Alcohol versus mechanical epithelial debridement: effect on underlying cornea before excimer laser surgery

PURPOSE: To determine the effects of 70% isopropyl alcohol used for corneal debridement on surface smoothness, stromal keratocytes, and ease of epithelial removal. SETTING: Cornea Research Laboratory, University of Rochester, and Excimer Laser Laboratory, Genesee Valley Eye Institute, Rochester, New York, USA. METHODS: Rabbit corneas were de-epithelialized mechanically or with 70% alcohol. The rabbits were split into groups and evaluated immediately or after a 50 microns deep excimer laser phototherapeutic keratectomy. All tissue was evaluated and compared in terms of surface smoothness parameters, loss of keratocytes, and inflammatory response to de-epithelialization. RESULTS: Computerized laser interferometric microscopy showed no between-group difference in the surface smoothness parameters. There was a marked absence of keratocytes in the superficial 25% of the corneal stroma. The loss of keratocytes was significantly higher (P < .001) in corneas treated with isopropyl alcohol. The inflammatory response 24 hours after epithelial removal was significantly higher (P < .001) in the corneas treated with alcohol. CONCLUSION: The use of 70% isopropyl alcohol applied for 2 minutes for epithelial removal did not enhance the quality of the subsequent excimer laser procedure. In contrast, isopropyl alcohol increased the inflammatory response, and it may have damaging effects on keratocytes. We would not advocate the use of 70% isopropyl alcohol as administered in our study to remove corneal epithelium before excimer laser surgery.     

241Am removal by DTPA vs. occurrence of skeletal malignancy

PURPOSE: The distribution of hyaluronan (HA) and the cellular response after photokeratitis induced by different ultraviolet (UV) wavelengths in the rabbit cornea was examined to help understand the mechanism of corneal injury and repair after UV damage. HA is a high molecular weight disaccharide polymer capable of binding considerable amounts of water. It is not normally found in the rabbit corneal stroma. The production of HA represents a generalized corneal response to injury. METHODS: Twenty-four albino rabbit corneas were exposed to 270, 290, and 310 nm of UV radiant energy in 8-nm full wavebands in doses producing biomicroscopically significant keratitis (three corneal thresholds for keratitis (Hc): 0.016 J/cm2 for 270 nm, 0.04 J/cm2 for 290 nm, and 0.14 J/cm2 for 310 nm) and in subkeratitis doses (0.7 Hc: 0.004 J/cm2 for 270 nm, 0.008 J/cm2 for 290 nm, and 0.03 J/cm2 for 310 nm). The rabbits exposed to 270 and 290 nm of UV radiation were sacrificed 3 days after exposure. The rabbits exposed to 310 nm of UV radiation were sacrificed 3, 7, and 14 days after exposure, respectively. The corneal tissue specimens were double stained and examined morphologically and histochemically for HA by light microscopy. RESULTS: Evaluation of corneas exposed to 270 and 290 nm of UV radiant energy in both subkeratitis and keratitis doses and those corneas exposed to 310 nm of radiant energy in subkeratitis dose showed neither stromal changes nor production of HA by corneal cells. Corneas exposed to 310 nm of UV radiant energy in keratitis dose at 3 days after exposure showed disappearance of keratocytes in entire thickness of central cornea. Cells bordering this damaged area were staining for HA. By 7 days after exposure almost the whole damaged area, except one fourth of anterior stroma, was repopulated by new keratocytes staining positive for HA. The corneal structures became normal and HA almost completely disappeared 14 days after exposure. CONCLUSIONS: A keratitis dose of 310 nm of UV light irradiation is needed to cause keratocyte damage. A keratitis dose of the shorter wavelengths does not cause keratocyte cell damage at the light microscopic level. The keratocyte production of HA appears to be a sign of cell readiness to repopulate the damaged stroma devoid of keratocytes.     

Keratocyte loss after corneal deepithelialization in primates and rabbits

PURPOSE: To evaluate the response of stromal keratocytes to central corneal deepithelialization. METHODS: Rabbits and monkeys underwent unilateral mechanical deepithelialization with a blunt instrument and were killed at intervals ranging from 15 minutes to 24 hours after surgery. Two rabbits underwent unilateral deepithelialization under a fluid bath containing corneal preservation medium. Two rabbits were treated unilaterally with corneal preservation medium topically applied every 15 minutes for 16 hours after epithelial removal. Four rabbits underwent linear keratotomy immediately after deepithelialization of the cornea or on normal unoperated corneas and were killed 1 day (two animals) and 14 days (two animals) after surgery. RESULTS: Deepithelialization resulted in severe ultrastructural changes in keratocytes within 30 minutes after surgery. After 24 hours, the number of keratocytes in the anterior stroma underneath the deepithelialized area had decreased significantly in rabbits (P = .0001) and in monkeys (P = .0007) compared with controls. The wound healing was altered and delayed when the epithelium was not present after keratotomy. The use of storage media during and after deepithelialization minimized the early keratocyte changes and appeared to stimulate reepithelialization. CONCLUSIONS: Removal of corneal epithelium causes loss of superficial stromal keratocytes in rabbits and monkeys. Keratocyte death may results from osmotic changes that alter the corneal wound healing response.     

Apolipoprotein E*3-Leiden transgenic mice as a test model for hypolipidaemic drugs

PURPOSE: We report an investigation into the distribution of proteoglycans (PGs) in normal, organ-cultured and dextran-treated human corneas. METHODS: Immunogold labeling was carried out at the electron microscope level to localize keratan sulphate (KS), chondroitin sulphate (CS), and heparan sulphate (HS) PGs. RESULTS: High levels of labeling for CS was found in the epithelium, endothelium, and keratocytes, with light labelling present in the basement membranes and the corneal stroma. Labeling for HS was present in the epithelium, endothelium, and keratocytes, with intense labeling present at the endothelium/Descemet's membrane interface and the epithelium/Bowman's layer interface. Large filaments were also observed in these regions in cuprolinic blue-stained specimens. Keratan sulphate was present at high levels in the stroma and the basement membranes with low levels present within the keratocytes, epithelium, and endothelium. The pattern of KS labeling along the collagen fibrils in the stroma sometimes showed evidence of periodicity. Organ-cultured corneas had extensive collagen-free "lakes," the interior of which immunolabeled positively for KS and showed staining with cuprolinic blue. The lakes were greatly reduced in the dextran-treated samples. CONCLUSION: This investigation determined the ultrastructural distribution of KS, CS, and HS PGs in human cornea and showed that organ culture is associated with a change in distribution of stromal PGs.     

Alternative paths of zinc transepithelial transport in the small intestine

PURPOSE: Neutrophil invasion is a primary event in the development of herpetic keratitis. It has been reported that HSV-1 infection of keratocytes induces the synthesis of IL-8, a potent neutrophil chemoattractant, while corneal epithelium does not. Nevertheless, little is known about the correlation between neutrophil migration and the production of chemotactic factors by HSV-1-infected corneal cells, especially in epithelial cells which form an initial barrier of the ocular surface. We examined whether human corneal epithelial cells as well as keratocytes could induce neutrophil chemotaxis in response to HSV-1 infection. METHODS: Human corneal epithelial cells immortalized with SV40 (HCE) and human keratocytes were infected with HSV-1. The culture fluids collected at 4, 12, 24 h after infection were assayed for human neutrophil chemotaxis using a modified Boyden chamber method. IL-8 levels in these supernatants were measured using enzyme-linked immunosorbent assay (ELISA). RESULTS: The chemotactic activity induced by HCE and keratocytes after MP strain of HSV-1 infection peaked as early as 4 h postinfection, then declined. Chemotactic activity induced by HSV-1-infected HCE and IL-8 levels on these supernatants paralleled with the infectious virus titer. It was inhibited by monoclonal anti-IL-8 antibody. UV-inactivation of MP strain abrogated neither the induction of chemotactic activity nor IL-8 secretion of infected HCE. CONCLUSIONS: At the early phase of HSV-1 infection, corneal epithelial cells play an important role in inducing neutrophil chemotaxis, which was mediated by IL-8.     

Expression of proliferating cell nuclear antigen in corneas kept in long term culture

PURPOSE: To investigate the proliferative activity of the donor corneal cells and to examine how this property changed during long term culture. METHOD: Fourteen human corneas from donors (ages from 50-91) were cultured in the medium (MEM+8% FBS with or without dextran). The proliferating status of corneal cells was evaluated by immunohistochemical staining of proliferating cell nuclear antigen (PCNA) in the cells. Three corneas at each time point were fixed in paraformalin at day 0, day 3 and after 3 weeks cultured in medium as well as 3 weeks plus 2 or 5 days in fresh medium with 8% dextran. Paraffin-embedded corneas were sectioned to 4 microm and stained with antibody PC 10 against PCNA. The number of PCNA positive cells was identified under light microscope. RESULT: Prior to organ culture only basal limbal epithelial cells stained positive for PCNA. After 3 days in culture 50 percent of the epithelial cells were positive as were several keratocytes and some endothelial cells in the peripheral corneas. After 21 days no cells showed proliferative activity. After 21 days in culture and 5 days in fresh deswelling medium the essentially monolayered epithelium stained positively in the limbal area. The proliferative activity of the keratocytes in the anterior stroma was extensive. Endothelial cells stained positive in the peripheral cornea. CONCLUSION: Limbal epithelial cells appear to survive in the organ culture. The corneas may be worth evaluating as sources of stem cells for grafting. Likewise, the keratocytes survive organ culture and can be induced to proliferate after a change to fresh medium. The endothelium is stimulated to proliferate in organ culture and in fresh medium after long term storage.     

Onchocerca volvulus-mediated keratitis: cytokine production by IL-4-deficient mice

Corneal inflammation similar to human onchocercal keratitis can be induced in mice by subcutaneous immunization of a soluble extract of Onchocerca volvulus (OvAg) followed by direct injection of OvAg into the corneal stroma. Previous studies have shown that corneal pathology is associated with increased systemic and corneal Th2 cytokine expression and that IL-4 gene knockout (IL-4-/-) mice develop less severe or no O. volvulus-mediated keratitis. The current study examined the contribution of Th2 cytokines to the diminished OvAg-induced corneal immunopathology observed in IL-4-/- mice. IL-4-/- mice (129Sv x C57B1/6), wild-type F2 littermates (IL-4+/+), and C57B1/6 mice were sensitized by repeated subcutaneous immunization with OvAg. Ten days after the final immunization, mice were sacrificed, spleens were removed, and cells were incubated with OvAg. Cells from immunocompetent C57B1/6 and IL-4+/+ mice produced IL-4 and IL-5, but no IFN-gamma, whereas cells from IL-4-/- mice had elevated IFN-gamma and no IL-4. Interestingly, cells from these animals produced levels of IL-5 protein equivalent to those of C57B1/6 and IL-4+/+ mice. To determine cytokine production in corneas during the onset of onchocercal keratitis, OvAg-immunized mice were injected intracorneally with OvAg, and cytokine gene expression in the cornea was determined by RT-PCR. Temporal analysis of cytokine gene expression in corneas of immunocompetent mice showed that the Th2-associated cytokines IL-4, IL-5, IL-10, and IL-13 were produced within 1 day of intrastromal injection, with sustained elevations for 10 days. Maximal IFN-gamma mRNA levels were not detected until Day 10. This was in contrast to IL-4-/- mice in which IFN-gamma appeared at Day 1 and remained elevated for at least 10 days. Moreover, in corneas from IL-4-/- mice, all Th2 cytokines with the exception of IL-4 were up-regulated and expressed with kinetics similar to that of IL-4+/+ littermates. Histologically, corneas from IL-4-/- mice were less edematous and contained fewer eosinophils and other inflammatory cells than those from immunocompetent controls. As there was no difference in peripheral eosinophil levels, these data indicate that the diminished severity of onchocercal keratitis in IL-4-/- mice is not due to failure to develop systemic or local Th2 cytokine responses or to produce eosinophils, but that IL-4 may be involved in recruitment of eosinophils and other inflammatory cells into the corneal stroma.     

Confocal microscopic characterization of wound repair after photorefractive keratectomy

PURPOSE: Development of postoperative corneal haze and regression of refractive effect are unfavorable clinical complications of excimer laser photorefractive keratectomy (PRK). Although exact mechanisms remain to be elucidated, these outcomes have been attributed to post-PRK corneal wound healing. The purpose of this study was to evaluate corneal wound repair quantitatively after PRK in a rabbit model using a newly developed in vivo technique, termed confocal microscopy through focusing (CMTF). METHODS: Twelve rabbit corneas received a monocular, 6-mm diameter, 9.0-diopter PRK myopic correction. Animals were evaluated sequentially up to 6 months after surgery by in vivo CMTF, which uses an image-intensity depth profile to measure epithelial and stromal thickness and uses corneal light reflectivity as an objective estimate of corneal haze. At differing temporal intervals, in vivo morphology was correlated with ex vivo histology using fluorescence microscopy. RESULTS: One week after PRK, an acellular layer of 86 +/- 24 microns was found anteriorly in the remaining stroma, which demonstrated surgically induced keratocyte death. Underlying keratocytes became activated and migrated toward the wound bed; repopulation was completed within 3 weeks. One week after PRK, there was a significant increase (P < 0.001) in light reflections detected from the photoablated stromal surface (1745 +/- 262 U) and from the underlying activated fibroblasts (713 +/- 607 U). Corneal reflectivity peaked at 3 weeks (4648 +/- 1263 U) and decreased linearly to 889 +/- 700 U by 6 months after the PRK; this corresponded to a reflectivity six times greater than the level seen in unoperated corneas. Two weeks after PRK, initial corneal edema had resolved, revealing an actual ablation depth (maximal stromal thinning) of 118 +/- 8 microns. Starting at 2 weeks after surgery, the stroma underwent gradual rethickening that reached 98% of the preoperative thickness at 6 months after PRK; at that time, only 6% of the initial photoablation depth persisted. By contrast, the central corneal epithelium showed no significant postoperative hyperplasia. CONCLUSIONS: Rabbit corneas treated by PRK showed a remarkable stromal wound-healing response that ultimately led to the restoration of the original stromal thickness by 6 months after surgery, demonstrating complete regression of the initial photoablative effect. Additionally, corneal wound healing was associated with increased light reflections from both the photoablated stromal surface and the activated wound-healing keratocytes underlying this area. Based on these findings, the authors hypothesize that the development of clinically observed corneal haze in PRK patients may be related, in part, to activation of corneal keratocytes and to putative changes in the extracellular matrix.     

Immunolocalisation of thrombospondin 1 in human, bovine and rabbit cornea

Light- and electron-microscopic immunohistochemical techniques were used to investigate the distribution of the matricellular protein thrombospondin 1 in normal human, bovine and rabbit cornea. Light-microscopic immunoreactivity for thrombospondin 1 was observed in the epithelial basement membrane, posterior Descemet's membrane and endothelium of human and bovine cornea. The bulk of the stroma, the stromal cells (keratocytes) and the anterior part of Descemet's membrane in human and bovine cornea were devoid of detectable thrombospondin 1 and the protein could not be demonstrated in any of the layers of the rabbit cornea. Electron-microscopic immunogold studies of human and bovine cornea revealed that thrombospondin 1 labelling of corneal endothelial (and basal epithelial) cells included focal deposits at cell membranes. It is postulated that thrombospondin 1 regulates interactions between cells and their basement membrane, and perhaps cell-to-cell interactions, in the normal human and bovine corneal endothelium and basal epithelium.     

Real-time confocal microscopic observations on human corneal nerves and wound healing after excimer laser photorefractive keratectomy

PURPOSE: Corneal wound healing after excimer laser photorefractive keratectomy (PRK) passes through a series of characteristic stages which have earlier been defined by means of histological, histochemical, and biochemical approaches. We investigated the potential of confocal microscopy to verify morphological changes in human corneas in vivo after PRK. METHODS: Ten corneas of eight patients that had earlier undergone PRK were examined at different postoperative time points (7 days-34 months). One of the PRK patients was examined sequentially three times. Three additional corneas, which had earlier undergone corneal grafting surgery and then were subjected to excimer laser photoastigmatic keratectomy (PARK), were studied as well. Seven healthy untreated corneas served as controls to define the normal morphology of human cornea. A tandem scanning confocal microscope (TSCM) was used to generate real-time images of the corneas on an S-VHS videotape. The images were either digitized and further processed or the individual video frames were produced with a video printer. RESULTS: Seven days post-PRK in vivo confocal microscopy revealed the presence of morphologically immature surface epithelial cells. Delicate nerves, activated keratocytes and deposition of extracellular light-reflecting scar tissue were perceived. The epithelium appeared normal one month post-PRK. Ongoing activation of the anterior stromal keratocytes along with extracellular scar tissue were detected. We also observed increasing numbers of regenerating subepithelial nerve leashes with somewhat twisted pattern. Highly reflective, presumably activated keratocytes were no longer detected 6-7 months post-PRK. Hypercellularity with scar tissue could still be found up to 30 months post-PRK. Only one cornea examined 34 months post-PRK showed normal keratocyte morphology and recovery of the anterior stroma. However, the morphology of subepithelial nerves was still somewhat abnormal. The two corneal grafts examined 11 or 32 months post-PARK exhibited a normal-appearing epithelium but considerable stromal hypercellularity and extracellular scar deposition. The subepithelial nerves were poorly regenerated in one eye and fairly well detectable in the other. The third graft examined 15 months post-PARK revealed the presence of enlarged surface epithelial cells and dense stromal scarring but no nerves. CONCLUSION: TSCM clinically confirms the earlier histological data on healing of excimer laser wounds. It offers a distinct improvement in the assessment of excimer laser-treated corneas, as it enables cellular details and nerves to be perceived in vivo. In addition the thickness of the stromal scar can be be measured for e.g. planning of phototherapeutic keratectomy.     

Corneal wound healing after laser in situ keratomileusis in rabbits

BACKGROUND: The aim of this study was to characterize the cell biology of wound healing in rabbit corneas subjected to laser in situ keratomileusis (LASIK). METHODS: Rabbit corneas underwent LASIK with various multizone photoablations or only a lamellar keratotomy followed by repositioning of the flap. We looked for indications for an active wound healing process. Immunohistochemistry for the extradomain A cellular fibronectin (EDA-cFn) or tenascin (Tn) and routine histology were examined. RESULTS: Four days after LASIK or lamellar keratotomy followed by repositioning of the flap, epithelial plugs and prominent keratocytes as well as Tn and EDA-cFn immunoreactions-indicative of a wound-healing process-appeared in the wound margins. Epithelial plugs were less conspicous, and prominent, presumably activated, keratocytes were no longer identified at the wound margin at 2.5 and 5 months after wounding. However, EDA-cFn and Tn immunoreactivities could still be observed. Only the stromal cells located in the periphery of the flap and in relatively close contact with the epithelium were surrounded by scar tissue expressing immunoreactivity for EDA-cFn or Tn. The central corneal stroma was devoid of scar tissue. CONCLUSION: Results indicate that the wound healing reaction after LASIK takes place only at the periphery of the microkeratome wound, leaving the central optical zone clear.     

Synthesis of corneal keratan sulfate proteoglycans by bovine keratocytes in vitro

Keratan sulfate proteoglycans (KSPGs) are the major proteoglycans of the cornea and are secreted by keratocytes in the corneal stroma. Previous studies have been able to show only transient secretion of KSPG in cell culture. In this study, cultures of bovine keratocytes were found to secrete the three previously characterized KSPG proteins into culture medium. Reactivity with monoclonal antibody I22 demonstrated substitution of these proteins with keratan sulfate chains. KSPG constituted 15% of the proteoglycan metabolically labeled with [35S]sulfate in keratocyte culture medium. This labeled KSPG contained keratan sulfate chains of 4700 Da compared to 21,000 Da for bovine corneal keratan sulfate. Labeled keratan sulfate from cultures contained nonsulfated, monosulfated, and disulfated disaccharides that were released by digestion with endo-beta-galactosidase or keratanase II. Nonsulfated disaccharides were relatively more abundant in keratan sulfate from culture than in corneal keratan sulfate. These results show that cultured bovine keratocytes maintain the ability to express all three of the known KSPG proteins, modified with keratan sulfate chains and sulfated on both N-acetylglucosamine and galactose moieties. KSPG made in vitro differs from that found in vivo in the length and sulfation of its keratan sulfate chains. The availability of cell cultures secreting corneal keratan sulfate proteoglycans provides an opportunity to examine biosynthesis and control of this important class of molecules.     

Pathogenic mechanisms in the rheumatoid nodule: comparison of proinflammatory cytokine production and cell adhesion molecule expression in rheumatoid nodules and synovial membranes from the same patient

OBJECTIVE: To investigate the production of proinflammatory cytokines and expression of cell adhesion molecules in the rheumatoid nodule. METHODS: Cytokine content (tumor necrosis factor alpha [TNFalpha], interleukin-1beta [IL-1beta], and IL-1 receptor antagonist [IL-1Ra]), at the messenger RNA (mRNA) and protein levels, and cell adhesion molecule expression were studied in 16 rheumatoid nodules and 6 synovial membranes. RESULTS: Macrophages in the rheumatoid nodules contained TNFalpha, IL-1beta, and IL-1Ra mRNA and protein, particularly in perivascular cells of the stroma and in the palisading layer. All cell adhesion molecules studied were expressed in both the rheumatoid nodules and synovial membranes, with increased expression of E-selectin in the rheumatoid nodule compared with the synovial membrane, and with the absence of vascular cell adhesion molecule 1 expression on cells of the palisading layer in the rheumatoid nodule. CONCLUSION: The presence of similar proinflammatory cytokines and cell adhesion molecules in the rheumatoid nodule and synovial membrane suggests that similar pathogenic processes result in the chronic inflammation and tissue destruction in these lesions.