PURPOSE

The purpose of this study was to describe the cellular architecture of normal human peripapillary sclera (PPS) and evaluate surface topography's role in fibroblast behavior.

METHODS

PPS cryosections from nonglaucomatous eyes were labelled for nuclei, fibrillar actin (FA), and alpha smooth muscle actin (αSMA) and imaged. Collagen fibrils were imaged using second harmonic generation. Nuclear density and aspect ratio of the internal PPS (iPPS), outer PPS (oPPS), and peripheral sclera were determined. FA and αSMA fibril alignment with collagen extracellular matrix (ECM) was determined. PPS fibroblasts were cultured on smooth or patterned membranes under mechanical strain and in the presence of TGFβ1 and 2.

RESULTS

The iPPS (7.1 ± 2.0 × 10-4, P < 0.0001) and oPPS (5.3 ± 1.4 × 10-4, P = 0.0013) had greater nuclei density (nuclei/µm2) than peripheral sclera (2.5 ± 0.8 × 10-4). The iPPS (2.0 ± 0.3, P = 0.002) but not oPPS (2.4 ± 0.4, P = 0.45) nuclei had smaller aspect ratios than peripheral (2.7 ± 0.5) nuclei. FA was present throughout the scleral stroma and was more aligned with oPPS collagen (9.6 ± 1.9 degrees) than in the peripheral sclera (15.9 ± 3.9 degrees, P =0.002). The αSMA fibers in the peripheral sclera were less aligned with collagen fibrils (26.4 ± 4.8 degrees) than were FA (15.9 ± 3.9 degrees, P = 0.0002). PPS fibroblasts cultured on smooth membranes shifted to an orientation perpendicular to the direction of cyclic uniaxial strain (1 Hz, 5% strain, 42.2 ± 7.1 degrees versus 62.0 ± 8.5 degrees, P < 0.0001), whereas aligned fibroblasts on patterned membranes were resistant to strain-induced reorientation (5.9 ± 1.4 degrees versus 10 ± 3.3 degrees, P = 0.21). Resistance to re-orientation was reduced by TGFβ treatment (10 ± 3.3 degrees without TGFβ1 compared to 23.1 ± 4.5 degrees with TGFβ1, P < 0.0001).

CONCLUSIONS

Regions of the posterior sclera differ in cellular density and nuclear morphology. Topography alters the cellular response to mechanical strain.