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From the Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, Texas.
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METHODS. 129SvEv/CD-1 mixed mice aged 6 to 8 weeks were treated with systemic scopolamine and exposure to an air draft for different lengths of time, from 4 hours to 10 days. Untreated mice were used as the control. The concentrations of IL-1ß and TNF-
in tear fluid washings and in corneal and conjunctival epithelia were measured by ELISA. MMP-9 in tear washings was evaluated by zymography, and gelatinase activity in the cornea and conjunctiva was determined by in situ zymography. Corneal and conjunctival epithelia were lysed in RIPA buffer for Western blot with MAPK antibodies, or they were lysed in 4 M guanidium thiocyanate solution for extraction of total RNA, which was used to determine gene expression by semiquantitative RT-PCR, real-time PCR, and gene array.
RESULTS. Compared with those in age-matched control subjects, the concentrations of IL-1ß and MMP-9 in tear fluid washings and the concentrations of IL-1ß and TNF- and gelatinolytic activity in the corneal and conjunctival epithelia were significantly increased in mice receiving treatments to induce dry eye after 5 or 10 days. The expression of IL-1ß, TNF-, and MMP-9 mRNA by the corneal and conjunctival epithelia was also stimulated in mice treated for 5 or 10 days. The levels of phosphorylated JNK1/2, ERK1/2, and p38 MAPKs in the corneal and conjunctival epithelia were markedly increased as early as 4 hours after treatment, and they remained elevated up to 5 days.
CONCLUSIONS. Experimental dry eye stimulates expression and production of IL-1ß, TNF-, and MMP-9 and activates MAPK signaling pathways on the ocular surface. MAPKs are known to stimulate the production of inflammatory cytokines and MMPs, and they could play an important role in the induction of these factors that have been implicated in the pathogenesis of dry eye disease.
activate JNK, ERK, and p38 MAPK signaling pathways in a variety of cell types, including epithelial cells,15 vascular endothelial cells,16 and fibroblasts.17 18 The activated kinases initiate a cascade of protein phosphorylation involving multiple other kinases and activate nuclear transcription factors such as NF
B, AP-1, and ATF,19 20 which stimulate expression of inflammatory cytokines, chemokines such as IL-8,21 and MMPs such as MMP-1, -9, and -13.22 23 The purpose of this study was to use a previously reported murine model of KCS to evaluate whether experimental dryness stimulates the expression and production of inflammatory molecules that have been identified in human KCS and activates MAPK signaling pathways. |
Animals and Methods |
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Tear Fluid Washings
Tear fluid washings were collected by a previously reported method.26 Briefly, 1.5 µL of PBS containing 0.1% bovine serum albumin (BSA) was instilled into the conjunctival sac. The tear fluid and buffer were collected with a 1-µL volume glass capillary tube (Drummond Scientific Co., Broomhall, PA) by capillary action from the tear meniscus in the lateral canthus. The 2-µL sample of tear washings was pooled from both eyes of each mouse and was stored at –80°C until zymography and ELISA were performed.
ELISA and Gelatin Zymography
The corneal and conjunctival epithelia from five groups of mice (12 eyes per group), including untreated control mice and mice treated with subcutaneous injections of scopolamine and placement in a blower hood for 1 day or 3, 5, or 10 days, were collected, lysed and subjected to total protein assay with a bicinchoninic protein assay kit (Micro BCA; Pierce, Rockford, IL). The concentrations of IL-1ß and TNF- in the cell lysates and in the tear fluid washings were determined by ELISA kits for mouse IL-1ß and TNF- (Quantikine M Murine; R&D Systems, Minneapolis, MN), according to the manufacturer’s protocol. The level of gelatinolytic enzymes in tear fluid washings was measured by SDS-PAGE gelatin zymography, according to a previously reported method.27 A 2-µL tear-washings sample (pooled from both eyes of each mouse) was added to SDS sample buffer and fractionated on an 8% polyacrylamide gel containing gelatin (0.5 mg/mL) by electrophoresis. The gels were soaked in 0.25% Triton X-100 for 30 minutes at RT to remove the SDS, then incubated in a digestion buffer containing 50 mM Tris-HCl, 150 mM NaCl, 10 mM CaCl2, 2 µM ZnSO4, 0.01% Brij-35, and 5 mM phenylmethylsulfonyl fluoride (PMSF), a serine protease inhibitor, at 37°C overnight to allow proteinase digestion of its substrate. The gels were rinsed in distilled water and stained with 0.25% Coomassie brilliant blue R-250 in 40% isopropanol for 2 hours and destained with 10% acetic acid.
In Situ Zymography
In situ zymography was performed to localize the gelatinase activity in the cornea and conjunctiva by a modification of a previous method.28 The fresh whole eyes with their eyelids and conjunctiva were embedded in a mixture of 75% (vol) OCT compound (Sakura Finetek USA. Inc., Torrance, CA) and 25% (vol) Immu-Mount (Thermo-Shandon, Pittsburgh, PA), then frozen in liquid nitrogen. Sections (10 µm) were cut on a cryostat (Leica, Wetzlar, Germany) and stored at –80°C until use. They were then thawed and incubated overnight with reaction buffer (0.05 M Tris-HCl [pH 7.6], 0.15 M NaCl, 5 mM CaCl2, and 0.2 mM NaN3), containing 40 µg/mL FITC-labeled DQ gelatin, which is available in a gelatinase-collagenase assay kit (EnzChek; Molecular Probes, Eugene, OR). As a negative control, 50 µM 1,10-phenanthroline, a inhibitor of metalloproteinases, was added to the reaction buffer before the FITC-labeled DQ gelatin was applied to frozen sections. Proteolysis of the FITC-labeled DQ gelatin substrate yields cleaved gelatin-FITC peptides that are fluorescent. The localization of fluorescence indicates the sites of net gelatinolytic activity. After incubation, the sections were washed three times with PBS for 5 minutes, counterstained with 1 µg/mL Hoechst 33342 dye (Sigma-Aldrich) in an anti-fade medium (Gel/Mount; Fisher, Atlanta, GA), and covered with 22 x 50 mm coverslip. The gelatinolytic activity of MMPs was localized and photographed by a fluorescence microscope (Eclipse E400; Nikon, Garden City, NY). Images were acquired by a digital camera (DMX 1200; Nikon).
Western Blot Analysis
The corneal and conjunctival epithelia collected and pooled from each group were lysed in RIPA lysis buffer containing 50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 2 mM sodium fluoride, 2 mM EDTA, 0.1% SDS, and an EDTA-free protease inhibitor cocktail tablet (Roche Applied Science, Indianapolis, IN). The cell extracts were centrifuged at 14,000g for 15 minutes at 4°C, and the supernatants were used for experiments. The total protein concentrations of the cell extracts were determined with a protein assay kit (Micro BCA; Pierce). The protein samples (50 µg per lane) were mixed with 6x SDS reducing sample buffer and boiled for 5 minutes before loading. Proteins were separated by SDS polyacrylamide gel electrophoresis (4%–15% Tris-HCl; gradient gels from Bio-Rad, Hercules, CA) and transferred electronically to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA). The membranes were blocked with 5% nonfat milk in TTBS (50 mM Tris [pH 7.5], 0.9% NaCl, and 0.1% Tween-20) for 1 hour at room temperature (RT) and then incubated 2 hours at RT with a 1:1000 dilution of rabbit antibody against phospho-p38 MAPK (Cell Signaling, Beverly, MA), 1:100 dilution of rabbit antibody against phospho-JNK, or 1:500 dilution of monoclonal antibody against phospho-p44/42 ERK (Santa Cruz Biotechnology, Santa Cruz, CA). After three washings with TTBS, the membranes were incubated for 1 hour at RT with the horseradish-peroxidase–conjugated secondary antibody goat anti-rabbit IgG (1:2000 dilution; Cell Signaling) or with goat anti-mouse IgG (1:5000 dilution; Pierce). After the membranes were washed four times, the signals were detected with an enhanced chemiluminescence reagent (ECL; Amersham, Piscataway, NJ) and the images were acquired (model 2000R; Eastman Kodak, New Haven, CT). The membranes were stripped in 62.5 mM Tris-HCl (pH 6.8), containing 2% SDS and 100 mM ß-mercaptoethanol at 60°C for 30 minutes and then were reprobed with 1:100 dilution of rabbit antibody against JNK (Santa Cruz Biotechnology) or 1:1000 dilution of rabbit antibodies against ERK or p38 MAPK (Cell Signaling). These three antibodies detect both the phosphorylated and unphosphorylated forms that represent the total levels of the MAPKs. The signals were detected and captured as described earlier.
RNA Isolation and Semiquantitative RT-PCR
Total RNA from the corneal and conjunctival epithelia collected and pooled from each group (10 eyes per group for each experiment) was isolated by an acid guanidium thiocyanate-phenol-chloroform extraction method,29 and stored at –80°C until use. Gene expression was analyzed by reverse transcription–polymerase chain reaction (RT-PCR)29 30 using a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as the internal control. In brief, first-strand cDNA was synthesized from 0.5 µg of total RNA with MuLV reverse transcriptase. PCR amplification of the first-strand cDNAs was performed with specific primer pairs for murine IL-1ß, TNF-, MMP-9, and GAPDH mRNA (Table 1) . Semiquantitative RT-PCR was established by terminating reactions at intervals of 24, 28, 32, 36, and 40 cycles for each primer pair to ensure that the PCR products formed were within the linear portion of the amplification curve.
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Statistical Analysis
Based on the normality of the data distribution, the t-test or Mann-Whitney test was used for statistical comparison of assay results between groups.
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Results |
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in the Corneal and Conjunctival Epithelia by ELISA in the corneal and conjunctival epithelia was not significantly higher after treatment for 1 to 5 days (P > 0.05); however, by 10 days the concentration in the corneal and conjunctival epithelia was significantly increased to 30.55 ± 2.71 pg/mg (P < 0.01) and 20.59 ± 11.40 pg/mg (P < 0.05), respectively, compared with 11.12 ± 6.89 and 5.90 ± 3.24 pg/mg in untreated eyes, respectively (Fig. 3B) .
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, and MMP-9 mRNA by Ocular Surface Epithelia, and MMP-9, as well as a housekeeping gene GAPDH, were evaluated by conventional semiquantitative RT-PCR with pooled total RNA samples of corneal or conjunctival epithelia obtained from four groups (10 eyes per group) of untreated mice and the mice treated with systemic scopolamine and placement in a blower hood for 1, 5, or 10 days (20 mice in each experiment). The levels of transcripts of IL-1ß, TNF-, and MMP-9 in the corneal and conjunctival epithelia of mice treated for 1 day were no different from untreated control eyes, but these were noted to increase at 5 and 10 days (Fig. 5) .
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, and MMP-9 mRNA by Ocular Surface Epithelia, and MMP-9 mRNA, as well as the housekeeping gene GAPDH, were evaluated by real-time PCR, using pooled total RNA samples of corneal or conjunctival epithelia (10 eyes per group) from untreated mice and mice treated for 1, 5, or 10 days (20 mice in each experiment). The use of real-time PCR confirmed the findings of conventional RT-PCR and provided a measure of the relative increase in mRNA levels of these factors after dry eye treatment. Quantitation of gene expression could be performed because the slope of the standard curve for each gene showed a similar efficiency of amplification (Fig. 6A) , and the real-time PCR produced a single specific product for each of the primer sets used (LUX; Invitrogen), proved by melting temperature analysis for each gene (Fig. 6B) . Nontemplate control experiments showed absence of DNA contamination. The comparative CT (threshold cycle) method was used to determine the difference (
CT) between the CT of each time point of the treatment and the CT of the untreated control. Before subtraction, the CT was normalized by the CT of an endogenous reference gene, GAPDH. The experiment was performed three times to ensure reproducibility of results. Because of a limited amount of RNA available from each experiment, not all sets of total RNA samples used for real-time PCR assays were from the same eyes that were used for the conventional RT-PCR shown in Figure 5 . Similar to the conventional RT-PCR, higher levels of IL-1ß, TNF-, and MMP-9 mRNAs were observed in the corneal (Fig. 7 , CE) and conjunctival (Fig. 7 , JE) epithelia after treatment for 5 or 10 days.
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. Because IL-1ß, TNF-, and MMP-9 mRNA were observed to increase after 5 days of treatment by conventional RT-PCR and real-time PCR (Figs. 5 7) , we performed the gene array analysis using total RNAs from the untreated and the 5-day–treated corneal epithelia. Several transcripts of inflammatory cytokines and receptors were found to be stimulated in the corneal epithelia by day 5 of treatment, including IL-1ß, IL-1 receptor 1 (IL-1R1), IL-1 receptor 2 (IL-1R2), TNF-, and TNF receptor 1 (TNFR1), as shown in Figure 8 .
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Discussion |
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, and MMP-9) and activates MAPK signaling pathways on the ocular surface epithelia.
Ocular Surface Inflammation in Experimental Dry Eye
Increased levels of proinflammatory cytokines and MMPs have been observed on the ocular surface of patients with KCS.2 34 35 36 Furthermore, inflammation has been observed to develop in neurturin-deficient mice that have naturally occurring and permanent dry eye. These mice have been observed to have increased concentrations of IL-1ß and MMP-9 in tear fluid washings and stimulated expression of IL-1ß, TNF-, macrophage inflammatory protein 2 (MIP-2), cytokine-induced neutrophil chemoattractant (KC), and MMP-9 mRNA by the corneal epithelia as the dry eye develops with age.26 The present study provides convincing evidence that ocular surface inflammation develops in response to experimentally induced dry eye in mice. The concentrations of IL-1ß and MMP-9 in the tear fluid washings and gelatinolytic activity on the ocular surface epithelia were observed to increase significantly in mice treated with subcutaneous injections of scopolamine and placement in a blower hood for 5 or 10 days (Figs. 1 2 4) . The increased IL-1ß concentration in the tear fluid washings could be due in part to a concentration effect caused by decreased tear volume. However, experimental evidence suggests that this is not the only cause. IL-1ß was measured in tear fluid washings, rather than in pure collected tear fluid. IL-1ß in the tear fluid was collected by placing a 1.5-µL drop of PBS on the ocular surface and collecting 1 µL. Therefore, all these samples were diluted tear washings, and the dilution factor was greater in the dry eyes. Using a fluorescein dilution method, we calculated the tear volume to be approximately 0.1 µL in normal eyes and 0.01 µL in dry eyes. Using these tear volumes, an approximate real tear concentration of IL-1ß was calculated. In normal eyes, the tear fluid is diluted approximately 16 times (0.1 µL tears + 1.5 µL PBS). In contrast, the tear fluid in dry eyes was diluted approximately 151 times (0.01 µL tears + 1.5 µL PBS). The measured IL-1ß concentrations of 20 and 80 pg/mL in the tear fluid washings of untreated mice and mice treated for 10 days, respectively (Fig. 1) , multiplied by these dilution factors, yields an estimated true tear fluid concentration of 320 pg/mL in normal eyes and 12,080 pg/mL in dry eyes. This estimated 38-fold difference is much higher than the 10-fold difference that would be expected from the difference in tear volume. Therefore, it is likely that the increased IL-1ß concentration in the tear washings of mice with dry eye is produced by the lacrimal glands and/or the stressed ocular surface epithelia. We also found the concentrations of IL-1ß and TNF- significantly increased in the corneal and conjunctival epithelia of the mice treated for 5 or 10 days (Fig. 3) . In addition, both conventional RT-PCR and real-time PCR showed that the levels of IL-1ß, TNF-, and MMP-9 mRNA in the corneal and conjunctival epithelia noticeably increased in mice treated for 5 or 10 days, compared with untreated mice (Figs. 5 7) . The gene array data further supported that dry eye induces inflammation at the transcriptional level by showing increased expression of inflammatory cytokines (IL-1ß, TNF-) and their receptors (IL-1R1, IL-1R2, and TNFR1) in the corneal epithelia of the mice treated for 5 days (Fig. 8) . It is likely that elevated levels of these cytokines in the corneal epithelium were due to stimulated production by the corneal epithelial cells, because we did not detect any infiltration of inflammatory cells into the corneal epithelium over a 2-week dry eye treatment period. In contrast, slight infiltration of the conjunctival epithelium and stroma with inflammatory cells has been observed in this murine dry eye model, and these cells could be responsible for some of the increased levels of inflammatory cytokines. IL-1 is a potent inducer of other inflammatory cytokines such as IL-6 and TNF-, and chemokines such as IL-8.37 In mice, IL-1 and TNF- stimulate the production of the key chemoattractants KC and MIP-2.38 39 IL-1 and TNF- also stimulate the production of MMPs by epithelial and inflammatory cells.30 40 The gelatinase MMP-9 is one of the most important MMPs on the ocular surface. Overexpression of MMP-9 by the corneal epithelia has been reported to impede re-epithelialization of the cornea after experimental thermal injury in animal models and has been associated with sterile corneal ulceration in humans.41 The concentration and activity of MMP-9 have been found to be significantly higher in the tear fluid of patients with KCS, with the highest levels observed in patients with sterile ulceration.5 MMP-9 is also an efficient activator of latent precursor cytokines, including TGF-ß1 and IL-1ß.42 We used multiple methods to demonstrate conclusively that ocular surface dryness induces the production of these key inflammatory factors and provides a model for studying their roles in the pathogenesis of KCS.
Effect of Dry Eye on MAPK Signaling Pathways
MAPKs are major cell-signaling mediators that play vital roles in the cellular response to stress. The JNK and p38 MAPK cascades are strongly activated by cellular stresses, as well as by proinflammatory agents such as endotoxin, IL-1, and TNF-.43 44 45 In contrast, ERK MAPK is strongly activated by growth factors such as platelet-derived growth factor, as well as many other stimuli that mediate cell proliferation, differentiation, and survival.46 Each MAPK pathway is activated by phosphorylation of threonine and tyrosine residues by upstream dual-specificity MAPK kinases (MKKs): ERK is activated by MKK1 and -2, p38 by MKK3 and -6, and JNK by MKK4 and -7. The mechanism of activation and the functional role of each MAPK cascade is dependent on the particular cell type and the type of stimuli used.47 In this study, immunoblot analysis was performed with antibodies specific for the activated or phosphorylated forms of these MAPKs (p-JNK, p-ERK, and p-p38). Our findings showed that compared with untreated mice, the phosphorylated forms of JNK1/2, ERK1/2, and p38 were markedly increased in the corneal and conjunctival epithelia of the mice treated for 4 hours or 1 day or 3 or 5 days (Fig. 9) . Scopolamine drops and/or the blower in a dry atmosphere or scopolamine injection alone did not activate MAPKs after 4 hours of treatment. In contrast, scopolamine injection combined with exposure to a blower for 4 hours was found to activate MAPKs (Fig. 10) . It is possible that scopolamine or dry atmosphere has some effect on activation of MAPKs with long-term treatment. However, the dry eye stress on the ocular surface appeared to be primarily responsible for MAPKs activation in our dry eye model. We found that the levels of phospho-JNK2 in response to dry eye were higher in the corneal epithelia than in the conjunctival epithelia (Fig. 9B) . This may indicate that the cornea and conjunctiva differ in their reactivity to dry eye stress. In mice, this could be because only the cornea, but not the conjunctiva, is exposed in the palpebral aperture.
MAPK intracellular signaling pathways have been demonstrated to play a central role in regulating a wide range of inflammatory responses in many different cell types. Activation of JNK/c-Jun and ERK1/2 MAPK signal transduction pathways leads to activation of murine peritoneal macrophages.48 p38 MAPK activity mediates TNF- and MIP-2 release, and migration of neutrophils and macrophages toward the chemokines MIP-2 and KC after lipopolysaccharide (LPS)-stimulation.49 Cross-talk between ERK and p38 MAPK mediates selective suppression of proinflammatory cytokine production by TGF-ß.50 p38 kinase appears to be involved in the stimulated release of IL-1ß and the sustained neutrophilic response in rat airway inflammation.51 In addition, MAPK cascades regulate the expression and activity of some MMPs (MMP-9, -1, -3, and -13), through activation of transcription factors such as NFB, AP-1, and ATF in different target cells.22 52 53 54 These findings suggest that these three activated MAPK pathways could mediate the production of proinflammatory cytokines and MMP-9 by stressed ocular surface epithelia in our murine dry eye model.
The mechanism of activation of MAPKs in this dry eye model is still unknown. The JNK, ERK, or p38 MAPK signaling pathways have been reported to be activated by inflammatory cytokines such as IL-1ß and TNF-,43 45 47 which are also known to stimulate expression and activity of MMPs in a variety of cell types.22 23 30 In this study, the production of IL-1ß and TNF- proteins in the corneal and conjunctival epithelia was not increased until treatment for 5 days (Fig. 3) , and the expression of their mRNA was not increased in 1-day treated mice (Figs. 5 7) . However, the levels of activated JNK, ERK, and p38 were observed to increase markedly within 4 hours of treatment (Fig. 9) . It suggests that the earlier activated MAPK signaling pathways in our dry eye model may play a role in the induction of inflammatory cytokines IL-1ß and TNF-, which could further trigger MAPK activation in turn and stimulate the expression of MMP-9. Further studies are needed to define the relationship between MAPK activation and stimulated production of IL-1ß and TNF- on the ocular surface. How are MAPKs activated in this model of dry eye? One of the original stimuli may come from the hyperosmotic stress of dry eye on the ocular surface. Hyperosmolarity of the tear film has been recognized as a key factor in the pathogenesis of dry eye and has been proposed as a gold standard for diagnosis of dry eye.14 In a preliminary study, we found that hyperosmotic stress can activate the JNK pathway and stimulate the expression of the inflammatory cytokines (IL-1ß and TNF-) and MMP-9 by cultured human corneal epithelial cells (Li, et al. IOVS 2002;43:ARVO E-Abstract 1981). The effect of the tear film’s hyperosmolarity on the activation of MAPKs on the ocular surface epithelia of mice remains to be determined.
In conclusion, our findings demonstrate that experimentally induced dry eye in mice stimulates ocular surface inflammation mimicking human KCS and activates three MAPK intracellular signaling pathways: JNKs, ERKs, and p38. These MAPKs may be relevant therapeutic targets in human KCS.
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Footnotes |
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Supported by National Eye Institute Grant EY11915 (SCP), an unrestricted Grant from Research to Prevent Blindness, the Oshman Foundation, and the William Stamps Farish Fund.
Submitted for publication October 17, 2003; revised March 30, June 14, and August 19, 2004; accepted September 1, 2004.
Disclosure: L. Luo, None; D.-Q. Li, None; A. Doshi, None; W. Farley, None; R.M. Corrales, None; S.C. Pflugfelder, None
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: Stephen C. Pflugfelder, Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, 6565 Fannin Street, NC-205, Houston, TX 77030; stevenp{at}bcm.tmc.edu.
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L. Tong, R. M. Corrales, Z. Chen, A. L. Villarreal, C. S. De Paiva, R. Beuerman, D.-Q. Li, and S. C. Pflugfelder Expression and Regulation of Cornified Envelope Proteins in Human Corneal Epithelium Invest. Ophthalmol. Vis. Sci., May 1, 2006; 47(5): 1938 - 1946. [Abstract] [Full Text] [PDF]
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 Z. Chen, W. H. Evans, S. C. Pflugfelder, and D.-Q. Li Gap Junction Protein Connexin 43 Serves as a Negative Marker for a Stem Cell-Containing Population of Human Limbal Epithelial Cells Stem Cells, May 1, 2006; 24(5): 1265 - 1273. [Abstract] [Full Text] [PDF]
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 J. Y. Niederkorn, M. E. Stern, S. C. Pflugfelder, C. S. De Paiva, R. M. Corrales, J. Gao, and K. Siemasko Desiccating Stress Induces T Cell-Mediated Sjogren's Syndrome-Like Lacrimal Keratoconjunctivitis J. Immunol., April 1, 2006; 176(7): 3950 - 3957. [Abstract] [Full Text] [PDF]
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J. He and H. E. P. Bazan Synergistic Effect of Platelet-Activating Factor and Tumor Necrosis Factor-{alpha} on Corneal Myofibroblast Apoptosis. Invest. Ophthalmol. Vis. Sci., March 1, 2006; 47(3): 883 - 891. [Abstract] [Full Text] [PDF]
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C. S. De Paiva, S. C. Pflugfelder, and D.-Q. Li Cell Size Correlates with Phenotype and Proliferative Capacity in Human Corneal Epithelial Cells Stem Cells, February 1, 2006; 24(2): 368 - 375. [Abstract] [Full Text] [PDF]
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 X. Zhou, F. Li, L. Kong, H. Tomita, C. Li, and W. Cao Involvement of Inflammation, Degradation, and Apoptosis in a Mouse Model of Glaucoma J. Biol. Chem., September 2, 2005; 280(35): 31240 - 31248. [Abstract] [Full Text] [PDF]
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