Tive cells per mm2. Isolation of Cervical Lymph Nodes Superficial cervical lymph nodes from each group were surgically excised, compresssed between two sterile frosted glass slides, and made into a Actimid Single-cell INCB024360 suspension. Cell populations were individually collected, centrifuged at 1000 rpm for 5 minutes, filtered, and resuspended. Single cells were processed for flow cytometry as described below. Flow Cytometry Analysis Single-cell suspensions from cervical lymph nodes were cell surface stained with PEanti-CD4, negative controls consisted of cells stained with PEisotype antibody. FITC-anti-CD8, negative 
controls consisted of cells stained with FITC-isotype antibody. Cells were then resuspended in fixation-permeabilization solution. A BD FACS Calibur was used for flow cytometry, and data were analyzed using BD Diva Software. These experiments were repeated twice. Statistical Analysis Statistical analyses were performed using SPSS 13.0 software. One-way ANOVA with Bonferroni correction was used for comparison among groups with Gaussian distributed values. The Mann-Whitney U test was used to compare non-normally distributed values between groups. p<0.05 was considered statistically significant. Results ICES Induces Corneal Epithelial Disruption Fluorescein staining assessed changes in corneal epithelial integrity. In the ICES group, after 1 week there was a slight increase in the staining score which peaked at 2 weeks and was invariant for the next 4 weeks. To evaluate if losses in the ICES group of tight junctional barrier function and epithelial integrity were accompanied by increases in MMP-9 5 / 18 Dynamic Changes Induced in Experimental Murine Dry Eye expression, its staining pattern was also evaluated. In parallel with the development of corneal fluorescein staining, MMP-9 became the most intense after 2 weeks of ICES, which was unchanged during the following 4 and 6 weeks. We also compared if there were differences in the development of MMP-9 expression in the ICES and SCOP groups. In the SCOP group, the MMP-9 expression was greater at all times than in the ICES group. 6 / 18 Dynamic Changes Induced in Experimental Murine Dry Eye ICES Induces Corneal Epithelium Apoptosis Caspase-3 immunofluorescence and TUNEL analyses were performed to evaluate the effect of ICES exposure on corneal epithelial apoptosis. Fig. 3A shows that caspase-3 expression increased after the first 2 weeks. It peaked already at 2-weeks and remained invariant at the 4-week and 6-week time points. Similarly, PubMed ID:http://jpet.aspetjournals.org/content/123/3/180 TUNEL results also demonstrated that the apoptotic cells increased in the ICES group, but remained unchanged at 2-, 4- and 6-weeks.. On the other hand, corneal epithelial 
apoptosis in the SCOP group seemed more pronounced at all of the same time points as those in the ICES group expect for a lack of a difference after 6 weeks. ICES Stimulates Inflammatory Cytokine Production in the Conjunctiva and Lacrimal Gland Levels of conjunctival IL-17, IL-23, IL-6, IL-1, TNF- mRNA transcripts peaked after 2 weeks in the ICES group without any further change during the subsequent 4 weeks. In contrast, conjunctival IFN- and TGF-2 levels increased in the ICES group and peaked at 6-weeks. However, the gene transcript levels of all of these cytokines in the SCOP group were higher than those in the ICES group at all the time points. 7 / 18 Dynamic Changes Induced in Experimental Murine Dry Eye The pattern changes in the transcript levels of all of these cytokines in the lacri.Tive cells per mm2. Isolation of Cervical Lymph Nodes Superficial cervical lymph nodes from each group were surgically excised, compresssed between two sterile frosted glass slides, and made into a single-cell suspension. Cell populations were individually collected, centrifuged at 1000 rpm for 5 minutes, filtered, and resuspended. Single cells were processed for flow cytometry as described below. Flow Cytometry Analysis Single-cell suspensions from cervical lymph nodes were cell surface stained with PEanti-CD4, negative controls consisted of cells stained with PEisotype antibody. FITC-anti-CD8, negative controls consisted of cells stained with FITC-isotype antibody. Cells were then resuspended in fixation-permeabilization solution. A BD FACS Calibur was used for flow cytometry, and data were analyzed using BD Diva Software. These experiments were repeated twice. Statistical Analysis Statistical analyses were performed using SPSS 13.0 software. One-way ANOVA with Bonferroni correction was used for comparison among groups with Gaussian distributed values. The Mann-Whitney U test was used to compare non-normally distributed values between groups. p<0.05 was considered statistically significant. Results ICES Induces Corneal Epithelial Disruption Fluorescein staining assessed changes in corneal epithelial integrity. In the ICES group, after 1 week there was a slight increase in the staining score which peaked at 2 weeks and was invariant for the next 4 weeks. To evaluate if losses in the ICES group of tight junctional barrier function and epithelial integrity were accompanied by increases in MMP-9 5 / 18 Dynamic Changes Induced in Experimental Murine Dry Eye expression, its staining pattern was also evaluated. In parallel with the development of corneal fluorescein staining, MMP-9 became the most intense after 2 weeks of ICES, which was unchanged during the following 4 and 6 weeks. We also compared if there were differences in the development of MMP-9 expression in the ICES and SCOP groups. In the SCOP group, the MMP-9 expression was greater at all times than in the ICES group. 6 / 18 Dynamic Changes Induced in Experimental Murine Dry Eye ICES Induces Corneal Epithelium Apoptosis Caspase-3 immunofluorescence and TUNEL analyses were performed to evaluate the effect of ICES exposure on corneal epithelial apoptosis. Fig. 3A shows that caspase-3 expression increased after the first 2 weeks. It peaked already at 2-weeks and remained invariant at the 4-week and 6-week time points. Similarly, PubMed ID:http://jpet.aspetjournals.org/content/123/3/180 TUNEL results also demonstrated that the apoptotic cells increased in the ICES group, but remained unchanged at 2-, 4- and 6-weeks.. On the other hand, corneal epithelial apoptosis in the SCOP group seemed more pronounced at all of the same time points as those in the ICES group expect for a lack of a difference after 6 weeks. ICES Stimulates Inflammatory Cytokine Production in the Conjunctiva and Lacrimal Gland Levels of conjunctival IL-17, IL-23, IL-6, IL-1, TNF- mRNA transcripts peaked after 2 weeks in the ICES group without any further change during the subsequent 4 weeks. In contrast, conjunctival IFN- and TGF-2 levels increased in the ICES group and peaked at 6-weeks. However, the gene transcript levels of all of these cytokines in the SCOP group were higher than those in the ICES group at all the time points. 7 / 18 Dynamic Changes Induced in Experimental Murine Dry Eye The pattern changes in the transcript levels of all of these cytokines in the lacri.
