| Purpose To investigate the characteristics of reconstituted tight junctions after corneal epithelial wounds and ultrastructure alterations of corneas in a high-fat diet combined with streptozocin(STZ)-induced rat model of type 2 diabetes mellitus(DM).Materials and Methods One-hundred Sprague-Dawley male rats were randomly divided into diabetic and normal control groups(n = 50 each). A high-fat diet combined with intraperitoneal STZ injection were used to induce type 2 DM. Wound healing was determined using an in vivo corneal epithelial debridement model. Scanning electron microscopy(SEM) was used to study differences in corneal epithelium between diabetic and normal rats. The rate of corneal wound healing was observed by fluorescein and hematoxylin and eosin staining. Occludin expression and distribution were assessed by Western blot and immunofluorescence. Diabetic and healthy rats were sacrificed before debridement and 16, 48, 72, and 120 hours afterwards for Western blot and immunofluorescence.Results 1.The average NL rat bodyweight(370 g) was 7.5% lower than DM rats(400 g) at 4 weeks(before STZ injection; P < 0.01), but the DM rats(410 g) weighed 19.6% less than NL rats(510 g) at 16 weeks(p < 0.01). The fasting blood-glucose(FGB) levels were approximately 5 mmol/L in normal rats and 6 mmol/L in DM rats at 4 weeks(before STZ injection)(P < 0.05). At 16 weeks, FGB levels were approximately 20 mmol/L in DM rats while they were still 5 mmol/L in the normal rats(P < 0.01).The DM rats also exhibited adipose development and muscle tissue wasting, polyuria, polydypsia, and some exhibited diarrhea and intestinal bloating.2.We made a 4-mm wound in the center of cornea, examined the healing progress with fluorescein staining to the denuded area and photographed. The healing rate was calculated and is shown in. The normal rats exhibited 55% and 84% healing at 16 and 24 hours post-wound(hpw), while the DM rats healed 34% and 56% at the same time, respectively. Corneal epithelial wound healing was thus significantly delayed in DM rats compared with healing in normal rats(P < 0.01).3.In the DM group, a number of surface cells were exfoliating. Abnormal intercellular connections, fissures between cells, decrease in the number of microvilli, and dropsy were all seen on SEM. In the NL group, cell surfaces showed normal morphology; the microvillus of corneal epithelial cells were normal and no dropsy were seen under SEM.4.In the NL group, before debridement, corneal multilayer epithelial cells were observed. At 16 to 24 hours after debridement, a monolayer or multilayers of epithelium covered the periphery of the denuded surface and by 48 hours re-epithelialization was complete. In the DM group, corneal epithelial basal cells and the hypothallus were swollen. At 16 hours after debridement, the denuded surface was not yet covered. At 24 to 48 hours after debridement, these corneas showed fewer multilayers of epithelium covering the denuded surface compared to normal rats.5.The expression of occludin was detected with Western blot analysis. The expression levels of occludin between the unwounded NL and DM groups were not different(P > 0.05). Occludin levels of the DM group were significantly lower than the NL group at 16 to 48 hours after debridement(P < 0.05), and the occludin levels of the two groups were similar at 72 and 120 hours(P > 0.05).Conclusions Abnormal occludin expression may contribute to delayed epithelial wound healing in diabetic corneas. |
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