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Dynamic ocular surface and lacrimal gland changes induced in experimental murine dry eye.

Xiao B, Wang Y, Reinach PS, Ren Y, Li J, Hua S, Lu H, Chen W - PLoS ONE (2015)

Bottom Line: To determine if there is a correlation between severity effects in these models and underlying pathophysiological responses, we compared the time dependent changes in each of these parameters that occur during a 6 week period.Interleukin-17(IL-17), IL-23, IL-6, IL-1, TNF-α, IFN-γ and TGF-β2 levels were estimated by real-time PCR measurements of conjunctival and lacrimal gland samples (LGs).Subsequently, the disease process stabilized for the next four weeks.

View Article: PubMed Central - PubMed

Affiliation: School of Ophthalmology and Optometry, Wenzhou Medical University, Zhejiang, China.

ABSTRACT
Dry eye disease can be a consequence of lacrimal gland insufficiency in Sjögren's Syndrome or increased tear film evaporation despite normal lacrimal gland function. To determine if there is a correlation between severity effects in these models and underlying pathophysiological responses, we compared the time dependent changes in each of these parameters that occur during a 6 week period. Dry eye was induced in 6-week-old female C57BL/6 mice by exposing them to an Intelligently Controlled Environmental System (ICES). Sixty mice were housed in ICES for 1, 2, 4 and 6 weeks respectively. Twelve were raised in normal environment and received subcutaneous injections of scopolamine hydrobromide (SCOP) 3 times daily for 5 days. Another sixty mice were housed in a normal environment and received no treatment. Corneal fluorescein staining along with corneal MMP-9 and caspase-3 level measurements were performed in parallel with the TUNEL assay. Interleukin-17(IL-17), IL-23, IL-6, IL-1, TNF-α, IFN-γ and TGF-β2 levels were estimated by real-time PCR measurements of conjunctival and lacrimal gland samples (LGs). Immunohistochemistry of excised LGs along with flow cytometry in cervical lymph nodes evaluated immune cell infiltration. Light and transmission electron microscopy studies evaluated LGs cytoarchitectural changes. ICES induced corneal epithelial destruction and apoptosis peaked at 2 weeks and kept stable in the following 4 weeks. In the ICES group, lacrimal gland proinflammatory cytokine level increases were much lower than those in the SCOP group. In accord with the lower proinflammatory cytokine levels, in the ICES group, lacrimal gland cytosolic vesicular density and size exceeded that in the SCOP group. ICES and SCOP induced murine dry eye effects became progressively more severe over a two week period. Subsequently, the disease process stabilized for the next four weeks. ICES induced local effects in the ocular surface, but failed to elicit lacrimal gland inflammation and cytoarchitectural changes, which accounts for less dry eye severity in the ICES model than that in the SCOP model.

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Inflammatory cells infiltration of the Lacrimal Gland.A-E, immunostained for CD4(A), CD8(B), CD103(C), CD11b(D), CD45(E) in the lacrimal glands sections. F, Cell counts in mouse lacrimal glands stained by immunohistochemistry for CD4, CD8, CD103, CD11b, CD45 sections in the normal group (N), the scopolamine-treated group (SCOP), and after desiccating stress in ICES for 1 week, 2 week, 4 week and 6 week (E1,E2,E4,E6). # P < 0.01 versus the scopolamine-treated group (SCOP), * P<0.05 versus the normal group(N) (Mann-Whitney U test). Original magnification:x20;scale bars = 50 μm. Experiments were repeated three times with two mice per group per experiment.
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pone.0115333.g005: Inflammatory cells infiltration of the Lacrimal Gland.A-E, immunostained for CD4(A), CD8(B), CD103(C), CD11b(D), CD45(E) in the lacrimal glands sections. F, Cell counts in mouse lacrimal glands stained by immunohistochemistry for CD4, CD8, CD103, CD11b, CD45 sections in the normal group (N), the scopolamine-treated group (SCOP), and after desiccating stress in ICES for 1 week, 2 week, 4 week and 6 week (E1,E2,E4,E6). # P < 0.01 versus the scopolamine-treated group (SCOP), * P<0.05 versus the normal group(N) (Mann-Whitney U test). Original magnification:x20;scale bars = 50 μm. Experiments were repeated three times with two mice per group per experiment.

Mentions: In order to further characterize differences in LG inflammation between the ICES and SCOP groups, we determined if increases in different inflammatory immune cell numbers correspond with rises in proinflammatory cytokine gene expression. Accordingly, we compared in the ICES and N groups CD4, CD8α immunohistochemistry (predominantly expressed on the surface of cytotoxic T cells), CD11b (a marker of monocyte/macrophage lineage), CD103 (a marker of intraepithelial lymphocytes) and CD45 (a marker of all leukocytes and largely naive T lymphocytes) positive T lymphocyte cell numbers (Fig. 5). CD8α cells in the ICES group were less than in the normal group (N). On the other hand, CD103 increased significantly after 1 week in the ICES group and remained elevated at the same level after 2-, 4- and 6-weeks, while CD4 levels in the ICES group remained at the baseline level at all the times. ICES group CD11b cells were the only ones that increased after 2 weeks. In the 2-week group, CD45 cell levels rose after 2 and 4-weeks. However, the numbers of infiltrated CD4, CD11b, CD103, CD45 cells in the SCOP group were much larger than those in the ICES group at all the times (Fig. 5).


Dynamic ocular surface and lacrimal gland changes induced in experimental murine dry eye.

Xiao B, Wang Y, Reinach PS, Ren Y, Li J, Hua S, Lu H, Chen W - PLoS ONE (2015)

Inflammatory cells infiltration of the Lacrimal Gland.A-E, immunostained for CD4(A), CD8(B), CD103(C), CD11b(D), CD45(E) in the lacrimal glands sections. F, Cell counts in mouse lacrimal glands stained by immunohistochemistry for CD4, CD8, CD103, CD11b, CD45 sections in the normal group (N), the scopolamine-treated group (SCOP), and after desiccating stress in ICES for 1 week, 2 week, 4 week and 6 week (E1,E2,E4,E6). # P < 0.01 versus the scopolamine-treated group (SCOP), * P<0.05 versus the normal group(N) (Mann-Whitney U test). Original magnification:x20;scale bars = 50 μm. Experiments were repeated three times with two mice per group per experiment.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4295848&req=5

pone.0115333.g005: Inflammatory cells infiltration of the Lacrimal Gland.A-E, immunostained for CD4(A), CD8(B), CD103(C), CD11b(D), CD45(E) in the lacrimal glands sections. F, Cell counts in mouse lacrimal glands stained by immunohistochemistry for CD4, CD8, CD103, CD11b, CD45 sections in the normal group (N), the scopolamine-treated group (SCOP), and after desiccating stress in ICES for 1 week, 2 week, 4 week and 6 week (E1,E2,E4,E6). # P < 0.01 versus the scopolamine-treated group (SCOP), * P<0.05 versus the normal group(N) (Mann-Whitney U test). Original magnification:x20;scale bars = 50 μm. Experiments were repeated three times with two mice per group per experiment.
Mentions: In order to further characterize differences in LG inflammation between the ICES and SCOP groups, we determined if increases in different inflammatory immune cell numbers correspond with rises in proinflammatory cytokine gene expression. Accordingly, we compared in the ICES and N groups CD4, CD8α immunohistochemistry (predominantly expressed on the surface of cytotoxic T cells), CD11b (a marker of monocyte/macrophage lineage), CD103 (a marker of intraepithelial lymphocytes) and CD45 (a marker of all leukocytes and largely naive T lymphocytes) positive T lymphocyte cell numbers (Fig. 5). CD8α cells in the ICES group were less than in the normal group (N). On the other hand, CD103 increased significantly after 1 week in the ICES group and remained elevated at the same level after 2-, 4- and 6-weeks, while CD4 levels in the ICES group remained at the baseline level at all the times. ICES group CD11b cells were the only ones that increased after 2 weeks. In the 2-week group, CD45 cell levels rose after 2 and 4-weeks. However, the numbers of infiltrated CD4, CD11b, CD103, CD45 cells in the SCOP group were much larger than those in the ICES group at all the times (Fig. 5).

Bottom Line: To determine if there is a correlation between severity effects in these models and underlying pathophysiological responses, we compared the time dependent changes in each of these parameters that occur during a 6 week period.Interleukin-17(IL-17), IL-23, IL-6, IL-1, TNF-α, IFN-γ and TGF-β2 levels were estimated by real-time PCR measurements of conjunctival and lacrimal gland samples (LGs).Subsequently, the disease process stabilized for the next four weeks.

View Article: PubMed Central - PubMed

Affiliation: School of Ophthalmology and Optometry, Wenzhou Medical University, Zhejiang, China.

ABSTRACT
Dry eye disease can be a consequence of lacrimal gland insufficiency in Sjögren's Syndrome or increased tear film evaporation despite normal lacrimal gland function. To determine if there is a correlation between severity effects in these models and underlying pathophysiological responses, we compared the time dependent changes in each of these parameters that occur during a 6 week period. Dry eye was induced in 6-week-old female C57BL/6 mice by exposing them to an Intelligently Controlled Environmental System (ICES). Sixty mice were housed in ICES for 1, 2, 4 and 6 weeks respectively. Twelve were raised in normal environment and received subcutaneous injections of scopolamine hydrobromide (SCOP) 3 times daily for 5 days. Another sixty mice were housed in a normal environment and received no treatment. Corneal fluorescein staining along with corneal MMP-9 and caspase-3 level measurements were performed in parallel with the TUNEL assay. Interleukin-17(IL-17), IL-23, IL-6, IL-1, TNF-α, IFN-γ and TGF-β2 levels were estimated by real-time PCR measurements of conjunctival and lacrimal gland samples (LGs). Immunohistochemistry of excised LGs along with flow cytometry in cervical lymph nodes evaluated immune cell infiltration. Light and transmission electron microscopy studies evaluated LGs cytoarchitectural changes. ICES induced corneal epithelial destruction and apoptosis peaked at 2 weeks and kept stable in the following 4 weeks. In the ICES group, lacrimal gland proinflammatory cytokine level increases were much lower than those in the SCOP group. In accord with the lower proinflammatory cytokine levels, in the ICES group, lacrimal gland cytosolic vesicular density and size exceeded that in the SCOP group. ICES and SCOP induced murine dry eye effects became progressively more severe over a two week period. Subsequently, the disease process stabilized for the next four weeks. ICES induced local effects in the ocular surface, but failed to elicit lacrimal gland inflammation and cytoarchitectural changes, which accounts for less dry eye severity in the ICES model than that in the SCOP model.

Show MeSH
Related in: MedlinePlus