Glaucoma is the second most common cause of blindness worldwide, leading to irreversible loss of vision. Prior studies indicate that ocular pressure-induced displacement of the lamina cribrosa (LC) may be responsible for retinal ganglion cell axon damage inside the neural canal. We present a novel approach to imaging the entire lamina cribrosa and the scleral canal at high lateral and axial resolution by using a combination of array tomography and nonlinear optical imaging of serial ultrathin orthogonal sections to detect second harmonic generated (SHG) signals from collagen. The resulting images can be analyzed individually or combined to form a three-dimensional reconstruction of the lamina. Due to the specificity of SHG generated from collagen the density and distribution of collagen inside the scleral canal can be objectively quantified with a high degree of accuracy. The reconstruction shows a non-uniform distribution of collagen along both the longitudinal and orthogonal axes. Mapping the collagen density by geographic region reveals significant differences in collagen content that result in “thin spots” with low collagen density as well as areas of very high collagen content. This suggests a non-uniform mechanical stiffness across the lamina that may account for increased axon damage observed in glaucoma patients. The inferior temporal region of the ONH in particular is marked by low collagen density, which corresponds with clinical observations identifying this region as being more susceptible to damage during the onset of glaucoma. Further application of this technique will help characterize the relationship of age, race and gender on the morphology of the LC.

Purpose

The purpose of this study was to characterize the natural history of meibomian gland morphogenesis in the mouse.

Methods

Embryonic (E) and post natal (P) C57Bl/6 mouse pups were obtained at E18.5, P0, P1, P3, P5, P8, P15, and P60. Eyelids were fixed and processed for en bloc staining with Phalloidin/DAPI to identify gland morphogenesis, or frozen for immunohistochemistry staining with Oil red O (ORO) to identify lipid and antibodies specific against peroxisome proliferator-activated receptor gamma (PPARγ) to identify meibocyte differentiation. Samples were then evaluated using a Zeiss 510 Meta laser scanning confocal microscope or Nikon epi-fluorescent microscope. Tissues from adult mice (2 month-old) were also collected for western blotting.

Results

Meibomian gland morphogenesis was first detected at E18.5 with the formation of an epithelial placode within the fused eyelid margin. Invagination of the epithelium into the eyelid was detected at P0. From P1 to P3 there was continued extension of the epithelium into the eyelid. ORO and PPARγ staining was first detected at P3, localized to the central core of the epithelial cord thus forming the presumptive ductal lumen. Ductal branching was first detected at P5 associated with acinar differentiation identified by ORO and PPARγ staining. Adult meibomian glands were observed by P15. Western blotting of meibomian gland proteins identified a 50 kDa and a 72 kDa band that stained with antibodies specific to PPARγ.

Conclusions

We have demonstrated that meibomian gland development bears distinct similarities to hair development with the formation of an epithelial placode and expression of PPARγ co-incident with lipid synthesis and meibocyte differentiation.