Hydrogen peroxide causes mitochondrial DNA damage in corneal epithelial cells.

PURPOSE: To study the effects of hydrogen peroxide exposure on mitochondrial DNA (mtDNA) in cultured human corneal epithelial cells. In addition, we compared the integrity of mtDNA found in epithelial cells isolated from keratoconus (KC) and normal (NL) corneas. METHODS: Telomerase immortalized human corneal epithelial cell line (hTCEpi) were cultured at pH 7.0 or pH 5.0 with or without 200 microM hydrogen peroxide (H2O2). Immunohistochemistry with a marker for oxidative damage, 8-hydroxy-2'-deoxyguanosine (8-OH-dG), was performed on KC and NL corneas (n = 10). Epithelial cells were isolated from KC corneas (n = 5) and NL corneas (n = 7). Total DNA was extracted, and the mtDNA was analyzed by long extension polymerase chain reaction (LX-PCR). The ratios of mtDNA to nuclear DNA were measured by PCR. The mtDNA control regions were PCR amplified and sequenced. RESULTS: In the epithelial cell cultures, the full-length LX-PCR mtDNA decreased 54% and 44% in the H2O2 + pH7 cultures and H2O2 + pH5 cultures, respectively. 8-OH-dG was present in all layers of KC epithelial cells but only in superficial layers of NL epithelial cells. The isolated KC and NL epithelial cells had comparable levels of full-length LX-PCR mtDNA (16.2 kb) and smaller sized mtDNA bands (4.3 +/- 0.99 vs 4.0 +/- 0.83 bands per individual, respectively). There were no significant differences in the control region nucleotide sequences in KC and NL epithelia. CONCLUSIONS: Hydrogen peroxide can significantly degrade LX-PCR mtDNA in vitro. Although the KC epithelium showed a higher degree of oxidative damage, the levels of mtDNA damage in NL and KC epithelial cells were similar to each other.

Epitope variability of Bcl-2 immunolocalization in the human corneal epithelium.

PURPOSE: To further study the immunological localization of Bcl-2 protein in human corneal epithelium. METHODS: Three anti-human Bcl-2 antibodies, generated against amino acid residues (aa) 4-21 (polyclonal), 1-205 (monoclonal), and 41-54 (monoclonal), were used to localize Bcl-2 protein immunocytochemically in fresh eye bank donor human corneas. RESULTS: In the central corneal epithelium, two anti-Bcl-2 antibodies (aa 4-21 and aa 1-205) showed intense cytoplasmic staining of basal epithelial cells. These antibodies produced similar staining in the limbal epithelium, with gradually less intense staining of wing and superficial cells. By contrast, as previously reported, a monoclonal antibody to aa 41-54 stained nuclei of all epithelial cell layers with the exception of some surface corneal epithelial cells; this antibody also demonstrated very bright anti-Bcl-2 staining of Langerhans cells localized in the peripheral corneal epithelium. CONCLUSION: In our previous study, Bcl-2 protein was immunocytochemically localized to the nuclear compartment of all corneal epithelial cell layers with the use of antibodies specific for the regulatory flexible loop domain of Bcl-2. However, Bcl-2 can also be uniquely localized to the cytoplasm of the corneal epithelium with the use of antibodies generated against aa 4-21 and aa 1-205. Taken together, these results using epitope specific antibodies indicate that different epitopes on the Bcl-2 protein are available for antibody binding within different cells and cellular compartments, suggesting that proliferation and differentiation may lead to changes in the Bcl-2 structure and conformation within different compartments of the epithelial cells themselves.

Proliferation rate of rabbit corneal epithelium during overnight rigid contact lens wear.

PURPOSE: To examine cell proliferation of the normal corneal epithelium and during extended rigid gas-permeable (RGP) lens wear. METHODS: Twenty-three New Zealand White rabbits were fitted unilaterally with either a low oxygen transmissible (Dk/t) or hyper-Dk/t RGP lens, with the other eye serving as a control. The rabbits were injected with 5-bromo-2-deoxyuridine (BrdU) 24-hours later and killed at three time points: 1, 3, and 7 days after injection. Corneas were processed for immunocytochemistry, and sequential digital images were taken from the superior limbus to the central epithelium with an epifluorescence microscope. The total number of BrdU-labeled cell pairs was quantified. RESULTS: The limbus in normal corneas was significantly less populated with BrdU-labeled cells than the central and peripheral epithelium (P < 0.05). The peripheral epithelium adjacent to the limbus was marked by a peak of labeled cells (P < 0.05). Both types of RGP lenses produced an increase in BrdU labeling in the limbus and a dramatic decrease in the central epithelium (80% for low Dk/t, 37% for hyper Dk/t). At day 3 and 7 after BrdU injection, the low-Dk/t lens continued to show decreased BrdU labeling centrally, whereas the limbus remained increased. Hyper-Dk/t lens wear however, showed persistent limbal elevation but equivalent numbers of BrdU-labeled cells centrally at days 3 and 7, compared with control corneas. Keratocytes unexpectedly showed BrdU labeling during RGP lens wear. CONCLUSIONS: Limbus, peripheral, and central epithelium were characterized by different proliferation rates in the normal rabbit cornea. RGP lens wear significantly altered the homeostatic proliferation pattern of the epithelium with the low-Dk/t lens having the most dramatic effect. RGP contact lens wear appears to stimulate proliferation of keratocytes.

Effects of low and hyper Dk rigid gas permeable contact lenses on Bcl-2 expression and apoptosis in the rabbit corneal epithelium.

PURPOSE: To study Bcl-2 expression and apoptotic cell shedding of the rabbit corneal epithelium during extended wear of low and hyper Dk rigid gas permeable (RGP) contact lenses. METHODS: Rabbits were fit with either a low or a hyper Dk RGP lens (Dk/Ltotal= 10 and 97). The rabbits wore the lenses for either 24 hours, 3 days, or 1 week at which point they were humanely sacrificed. Immunocytochemistry and western blot analyses were performed to detect Bcl-2 in the corneal epithelium; TUNEL assay (TdT-mediated dUTP nick-end labeling) was used to identify apoptotic epithelial cells. RESULTS: 1) Immunocytochemistry: In the normal cornea, antibodies to Bcl-2 uniformly stained nuclei of all epithelial cell layers. Occasional surface epithelial cells, however, showed no anti-Bcl-2 nuclear staining; concomitant TUNEL assay revealed that all TUNEL-labeled-surface cells were Bcl-2 negative. By contrast, RGP contact lens wear, regardless of test lens oxygen transmissibility or lens wearing interval, significantly decreased both the total number of Bcl-2 negative and TUNEL-labeled cells on the epithelial surface (P < 0.05). In addition, contact lens wear was associated with labeling of keratocytes with TUNEL assay in the anterior stroma. 2) Western blot analysis: Total epithelial layer Bcl-2 expression was markedly decreased in the low Dk lens test group but was similar to control values in the hyper Dk lens test group. CONCLUSION: Bcl-2 protein seems to play an important role in the regulation of apoptotic cell shedding in the normal rabbit corneal epithelium. The identical staining pattern was seen in previous studies of the normal human cornea. RGP contact lens wear, however, appears to block the changes in Bcl-2 protein prior to apoptotic surface cell shedding, suggesting a lens-related anti-apoptotic effect. Taken together, these findings may explain why contact lens wear reduces surface cell exfoliation as previously reported in human studies.

Pathology of ocular irritation with bleaching agents in the rabbit low-volume eye test.

Despite differences in the processes leading to tissue damage, the ocular irritation response to various surfactants, two concentrations of an acid and an alkali, and an acetone, alcohol, aromatic amine, and aldehyde has been shown to depend on the extent of initial injury. The purpose of this study was to assess the extent to which this fundamental relationship exists for bleaching agents in the rabbit low-volume eye test. Ten microl of sodium perborate monohydrate (NaBO3), sodium hypochlorite (NaOCl), 10% hydrogen peroxide (H2O2), and 15% H2O2 was applied directly to the cornea of the right eye of each rabbit. Macroscopic assessments for irritation were made 3 hours after dosing and periodically until 35 days. Light microscopic examinations were conducted on tissues obtained at 3 hr and on 1, 3, and 35 days. In vivo confocal microscopy (CM) and measurements of dead corneal epithelial cells and keratocytes at 3 hours and 1 day were used to characterize quantitatively initial corneal injury, while in vivo CM performed at 3 hours and 1, 3, 7, 14, and 35 days was used to characterize quantitatively the corneal changes over time. The changes with NaBO3 and NaOCl were consistent with mild irritancy. For both, corneal injury was limited to the epithelium and superficial stroma. The changes with 10% H202 and 15% H2O2 were consistent with severe irritation. Both concentrations affected the epithelium and deep stroma, with 15% H2O2 also at times affecting the endothelium. However, unlike other irritants previously studied, with 10% H2O2 and 15% H2O2 there was an incongruity between the extent of epithelial and stromal injury, with stromal injury being more extensive than epithelial injury. A similar, although less dramatic, effect was observed with NaBO3. Additionally, there was still significant keratocyte loss at 35 days with 10% H2O2 and 15% H2O2 even though the eyes at times were considered to be macroscopically normal. These observations highlight the need to include both epithelial and stromal components in an ex vivo or in vitro alternative assay. In conclusion, these results continue to support and extend our hypothesis that ocular irritation is principally defined by the extent of initial injury despite clear differences in the means by which irritants cause tissue damage. Importantly, we have identified unique differences in the ocular injury and responses occurring with bleaching agents that are important to consider in the development and validation of alternative ocular irritation tests to characterize a broad range of materials differing in type and irritancy.

Bcl-2 expression in the human cornea.

The purpose of this study was to determine the localization of Bcl-2 protein in the human cornea. Anti-human Bcl-2 monoclonal antibodies (MAbs) against selective Bcl-2 peptide sequences were used to localize Bcl-2 protein immunocytochemically in fresh eye bank donor human corneas (n = 4). Specificity of each MAb was determined by Western blot analysis of pooled protein extracted from human corneal epithelium (n = 3). Expression of Bcl-2 protein in apoptotic surface epithelial cells was detected by co-labeling with TUNEL assay and anti-Bcl-2 antibody staining. Two MAbs specific for amino acids residues (aa) 41-54 within the loop domain of Bcl-2 protein stained nuclei of all corneal epithelial cell layers. MAb specific for aa 61-76, also within the loop domain, produced faint nuclei and nuclear envelope staining. Occasional corneal surface epithelial cells however, consistently lacked anti-Bcl-2 nuclear staining with these three MAbs; concomitant TUNEL assay revealed that all TUNEL positive-surface cells were Bcl-2 negative. In the stroma, keratocytes showed similar but weak anti-Bcl-2 staining. All corneal endothelial cells showed intense nuclear staining with MAbs, with no gradient or absence of staining. In summary, Bcl-2 protein can be localized to the nuclei and nuclear envelope of corneal epithelial cells, keratocytes and endothelial cells with the use of MAbs specific for the loop domain of Bcl-2. TUNEL-labeled surface epithelial cells did not stain with MAbs to Bcl-2, suggesting degradation or epitope masking perhaps by specific phosphorylation of the loop domain during apoptosis. Taken together, these findings suggest that Bcl-2 protein may play a critical role in modulating apoptotic cell desquamation in the human corneal epithelium.

Effects of rigid and soft contact lens daily wear on corneal epithelium, tear lactate dehydrogenase, and bacterial binding to exfoliated epithelial cells.

OBJECTIVE: To determine the effects of lens type and oxygen transmissibility on human corneal epithelium during daily lens wear (DW). DESIGN: Prospective, randomized, double-masked, single-center, parallel treatment groups clinical trial. PARTICIPANTS: Two hundred forty-six patients fitted with: (1) high oxygen-transmissible soft lenses (n = 36), (2) hyper oxygen-transmissible soft lenses (n = 135), and (3) hyper oxygen-transmissible rigid gas-permeable (RGP) lenses (n = 75). INTERVENTION: Irrigation chamber to collect exfoliated epithelial surface cells, confocal microscopy, and tear collection at baseline, 2 weeks, and 4 weeks of DW. MAIN OUTCOME MEASURES: (1) Pseudomonas aeruginosa (PA) binding to exfoliated corneal epithelial surface cells, (2) central epithelial thickness, (3) superficial epithelial cell area, (4) epithelial surface cell exfoliation, and (5) tear lactate dehydrogenase (LDH). RESULTS: Four weeks of DW with the high oxygen-transmissible soft lens significantly increased PA binding from baseline 6.55 +/- 3.01 to 8.75 +/- 3.05 bacteria per epithelial cell (P < 0.01). By contrast, hyper oxygen-transmissible soft lens wear increased binding significantly less (6.13 +/- 2.45 to 7.62 +/- 3.06; P < 0.01), whereas hyper oxygen-transmissible RGP lens wear demonstrated no significant changes (5.91 +/- 2.40 to 6.13 +/- 2.17; P = 0.533). No significant change in central epithelial thickness was found after 4 weeks of DW in either soft lens; however, the epithelial thickness decreased by 9.8% (P < 0.001) with RGP lens wear. Epithelial cell surface area increased 3.3% and 4.1% with the high and hyper oxygen-transmissible soft lenses, respectively, and 10.5% with the hyper oxygen-transmissible RGP lens (P < 0.001). Epithelial desquamation significantly decreased in all groups (P < 0.001). Tear LDH levels increased for all test lenses (P < 0.001). CONCLUSIONS: Increased PA binding induced by wear of a conventional soft lens material is significantly greater than that induced by the new hyper oxygen-transmissible soft silicone hydrogel lens during DW. However, both soft materials showed significant increases in PA binding as compared with baseline controls. By contrast, hyper oxygen-transmissible RGP lens DW did not increase PA binding significantly. Taken together, these findings suggest for the first time both an oxygen effect as well as a difference between soft and rigid lens types on PA binding in DW.

Pathology of ocular irritation with acetone, cyclohexanol, parafluoroaniline, and formaldehyde in the rabbit low-volume eye test.

The ocular irritation responses to 11 different surfactants and two concentrations of acetic acid and sodium hydroxide have been shown to depend on the extent of initial injury, despite marked differences in the processes leading to tissue damage. The purpose of these studies was to determine the extent to which this fundamental relationship applies to other nonsurfactants. Ten microl of acetone (ACT). cyclohexanol (CY), parafluoroaniline (PF), or 37% formaldehyde (FA) was directly applied to the cornea of the right eye of each rabbit. Eyes and eyelids were macroscopically scored for signs of irritation beginning 3 hours after dosing and periodically until recovery or 35 days. Tissues were obtained for light microscopic examination after 3 hours and on days 1, 3, and 35. Initial corneal injury was characterized quantitatively at 3 hours and I day using in vivo confocal microscopy (CM) and by postmortem quantitation of dead corneal epithelial cells and keratocytes using a Live Dead Assay (L/D, Molecular Probes) and scanning laser CM. Corneal changes over time were characterized quantitatively using in vivo CM performed at 3 hours and 1, 3, 7, 14, and 35 days. The changes with ACT were consistent with mild irritation. Corneal injury was limited to the epithelium and superficial stroma, with the mean normalized depth of injury (NDI) being less than 10% with the majority of regions showing no stromal injury. Changes with CY and PF were consistent with moderate to severe irritation, and FA caused severe irritation. Specifically, corneal injury by CY and PF tended to involve the epithelium and anterior stroma, with the mean NDI being 10.4% to 23.8%, while injury with FA involved the epithelium, deep stroma, and at times the endothelium. Interestingly, with FA significantly less injury was observed at 3 hours with a dramatic increase in injury observed at 1 day and thereafter. In conclusion, these results continue to support and extend our hypothesis that ocular irritation is principally defined by the extent of initial injury despite clear differences in the means by which irritants cause tissue damage. We believe this approach can be applied to developing alternative assays based on injury to ex vivo eyes or injury to an in vitro corneal equivalent system.

Measurement of corneal sublayer thickness and transparency in transgenic mice with altered corneal clarity using in vivo confocal microscopy.

Measurement of sublayer thickness and transparency at cellular level in the living animal are critical to understanding the role of specific transgenes and transgene products in controlling corneal development and maintenance of transparency. Using two different transgenic mouse strains having altered corneal clarity, we have evaluated the ability of in vivo confocal microscopy to measure corneal haze and localize light scattering structures. Projection of 2-D and 3-D image information identified the nature and location of light scattering within the cornea and allowed correlation of unique structural differences to transgene expression. Our findings suggest that in vivo confocal microscopy can be used to identify the effects of transgene expression on mouse corneal transparency.

Extent of initial corneal injury as a basis for alternative eye irritation tests.

Based on studies that have characterized the extent of injury occurring with irritants of differing type and severity, we have proposed that extent of initial injury is the principal mechanism underlying ocular irritation. We report here our efforts to apply this hypothesis, as a mechanistic basis, to the development of an alternative eye irritation assay using an ex vivo rabbit corneal model. Rabbit eyes were obtained immediately after sacrifice or from an abattoir and 8.5-mm diameter corneal buttons were removed and cultured overnight at an air-liquid interface under serum-free conditions. Buttons were exposed to materials of differing type (surfactant, acid, base, alcohol and aldehyde) and irritancy (slight to severe) that had been previously characterized microscopically in the rabbit low-volume eye test. Exposure was accomplished by applying 1.5 microl of an irritant to a sterile, 3 mm diameter, filter paper disk and then placing the disk on the center of the corneal button for 10 s. After removal of the disk, buttons were washed and cultured for 3, 24 or 48 h. Buttons were then evaluated for extent of injury using a Live/Dead staining kit and fluorescent microscopy to measure cell size of live surface epithelial cells, area of epithelial denudation and depth of stromal injury. Ex vivo exposure to slight irritants generally reduced surface epithelial cell size (i.e. erosion) while exposure to mild irritants produced epithelial denudation with variable injury to the corneal stroma. Severe irritants generally produced extensive epithelial denudation and damaged the corneal stroma and endothelium. Overall, ex vivo extent of injury significantly correlated with in vivo extent of injury as measured in previous animal tests (r=0.81, P<0.001). These findings indicate that extent of corneal injury, as shown to be associated with ocular irritation occurring in vivo, can be applied to the development of a mechanistically-based alternative eye irritation model. We believe that this approach may ultimately lead to an alternative assay to replace the use of animals in ocular irritation testing.

Stress fiber formation is required for matrix reorganization in a corneal myofibroblast cell line.

Corneal wound healing fibroblasts (myofibroblasts) develop a muscle-like contractile apparatus composed of prominent microfilament bundles (stress fibers) and express alpha-smooth muscle actin (alpha-SMA). In this study, gelsolin, an actin filament-severing protein, was overexpressed in a alpha-SMA-expressing corneal myofibroblast cell line (TRK43) to assess whether intact stress fibers are required for in vitro matrix organization and wound contraction. Stably integrated gelsolin was introduced by electroporation of an expression construct (pREPCG8) into cultured cells. Thirty-seven clones were isolated with half of the clones showing a fibroblastic phenotype while the remaining half appeared epithelioid. One fibroblastic clone, GS56, and one epithelioid clone, GS44, were selected for detailed characterization. The GS56 cells appeared highly elongated and spindle-shaped and had prominent stress fibers and focal adhesions. GS44 cells showed disruption of stress fibers and a cortical f-actin organization as well as the down regulation of alpha-SMA expression by immunocytochemistry and Western blotting. Both phenotypes showed enhanced gelsolin expression; however, fractionation of cell extracts demonstrated differences in the subcellular distribution of gelsolin with GS44 cells having markedly reduced and GS56 cells having markedly increased cytoskeletal gelsolin. In an in vitro wound contraction assay, epithelioid GS44 cells showed a significantly impaired ability to contract a collagen matrix compared to that of TRK43 cells, CT9 or GS56 transfectants. Loss of stress fibers in GS44 cells also correlated with enhanced cell motility. Together, these results demonstrate that the ability to form microfilament bundles or stress fibers is required for matrix organization and contraction by corneal myofibroblasts. Although no clear explanation is available, we suspect that differences in gene insertion of the gelsolin overexpression vector may have led to differential intercellular localization of gelsolin and its effect on stress fiber formation in the two cell lines.

Organization of junctional proteins in proliferating cat corneal endothelium during wound healing.

PURPOSE: To evaluate for the first time cell junctional protein organization in proliferating corneal endothelial cells during in vivo wound healing. METHODS: A total of 16 cats (32 eyes) were used in this study. A single 3-mm diameter (n = 24) or 1- to 2-mm diameter (n = 8) scrape injury was created in the central corneal endothelium of each eye. Twenty-four, 48, 72 hours or 5 days after scrape injury, eyes were collected for in situ double- or triple-labeling with phalloidin, anti-ZO-1, alpha-catenin, beta-catenin, and MIB-1 (monoclonal antibody to Ki67, a marker for actively cycling cells) and were imaged using confocal laser microscopy. RESULTS: In 3-mm diameter injuries, endothelial cells completely resurfaced the wound 48 to 72 hours after scrape injury; smaller wounds resurfaced by 48 hours. Ki67 staining was negative 24 hours after scrape injury in all cases. Ki67-positive cells were observed in the central region of the wounds after 48 and 72 hours, and mitotic figures and pairs of postmitotic cells were observed. On day 5, Ki67-positive cells were rarely detected, and no mitotic figures were observed. In the wound area, a significant increase in cell area and a reduction in hexagonality were observed in cycling cells after 48 and 72 hours. Normal apical, pericellular staining of f-actin, ZO-1, alpha-catenin, and beta-catenin was partially maintained at all times during wound healing of small and large wounds. Double-labeling confirmed that these proteins were also present along the apical cell border in Ki67-positive cells. CONCLUSIONS: After in vivo scrape injury, proliferation is limited temporally and spatially to spreading endothelial cells within the wound. Cell junctional connections appear to be maintained in actively cycling cells during healing.

Corneal haze after photorefractive keratectomy using different epithelial removal techniques: mechanical debridement versus laser scrape.

PURPOSE: To determine differences of corneal wound healing and haze after photorefractive keratectomy (PRK) using either mechanical epithelial debridement or laser-scrape epithelial removal in human subjects. DESIGN: A 6-month randomized, masked, prospective, paired-eye clinical study. PARTICIPANTS: Twenty eyes in 10 myopic patients treated between March 1999 and May 1999. INTERVENTION: Photorefractive keratectomy treatments with two different epithelial removal techniques. Continuous z-scan of confocal image, termed confocal microscopy through focusing (CMTF), was performed before surgery and at 3 weeks, 6 weeks, 3 months, and 6 months after surgery. MAIN OUTCOMES MEASURES: Epithelial and stromal thickness measurement, achieved stromal ablation depth, and objective assessment of corneal light-backscattering (corneal haze) were obtained from digital image analysis of the CMTF scans. Manifest refraction was also measured. Student's paired t test or two-way repeated-measures analysis of variance after rank transformation were performed to evaluate statistical differences between groups. RESULTS: Comparison of the mean posttreatment spherical equivalent between the two techniques showed no statistically significant difference. In preoperative corneas, mean epithelial thickness was 50.08+/-3.70 microm in the mechanical debridement group and 50.49+/-4.01 microm in laser-scrape group (not significant). For both groups, the epithelium was significantly thinner at 3 weeks, but returned to preoperative values by 6 months, with no difference between groups. Planned stromal ablation depth by PRK was 59.38+/-11.48 microm (39-73 microm; n = 8) in the mechanical group and 57.75 +/- 7.21 microm (48-70 microm; n = 8) in the laser-scrape group. Achieved stromal ablation depth was not significantly different between the two groups. Most importantly, in both groups CMTF-measured corneal haze increased significantly after surgery, peaked at 3 months, and then decreased at 6 months, with no significant difference between groups. (Power = 0.96). CONCLUSIONS: There is no significant difference in the corneal wound healing response between mechanical epithelial debridement versus laser-scrape technique in human myopic eyes undergoing PRK.

Development of a Langerhans cell-targeted gene therapy format using a dendritic cell-specific promoter.

Langerhans cells (LC), which are a skin-specific member of the dendritic cell (DC) family of antigen presenting cells, play critical roles in the initiation of cellular immune responses in the skin. We developed a LC-targeted gene therapy format in this study, aimed at the establishment of in situ protocols for genetic manipulation of LC function. Dectin-2 is a unique C-type lectin that is expressed selectively by DC, including epidermal LC. A 3.2 kb 5' flanking fragment isolated from the mouse dectin-2 gene, termed the dectin-2 promoter (pDec2), exhibited significant transcriptional activities in epidermal-derived DC lines of the XS series, but not in any of the tested non-DC lines. When pDec2-driven luciferase gene (pDec2-Luc) or enhanced green fluorescence protein gene (pDec2-EGFP) was delivered to mouse skin using the gene gun, expression of the corresponding gene product was observed in the epidermal compartment almost exclusively by the IA+ population (ie LC). LC in the gene gun-treated sites showed features of mature DC and they migrated to the draining lymph node, suggesting that LC-targeted gene expression may lead to the development of immune responses. In fact, EGFP-specific cellular immune responses became detectable after gene gun-mediated delivery of pDec2-EGFP plasmid. These results introduce a new concept that LC function can be genetically manipulated in situ by the combination of gene gun-mediated DNA delivery and a DC-specific promoter.

Quantitative characterization of acid- and alkali-induced corneal injury in the low-volume eye test.

Defining the extent of initial injury has proven to be a useful basis for differentiating the ocular irritation potential of surfactants; however, the applicability of this method to other types of irritants has not been demonstrated. In the following studies we characterized the extent of corneal injury following exposure to different concentrations of acetic acid and sodium hydroxide (NaOH) in the rabbit low-volume eye test. Groups of rabbits received 3% acetic acid, 10% acetic acid, 2% NaOH, or 8% NaOH and were evaluated in vivo by macroscopic and in vivo confocal microscopic examination and postmortem using a live/dead staining kit and scanning laser confocal microscopic examination. Quantitative assessment of macroscopic scores, corneal surface epithelial cell size, corneal epithelial thickness, corneal thickness, depth of stromal injury, corneal light scattering (confocal microscopy through focusing, CMTF), and number of dead cells was conducted at various times, including the following: at 3 hours and at 1, 3, 7, 14, and 35 days. Based on macroscopic scores, the order of ocular irritancy potential was 3% acetic acid < 2% NaOH < 10% acetic acid < 8% NaOH. Evaluation of the quantitative in vivo and postmortem microscopic live/dead data revealed a slight decrease in epithelial thickness and an increase in dead epithelial cell numbers with 3% acetic acid. With 2% NaOH, significant focal changes in epithelial cell size, epithelial thickness, corneal thickness, and number of dead surface epithelial cells occurred at 3 hours and at 1 day, with injury to only a very small number of corneal stromal keratocytes, despite the presence of epithelial denudation. Changes with 10% acetic acid were similar to those noted with 2% NaOH at 3 hours and 1 day, but these changes were more diffuse and included stromal injury to a depth of 7.2 +/- 9.3% of the corneal thickness, with significant numbers of dead keratocytes. Eight percent NaOH, on the other hand, caused focally extensive injury that averaged 26.3 +/- 18.4% of the corneal thickness at 1 day, with significant light scattering from the cornea, which did not return to normal by 35 days postinjury. Overall, these data indicate that ocular irritation as a result of acetic acid and NaOH was associated with changes similar to those observed with surfactants (ie, slight irritants damage the corneal epithelium, mild and moderate irritants damage the corneal epithelium and anterior stromal cells, and severe irritants damage the corneal epithelium and deep stroma). To our knowledge, this is the first time that the ocular irritation potential for different types of materials (acid/alkali, surfactants) has been shown to be primarily dependent on the initial area and depth of injury.

On-line 3-dimensional confocal imaging in vivo.

PURPOSE: In vivo confocal microscopy through focusing (CMTF) can provide a 3-D stack of high-resolution corneal images and allows objective measurements of corneal sublayer thickness and backscattering. However, current systems require time-consuming off-line image processing and analysis on multiple software platforms. Furthermore, there is a trade off between the CMTF speed and measurement precision. The purpose of this study was to develop a novel on-line system for in vivo corneal imaging and analysis that overcomes these limitations. METHODS: A tandem scanning confocal microscope (TSCM) was used for corneal imaging. The TSCM video camera was interfaced directly to a PC image acquisition board to implement real-time digitization. Software was developed to allow in vivo 2-D imaging, CMTF image acquisition, interactive 3-D reconstruction, and analysis of CMTF data to be performed on line in a single user-friendly environment. A procedure was also incorporated to separate the odd/even video fields, thereby doubling the CMTF sampling rate and theoretically improving the precision of CMTF thickness measurements by a factor of two. RESULTS: In vivo corneal examinations of a normal human and a photorefractive keratectomy patient are presented to demonstrate the capabilities of the new system. Improvements in the convenience, speed, and functionality of in vivo CMTF image acquisition, display, and analysis are demonstrated. CONCLUSIONS: This is the first full-featured software package designed for in vivo TSCM imaging of the cornea, which performs both 2-D and 3-D image acquisition, display, and processing as well as CMTF analysis. The use of a PC platform and incorporation of easy to use, on line, and interactive features should help to improve the clinical utility of this technology.

Corneal opacity in lumican-null mice: defects in collagen fibril structure and packing in the posterior stroma.

PURPOSE: Gene targeted lumican-null mutants (lum(tm1sc)/lum(tm1sc)) have cloudy corneas with abnormally thick collagen fibrils. The purpose of the present study was to analyze the loss of transparency quantitatively and to define the associated corneal collagen fibril and stromal defects. METHODS: Backscattering of light, a function of corneal haze and opacification, was determined regionally using in vivo confocal microscopy in lumican-deficient and wild-type control mice. Fibril organization and structure were analyzed using transmission electron microscopy. Biochemical approaches were used to quantify glycosaminoglycan contents. Lumican distribution in the cornea was elucidated immunohistochemically. RESULTS; Compared with control stromas, lumican-deficient stromas displayed a threefold increase in backscattered light with maximal increase confined to the posterior stroma. Confocal microscopy through-focusing (CMTF) measurement profiles also indicated a 40% reduction in stromal thickness in the lumican-null mice. Transmission electron microscopy indicated significant collagen fibril abnormalities in the posterior stroma, with the anterior stroma remaining relatively unremarkable. The lumican-deficient posterior stroma displayed a pronounced increase in fibril diameter, large fibril aggregates, altered fibril packing, and poor lamellar organization. Immunostaining of wild-type corneas demonstrated high concentrations of lumican in the posterior stroma. Biochemical assessment of keratan sulfate (KS) content of whole eyes revealed a 25% reduction in KS content in the lumican-deficient mice. CONCLUSIONS: The structural defects and maximum backscattering of light clearly localized to the posterior stroma of lumican-deficient mice. In normal mice, an enrichment of lumican was observed in the posterior stroma compared with that in the anterior stroma. Taken together, these observations indicate a key role for lumican in the posterior stroma in maintaining normal fibril architecture, most likely by regulating fibril assembly and maintaining optimal KS content required for transparency.

Specular microscopy, confocal microscopy, and ultrasound biomicroscopy: diagnostic tools of the past quarter century.

PURPOSE: To identify, characterize, and illustrate the most important past and future potential contributions of specular, confocal, and ultrasound biomicroscopy to clinical diagnosis and research applications in the cornea from the past 25 years. METHODS: Specular microscopy, in vivo tandem scanning confocal microscopy (TSCM), scanning slit confocal microscopy (SSCM), and high-frequency ultrasound biomicroscopy are examined. RESULTS AND CONCLUSIONS: This review demonstrates the abilities and limitations of three powerful new in vivo imaging modalities to resolve the cellular and structural layers of the cornea temporally and spatially in three or four dimensions, (x, y, z, t). Clinical pathological processes such as inflammation. infection, wound healing, toxicity, embryonic development, differentiation, and disease, which previously could be studied only under static ex vivo conditions, can now be dynamically evaluated over time. Thus, with continued development and application in vivo, noninvasive microscopic techniques should provide exciting new insights into understanding the structure and function of not only the eye, but also other multicellular organ systems in health and disease. These new imaging paradigms are in the first rank of advances in medical science in the past quarter century.

Stromal wound healing explains refractive instability and haze development after photorefractive keratectomy: a 1-year confocal microscopic study.

PURPOSE: To evaluate the mechanism(s) producing refractive instability and corneal haze development after photorefractive keratectomy (PRK). DESIGN: Prospective, nonrandomized, comparative case series, self-controlled. PARTICIPANTS: Seventeen eyes of 17 patients with low- to moderate-grade myopia (-2.88 to -9.13 diopters [D]) were included. METHODS: Surgical intervention was a standardized, 6-mm diameter PRK procedure using the Meditec MEL 60 excimer laser (Aesculap-Meditec, Heroldsberg, Germany). The photoablation center was evaluated before surgery and at 1, 3, 6, 9, and 12 months after PRK using rapid, continuous z-scans of confocal images, termed confocal microscopy through focusing (CMTF). MAIN OUTCOME MEASURES: Simultaneous epithelial and stromal thickness analysis and objective assessment of corneal light backscattering were obtained from digital image analysis of the CMTF scans. Corneal reinnervation and anterior stromal keratocyte density and wound healing morphologic features were evaluated on high resolution, in vivo confocal images. Manifest refraction was measured and corneal clarity was graded by slit-lamp biomicroscopy. RESULTS: Epithelial thickness averaged 45+/-10 microm at 1 month, 50+/-8 microm at 3 months, and 52+/-6 microm at 12 months after PRK, as compared with 51+/-4 microm before surgery, demonstrating complete restoration of the preoperative thickness without compensatory hyperplasia. Interestingly, epithelial rethickening had no significant correlation with refractive regression. By contrast, stromal regrowth (from 1-12 months) averaged 6+/-12 microm (range, 27 microm thinning-22 microm rethickening) and correlated closely (r = 0.84, P<0.001) with changes in refraction that averaged 0.84+/-1.23 D, ranging from -1.63 D (hyperopic shift) to +3.38 D (myopic regression). Stromal rethickening increased proportionally with the actual photoablation depth (r = 0.63, P<0.01); linear regression analysis suggested an average regrowth rate of 8% per year for the entire study group. Stromal rethickening was not associated with CMTF haze development over time, suggesting that haze and regression were caused by two independent wound healing mechanisms. In agreement with these findings, all "hazy" corneas showed increased numbers of anterior stromal wound healing keratocytes with increased reflectivity of both nuclei and cell bodies, suggesting that cellular-based reflections, as opposed to extracellular matrix deposition, are the major origin of increased corneal light scattering after PRK. CONCLUSIONS: Taken together, these data indicate that keratocyte-mediated regrowth of the photoablated stroma appears to be the main cause of myopic regression in humans treated with a 6-mm diameter PRK, whereas hyperopic shifts appear to be a direct consequence of stromal thinning. By contrast, the corneal epithelium appeared to restore its preoperative thickness without contributing significantly to the refractive changes after PRK. Finally, this study also provides strong evidence that the development of haze after PRK is directly associated with increased cellular reflectivity from high numbers of wound healing keratocytes.

A role for NF-kappaB-dependent gene transactivation in sunburn.

Exposure of skin to ultraviolet (UV) radiation is known to induce NF-kappaB activation, but the functional role for this pathway in UV-induced cutaneous inflammation remains uncertain. In this study, we examined whether experimentally induced sunburn reactions in mice could be prevented by blocking UV-induced, NF-kappaB-dependent gene transactivation with oligodeoxynucleotides (ODNs) containing the NF-kappaB cis element (NF-kappaB decoy ODNs). UV-induced secretion of IL-1, IL-6, TNF-alpha, and VEGF by skin-derived cell lines was inhibited by the decoy ODNs, but not by the scrambled control ODNs. Systemic or local injection of NF-kappaB decoy ODNs also inhibited cutaneous swelling responses to UV irradiation. Moreover, local UV-induced inflammatory changes (swelling, leukocyte infiltration, epidermal hyperplasia, and accumulation of proinflammatory cytokines) were all inhibited specifically by topically applied decoy ODNs. Importantly, these ODNs had no effect on alternative types of cutaneous inflammation caused by irritant or allergic chemicals. These results indicate that sunburn reactions culminate from inflammatory events that are triggered by UV-activated transcription of NF-kappaB target genes, rather than from nonspecific changes associated with tissue damage.