Age-related changes in the meibomian gland.

The purpose of this study was to characterize the age-related changes of the mouse meibomian gland. Eyelids from adult C57Bl/6 mice at 2, 6, 12 and 24 months of age were stained with specific antibodies against peroxisome proliferator activated receptor gamma (PPARgamma) to identify differentiating meibocytes, Oil Red O (ORO) to identify lipid, Ki67 nuclear antigen to identify cycling cells, B-lymphocyte-induced maturation protein-1 (Blimp1) to identify potential stem cells and CD45 to identify immune cells. Meibomian glands from younger mice (2 and 6 months) showed cytoplasmic and perinuclear staining with anti-PPARgamma antibodies with abundant ORO staining of small, intracellular lipid droplets. Meibomian glands from older mice (12 and 24 months) showed only nuclear PPARgamma localization with less ORO staining and significantly reduced acinar tissue (p < 0.04). Acini of older mice also showed significantly reduced (p < 0.004) numbers of Ki67 stained nuclei. While Blimp1 appeared to diffusely stain the superficial ductal epithelium, isolated cells were occasionally stained within the meibomian gland duct and acini of older mice that also stained with CD45 antibodies, suggesting the presence of infiltrating plasmacytoid cells. These findings suggest that there is altered PPARgamma receptor signaling in older mice that may underlie changes in cell cycle entry/proliferation, lipid synthesis and gland atrophy during aging. These results are consistent with the hypothesis that mouse meibomian glands undergo age-related changes similar to those identified in humans and may be used as a model for age-related meibomian gland dysfunction.

Second-harmonic imaging microscopy of normal human and keratoconus cornea.

PURPOSE: The purpose of this study was to evaluate the ability of second-harmonic imaging to identify differences in corneal stromal collagen organization between normal human and keratoconus corneas. METHODS: Six normal corneas from eye bank donors and 13 corneas of patients with keratoconus, obtained after penetrating keratoplasty were examined. A femtosecond titanium-sapphire laser with 800-nm output was used to generate second-harmonic signals collected at 400 nm from central and paracentral corneal tissue blocks. Three-dimensional (3-D) data sets were collected and reconstructed to evaluate the location and orientation of stromal collagen lamellae. RESULTS: Imaging of second-harmonic signals combined with 3-D reconstruction of the normal cornea identified a high degree of lamellar interweaving, particularly in the anterior cornea. Of note was the detection of lamellae that inserted into Bowman's layer and were oriented transverse to the corneal surface, penetrating posteriorly approximately 120 mum. In keratoconus corneas, imaging second-harmonic signals identified less lamellar interweaving and a marked reduction or loss of lamellae inserting into Bowman's layer in 12 of 13 cases, particularly in regions associated with cone development without breaks in Bowman's layer or scarring. CONCLUSIONS: Compared with normal adult corneas, marked abnormalities were detected in the organization of the anterior corneal collagen lamellae of keratoconus corneas by second harmonic imaging. These structural abnormalities are consistent with the known changes in collagen organization and biomechanical strength of keratoconus.

Corneal response to femtosecond laser photodisruption in the rabbit.

In this report we evaluated the effect of femtosecond laser energy on the development of corneal haze and keratocyte activation in rabbits following intra-stromal photodisruption to create LASIK flaps using a modified commercial femtosecond surgical laser. Three groups of flap parameters were studied: 1.5 microJ/pulse with 10 microm spot separation and complete side cut (Group 1); 3.5 microJ/pulse with 14 microm spot separation and complete side cut (Group 2); 3.5 microJ/pulse with 14 microm spot separation and partial (50 microm) side cut (Group 3). All flaps were left attached without lifting to avoid epithelial contamination. Rabbits were then evaluated pre- and post-operatively by quantitative in vivo and ex vivo confocal microscopy. The achieved flap thickness 1 week after surgery averaged 88.9+/-12.8, 90.8+/-6.9 and 86.5+/-6.8 microm for Groups 1-3 respectively (p=NS). Interface thickness was significantly greater (p<0.05) in the higher energy groups averaging 40.0+/-11.2 and 37.7+/-5.7 microm for Groups 2-3 compared to 28.6+/-4.5 microm for Group 1. Corneal haze was barely detectible and not significantly different between groups, although haze was detected in the region of the side-cuts in Groups 1 and 2. No clinically significant changes in stromal or epithelial thickness were noted. Laser confocal microscopy showed the presence of small diameter cells within the flap interface that resided within disrupted regions of the corneal collagen lamellae. Keratocyte activation was only detected in regions of the 100% side cut and not over the flap interface. In conclusion, the results of this study indicate that photodisruption of the corneal stroma alone without flap elevation regardless of laser energy does not induce significant corneal haze in the rabbit. However, a thicker stromal interface was seen with the higher energy suggesting greater stromal damage.

Functional Foxp3+ CD4+ CD25(Bright+) "natural" regulatory T cells are abundant in rabbit conjunctiva and suppress virus-specific CD4+ and CD8+ effector T cells during ocular herpes infection.

We studied the phenotype and distribution of "naturally" occurring CD4(+) CD25(+) T regulatory cells (CD4(+) CD25(+) nT(reg) cells) resident in rabbit conjunctiva, the main T-cell inductive site of the ocular mucosal immune system, and we investigated their suppressive capacities using herpes simplex virus type 1 (HSV-1)-specific effector T (T(eff)) cells induced during ocular infection. The expression of CD4, CD25, CTLA4, GITR, and Foxp3 was examined by reverse transcription-PCR, Western blotting, and fluorescence-activated cell sorter analysis in CD45(+) pan-leukocytes isolated from conjunctiva, spleen, and peripheral blood monocyte cells (PBMC) of HSV-1-infected and uninfected rabbits. Normal conjunctiva showed a higher frequency of CD4(+) CD25((Bright+)) T cells than did spleen and PBMC. These cells expressed high levels of Foxp3, GITR, and CTLA4 molecules. CD4(+) CD25((Bright+)) T cells were localized continuously along the upper and lower palpebral and bulbar conjunctiva, throughout the epithelium and substantia propria. Conjunctiva-derived CD4(+) CD25((Bright+)) T cells, but not CD4(+) CD25((low)) T cells, efficiently suppressed HSV-specific CD4(+) and CD8(+) T(eff) cells. The CD4(+) CD25((Bright+)) T-cell-mediated suppression was effective on both peripheral blood and conjunctiva infiltrating T(eff) cells and was cell-cell contact dependent but independent of interleukin-10 and transforming growth factor beta. Interestingly, during an ocular herpes infection, there was a selective increase in the frequency and suppressive capacity of Foxp3(+) CD4(+) CD25((Bright+)) T cells in conjunctiva but not in the spleen or in peripheral blood. Altogether, these results provide the first evidence that functional Foxp3(+) CD4(+) CD25((Bright+)) T(reg) cells accumulate in the conjunctiva. It remains to be determined whether conjunctiva CD4(+) CD25(+) nT(reg) cells affect the topical/mucosal delivery of subunit vaccines that stimulate the ocular mucosal immune system.