Delay of corneal epithelial wound healing and induction of keratocyte apoptosis by platelet-activating factor

PURPOSE: To examine the role of the lipid mediator platelet-activating factor (PAF) in epithelial wound healing. METHODS: A 7-mm central de-epithelializing wound was produced in rabbit corneas, and the tissue was incubated with 125 nM carbamyl PAF (cPAF), an analogue of PAF. Rabbit corneal epithelial and stromal cells were also cultured in the presence of cPAF. Cell adhesion, proliferation, and migration assays were conducted. Apoptosis was assayed by TUNEL staining on preparations of corneal tissue sections and in cells in culture. RESULTS: Twenty-four hours after injury, 50% of the wounded area was covered by new epithelium, whereas only 30% was covered in the presence of cPAF. At 48 hours, the epithelium completely closed the wound, but only 45% of the original wound was covered in corneas treated with cPAF. Similar inhibition of epithelial wound closure was found with human corneas incubated with PAF in organ culture. Moreover, addition of several growth factors involved in corneal wound healing, such as epidermal growth factor, hepatocyte growth factor, and keratinocyte growth factor, could not overcome the inhibitory action of PAF in wound closure. Three PAF antagonists, BN50727, BN50730, and BN50739, abolished the effect of PAF. A significant increase in TUNEL-positive staining occurred in corneal stromal cells (keratocytes), which was inhibited by preincubating the corneas with PAF antagonists. However, no TUNEL-positive staining was found in epithelial cells. TUNEL-staining results in cultured stromal cells (keratocytes) and epithelial cells in first-passage cell culture were similar to those in organ-cultured corneas. In addition, PAF caused 35% to 56% inhibition of adhesion of epithelial cells to proteins of the extracellular matrix: collagen I and IV, fibronectin, and laminin. There were no significant changes in proliferation or migration of epithelial cells induced by the lipid mediator. CONCLUSIONS: The results suggest PAF plays an important role in preventing corneal wound healing by affecting adhesion of epithelial cells and increasing apoptosis in stromal cells. PAF antagonists could be of therapeutic importance during prolonged ocular inflammation, helping to avoid loss of corneal transparency and visual acuity.     

Initial characterization of whole-cell currents from freshly dissociated corneal keratocytes.

The perforated patch technique was utilized to obtain whole-cell currents from freshly dissociated rabbit corneal keratocytes. We describe and provide the initial characterization of two distinct whole cell currents in rabbit keratocytes: a K(+)-selective delayed rectifier and a voltage-sensitive, tetrodotoxin blockable Na+ current. The voltage-sensitive Na+ current is of sufficient magnitude to allow us to initiate action potentials when current-clamping the cells. This is the first detailed electrophysiological study of corneal keratocytes.     

Increased apoptosis and abnormal wound-healing responses in the heterozygous Pax6+/- mouse cornea

PURPOSE: Corneal wound healing involves a cascade of interactions between the epithelium and stroma. Pax6 is upregulated, and early events include epithelial cell migration and apoptosis of superficial keratocytes. The mouse heterozygous Pax6 (Pax6+/-) corneal phenotype mimics human aniridia-related keratopathy (ARK), and some aspects of wound healing have been shown to be abnormal, including matrix metalloproteinase (MMP)-9 expression. The purpose of this study was to test whether the Pax6+/- genotype affects corneal wound-healing responses, including stromal cell apoptosis, epithelial cell migration rate, and MMP secretion in culture. METHOD: Pax6+/- and wild-type (Pax6+/+) mice were killed and their corneas wounded by epithelial debridement. Whole eyes were cultured in organ culture and corneal epithelial healing rates and keratocyte apoptosis were quantified by topical fluorescein staining and TUNEL, respectively. Dissociated corneal epithelial cells from Pax6+/- and wild-type mice were cultured, and the activities of secreted MMP-9 were determined by zymography. RESULTS: Wound-healing rates during the first 6 hours were significantly faster for larger wounds and for Pax6+/- corneas. Compared with wild-type, wounded Pax6+/- eyes showed significantly more stromal cell apoptosis, and cultured Pax6+/- corneal epithelial cells produced lower MMP-9 activity. CONCLUSIONS: The cumulative effect of abnormal wound-healing responses, characterized by increased stromal cell apoptosis and reduced levels of MMP-9 secretion may contribute to the corneal changes in the Pax6+/- mice. Possible contributions of elevated stromal cell apoptosis and other abnormal wound-healing responses to ARK are discussed.     

Expression of basic fibroblast growth factor in rabbit corneal alkali wounds in the presence and absence of granulocytes

PURPOSE: To study the expression of basic fibroblast growth factor (bFGF) in the early phases of corneal wound healing in the presence or absence of granulocytes. METHODS: A central penetrating corneal alkali wound was inflicted to one eye in each of 14 rabbits under general anaesthesia. Subsequently, seven of the rabbits were given fucoidin i.v. for 36 hours in order to block the selectins on the vascular endothelium, thus preventing blood granulocytes from entering the tissues. Then, corneas were prepared, stained for bFGF and evaluated by light microscopy. RESULTS: Whereas normal corneal epithelium expressed bFGF weakly, conjunctival epithelium did so strongly, particularly the goblet cells. The corneal endothelium showed medium staining, while keratocytes and vascular endothelial cells did not consistently express bFGF. After 36 hours of wound healing, a marked up-regulation of bFGF expression was observed in the corneal epithelial and endothelial cells, as well as in the keratocytes, that were migrating into the wound. No other changes were noted. None of these features were modulated when granulocyte emigration was prevented by fucoidin administration. CONCLUSIONS: The difference in bFGF expression between the corneal and conjunctival epithelium suggests a role for this growth factor in the barrier function at the limbus. Moreover, the specific presence of bFGF in cells migrating into the wound indicates the participation of bFGF in corneal wound healing. Expression of bFGF was independent of granulocytes.     

Expression of HGF, KGF, EGF and receptor messenger RNAs following corneal epithelial wounding

Hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), epidermal growth factor (EGF), and their receptors have been associated with homeostasis and wound healing in the cornea. The purpose of this study was to examine the expression of the messenger RNAs for these growth factors and receptors in a wounded series of mouse corneas using in situ hybridization. In situ hybridization was performed with 3H-labeled riboprobes on unwounded corneas and corneas at 30 minutes, 4, 12, 24, 48 and 72 hr, and 7 days after epithelial scrape wounds in Balb/C mice. Qualitative and semi-quantitative analyses were performed. Expression of HGF, KGF and EGF mRNAs in keratocytes in the unwounded cornea was low. EGF mRNA was also expressed in unwounded corneal epithelium. Following wounding, however, these growth factor mRNAs were markedly upregulated in keratocytes. EGF mRNA expression in the epithelium appeared unaffected by wounding. At seven days after wounding and several days following closure of the epithelial defect, HGF mRNA and KGF mRNA were still expressed at higher levels in keratocytes compared with unwounded corneas. No difference in expression of HGF or KGF mRNAs between limbal, peripheral corneal, or central corneal keratocytes was noted in the unwounded cornea, KGF receptor mRNA was prominently expressed throughout the unwounded corneal epithelium. HGF receptor mRNA and EGF receptor mRNAs were expressed at low levels in unwounded cornea epithelium. Following scrape injury, expression of HGF receptor mRNA and KGF receptor mRNA were markedly upregulated in the corneal epithelium, while no significant increase in EGF receptor mRNA expression was noted. These studies suggest a prominent role for HGF and KGF in modulating corneal epithelial wound healing following injury. Less prominent changes in EGF mRNA and EGF receptor mRNA in the corneal epithelium following wounding may suggest that EGF has more of a role in homeostasis in the mouse corneal epithelium.     

Corneal epithelial wound healing increases the expression but not long lasting activation of the p85alpha subunit of phosphatidylinositol-3 kinase.

PURPOSE: Corneal epithelial wound healing is a complex process involving several growth factors whose interaction with tyrosine kinase receptors (RTK) leads to the recruitment of enzymes coupled to second messengers that propagate and amplify growth factor-induced signals inside the cells. Phosphatidylinositol-3 kinase (PI-3K) is one such enzyme. Here we have investigated changes in PI-3K activity and expression during re-epithelialization after in vivo and in vitro corneal injury. METHODS: For the in vivo model, epithelium was collected from rabbit corneas at different stages of wound healing after complete de-epithelialization. For in vitro studies, after 7 mm central scrape wounds were applied, rabbit corneas were maintained in organ culture. Immunoprecipitation and Western blot using anti-p85alpha antibodies were employed to determine PI-3K activity and expression of the p85alpha regulatory subunit of PI-3K. Two specific PI-3K inhibitors, Wortmannin and LY 294002 were used to study the effect of PI-3K activity on corneal epithelial wound healing. RESULTS: Two to four days after in vivo corneal epithelial wound healing, there was a 6-8 fold increase in the expression of the p85alpha subunit of PI-3K. By 8 days, the expression of p85alpha was similar to non-injured tissue. Increased expression of the 85kDa protein was observed mainly in the membrane fraction. Similarly, the expression of PI-3K was increased 24h after injured corneas were maintained in organ culture. Increase of p85alpha was confined to the wound region and surrounding area. No concomitant increase in PI-3K activity was observed in any of the wound models. Forty-eight hours after the central injury, Wortmannin and LY294002 inhibited wound healing by about 50%. CONCLUSIONS: Association of most of the increased p85alpha with the membrane fraction and no detectable increase in PI-3K activity during corneal re-epithelialization indicates that PI-3K activation is transitory. The results also suggest a mechanism of down regulation of the enzyme to avoid uncontrollable growth and cellular hypertrophy after growth factor stimulation during wound healing.     

Role of p38 MAP kinase in regulation of cell migration and proliferation in healing corneal epithelium

PURPOSE: The purpose of the present study was to examine the roles of signaling pathways potentially activated by TGFbeta (i.e., Smad and p38 mitogen-activated kinase [MAPK]) in regulation of cell migration and proliferation of healing mouse corneal epithelium. METHODS: Activation of Smads or p38MAPK was evaluated by immunohistochemistry in healing mouse corneal epithelium after debridement. The role of endogenous TGFbeta or p38MAPK in epithelial healing was determined in organ-cultured mouse corneas with an epithelial defect, in the presence or absence of a TGFbeta-neutralizing antibody or p38MAPK inhibitors, respectively. Cell proliferation was evaluated by incorporation of bromodeoxyuridine. RESULTS: Migrating mouse corneal epithelium had minimal cell proliferation. Smad3 and -4 were found in nuclei of normal corneal epithelium, whereas they were absent in nuclei of migrating cells in association with Smad7 upregulation on epithelial debridement. Administration of TGFbeta-neutralizing antibody reduced the protein expression of Smad7 in vivo after a corneal injury. In contrast, phosphorylation and nuclear translocation of p38MAPK were markedly evident in migrating epithelium during healing, but not in uninjured epithelium. In organ culture, addition of p38MAPK inhibitors blocked cell migration more markedly than neutralizing TGFbeta-antibody and enhanced cell proliferation in the injured corneal epithelium, in association with phosphorylation of Erk. CONCLUSIONS: Endogenous TGFbeta enhances migration of corneal epithelium during wound healing in mice. The p38MAPK, but not the Smad, cascade plays a major role in promoting cell migration and in suppressing cell proliferation in migrating epithelium.     

Expression of K12 keratin in alkali-burned rabbit corneas.

The healing of alkali-injured corneas is characterized by the persistence of polymorphonuclear leukocytes (PMN) in tissues and recurrent corneal epithelial defects. It has been suggested that the proteolytic enzymes secreted by PMN may account in part for the recurrent epithelial defects in the alkali-burned corneas. Cytoplasmic keratins, which form intracellular intermediate filaments, participate in the formation of hemidesmosomes and play a key role in the focal adhesion of epithelial cells to the basement membranes. The K3/K12 keratin pair is a major constituent of differentiated and stratified corneal epithelium. We have recently cloned the cDNA encoding the rabbit K12 keratin. In the present study we examined the expression of K12 keratin during the healing of alkali-burned rabbit corneas by slot-blot and in situ hybridization. Our results indicate that in normal cornea K12 keratin is equally expressed in all cell layers of stratified corneal epithelium and suprabasal layers of limbal epithelium, but not in bulbar conjunctival and other epithelia, i.e., lens, iris, and retinal pigment epithelium. The basal cells of the detached regenerating epithelium of the injured cornea express a very low level of K12 keratin. These observations are consistent with the notion that defective expression of K3/K12 keratins may play a role in the abnormal attachment of the regenerating epithelium to the basement membrane.     

Two types of K+ currents modulated by arachidonic acid in bovine corneal epithelial cells.

PURPOSE. Fenamate sensitivity of the large-conductance K+ current in the corneal epithelium suggests that K+ transport could be modulated by arachidonic acid (AA) and/or its metabolites, which also regulate corneal epithelial migration. The main purpose of this study was to investigate AA-induced modulation of K+ currents expressed in the bovine corneal epithelium. METHODS. Freshly isolated bovine corneal epithelial cells were perfused with Ringer solution. Whole-cell currents were recorded by using either the conventional whole-cell-patch or the perforated-patch configuration. RESULTS. Two distinct types of K+ currents dominated the whole-cell current. The first was a voltage-gated K+ current that was inactivated completely by membrane depolarization. The inactivating voltage-gated K+ current was largest in presumptive basal cells. The second was a noisy, sustained K+ current that was never inactivated and seemed to be a counterpart of the large-conductance K+ current reported in the rabbit corneal epithelium. External application of AA (5-20 microm) inhibited the inactivating voltage-gated K+ current and augmented the noisy, sustained K+ current. Identical dual modulation was induced by other fatty acids (e.g., palmitoleic acid) that are not substrates for enzymes in the AA cascade. CONCLUSIONS. An inactivating voltage-gated K+ channel was identified for the first time in the corneal epithelium. AA and some fatty acids may directly activate the large-conductance K+ channel to augment its housekeeping functions in corneal epithelial cells.     

Receptor-mediated activation of a Cl(-) current by LPA and S1P in cultured corneal keratocytes

PURPOSE: This study was designed to examine the effects of lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) on Cl(-) currents (ICl(LPA)) in cultured corneal keratocytes isolated from the corneas of New Zealand White rabbits. METHODS: ICl(LPA) and resting voltages were recorded with the amphotericin perforated-patch technique. Phenotype was determined with antibodies to alpha-smooth muscle actin. RESULTS: Keratocytes cultured in serum have a phenotype (myofibroblast) and ionic currents similar to those of keratocytes isolated directly from corneas during wound healing. LPA and S1P both activated ICl(LPA) in a dose-dependent manner, and the LPA receptor-specific antagonist dioctyl-glycerol pyrophosphate (DGPP) blocked the LPA response, but not the S1P response. In addition, a relatively inactive form of LPA (LPA 8:0) was relatively ineffective in activating ICl(LPA). Activation of ICl(LPA) significantly depolarized the cells, and this depolarization was reversed by blocking ICl(LPA) with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) or 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). CONCLUSIONS: These results demonstrate that activation of ICl(LPA) by LPA in cultured corneal keratocytes is receptor mediated and that ICl(LPA) can also be activated by S1P. From a functional standpoint, this work confirms that the current, which is typically thought of as purely volume-activated, can be activated through a receptor. In addition, activation of ICl(LPA) results in depolarization of the keratocyte. Finally, this work demonstrates that cultured corneal keratocytes can act as a model for the study of ion channel function in keratocytes during corneal wound healing.     

RANK, RANKL, OPG, and M-CSF expression in stromal cells during corneal wound healing

PURPOSE: To examine the influx of monocytes into the cornea after epithelial scrape injury and the expression of chemokines that potentially regulate monocyte phenotype in cultured corneal fibroblasts and keratocytes in situ. METHODS: Monocytes were detected by immunocytochemistry for the monocyte-specific antigen CD11b, in unwounded and epithelial scrape-wounded mouse corneas. The receptor activator of NF-kappa B ligand (RANKL), osteoprotegerin (OPG), and monocyte chemotactic and stimulating factor (M-CSF) mRNAs were detected in cultured mouse stromal fibroblasts by RT-PCR and RNase protection assay. RANKL, OPG, and M-CSF proteins were detected in cultured mouse stromal fibroblasts by immunoprecipitation and Western blot analysis. RANKL, RANK, M-CSF, and OPG proteins were detected in unwounded and wounded mouse corneas by immunocytochemistry. Chimeric mice with green fluorescent protein-labeled bone marrow-derived cells underwent corneal scrape injury and were monitored by fluorescence microscopy and immunocytochemistry. RESULTS: A small number of cells expressing the monocyte-specific CD11b antigen were detected in the stromas of unwounded mouse corneas. A larger number of CD11b-positive cells was detected in the stroma at 24 or 48 hours after epithelial scraping injury. Experiments with chimeric mice with fluorescent green protein-labeled, bone marrow-derived cells demonstrated conclusively the origin of these CD11b(+) cells. RANKL, OPG, and M-CSF mRNAs and proteins were detected in cultured mouse stromal fibroblasts. RANKL, M-CSF, and OPG proteins were detected in unwounded corneas, but were expressed at higher levels in stromal cells during the 24- to 48-hour interval after epithelial scrape injury. RANK was detected in stromal cells presumed to be monocytes at 24 and 48 hours after epithelial injury. CONCLUSIONS: Cells expressing the CD11b monocyte-specific antigen appear in the corneal stroma in high numbers by 24 hours after epithelial injury and persist beyond 10 days after wounding. Cultured corneal fibroblasts and keratocytes in situ express RANKL, OPG, and M-CSF cytokines involved in regulating osteoclast differentiation from monocytes in bone. Cells expressing RANK were detected in the stroma at 24 and 48 hours after epithelial injury. The cytokine systems that regulate monocyte transition to osteoclast in bone are upregulated in the cornea in response to epithelial injury and may participate in regulating monocyte phenotype during corneal stromal wound healing.     

Keratocyte activation and apoptosis in transplanted human corneas in a xenograft model

PURPOSE: To study keratocyte activation and cellular apoptosis in transplanted human corneas during the early postoperative period. METHODS: Ten human donor corneas preserved for 6 days at 4 degrees C were transplanted into the eyes of 10 adult cats. After confocal and specular microscopy in vivo 1 week after keratoplasty, the cats were killed, and the fixed corneas were examined by TUNEL assay and by scanning (SEM) and transmission electron microscopy (TEM). RESULTS: Abnormal keratocytes, in which portions of cell bodies and processes as well as nuclei were visible, were present in all corneas and occupied the anterior 16 to 562 microm of the stroma. By TEM in the same corneas, these abnormalities represented keratocytes that were activated to a repair phenotype. Only 0% to 1% of all corneal cells were apoptotic by TUNEL assay, except for the donor keratocytes near the wound, where 7% were apoptotic. The midstromal keratocyte density was decreased at 13,936 +/- 5,910 cells/mm(3) (mean +/- SD), and the endothelial cell density was 2,298 +/- 688 cells/mm(2), representing an endothelial cell loss of 7% +/- 16%. CONCLUSIONS: Substantial keratocyte activation and low levels of cellular apoptosis occur 1 week after human corneal transplantation. The human-to-cat xenograft model of corneal transplantation demonstrated endothelial cell loss and other clinical findings similar to human allografts. The model will be useful for preclinical testing of new methods of long-term corneal preservation and of donor endothelial cell augmentation, as well as the study of human corneal wound healing and keratocyte replacement during the early postoperative period.     

Delayed wound closure and phenotypic changes in corneal epithelium of the spontaneously diabetic Goto-Kakizaki rat

PURPOSE: To characterize wound closure and phenotypic changes in the corneal epithelium of the Goto-Kakizaki (GK) rat, a spontaneous model of type 2 diabetes. METHODS: Corneal wound healing was monitored by fluorescein staining after epithelial debridement. Tear secretion was measured with the Schirmer test, and corneal sensation was evaluated with an esthesiometer in 13- to 15-week-old GK and Wistar (control) rats. The distributions of cytokeratin 12 (K12), K14, and connexin43 in the corneal epithelium were examined by immunohistofluorescence analysis. The proliferation capacity of epithelial cells in the intact cornea and during wound healing was evaluated by immunostaining for Ki-67. RESULTS: Tear secretion, corneal sensation, and corneal epithelial wound closure rate were all decreased in GK rats compared with those in Wistar rats. Whereas connexin43, K14, and Ki-67 were all restricted to the single layer of basal cells in the corneal epithelium of Wistar rats, they were detected in the two layers of cells closest to the basement membrane in that of GK rats. The frequency of Ki-67-positive cells in the intact corneal epithelium was greater in GK rats than in Wistar rats, and it was increased to a greater extent in the peripheral cornea of GK rats than in that of Wistar rats during wound healing. CONCLUSIONS: Spontaneously diabetic GK rats manifest characteristics similar to those of diabetic keratopathy in humans, including delayed wound closure, and they exhibit phenotypic changes in corneal epithelial cells.     

Wound healing following anterior keratectomy and lamellar keratoplasty in the pig

PURPOSE: To examine corneal wound healing in an animal model of two types of mechanical lamellar keratectomy. METHODS: One eye from each of 28 pigs was studied. Using a motorized keratome, corneas were subjected to an anterior lamellar keratectomy with removal of anterior stroma and epithelium, or to automated lamellar keratoplasty (ALK) with reapposition of a corneal flap. The exposed stromal surfaces were labeled intraoperatively with a fluorescent dye (DTAF) to assess deposition of stromal components during subsequent wound healing. Examination before surgery and enucleation included measurement of corneal curvature and intraocular pressure, and assessment of corneal haze. Eyes were prepared for histological examination, fluorescence microscopy, and for fibronectin immunohistochemistry. RESULTS: Both keratectomy procedures produced flattening of corneas by up to 3.80 diopters, 28 days after surgery. Corneal haze was more pronounced in eyes from which epithelium was removed (anterior lamellar keratectomy group). The increased haze in this group was associated histologically with appearance of many reactive keratocytes and inflammatory cells, deposition of new stromal material, and more widespread appearance of fibronectin immunoreactivity. In the lamellar keratoplasty group, only the edges of the corneal wound showed significant reactivity, and included keratocyte activation and epithelial ingrowth. CONCLUSIONS: The pig provides a useful model for studies of refractive surgical techniques using procedures and instruments designed for use in humans. Mechanized keratectomy procedures that minimize disruption of the epithelium and Bowman's layer produce a less reactive corneal wound than procedures in which an expanse of epithelium and anterior stroma are removed.     

Identification of a Kv3.4 channel in corneal epithelial cells

PURPOSE: Voltage-gated K(+) channels maintain salt and water balance and normal function of corneal epithelial cells. To determine their identity, Kv channel types were sought in cultured rabbit corneal epithelial cells and in the intact rat corneal epithelium. METHODS: Immunohistochemistry and Western blot analysis were performed to detect K(+) channels in the membrane and cell lysates of rat and SV-40-transformed rabbit corneal epithelial (RCE) cells, using specific antibodies. The whole-cell patch clamp was used to characterize the biophysical and pharmacologic properties of the K(+) current in RCE cells. RESULTS: Expressions of K(+) channel types in corneal epithelial cells were detected by using a panel of specific anti-K(+) channel antibodies. Western blot analysis, using specific anti-K(+) channel antibodies including anti-Kv1.1, -2.1, -3.1, -3.2, -3.4, -4.2, and -4.3, demonstrated that in corneal epithelial cells Kv3.4 channel was highly expressed in whole-cell lysates and in cell membrane preparations. The anti-Kv3.4 channel antibody produced intense immunoreactivity in both RCE cells and rat corneal epithelium. Fluorescence immunostaining and avidin-biotin-peroxidase complex immunostaining confirmed localization of Kv3.4 channels in the cell membrane of both RCE and rat corneal epithelial cells. Voltage depolarization-activated K(+) currents in RCE cells were inhibited by applications of either 4-aminopyridine (4-AP, at micromolar levels), alpha-dendrotoxin at nanomolar levels, or blood-depressing substance-I at nanomolar levels. CONCLUSIONS: Biochemical and pharmacological profiles of the voltage-gated, 4-AP-sensitive K(+) channel in rat and RCE cells resemble characteristics of a Kv3.4 channel, a member of the Shaw subfamily. This channel may play important roles in maintaining normal function of corneal epithelium.     

Expression of keratin 12 and maturation of corneal epithelium during development and postnatal growth

PURPOSE: To determine the kinetics of corneal epithelial maturation during embryonic development and postnatal growth. METHODS: Expression patterns of keratin (K)12 and K14 were determined in mouse embryos (embryonic days [E]15.5-19.5), corneas of postnatal day (P)0 to 10 months, and healing corneas after epithelial debridement in P30 and P90 mice. The expression of alkaline phosphatase (AP) was determined during postnatal growth and healing of epithelial debridement of Krt12(Cre/Cre)/ZAP bitransgenic mice. RESULTS: During embryonic development, K12 expression by corneal peridermal epithelium commenced at E15.5. In the period from E15.5 to P10, the expression of K12 was restricted to the suprabasal and/or superficial cells of the corneal epithelium, whereas the K14 expression was restricted to the basal cells. After P30, K12 expression was sporadically detected in the basal corneal epithelium, and the number of K12-positive basal cells increased as the mice grew older. The number of K14-positive cells that coexpressed K12 increased with age and reached a plateau after P180. Healing of the debrided epithelium facilitated the increase in K14-positive cells that coexpressed K12. Many basal cells of Krt12(Cre/Cre)/ZAP mice remained undifferentiated and expressed LacZ at P15, and they then differentiated to express Cre, which leads to excision of LacZ and AP expression. CONCLUSIONS: In the mouse, the corneal epithelium does not become fully mature until 3 to 6 months after birth, in that a significant number of corneal basal epithelial cells of young mice (相似文献