data and observations can tered light at the temporal limbus to induce a predicted emerge from future protocols assessing the histological shaped and sized pterygium-like growth. Although this characteristics between the lesions in different post injec - model reproduces the real UV light stimulation that is tion times. hypothesized to play a biologic key role in pterygium Although both Matrigel and chondrocyte derived pathogenesis, the conjunctival/limbal stimulation with extracellular matrix are used to promote cell prolifera- UV light required to produce significant conjunctival tion, the former is known for its gel composition at room epithelial proliferation would be extensive, hence the temperature containing bFGF, IGF, EGF, laminin, colla- need of a computational algorithm to simulate the pre- gen 1, and heparin sulfate proteoglycan , which exerts dicted growth and final shape. Moreover, no histological a slow release characteristic that favors angiogenesis [13– analysis was reported to register the characteristics of the 16]. In the mice model injected with Matrigel reported by induced tissue growth. Cox, there was no tissue growth at day 6. Our model sug- In a different research, 6 week old athymic nude mice gests that the combination of NIH3T3 cells (an immor- were used to induce pterygium with an injection of talized mouse fibroblast line) with Matrigel, enhances the 1 × 10 human epithelial pterygium cells followed by an growth of the pterygium-like tissue. This was confirmed injection of chondrocyte derived extracellular matrix 7, by the macroscopic clinical analysis performed to assess 10 or 14 days after the epithelial cell injection . At day red-pixel saturation, which can correlate to hyperemia, 17, a lesion growth was observed with higher significant neovascularization and other histological events; and tis- size in the eyes injected with only epithelial cells than that sue growth area measured in square millimeters. observed in the eyes injected with cells and extracellular A histological analysis using a scale of 6 parameters matrix. Their histological analysis revealed epithelial cells commonly observed in human pterygium [17–20] was extending into the superficial stroma, neo-vessels, and carried out. The most distinctive feature of human extracellular matrix breakdown in the eyes injected with pterygium is neovascularization [19, 21]. In our analysis, epithelial cells, but not in those injected with extracellu- tissues of both groups showed neovascularization with a lar matrix. Similarly, Cox et al. used conjunctival injec- slight increased level in right eyes, but in a lower grade tion with 1 × 10 human epithelial pterygium cells mixed than that observed in human pterygium . Nonethe- with 5 μL of Matrigel on nude athymic mice, observing a less, the number of new vessels in human pterygium has lesion growth on the injection site after 6 days . They been reported as extremely variable [19, 20]. In human reported no lesion growth in mice injected with only pterygium, redness and fleshiness are correlated with the Matrigel. In the histological analysis, neovascularization vascular density and stromal fibrosis, while the extent and migrating cells could be seen only in the epithelial of the pterygium over the cornea has an inverse relation cell injected mice conjunctivas. with the stromal elastosis . This is in accordance with Our model was conducted on young White New Zea- our results, in which higher redness and vascularization land rabbits, an animal model that shares similarities was observed in right eyes. No invasion over the cornea with human ocular structure including size, thus pro- was observed after 7 days, further experiments analyzing viding an advantage over the surgical manipulation and the growth of the tissue over a longer period will provide the extent of pterygium-like tissue developed useful for information about the correlation of this parameter and further analysis. In our study, we compared the effect of the stromal elastosis level observed. the conjunctival injection of only Matrigel, in left eyes, No epithelial inflammation was seen in the tissue and Matrigel with NIH3T3 fibroblasts, in right eyes. The induced in right or left eyes, paralleling the histopa- concentration of injected cells and volume of Matrigel thology of primary human pterygium. Elastosis was was the same used by Yang and Cox [10, 11]. Although seen only in one eye of each treatment group. The latter following similar methods, Yang et al.  decided to parameter is highly associated to chronic UV radiation wait until day 17 after injection to perform the histologic exposure , and it is reported as a key histological examination since it was only then when they observed characteristic in primary pterygium and minimal in a significant difference in the lesion size between their recurrent pterygium . The absence of significant groups (eyes with only epithelial fibroblasts cells com - elastotic changes in the analyzed tissues could be asso- pared to the injection of extracellular matrix and cells). ciated with the method used to induce the pterygium- On the other hand, as evidenced on Fig. 2, we observed like tissue formation (eliciting inflammatory immune a significant difference in the lesion size since day 3 post response and/or fibroblastic cell proliferation, not asso - injection between right eyes (NIH 3T3 cell + Matrigel) ciated with UV light exposure) and to the short period Zavala et al. Biol Res (2018) 51:15 Page 6 of 7 after which histological analysis was performed. Oth- Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub‑ erwise, cellular edema was similar between right eyes lished maps and institutional affiliations. and human pterygium, comparable with the observa- tions of previous reports . 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