In vivo behaviors of epithelial cells under tissue repair process are modulated by multiple factors. corneas, with reduction of malondialdehyde levels in the epithelial cells. Finally, we showed that a chemical scavenging reactive oxygen species reversed the impairment of attenuation of epithelial repair with a reduction of tissue levels of malondialdehyde. In conclusion, loss of tenascin X prolonged corneal epithelial wound healing and increased neutrophilic inflammatory response to debridement in mice. Tenascin X contributes to the control of neutrophil infiltration needed to support the regenerative response to injury and prevent the oxidative stress mediators from rising to cytotoxic levels. Interleukin, proliferating cell nuclear antigen, matrix metalloproteinase. Western blotting We then quantified the accumulation level of malondialdehjyde, a product related to ROS activity, in healing corneal epithelium of both genotypes of mice. Corneal epithelium of an uninjured cornea (Transforming Growth Factor , Myeloperoxidase, Glyceraldehyde-3-phosphate dehydrogenase. Effect of systemic neutrophil depletion of on epithelial debridement healing To examine if accelerated infiltration of neutrophils in tissue impairs healing of an epithelial defect in a KO cornea, neutrophils were systemically depleted by administration of a specific antibody. KO mice ( em n /em ?=?6 in each group) received rat anti-mouse Ly6G/Ly6C (Gr-1) antibody (50?mg/100?ml PBS, Bio X Cell, Lebanon, NH, USA) or rat anti-mouse IgG2b antibody as the control (50?mg/100?ml PBS, Bio X Cell, Lebanon, NH, USA) as Shikimic acid (Shikimate) previously Shikimic acid (Shikimate) reported . After 3 days a round epithelial defect was created in a central cornea of the KO and WT mice ( em n /em ?=?6) and allowed to heal. At 18?h post-debridement the remaining defect was stained with fluorescein green and photographed. The size of the defect was statistically analyzed as explained above. The mice were sacrificed and Giemsa staining examined blood samples to check neutrophil depletion. Immunohistochemistry evaluated malondialdehyde formation in eyes at 24?h post debridement. Epithelial healing in a KO mouse with systemic administration of N-Acetyl-L-cysteine (NAC) NAC was used to scavenge ROS in mice. Nos2 Twelve KO mice of 8-week-old with an epithelial defect was treated with either of NAC (Sigma Aldrich, St. Louis, MO, em i. p /em ., 200?mg/kg in 0.1?ml/10?g solution/ body weight, em n /em ?=?6) or saline ( em n /em ?=?6). Immediately after the treatment, debridement of corneal epithelium was produced as the way above mentioned in one eye of these 12 KO mice [33C35]. Six WT mice were also processed for epithelial debridement. At 18?h post-debridement the size of the remaining epithelial defect was evaluated in photographs of fluorescein-stained corneas. Then, the animals were killed and each vision was processed for immunohistochemistry for malondialdehyde. Results TNX expression pattern in cornea Immunostaining detected TNX expression in the peripheral stroma, but not in the central, cornea of an uninjured animal as previously reported (data not shown) . However, during the epithelial healing process TNX protein expression was obvious up to 36?h (Fig.?1). Stromal cells transiently upregulated TNX and they reached a peak at 24?h post-debridement (Fig.?1D). Open in a separate windows Fig. 1 Immunohistochemical detection of expression pattern of tenascin X protein in a mouse cornea.A Tenascin X was not detected in the epithelium and stroma of central cornea. Frames (BCE) shows immunohistochemical detection of tenascin X at 6, 12, 24 and 36?h, respectively. Healing epithelium upregulated expression of tenascin X protein Shikimic acid (Shikimate) up to 36?h post-debridement (arrows). Cells in the stroma (arrowheads) transiently upregulated tenascin X with the peak at 24?h post-debridement (D), and then declined the expression at 36?h (E). Bar, 100?m; epi epithelium, st stroma. Healing of an epithelial debridement A round corneal epithelial defect gradually waned after debridement and completely resurfaced itself at around 24?h in the WT mice. Reepithelialization was significantly delayed in a KO mouse from 12? h and up until 30?h. (Fig.?2a, b, Supplementary Fig.?1). Open in a separate windows Fig. 2 Healing of an epithelial debridement in a mouse cornea.a Green fluorescein staining detects an epithelial defect in each cornea. A round defect of 2.0?mm in diameter in corneal epithelium gradually became smaller and resurfaced around at 24?h in WT mice. The area of the remaining epithelial defect was indicated by the white dotted lines in Supplememtary Fig.?1. Reepithelialization was significantly delated in a KO mouse from at 12?h until 30?h post-epithelium debridement. b Statistical analysis detected statistical difference in the % remaining defect between two genotypes of mice at 12, 18, 24 and 30?h post-debridement. c Hematoxylin-eosin staining histology showed no obvious difference.