Wound healing of corneal epithelium in mice affected by reversed circadian rhythms

Objective To observe the effect of reversed circadian rhythms on wound healing of mouse corneal epithelium. Methods Ninety male C57BL/6 mice were divided into LD group (12 hours light/12 hours dark) and DL group (12 hours dark/12 hours light) by random number table, and then were placed in circadian rhythm box for 12 days.The circular area was scarped and marked as 2 mm diameter area in the center of the mouse's cornea with a golf-like knife.The dynamics of epithelial healing in the wound area were observed under microscope by fluorescein staining and hematoxylin-eosin staining.Besides, being marked antibodies of anti-Ly6G-FITC, anti-γδT-PE and DAPI, dynamic changes of the dividing cells, neutrophils and γδT cells were also investigated for every 6 hours until 42 hours.All mice were treated in accordance with the Association for Research in Vision and Ophthalmology's Statement for the Use of Animals in Ophthalmology and Vision Research and the guidelines of the Animal Experimental Committee at Jinan University (JN-A-2002-01). Results In LD group, percentage of corneal epithelial defective area were (100.000±0.000)%, (37.677±5.243)%, (14.959±1.739)% and (0.000±0.000)% after wounding 0 hour, 6, 12, 18 and 24 hours.In DL group, percentage of the corneal epithelial defective area were (100.000±0.000)%, (10.967±1.065%)%, (1.985±0.106)% and (0.000±0.000)% after wounding 0 hour, 6, 12, 18 and 24 hours.The healing rate in DL group was higher than that in LD group, with a significant difference between them (P 0.05). After 24 hours, thickness of corneal epithelium in DL group was (19.473±0.856)μm, and was more than that in LD group [(17.485±0.718)μm], with a significant difference between them (P<0.05). Paraffin section of wounded corneal epithelium after 24 hours by hematoxylin and eosin staining showed that corneal epithelium cells arranged loosely and disorderly and were in irregular shape in both groups.The epithelium were mainly basal cells in LD group, while epithelium included basal cell and a few pinacocytes in DL group.After corneal epithelium wounded, the number of cell division, neutrophils and corneal limbus γδT cells in two groups were statistically significant difference, respectively(P<0.05). Conclusions Reversed circadian rhythms can significantly regulate the wound healing of corneal epithelium. Key words: Cornea; Reversed circadian rhythms; Wound healing; Inflammation