Efficacy of bakuchiol in corneal alkali injury model in rats

Objective: This study aimed to investigate the effects of hydrogel-loaded bakuchiol as a therapeutic method on epithelial defect sizes; corneal neovascularization, corneal opacity severity; histopathological sections, and immunohistochemical markers (TNF-α, VEGF) in a corneal alkali burn model in rats. Materials and Methods: A corneal alkali burn was created in the central cornea of 35 male Wistar rats by placing a 3mm diameter filter paper soaked in 1 N NaOH for 30 seconds under topical and general anesthesia. The randomized rats were divided into 5 groups (control, visco, steroid, visco loaded with 250 IU/g bakuchiol, and visco loaded with 500 IU/g bakuchiol). All groups received eye drops containing ciprofloxacin every 12 hours for infection prevention. The control group received no antibiotic treatment. The visco group received ophthalmic hydrogel application four times a day. The steroid group received eye drops containing dexamethasone four times a day. The groups with 250 IU/g and 500 IU/g bakuchiol loaded visco received ophthalmic hydrogel applications at the specified doses four times a day. Rats were examined and photographed on days 0, 3, 7, and 14 under intraperitoneal ketamine general anesthesia using a slit lamp microscope. On day 14, rats were sacrificed under high-dose intraperitoneal general anesthesia through decapitation. Enucleation was performed, and the eyes were fixed in 10% formalin. Histopathological evaluations were conducted using Hematoxylin-Eosin staining. Immunohistochemical evaluations were conducted using TNF Alpha and VEGF antibodies. Results: Significant differences were observed in the Mean Resistant Corneal Vascularization Ratios between the 500 IU/g bakuchiol-loaded visco group and the control, visco, and steroid groups (p<0,05). Significant differences were also observed between the control group and the 250 IU/g bakuchiol-loaded visco group (p<0,05). The 500 IU/g bakuchiol-loaded visco group had the lowest corneal neovascularization ratio followed by the 250 IU/g bakuchiol-loaded visco group, steroid group, visco group, and control group, respectively. Significant differences were observed in the Corneal Opacity Score between the control group and the 500 IU/g bakuchiol-loaded visco group (p<0,05). The 500 IU/g bakuchiol-loaded visco group had the lowest corneal opacity average followed by the 250 IU/g bakuchiol loaded visco group, steroid group-visco group, and control group, respectively. Significant differences were observed in the "3rd Day Corneal Epithelial Defect Percentage" averages among groups (p†=0.004). The highest epithelial defect was observed in the steroid group on the 3rd day, followed by the 250 IU/g bakuchiol loaded visco group and the control group. The 500 IU/g bakuchiol-loaded visco group and the visco group showed completely closed epithelial defects on the 3rd day. The Corneal Epithelial Defect Percentage was zero in all groups on days 7 and 14. Conclusion: Bakuchiol demonstrated a dose-dependent increasing effect on reducing corneal vascularization, preventing corneal scar formation, and reducing corneal hemorrhage and stromal edema in corneal alkali injuries. It was more effective than steroids, a part of classical treatment. Our data suggest that hydrogel loaded bakuchiol has more effective anti-TNF alpha and anti-VEGF effects than topical steroids. Both the defined organ-protective effects and anti-inflammatory effects of bakuchiol might have activated the mechanisms leading to these positive results. Further detailed studies at the molecular level are required to fully understand the mechanism. We believe bakuchiol could be an effective treatment method for corneal chemical injuries. Advanced preclinical and clinical studies are needed for dose adjustment, application timing, and application technique. Keywords: Bakuchiol, corneal alkali injury, VEGF, TNF Alpha, hydrogel