Regenerated Corneal Epithelium Expresses More ßIII-Tubulin After Chemical Injuries Compared to Mechanical Injuries.

Purpose: Defining the regenerative response following various types of corneal chemical and mechanical injuries is important for understanding the pathophysiology of the injury and evaluating the effectiveness of the therapies. This study characterizes corneal epithelial healing in a murine chemical and mechanical injury model. Methods: Four groups of 10 mice each received complete corneolimbal injuries by AlgerBrush, AlgerBrush/thermal, NaOH (0.5 N), or ethanol. Slit-lamp and optical coherence tomography examinations were performed daily for 14 days. Corneal opacity (CO) and neovascularization (NV) were evaluated. The origin of the regenerated epithelium was illustrated by anti-cytokeratin 12 (K12) and anti-K13. The height of regenerated corneal epithelium and intraepithelial free nerve endings (FNEs) stained with anti-ßIII-tubulin were measured. The amount of fibrosis was measured by anti-α-smooth muscle actin (α-SMA) monoclonal antibody in the different groups. Statistical analysis was performed by ANOVA and t-test. Results: Corneal opacity and neovascularization were markedly higher in the NaOH and AlgerBrush/thermal groups. Molecular studies revealed the following: Regenerated corneal epithelium thickness was less than normal in all groups, the AlgerBrush group had the shortest height of the regenerated epithelium, ßIII-tubulin was expressed in the entire height of corneal epithelium in all groups except in the AlgerBrush group, and K12 was replaced by K13 in all groups. Conclusions: Corneal wound healing is more effective following chemical injuries in terms of epithelial thickness. Inflammation may play an important role in the outcome. Translational Relevance: Inflammation following different injuries may be redirected to be more effective in corneal regeneration and clarity.

Corneal and Limbal Alkali Injury Induction Using a Punch-Trephine Technique in a Mouse Model.

The cornea is critical for vision, and corneal healing after trauma is fundamental in maintaining its transparency and function. Through the study of corneal injury models, researchers aim to enhance their understanding of how the cornea heals and develop strategies to prevent and manage corneal opacities. Chemical injury is one of the most popular injury models that has extensively been studied on mice. Most previous investigators have used a flat paper soaked in sodium hydroxide to induce corneal injury. However, inducing corneal and limbal injury using flat filter paper is unreliable, since the mouse cornea is highly curved. Here, we present a new instrument, a modified biopsy punch, that enables the researchers to create a well-circumscribed, localized, and evenly distributed alkali injury to the murine cornea and limbus. This punch-trephine method enables researchers to induce an accurate and reproducible chemical burn to the entire murine cornea and limbus while leaving other structures, such as the eyelids, unaffected by the chemical. Moreover, this study introduces an enucleation technique that preserves the medial caruncle as a landmark for identifying the nasal side of the globe. The bulbar and palpebral conjunctiva, and lacrimal gland are also kept intact using this technique. Ophthalmologic examinations were performed via slit lamp biomicroscope and fluorescein staining on days 0, 1, 2, 6, 8, and 14 post-injury. Clinical, histological, and immunohistochemical findings confirmed limbal stem cell deficiency and ocular surface regeneration failure in all experimental mice. The presented alkali corneal injury model is ideal for studying limbal stem cell deficiency, corneal inflammation, and fibrosis. This method is also suitable for investigating pre-clinical and clinical efficacies of topical ophthalmologic medications on the murine corneal surface.

Evaluation of the Algerbrush II rotating burr as a tool for inducing ocular surface failure in a mouse model.

Purpose: The Algerbrush II has been widely used to induce corneal and limbal injuries in animal models. The extent of injury varies with the duration of exposure, pressure from the placement of the burr, and the size of the burr. However, no study has explored the correlation between the duration of exposure and the severity of injury in mouse model with corneal and limbal stem cell deficiency (LSCD) induced using the Algerbrush II. Therefore, this study aimed to evaluate the variations in the severity of corneal and limbal injury with different durations of the Algerbrush II application. Methods: The entire cornea and limbus of C57BL/6 mice were injured for 30-45 s, 60-75 s, 90-120 s, and 3-4 min. Photography and slit-lamp examination was performed on days 0, 2, 4, and 7, followed by hematoxylin & eosin, periodic acid-Schiff, and immunohistochemical staining. Statistical analysis was performed using one way ANOVA analysis. Results: A duration of 30-45 s of injury was found to be sufficient to induce superficial corneal and limbal epithelial debridement and re-epithelialization was completed in all eyes by day 7; however, clinical signs of LSCD were not observed in all mice. Increasing the exposure time to 90-120 s resulted in central 2+ corneal opacity with limbal and paracentral corneal neovascularization. All eyes injured for 3-4 min displayed clinical signs of LSCD, such as persistent epithelial defects on day 7 after the injury, central corneal neovascularization, and 2.2+ diffuse corneal opacity. Histological signs of LSCD, including goblet cell metaplasia and K13 expression on the corneal surface, were observed in all injured eyes. Conclusions: Our findings suggest that the duration of injury is an important factor influencing the severity of LSCD in a murine model of injury. A 1-mm rotating burr was found to be more effective for keratectomy and pigment release, whereas a 0.5-mm burr was more suitable for corneal epithelial debridement.

Autologous Activated Omental versus Allogeneic Adipose Tissue-Derived Mesenchymal Stem Cells in Corneal Alkaline Injury: An Experimental Study.

PURPOSE: To compare the effects of two types of mesenchymal stem cells (MSCs), activated omental cells (AOCs), and adipose tissue-derived stem cells (ADSCs) in the healing process of animal model of ocular surface alkali injury. METHODS: An alkaline burn was induced on the ocular surfaces of eighteen rats divided randomly into three groups. The first and second groups received subconjunctival AOCs and ADSCs, respectively. The control group received normal saline subconjunctival injection. On the 90th day after the injury, the eyes were examined using slit-lamp biomicroscopy. Corneal neovascularization and scarring were graded in a masked fashion. Histological evaluation of the corneal scar was performed, and the number of inflammatory cells was evaluated. RESULTS: Corneal neovascularization scores revealed higher neovascularization in the control (0.49 ± 0.12) than the AOC (0.80 ± 0.20, P = 0.01) and ADSC groups (0.84 ± 0.24, P = 0.007). There were no statistically significant differences between the neovascularization score of the AOC and ADSC groups (P > 0.05). According to histologic evaluation, stromal infiltration was significantly more in the control group compared to AOC and ADSC groups (P < 0.05). CONCLUSIONS: Our results suggest that MSCs, even with different sources, can be used to promote wound healing after corneal chemical burns. However, the ease of harvesting ADSC from more superficial fat sources makes this method more clinically applicable.

Omental transposition in treatment of severe ocular surface alkaline burn: an experimental study.

Since alkaline substances can rapidly penetrate into the cornea and subsequently damage limbal stem cells, another source of stem cells may be necessary to reconstruct the ocular surface. Omentum has some such characteristics like ability to regenerate tissue as well as anti-inflammatory capacity. Presence of adult stem cells and pluripotent embryonic cell markers make it suitable in wound healing; therefore, it seems reasonable to evaluate whether omentum can be helpful to restoration of ocular surface in severe alkaline burn. In this experimental trial, two groups of dogs (5 in each) were assigned. Following ethics approval, ocular surface alkaline burn was induced in both groups by placing filter papers soaked with NaOH (0.5 mol/l) on the cornea of one eye. Subsequently, group 1 (n=5) was treated only by conventional therapy; group 2 (n=5) was treated with omental elongation and transposition to the injured eye immediately following injury. Both groups were followed for six months. Ocular surface was evaluated by slit lamp microscope and corneal clarity was assessed and graded. At the end of six months, corneal opacity and vascularization were significantly reduced in group 2 (p-values of 0.009, 0.049, and 0.032 for corneal opacity, fluorescein staining, and vascularization grades, respectively). We have concluded that transposition of omental pedicle may be an effective treatment for severe ocular surface alkaline burn although more studies might be required.