Salubrinal Ameliorates Inflammation and Neovascularization via the Caspase 3/Enos Signaling in an Alkaline-Induced Rat Corneal Neovascularization Model

Abstract

Background and Objectives: Ocular alkaline burn is a clinical emergency that can cause permanent vision loss due to limbal stem cell deficiency and corneal neovascularization (CNV). Although the basic pathogenetic mechanisms are considered to be acute oxidative stress and corneal neovascularization triggered by inflammation, the underlying intracellular mechanisms have not been clearly elucidated. The aim of this study was to investigate the role of endoplasmic reticulum (ER) stress on inflammation and neovascularization, and the effect of the ER stress inhibitor salubrinal (SLB), as a novel treatment in a corneal alkaline burn model in rats. Methods: Chemical burns were created by cautery for 4 s using a rod coated with 75% silver nitrate and 25% potassium nitrate in the corneal center for the corneal neovascularization (CNV) model. Twenty-eight Wistar albino rats were divided into four groups: SHAM, CNV, CNV + SLB, and CNV + bevacizumab (BVC). After the CNV model was applied to the right eye, a single subconjunctival dose (0.05 mL) of 1 mg/kg salubrinal was injected into both eyes in the CNV + SLB group. A total of 1.25 mg/mL of subconjunctival BVC was administered to the CNV + BVC group. Fourteen days after experimental modeling and drug administration, half of the globes were placed in liquid nitrogen and stored at -20 °C until biochemical analysis. The remaining tissues were collected and fixed in 10% buffered formalin for histopathological and immunohistochemical analysis. Three qualitative agents from three different pathways were chosen: TNFR for inflammation, endothelial nitric oxide synthase (e-NOS) for vascular endothelial growth factor (VEGF)-mediated vascular permeability, and caspase-3 for cellular apoptosis. Results: Significantly lower caspase-3 and eNOS levels were detected in the CNV + SLB and CNV + BVC groups than in the CNV group. Additionally, histopathological evaluation revealed a significant decrease in neovascularization, inflammatory cell infiltration, and fibroblast activity in the CNV + SLB and CNV + BVC groups. The endoplasmic reticulum stress inhibitor, salubrinal, administered to the treatment group, attenuated apoptosis (caspase-3) and inflammation (e-NOS). In the control group (left eyes of the SLB group), salubrinal did not have a toxic effect on the healthy corneas. Conclusion: The ER stress pathway plays an important role in angiogenesis after alkaline corneal burns, and treatment with SLB modulates this pathway, reducing caspase-3 and eNOS levels. Further studies are needed to understand the molecular mechanisms altered by SLB-mediated therapy. The fact that more than one mechanism plays a role in the pathogenesis of CNV may require the use of more than one molecule in treatment. SLB has the potential to affect multiple steps in CNV pathogenesis, both in terms of reducing ER stress and regulating cellular homeostasis by inhibiting the core event of integrated stress response (ISR). Therefore, it can be used as a new treatment option and as a strengthening agent for existing treatments. Although blockade of intracellular organelle stress pathways has shown promising results in experimental studies, more in-depth research is needed before it can be used in routine practice. To the best of our knowledge, this study is the first to report the role of ER stress in corneal injury.

Keywords: apoptosis; corneal injury; eNOS; endoplasmic reticulum stress; inflammation.

Figure 1

ER stress-mediated apoptosis pathway under corneal injury. Under hypoxic state, PERK involve in apoptosis via eIF2α. The hallmark event is the progressive accumulation of misfolded protein aggregates. Apoptosis is a genetically programmed process aimed at eliminating damaged cells by activation of caspases. The process is activated either by TNFR stimulus extracellularly or intracellularly mainly by nonreceptor stimuli such as DNA damage, ER stress, metabolic stress, UV radiation, or growth factor deprivation. Autophagy, on the other hand, is a cellular catabolic pathway involving protein degradation, organelle cycling, and nonselective degradation of cytoplasmic components.

Figure 2

Rat corneal sections. (A,C,E,G) Normal corneal histology in CNV-L, SHM-L, CNV + B-L, and CNV + S-L groups, respectively. (B,D) Extensive and intense neovascularization, significant inflammatory cell infiltrations, and increased fibroblast activity in the subepithelial area in CNV-R and SHM-R groups. (F,H) A significant decrease in histopathological findings in CNV + B-R and CNV + S-R groups. (H,E) Staining, ×40, scale bar = 50 µm (arrows show inflammatory cell infiltrations, asterisk shows neovascularization areas).

Figure 3

Immunohistochemical stainings. No immunoreactivity was observed in CNV-L (A), SHM-L (E), CNV + B-L (I), or CNV + S-L (M) groups with caspase-3, e-NOS, and TNFR. Increased caspase-3 immunoreactivity in CNV-R (B) and SHM-R (F) groups and negative capase-3 immunoreactivity in CNV + B-R (J) and CNV + S-R (N) groups. A slight increase in e-NOS immunoreactivity in CNV-R (C) and SHM-R (G) groups and negative e-NOS immunoreactivity in CNV + B-R (K) and CNV + S-R (O) groups. Negative immunoreactivity with TNFR in CNV-R (D), SHM-r (H), CNV + B-R (L), and CNV + S-R (P) groups. ×40, scale bar = 50 µm.