Insulin eye drops improve corneal wound healing in STZ-induced diabetic mice by regulating corneal inflammation and neuropeptide release

Abstract

Introduction: In recent years, insulin eye drops have attracted increasing attention from researchers and ophthalmologists. The aim of this study was to investigate the efficacy and possible mechanism of action of insulin eye drops in diabetic mice with corneal wounds.

Methods: A type 1 diabetes model was induced, and a corneal epithelial injury model of 2.5 mm was established. We used corneal fluorescein staining, hematoxylin-eosin (H-E) staining and the Cochet-Bonnet esthesiometer to examine the process of wound healing. Subsequently, the expression levels of Ki-67, IL-1β, β3-tubulin and neuropeptides, including substance P (SP) and calcitonin gene-related peptide (CGRP), were examined at 72 h after corneal injury.

Results: Fluorescein staining demonstrated an acceleration of the recovery of corneal epithelial injury in diabetic mice compared with the saline treatment, which was further evidenced by the overexpression of Ki-67. Moreover, 72 h of insulin application attenuated the expression of inflammatory cytokines and neutrophil infiltration. Remarkably, the results demonstrated that topical insulin treatment enhanced the density of corneal epithelial nerves, as well as neuropeptide SP and CGRP release, in the healing cornea via immunofluorescence staining.

Conclusions: Our results indicated that insulin eye drops may accelerate corneal wound healing and decrease inflammatory responses in diabetic mice by promoting nerve regeneration and increasing levels of neuropeptides SP and CGRP.

Keywords: Diabetic keratopathy; Inflammation; Insulin eye drops; Nerve regeneration; Neuropeptides.

Fig. 1

Insulin eye drops promote corneal wound healing and corneal epithelial regeneration. (A) Schematic illustration of the experimental protocol. (B)Representative images of corneal fluorescein staining of diabetic mice in DM-In group and DM-Con group after the removal of corneal epithelium at 0 h, 12 h, 24 h, 48 h and 72 h. (C) The percentage of corneal epithelial defect area at different time points and original wound area in two groups. (n = 11 in DM-In group; n = 14 in DM-Con group). Data are expressed as mean ± SD. (D) Representative immunofluorescence images of Ki67 (red, white arrows) costaining with DAPI (blue) in diabetic cornea between two groups. Scale bars: 20 μm. STZ: streptozotocin; DM: Diabetes mellitus; DM-In, diabetic mice with topical insulin treatment; DM-Con, diabetic mice with topical physiological saline treatment; Ep, corneal epithelium; St, corneal stroma

Fig. 2

Insulin eye drops attenuate inflammatory responses. (A) The typical histopathological changes of diabetic cornea by HE staining in DM-In group mice and DM-Con mice at 72 h (n = 3). Asterisk mark: lymphocytes; black dotted circle: segmented granulocytes. Scale bars: 20 μm. (B) Representative Il-1β (red) expression in corneal epithelium was examined by immunofluorescent techniques at 72 h (n = 3). In the sagittal frozen sections of corneas, Il-1β expression was found mostly within corneal epithelium. Scale bars: 20 μm. DM-In, diabetic mice with topical insulin treatment; DM-Con, diabetic mice with topical physiological saline treatment; Ep, corneal epithelium; St, corneal stroma

Fig. 3

Insulin eye drops improve corneal nerve regeneration. (A) The typical changes of regenerated nerve fibers in the cornea after the administration of insulin by using β3-tubulin (red), a pan-neuronal marker, immunofluorescence in the whole-mount cornea (n = 3). (B) Representative corneal cross-sections further show the locations and the regeneration (white arrows) of the central corneal epithelial nerve costaining with β3-tubulin (red) and DAPI (blue), (n = 3). (C) The length of corneal nerve fibers (µm/µm2) in the central and peripheral areas was calculated using Neuron J software. (D) The dynamic changes of corneal sensation (mm) in two groups at 0 h and 72 h. Data were exhibited as mean ± SD; *P < 0.05. Scale bars: 500 μm, 100 μm, and10µm. DM-In, diabetic mice with topical insulin treatment; DM-Con, diabetic mice with topical physiological saline treatment; Ep, corneal epithelium; St, corneal stroma

Fig. 4

Insulin eye drops enhance expression levels of the neuropeptides. (A) Representative images of immunofluorescence staining of the whole cornea labeled with CGRP (red), SP (green) and DAPI (blue) (n = 3). The intensity of neurotrophic factors in the whole-mount cornea were quantified by Image J software. Data were exhibited as average ± SD; *P < 0.05. (B) and (C) are representative images of the corneal sections, showing the positions of CGRP (red) and SP (green), as indicated by the white arrows. Scale bars: 500 μm, 100 μm, and10µm. DM-In, diabetic mice with topical insulin treatment; DM-Con, diabetic mice with topical physiological saline treatment; Ep, corneal epithelium; St, corneal stroma