Regional Differences and Physiologic Behaviors in Peripapillary Scleral Fibroblasts

Abstract

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.

Figure 1.

Greater nuclear density and rounder nuclei in PPS than peripheral sclera. (A) Schematic of scleral section indicating different regions, including optic nerve (ON, white), iPPS (yellow), oPPS (blue), and peripheral sclera (grey). (B) Image of scleral section stained for nuclei (Sytox Green, white) with ON, iPPS, oPPS, and peripheral sclera depicted (scale bar = 500 µm). (C) Nuclear density is increased, and aspect ratio is decreased in peripapillary regions compared to peripheral sclera (n = 5 eyes). *P < 0.05, **P < 0.01, ****P < 0.0001.

Figure 2.

Fibrillar actin processes permeate scleral stroma. (A) FA staining (phalloidin, red) of scleral section including ON, iPPS, oPPS, and peripheral sclera (scale bar = 500 µm; + = fold; * = penetrating nerve or vessel). Higher magnification view of FA staining of (B) peripheral sclera and (C) oPPS shows basket weave and circumferential arrangements, respectively (scale bar = 50 µm).

Figure 3.

Greater FA-collagen alignment in oPPS than peripheral sclera. Examples of alignment analysis of (A) peripheral sclera and (B) oPPS with FA and SHG images, their corresponding vector fields, and alignment heat maps. Darker blue indicates poorer alignment and black boxes indicate points in which sufficient image signal was not present for analysis in either FA or SHG images. Reduced FA-collagen alignment in peripheral sclera versus oPPS in image sets that included (C) and excluded (D) areas adjacent to penetrated vessels and nerves.

Figure 4.

Scleral αSMA expression occurs in a small number of stromal fibroblasts. FA (A) and αSMA (B) in the same scleral section. The αSMA expression is highlighted in vessels (*), fibrils (arrows), and stromal cells (arrowheads; scale bar = 500 µm). The central retinal artery is far right of A and B. Higher magnification view of αSMA-expression cell body (C) and fibril (D) (scale bar = 25 µm).

Figure 5.

The αSMA-collagen alignment is reduced in peripheral sclera but not PPS. (A) Alignment analysis of FA and αSMA with collagen (SHG) with mean alignment values placed under each heatmap. (B) αSMA and FA alignment with collagen in peripheral sclera (left) and PPS (right).

Figure 6.

Topographic cues align cells. Immunofluorescence imaging of PPS fibroblasts on smooth membrane (A) and on of fibroblasts on patterned surface topography with horizontally oriented grooves (B) (scale bar = 275 µm; red = FA, blue = DAPI). Frequency distribution of cellular orientation of cells on flat (red) and patterned (purple) membranes.

Figure 7.

Topographic cues and TGFβ-treatment alter response to mechanical strain. (A) Cellular orientation on flat smooth membranes with and without cellular strain. (B) Cellular strain orientation on patterned membranes with and without TGFβ1 or 2 (2 ng/mL for 24 hours) treatment.