A mouse model of lamellar intrastromal femtosecond laser keratotomy: ultra-structural, inflammatory, and wound healing responses.

PURPOSE: The availability of knockout mouse species provide a highly versatile platform for critically examining the corneal wound healing response. We aimed to develop and characterize the wound healing response in a mouse model of intrastromal femtosecond laser (FSL) keratotomy. METHODS: An intrastromal lamellar dissection using a Visumax FSL was performed on 16 wild type mice (C57BL6) . The energy level was optimized at 150nJ. The FSL was programmed to perform a lamellar dissection at 50 µM depth without sidecut. The flap was not lifted. Fellow eyes were used as controls. Slit lamp photography and confocal microscopy were performed immediately before the mice were sacrificed 4 h, 1, 3, and 7 days post surgery. Corneas were harvested for immunocytochemistry, transmission electron microscopy (TEM) and light microscopy (LM). RESULTS: Confocal microscopy showed an absence of keratocytes in the area immediately surrounding the dissection plane. The dissection plane and individual FSL plasma cavitation bubbles were clearly evident on TEM. There was evidence of Keratocyte cell death along the laser resection plane on TEM. LM revealed the dissection plane at a 20 µM depth, although not all epithelial cell layers were intact. Staining for monocytes using antibodies for CD11b (cluster of differentiation 11b) showed early migration at the peripheries at 4 h that increased at 24 h and became more central in treated corneas (p<0.001). Apoptotic cells were evident on TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay in the immediate ablation zone and were significantly raised at 4 and 24 h (p<0.001). Ki67 (Kiel 67 protein) positive proliferating keratocytes are evident at 3 days and increased significantly by 7 days (p<0.001). Minimal fibroblast (cluster of differentiation 90, CD90) transformation was seen at 1 week. No myofibroblasts were detected. DISCUSSION: We have demonstrated that FSL lamellar cuts can be effectively performed on mice and that this model exhibits typical signs of the corneal wound healing response. This model could provide a ubiquitous platform in which to study corneal wound healing responses in both wild type and knockout mice species. The ability to create such a lamellar pocket may be utilizzd for intrastromal drug delivery.

Stromal interleukin-1 expression in the cornea after haze-associated injury.

The purpose of this study was to determine whether myofibroblasts or other cells in the stroma in the cornea produce interleukin (IL)-1alpha or IL-1beta that could modulate myofibroblast viability in corneas with haze after photorefractive keratectomy (PRK). Twenty-four female rabbits had haze-generating PRK for 9 diopters of myopia and were sacrificed at 1 week, 2 weeks, 3 weeks or 4 weeks after surgery. Corneal rims were removed, frozen in OCT at -80 degrees C, and analyzed by immunocytochemistry using primary antibodies to IL-1alpha, IL-1beta and alpha smooth muscle actin (SMA). Double immunostaining was performed for the co-localization of SMA with IL-1alpha or IL-1beta. Central dense haze and peripheral slight haze regions of each cornea were analyzed. SMA+ cells that expressed IL-1alpha protein were detected in both regions of the corneas at most time points following PRK. However, in the haze region at the 1, 3 and 4 week time points, significantly more (p<0.01) SMA+ cells did not express IL-1alpha. Also, in the haze region at all three time points, significantly more (p<0.01) SMA- cells than SMA+ cells expressed interleukin-1alpha protein. IL-1beta expression patterns in SMA+ and SMA- stromal cells was similar to that of IL-1alpha after PRK. Previous studies have demonstrated that IL-1alpha or IL-1beta triggers myofibroblast apoptosis in vitro, depending on the available concentration of apoptosis-suppressive TGFbeta. This study demonstrates that SMA- cells such as corneal fibroblasts, keratocytes, or inflammatory cells may produce IL-1alpha and/or IL-1beta that could act in paracrine fashion to regulate myofibroblast apoptosis--especially in the region where there is haze in the cornea after PRK was performed and SMA+ myofibroblasts are present at higher density. However, some SMA+ myofibroblasts themselves produce IL-1alpha and/or IL-1beta, suggesting that myofibroblast viability could also be regulated via autocrine mechanisms.

Molecular cloning, localization and circadian expression of chicken melanopsin (Opn4): differential regulation of expression in pineal and retinal cell types.

The avian retina and pineal gland contain autonomous circadian oscillators and photo-entrainment pathways, but the photopigment(s) that mediate entrainment have not been definitively identified. Melanopsin (Opn4) is a novel opsin involved in entrainment of circadian rhythms in mammals. Here, we report the cDNA cloning of chicken melanopsin and show its expression in retina, brain and pineal gland. Like the melanopsins identified in amphibians and mammals, chicken melanopsin is more similar to the invertebrate retinaldehyde-based photopigments than the retinaldehyde-based photopigments typically found in vertebrates. In retina, melanopsin mRNA is expressed in cells of all retinal layers. In pineal gland, expression was strong throughout the parenchyma of the gland. In brain, expression was observed in a few discrete nuclei, including the lateral septal area and medial preoptic nucleus. The retina and pineal gland showed distinct diurnal expression patterns. In pineal gland, melanopsin mRNA levels were highest at night at Zeitgeber time (ZT) 16. In contrast, transcript levels in the whole retina reached their highest levels in the early morning (ZT 0-4). Further analysis of melanopsin mRNA expression in retinal layers isolated by laser capture microdissection revealed different patterns in different layers. There was diurnal expression in all retinal layers except the ganglion cell layer, where heavy expression was localized to a small number of cells. Expression of melanopsin mRNA peaked during the daytime in the retinal pigment epithelium and inner nuclear layer but, like in the pineal, at night in the photoreceptors. Localization and regulation of melanopsin mRNA in the retina and pineal gland is consistent with the hypothesis that this novel photopigment plays a role in photic regulation of circadian function in these tissues.

Withania somnifera root extract in the regulation of lead-induced oxidative damage in male mouse.

The importance of Withania somnifera root extract in the regulation of lead toxicity with special reference to lipid peroxidative process has been investigated in liver and kidney tissues. While lead treatment (0.5 mg kg(-1)body wt. day(-1)for 20 days) enhanced hepatic and renal lipid peroxidation (LPO), administration of plant extract in the doses of 0.7 g kg(-1)and 1.4 g kg(-1)body wt. day(-1)along with equivalent doses of lead acetate for 20 days significantly decreased LPO and increased the activities of antioxidant enzymes, viz., superoxide dismutase (SOD) and catalase (CAT), thus retaining normal peroxidative status of the tissues. We suggest that the ameliorating role of root extract of W. somnifera in the lead intoxicated mice could be the result of its antiperoxidative action.

Possible involvement of lipid peroxidation in the inhibition of type I iodothyronine 5'-monodeiodinase activity by lead in chicken liver.

The possible involvement of lipid peroxidation (LPO) in the lead-induced inhibition of type I iodothyronine 5'-monodeiodinase activity (5'-D) has been worked out in chicken liver. Lead nitrate (1.5 mg per bird per day) for 30 days increased the lipid peroxidative process with a concomitant decrease in 5'-D activity in chicken liver. Also, a significant decrease in serum triiodothyronine (T3) concentration and an increase in serum thyroxine (T4) concentration were observed. The data suggest that lead-induced inhibition of type I 5'-D activity in chicken liver is mediated through the lipid peroxidative process.

Protective effects of vitamin E against lead-induced deterioration of membrane associated type-I iodothyronine 5'-monodeiodinase (5'D-I) activity in male mice.

The protective role of vitamin E (vit E) on lead-induced thyroid dysfunction with special reference to type-I iodothyronine 5'-monodeiodinase (5'D-I) activity in mice liver was investigated. Daily intraperitoneal (i.p.) injection of lead acetate (0.5 mg/kg body weight) for 30 days significantly decreased serum 3,3',5-triiodothyronine (T3) concentration and hepatic 5'D-I activity. Furthermore, lead significantly increased peroxidative reactions involving membrane components (lipid peroxidation, LPO) while the activities of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) were decreased in mouse liver. Simultaneous administration of vit E (5 mg/kg body weight) and 0.5 mg/kg body weight of lead restored thyroid function in mice by maintaining normal hepatic 5'D-I activity and serum thyroid hormone concentrations. It also prevented increase in LPO and inhibition of SOD and CAT activities in liver. We suggest that the intact membrane structure is a must for 5'D-I activity and the administration of vit E may prevent the lead induced thyroid dysfunction by maintaining membrane architecture.

Free radical mediated membrane perturbation and inhibition of type-I iodothyronine 5'-monodeiodinase activity by lead and cadmium in rat liver homogenate.

The possible involvement of lipid peroxidation (LPO) in the lead (Pb) and cadmium (Cd) induced thyroid dysfunction with special reference to type-I iodothyronine 5'-monodeiodinase (5'-D) activity was studied in rat liver homogenate. Peroxidative reactions involving membrane components were found to be markedly stimulated by chronic administration of Pb and Cd in rats. Metal induced inhibition in 5'-D activity was also observed. Since LPO is primarily an outcome of free radical generation, we suggest metal induced free radical mediated inhibition of 5'-D activity in rat liver homogenate. In addition, serum triiodothyronine (T3) and thyroxine (T4) concentrations were also decreased by metals.

Loss of membrane integrity and inhibition of type-I iodothyronine 5'-monodeiodinase activity by fenvalerate in female mouse.

The possible involvement of lipid peroxidation (LPO) in the fenvalerate-induced thyroid dysfunction with special reference to type I 5'-monodeiodinase (5'-D) activity has been worked out. Fenvalerate (40, 80 and 120 mg/kg body weight) enhanced LPO in biomembranes of liver and kidney leading to a decrease in membrane integrity. 5'-D activity and serum concentration of triiodothyronine (T3) were reduced by the highest dose. Serum thyroxine (T4) concentration was decreased in all the three fenvalerate-treated groups, indicating the sensitivity of thyroid gland to this pesticide. A marginal increase in T4 concentration in highest dose-treated group compared to that of the lower one supports the view that the monodeiodination of the phenolic ring of T4 is inhibited by fenvalerate. We suggest the possible inactivation of 5'-D by the generated free radicals in pesticide-treated animals.