Moesin as a key cytoskeleton regulator in corneal fibrosis.

PURPOSE: : Corneal fibrosis is the third leading cause of blindness worldwide. α-Smooth muscle actin (SMA), a marker of fibrosis, is closely regulated through an intermediate group of submembrane molecules - cytoskeleton regulators. The purpose of this study was to elucidate the role of specific cytoskeleton regulators in a mouse model of corneal fibrosis. METHODS: : A mouse model of corneal fibrosis was developed using anterior keratectomy (AK) and the topical application of transforming growth factor (TGF)-ß1 (1 µg/ml). The RT² Profiler™ PCR Array for cytoskeleton regulators was used to assay changes in levels of specific members of this class of proteins. Moesin siRNA was delivered into the corneal stroma by iontophoresis in vivo. Transformation of the corneal keratocyte-to-myofibroblast in corneal fibrosis, as defined by the expression of α-SMA, was determined by Western blot. RESULTS: : After AK and topical application of TGF-ß1, moesin was the most highly upregulated gene among 84 cytoskeleton regulator genes; iontophoresing moesin siRNA into the corneal stroma reduced the expression of α-SMA to 0.22-, 0.52-, and 0.31-fold of control at postoperative (PO) day 1, 3, and 5, respectively; also, upregulation of phospho-Smad 2 induced by TGF-ß1 was reduced by moesin siRNA to 0.59-, 0.56-, and 0.31-fold of control and expression of phospho-Smad 3 was reduced to 0.58-, 0.53-, and 0.47-fold of control at the same PO days. CONCLUSIONS: : Moesin may be a potential drug target for inhibiting corneal fibrosis, and the details of moesin-related signaling pathways would be critical for understanding corneal fibrosis.

The structural parameters for antimicrobial activity, human epithelial cell cytotoxicity and killing mechanism of synthetic monomer and dimer analogues derived from hBD3 C-terminal region.

Understanding the molecular mechanisms of antimicrobial peptide-membrane interactions is crucial in predicting the design of useful synthetic antimicrobial peptide analogues. Defensins are small (3-5 kDa) cysteine-rich cationic proteins which constitute the front line of host innate immunity. In this study, a series of eight 10 AA C-terminal analogues of hBD3 [sequence: RGRKXXRRKK, X = W, F, Y, V, L, I, H, C(Acm); net charge = +7, coded as W2, F2, Y2, V2, L2, I2, H2, and C2] and covalent V2-dimer [(RGRKVVRR)(2)KK] (18 AA, net charge = +11) were synthesized using solid phase peptide synthesis (SPPS) in Fmoc chemistry. Wild-type hBD3 was used as a control in all analyses. W2, V2, and especially Y2 showed high activity selectively against Gram-negative bacteria Pseudomonas aeruginosa in the concentration range of 4.3-9.7 microM. The covalent dimeric form of V2-monomer, V2-dimer, showed increased antibacterial killing compared to the monomeric form, V2-monomer. Cytotoxicity assays on a human conjunctival epithelial cell line (IOBA-NHC cells) showed that no change in viable cell number 24 h after constant exposure to all the eight peptide analogues even at concentrations up to 200 microg/ml. Fluorescence correlation spectroscopy (FCS) was used to study the interaction of these peptides against POPC vesicles (neutral; mammalian cell membrane mimic) and POPG vesicles (negatively charged; bacterial cell membrane mimic). Using FCS, significant aggregation and some leakage of Rhodamine dye were observed with POPG with Y2, W2 and V2 at the concentration of 5-10 mmicroM and no significant aggregation or disruption of vesicles was observed for all peptide analogues tested against POPC. V2-dimer induced more leakage and aggregation than the monomeric form. Overall, V2-dimer is the most effective antimicrobial peptide, with aggregation of POPG vesicles observed at concentrations as low as 1 microM. The concentration of 5-10 microM for Y2 from FCS correlated with the concentration of 5 microM (6.25 microg/ml), at which Y2 showed a cooperative increase in the activity. This suggests a structural transition of Y2 in the 2.5-5 microM concentration range resulting in the correlated increased antimicrobial activity. These results and the FCS together with previous NMR and molecular dynamics (MD) suggested that the charge density-based binding affinity, stable covalent dimerization, the ability to dimerize or even oligomerize and adopt a well-defined structure are important physicochemical properties distinguishing more effective cationic antimicrobial peptides.

Epithelial microfilament regulators show regional distribution in mouse conjunctiva.

PURPOSE: The conjunctival epithelium is a continuous sheet of cells with regional characteristics that appear to be similar. This study was designed to investigate the distribution and levels of expression of a subset of microfilament regulators in the forniceal, palpebral, and bulbar conjunctival epithelia. METHODS: Balb/C mice were used. The localizations of paxillin, focal adhesion kinase, vinculin, talin1, cofilin, profilin, gelsolin, integrin ß1, and integrin α6 were studied with the use of cross-sectional immunofluorescent staining. For a detailed cellular analysis, positioning and ablation with the laser microbeam (PALM) Combi System was used to obtain forniceal, bulbar, and palpebral conjunctival epithelia for expression comparison with the use of western blot analysis and quantitative real-time polymerase chain reaction. RESULTS: Immunostaining showed that focal adhesion kinase, cofilin, profilin, gelsolin, talin1, and vinculin were expressed in all layers of the forniceal, palpebral, and bulbar conjunctival epithelia. Paxillin, integrin ß1, and α6 was found to be located in the basal cell layer in all three of these areas. Quantitative real-time polymerase chain reaction showed that the transcript levels of these microfilament regulators in the forniceal conjunctivae were higher than those levels found in the bulbar and palpebral conjunctivae. Western blot analysis confirmed the differential expression levels of these microfilament regulators in the forniceal, bulbar, and palpebral conjunctivae. CONCLUSIONS: Differences in the levels of microfilament regulators in the forniceal, bulbar, and palpebral conjunctivae suggest different modes of interaction with their microenvironment and within cell layers.

Expression of muscarinic receptors in human and mouse sclera and their role in the regulation of scleral fibroblasts proliferation.

PURPOSE: To determine the expression of muscarinic receptor subtypes (mAChRs) in human and mouse scleral fibroblasts (SFs), to investigate the mechanism that mediate the role mAChRs play in cell proliferation, and to explore the underlying intracellular signaling pathways involved in mouse SFs with treatment of muscarinic agents. METHODS: Reverse transcription polymerase chain reaction (RT-PCR) was used to detect mRNA expression of mAChRs in the human and mouse sclera. Western blot analysis and immunocytochemistry were used to detect proteins of mAChRs in the cultured SFs. An immunohistochemical study was used to further detect the presence of mAChR proteins in frozen scleral sections. BrdU (5-bromo-2-deoxyuridine ) cell proliferation assay was performed to measure DNA synthesis. Enzyme linked immunosorbent assay (ELISA) was used to measure in vitro kinase activity for epidermal growth factor receptor (EGF-R), fibroblast growth factor (FGF-2), transforming growth factor (TGF)-beta1, and extracellular signal-regulated kinase (ERK)1/2. Expressions of epidermal growth factor-receptor (EGF-R); protein kinase C (PKC); Proline-rich tyrosine kinase 2 (Pyk-2), v-raf murine sarcoma viral oncogene homolog B1 (B-Raf), Rat Sarcoma (Ras), c-Jun N-terminal kinases (JNK1/2), and ERK1/2 were detected by immunoblot. RESULTS: mAChR for subtypes M(1)-M(5) were detected in both mouse and human SFs by protein, cellular, and mRNA analysis. EGF-R, PKC, Pyk-2, B-Raf, Ras, JNK1/2, and ERK1/2 were activated after treatment by agonists and antagonists, indicated by changes in phosphorylation of these proteins. Atropine abolished the carbachol-induced activation of SF cell proliferation in a concentration-dependent manner. Carbachol also activated p42/44 mitogen-activated protein kinase (MAPK) and Ras in a time-dependent manner. Muscarinic agents also modulated fibroblast growth factor expression in these cells. CONCLUSIONS: This study confirms the presence and functional role of all five mAChRs in human and mouse SFs. These results show that proliferative responses of SFs to muscarinic receptor stimulation are mediated via the activation of the classical MEK-ERK-MAPK cascade.

Effect of dispase denudation on amniotic membrane.

PURPOSE: To describe the cellular components, biochemical composition, and membrane surface characteristics of denuded human amniotic membrane (DHAM) treated with Dispase II. METHODS: DHAM was incubated with Dispase II (1.2 U/ml) for 30 min, 60 min, or 120 min. This was followed by gentle scraping to remove any remaining epithelial cells using a cell scraper. Histology, immunohistochemistry for extracellular matrix molecules and growth factors, and transmission (TEM) and scanning electron microscopy (SEM) were performed to assess the effects of increasing durations of incubation on DHAM structure. RESULTS: Dispase II treatment was associated with the digestion of several ECM molecules, particularly those in the basement membrane including collagen VI, fibronectin, and laminin. FGF-2 and PDGF-B expression were unaffected by Dispase II, but TGF-alpha, TGF-beta1, TGF-beta 2R, PDGF-A, VEGF, and EGFR expression were all reduced by Dispase II incubation. TEM confirmed the disruption of DHAM ultrastructure with increasing duration of Dispase II incubation, beginning with disruption of the basal lamina and progressing to loosening of the stromal collagen network as well. CONCLUSIONS: The use of Dispase II in the preparation of DHAM causes significant changes to the ultrastructure of the membrane, particularly the BM. Prolonged incubation with dispase may cause significant disruption in DHAM structure which may affect cell growth in cultured explants.

Expression of insulin-like growth factor binding protein-3 in pterygium tissue.

BACKGROUND: Pterygium is a fibrovascular overgrowth from the conjunctiva onto the cornea. The pathogenesis of this common ocular surface disorder is not well understood and the only treatment is surgical removal. METHODS: DNA microarray analysis of primary pterygium tissue was carried out using uninvolved conjunctiva tissue as a comparison for gene expression levels. Real time polymerase chain reaction (PCR) was used to verify the mRNA level of expression for genes changed in pterygium. Western blot analysis and immunohistochemistry showed protein expression levels and the tissue distribution. RESULTS: Microarray analysis revealed that mRNA levels of a number of genes were changed in primary pterygium. In particular the gene for insulin-like growth factor binding protein-3, (IGFBP3), which modulates the effects of insulin-like growth factor on cells was significantly decreased. Both the message and protein expression of IGFBP3 in pterygium were decreased compared to normal, uninvolved conjunctiva. CONCLUSION: Decreased levels of IGFBP3 protein have been strongly correlated with the presence of cancer. Identification of the low level of expression of IGFBP3 in pterygium suggests that the pathway controlling cell proliferation has lost an important control mechanism, which may explain the continued growth of pterygium.

The use of human serum in supporting the in vitro and in vivo proliferation of human conjunctival epithelial cells.

AIM: To evaluate the use of human serum (HS) in supporting the in vitro and in vivo proliferation of human conjunctival epithelial cells, and compare it with fetal bovine serum (FBS) and bovine pituitary extract (BPE). METHODS: Conjunctival epithelial cells were cultivated in media supplemented with HS (5%, 10%), FBS (5%, 10%), and BPE (70 microg/ml, 140 microg/ml). The colony forming efficiency (CFE), bromodeoxyuridine (BrdU) ELISA proliferation assay, and cell generations were analysed. Cells were evaluated for keratin (K4, K19, and K3) and MUC5AC expression by immunostaining and RT-PCR. Conjunctival equivalents constructed on amniotic membranes were transplanted onto severe combined immune deficient (SCID) mice for 10 days and analysed histologically. RESULTS: The proliferation assays of HS supplemented cultures (CFE, 6.7% (SD 1.8%); BrdU absorbance, 0.86 (0.16)) were comparable to FBS supplemented (CFE, 9.3% (1.8%); BrdU absorbance, 1.11 (0.18)) and BPE supplemented cultures (CFE, 5.9 (1.5); BrdU absorbance, 0.65 (0.12)). Goblet cell densities for HS, FBS, and BPE supplemented media were 52 cells/cm(2), 60 cells/cm(2), and 50 cells/cm(2), respectively. HS supplemented cultures formed stratified epithelial sheets in vivo following transplantation. CONCLUSIONS: The proliferative capacity of conjunctival epithelial cells cultivated in HS supplemented cultures was comparable to FBS and BPE supplemented cultures. The elimination of animal material from the culture system is advantageous when cultivating cells for clinical transplantation.

Corneal pain evoked by thermal stimulation.

The thermal sensitivity of the eyelid and cornea was compared using an automated apparatus to produce stimulus pulses of known magnitude and duration over the range 33--45 degrees C. Subjects reported only temperature sensation when the skin of the upper eyelid was tested; however, corneal stimulation in the same subjects was always perceived as nociceptive. The possibility that other ocular tissues may be involved in the pain responses was shown to be unlikely by direct experimentation or by calculation of heat flow in those tissues. Cornea and eyelid thresholds were compared in relationship to the structural and physical properties of these tissues. It was found that the nerve endings of the corneal epithelium are less sensitive to temperature change when compared to the thermal receptors of the eyelid. It is concluded that the cornea is useful for the experimental study of pain.

Recovery of corneal sensation following hard contact lens wear and the implication for adaptation.

Recovery of corneal sensitivity was determined in hard contact lens wearers 2, 4, 6, 12, and 24 hr after removing the lenses. The extent of recovery was shown by the statistical similarity of the threshold values for the contact lens group and the thresholds of a group of subjects who had never worn contact lenses. Consideration of the time course of recovery and of the thermal modality of the stimulus suggest that the sensory decrement induced by contact lens wear cannot be attributed to simple adaptation.

Neural remodeling following experimental surgery of the rabbit cornea.

Wounding of the cornea results in extensive damage to the innervation that must be repaired to restore its normal structure and function. Four types of experimental wounds were produced in the corneas of young albino rabbits: 180 degrees penetrating perilimbal incisions, 4-mm central circular keratectomies and keratotomies, and radial keratotomies. Following perilimbal incisions, the denervated half of the cornea was reinnervated primarily by regenerating nerves that penetrated the limbal scar tissue. New neural growth from the innervated portion of the cornea provided a minor contribution to the denervated area. The regenerative response of the nerves following nonpenetrating procedures was found to be a biphasic process. In the first phase, a short period of degeneration of all nerves within the area enclosed by the wound boundary overlapped in time with the appearance of long, large caliber, dense neurites that coursed perpendicularly to the wound margins. The neurites originated from the intact subepithelial plexus at some distance from the wound margins. The second phase was initiated by the degeneration of the wound-oriented neurites and the concomitant appearance of a second generation of neurites. These new neurites originated from the transected stumps of the regenerating subepithelial axons at or near the wound margins. The oblique disposition of the second wave of neurites was similar to that of basal leashes in normal corneas. Nonpenetrating wounding procedures exhibited similar neural remodeling principles. In both types of keratotomies, nerve endings terminated within the wound in enduring and densely packed neuroma-like arrangements, while in keratectomies, nerve endings continued to grow toward the center of the cornea.

Alteration of corneal epithelial ion transport by sympathectomy.

The cornea is dually innervated, receiving afferent nerves from the trigeminal ganglion and efferent nerves from the superior cervical ganglion. This study examines the specific effects of superior cervical ganglionectomy (SCGX) on the in vitro ion transport characteristics of the rabbit corneal epithelium. Two weeks after SCGX, epithelial Cl--dependent transport and total ionic conductance were increased in comparison to values obtained in paired control eyes. This increased transport level appeared to be independent of membrane receptor activity as demonstrated by lack of responsiveness to alpha-adrenergic, beta-adrenergic, serotonergic, dopaminergic, nicotinic cholinergic, or muscarinic cholinergic blockade. Nevertheless, SCGX produced a supersensitivity to epinephrine-stimulated transport as measured by the responsiveness of the ion transport current. Furthermore, SCGX abolished the responsiveness of the epithelium to serotonin. On the basis of these and earlier findings, the authors conclude that corneal sympathetic innervation influences membrane and receptor properties. Autonomic neurotrophic effects in the corneal epithelium include suppression of apical membrane Cl- permeability and of beta-adrenoreceptor sensitivity to biogenic amines. It is proposed that the corneal serotonergic receptors that activate Cl- transport lie on the sympathetic nerve terminals and stimulate this transport process by causing the neural release of a catecholamine.

Cryogenic lesion alters the metabolism of arachidonic acid in rabbit cornea layers.

The metabolism of radiolabeled arachidonic acid in epithelium, stroma, and endothelium was studied in normal and cryogenically lesioned rabbit corneas. The synthesis of cyclooxygenase- and lipoxygenase-reaction products, as well as the incorporation of arachidonic acid in phospholipids and neutral lipids, was followed by in vitro incubation (60 min) of corneas obtained 2 hr, 5 days, and 15 days after injury. In unwounded controls, prostaglandin E2 (PGE2) was the major cyclooxygenase product formed in the stroma, whereas thromboxane B2 predominated in the endothelium and epithelium. The major lipoxygenase product detected under these conditions in the epithelium was mono-hydroxyeicosatetraenoic acid (mono-HETE) and in the stroma, 12-HETE. In contrast, lipoxygenase products could not be detected in control endothelium. Two hours after injury, the labeling of lipids in epithelium and endothelium decreased; the largest decrease was in phosphatidylinositol, followed by phosphatidylcholine and phosphatidylethanolamine. At the same time, cyclooxygenase-reaction products in the epithelium increased, particularly PGF2 alpha. Prostaglandin levels in the stroma rose rapidly after injury and remained elevated for 15 days. In the endothelium, increases in PGF2 alpha and PGE2 were the most prominent effects of injury. After wounding, lipoxygenase products appeared for the first time in the endothelium and increased in the stroma and epithelium. Within 2 hr after lesioning, 12-HETE and 5-HETE increased in stroma. These studies show that the metabolism of arachidonic acid is altered by cryogenic injury and that the resulting changes differ in the three layers of the cornea. These changes involve arachidonoyl groups of phospholipids and cyclooxygenase and lipoxygenase products, and it is suggested that they are at least partly due to the migration of inflammatory cells to the wound site.

Dopamine modulation of active ion transport in rabbit corneal epithelium.

The addition of micromolar quantities of dopamine stimulated ion transport in the isolated rabbit corneal epithelium. This response was blocked by pretreatment with the dopamine antagonist, haloperidol, and by the elimination of Cl- from the bathing solutions. The beta-adrenergic antagonist, timolol, was also a potent inhibitor of the epithelial response to dopamine. The presence of the serotonin antagonist, methysergide, or the dopamine beta-hydroxylase inhibitor, FLA-63, did not significantly alter the corneal response to dopamine. Following superior cervical ganglionectomy, the epithelial response to dopamine was abolished. These findings are consistent with the idea that Cl- secretion in the rabbit corneal epithelium can be modulated by preterminal dopamine receptors located on the sympathetic nerve fibers; therefore, dopamine stimulation appears to be a serial process mediated by the release of norepinephrine from sympathetic nerve terminals in the epithelium.

Effect of growth factors on collagen lattice contraction by human keratocytes.

A three-dimensional gel contraction model was used to evaluate interactions between human keratocytes and different kinds of collagen in the presence or absence of various growth factors. Bovine collagen type I or human placental copolymerized collagen type I/III was used to create the lattices. Normal keratocytes from neonatal, aged, and insulin-dependent diabetic donors, as well as abnormal keratocytes from a donor with macular corneal dystrophy, were cultured. Growth factors included epidermal growth factor (EGF), basic fibroblastic growth factor (FGF), insulin-like growth factor (IGF-I), and platelet-derived growth factor homodimer beta beta (PDGF). Gel area and optical transmittance were determined from computerized measurements. Dose-response experiments (0.01-100 ng/ml) demonstrated that PDGF at 10 ng/ml (P less than 0.005) and EGF at 1 and 10 ng/ml (P less than 0.0001) were the most effective in promoting gel contraction, compared to IGF-I and FGF. Comparison of cell strains revealed different dose-response profiles. Cells from insulin-dependent diabetics and cells from a donor with macular dystrophy contracted lattices more rapidly than cells from normal neonates (P less than 0.0001). Lattices of copolymerized human collagen type III/I demonstrated significantly reduced contraction rates (P less than 0.0001) and increased optical transmittance, compared to bovine collagen type I lattices. Ultrastructural studies revealed that keratocytes extend processes to form a network within the collagen lattice. Specialized intercellular junctional complexes were observed by transmission electron microscopy. This model provides a useful in vitro corneal stroma-equivalent for the study of keratocyte, extracellular matrix, and growth factor interactions.

Epithelial wound closure in the rabbit cornea. A biphasic process.

The rapid and complete repair of the corneal epithelium following ocular surgery or trauma is essential for the maintenance of normal visual acuity. In this study the authors examined epithelial wound healing in the rabbit after cells were mechanically removed leaving the basal lamina intact. The decrease in wound area (mm2/hr) was neither linear nor amenable to simple kinetic analysis. However, analysis of the data in terms of the decrease in wound radius (mm/hr) revealed a biphasic process consisting of an initial latent phase with no epithelial movement (5.5 +/- .3 hr), followed by a linear healing phase. The rate of epithelial movement in the linear healing phase was 64 +/- 2 microns/hr. Neither the latent phase nor the rate of epithelial migration during the healing phase was affected by variations in initial wound size. Ultrastructural studies demonstrated that during the latent phase there was an increased desquamation of surface cells as well as cellular and subcellular reorganization of the basal cells. At the end of the latent phase, the leading edge of the wound was composed of a single cell layer. The onset of epithelial migration coincided with the first ultrastructural observation of typical ruffled membranes and filopodia. This work demonstrates that the analysis of the decrease in wound radius provides a straightforward and accurate means to assess the kinetics and therapeutic modulation of epithelial wound healing.

Human recombinant epidermal growth factor in experimental corneal wound healing.

Human recombinant epidermal growth factor (hEGF) was evaluated in various corneal wound healing models in the rabbit. Human EGF accelerated epithelial wound healing in corneal reepithelialization, anterior-keratectomy, and alkali-burn models at concentrations of 10-500 micrograms/ml given four times daily (qid). In the corneal reepithelialization model, 100 micrograms/ml of hEGF qid produced a 45% increase in the wound-healing rate compared with control (0.13 versus 0.09 mm/hr) with a similar response at 500 micrograms/ml qid. In the anterior-keratectomy model, 500 micrograms/ml of hEGF qid accelerated healing by 40% (0.07 versus 0.05 mm/hr), although the 100 micrograms/ml dose was not active in this model, and 1 microgram/ml of hEGF actually slowed the healing rate. In the alkali-burn model, 10 and 100 micrograms/ml of hEGF qid for 32 days appeared to produce faster initial healing of the wound compared with control, although the wound recurred in both hEGF and control groups. These results suggest that hEGF may be helpful in some epithelial disorders in humans, although considerations of dose response and optimal dosing regimens must be addressed.

Flow cytometry measurements of the DNA content of corneal epithelial cells during wound healing.

This study presents the first application of flow cytometry (FCM) techniques to the assessment of cell cycle dynamics in the corneal epithelium after experimental wounding. Anterior keratectomies 6 mm in diameter were created in the central corneas of albino rabbits. The authors sampled the epithelial tissue obtained outside the wound at 12-hr intervals until wound closure at 72 hr. Regenerated epithelium from the surface of the wounded area was collected at 78 hr. The percentages of nuclei in the G0/G1 (growth), S (DNA synthesis), and G2/M (tetraploid/mitosis) phases were determined by FCM. An increase in the percentage of nuclei in the G2/M phase at 36 hr was seen, compared with cell populations in samples from unwounded control corneas. The authors found an increase in mitotic activity in the corneal epithelium during the period of cell migration before wound closure.

Corneal nerve disruption reactivates virus in rabbits latently infected with HSV-1.

Trauma, inflammation, and neuronal stimulation or damage can reactive latent herpes simplex virus type 1 (HSV-1). The innervation density of the corneal epithelium is 300-600 times that of skin and, therefore, corneal nerve disruption could provide a strong stimulus for HSV-1 reactivation. This study has documented HSV-1 ocular reactivation following three methods of corneal nerve disruption in rabbits. Twenty HSV-1 latently infected rabbits (26 eyes) were divided into three groups: 7 rabbits received uniocular cryogenic injury, 7 rabbits underwent uniocular anterior superficial keratectomy, and 6 rabbits had binocular transection of the corneal nerves at the corneoscleral limbus which, in contrast to the other treatments, produced minimal epithelial change. Opposite eyes in the first two groups of rabbits were left undisturbed to serve as HSV-1 infected controls. Three additional rabbits, not infected with HSV-1, underwent gold chloride impregnation of the corneal nerves for light microscopic documentation of corneal nerve damage induced by each procedure. On all HSV-1 infected eyes, daily HSV-1 ocular cultures were obtained for 7 consecutive days. All three procedures resulted in marked corneal nerve destruction and degeneration. HSV-1 shedding occurred in 5/7 (71%) of the eyes that underwent cryogenic lesioning; in 5/7 (71%) of the eyes that underwent anterior keratectomy; and in 8/12 (67%) of the eyes that had the corneal nerves transected at the corneoscleral limbus. Only 4 (29%) of the 14 control eyes had positive HSV-1 ocular cultures. This investigation provides strong evidence that corneal nerve disruption is correlated with ocular HSV-1 reactivation.

Expression of tenascin and cellular fibronectin in the rabbit cornea after anterior keratectomy. Immunohistochemical study of wound healing dynamics.

Anterior keratectomy (AKE) was done on rabbits, and the appearance of immunohistochemically demonstrable tenascin (TN) or cellular fibronectin (cFN) was studied at different times (5 min to 14 months) after the operation. The substance TN was first observed 12 hr after wounding in the posterior stroma; cFN appeared with the same localization 12 hr later. During postoperative week 1, both TN and cFN immunoreactions shifted to more anterior parts of the cornea, and 9 days after wounding, they were localized in the most anterior part of the stroma only. Thereafter the reactions gradually decreased in intensity but still were visible 3 months after AKE. No reaction for TN or cFN was present 14 months postoperatively.

Beta-adrenergic and serotonergic stimulation of rabbit corneal tissues and cultured cells.

The adult rabbit cornea synthesizes cyclic AMP in response to both serotonin and isoproterenol. The authors have examined the postnatal development of these pathways and attempted to localize the responsive cell type(s) by dissection, cell culture, and surgical denervation. Full thickness corneas of neonatal rabbits have beta-adrenergic responses similar to the adult but fail to respond to serotonin until the animals are 9-12 weeks old. When adult corneas are separated into epithelia, stromal, and endothelial layers, only the stromal layer synthesizes cyclic AMP in response to serotonin, whereas all layers respond to isoproterenol. When grown in tissue culture, keratocytes, epithelial, and endothelial cells are unresponsive to serotonin but respond to isoproterenol. Neither adrenergic nor sensory denervation abolishes the corneal adrenergic or serotonergic response pathways. These results indicate that the epithelial cells do not contain the serotonin stimulated, cyclic AMP-mediated pathway as originally postulated. The cell population that does contain this pathway is within the stroma and may be the Schwann cells.