Scleral ultrastructure and biomechanical changes in rabbits after negative lens application

Abstract

Aim: To address the microstructure and biomechanical changes of the sclera of rabbits after negative lens application by spectacle frame apparatus.

Methods: Five New Zealand rabbits of seven weeks post-natal were treated with -8 D lens monocularly over the course of two weeks. Refractive errors and axial length (AXL) were measured at the 1^st, 7^th and 14^th days of the induction period. Ultrastructure of sclera was determined with electron microscopy. Biomechanical properties were tested by an Instron 5565 universal testing machine.

Results: Lens-induced (LI) eyes elongated more rapidly compared with fellow eyes with AXL values of 15.56±0.14 and 15.21±0.14 mm (P<0.01). Fibril diameter was significantly smaller in the LI eyes compared with control ones in the inner, middle, and outer layers (inner layer, 63.533 vs 76.467 nm; middle layer, 92.647 vs 123.984 nm; outer layer, 86.999 vs 134.257 nm, P<0.01, respectively). In comparison with control eyes, macrophage-like cells that engulfed fibroblasts, dilated endoplasmic reticulum, and vacuoles in fibroblasts were observed in the inner and middle stroma in the LI eyes. Ultimate stress and Young's modulus were lower in the LI eyes compared with those in the control eyes.

Conclusion: Negative lens application alters eye growth, and results in axial elongation with changes in scleral ultrastructural and mechanical properties.

Keywords: biomechanics; negative lens; rabbit; sclera; ultrastructure.

Figure 1. Spectacle frame

Lenses were attached to arcs made of Velcro and sutured by strings to the soft frame also made from soft Velcro, which circled the ear and neck of the rabbit.

Figure 2. Illustration of scleral sample processing for biomechanical test and microscopy

A: Nasal half of the eyeball; B: Temporal half of the eyeball.

Figure 3. Light microscopy of a semi-thin stained with toluidine blue from a strip excised from the edge of the ONH in the nasal direction of the nasal half

Image (×40) shows perpendicularly arranged photoreceptors, suggesting the strips were cut squarely, as per Figure 2.

Figure 4. Results of lens-induced anisometropia in rabbits

A, B, C: Anterior chamber, lens thickness and axial length via A-scan ultrasonography, respectively; D: Refractive error via retinoscopy. Solid lines and dashed lines stands for control eyes and LI eyes, respectively. Error bars represent standard error of the mean. LI: Lens-induced.

Figure 5. Collagen fibril diameter taken from representative electron micrographs (×42 000) of the LI and control eyes

The results showed sections taken from the outer, middle and inner layers of scleral stroma and the corresponding distribution of the diameter of collagen fibrils (shown as percentage). Small fibrils have diameters ranging from 10 to 100 nm, whereas large ones ranging from 110 to 380 nm. LI: Lens-induced.

Figure 6. Electronic micrographs representing the microstructure of the rabbit sclera in control eyes (A-C) and the LI eyes (D-I)

The collagen fibril bundles increased in size from the inner to outer stroma of the sclera. The number of cells decreased from inner to outer layers. In the outer scleral stroma (Sco), fibroblasts (fb) with thin processes (arrowhead) and elastic fibers (e) were scatterd within or between collagen fibril bundles. The inner scleral stroma (Sci) contain fibroblasts (fb) elastic fibers (e). Fibroblasts were observed in the outer scleral stroma of the LI eyes, but no fibers were observed (D). A fibroblast (fb) was surrounded by a macrophage-like cell (mp) (E). The phagocytes may contain collagen fibrils (triple arrow) (F). The cytoplasm of an abnormal fibroblast contained vacuoles (double arrow) and a dilated endoplasmic reticulum (G, I). In the LI eyes, some macrophage-like cells (mp) and lysosomes (l) were found in the middle scleral stroma (Scm). In the LI eyes, no consistency was observed in elastic fibers in the lamina fusca (Lf) (arrow) (I). LI: Lens-induced.

Figure 7. Illustration of biomechanical test

A: Biomechanical test of scleral strips on an Instron 5565 universal testing machine; B: Stress-strain curve of tensile test. Two lines were chosen from one subject in this study. Ultimate stress of sclera in the LI eyes was lower than that in contralateral eyes (13.53 vs 18.65 MPa, P=0.021). Young's modulus was lower in the LI eyes than that in control eyes (0.4740 vs 0.8145 MPa, P=0.043). LI: Lens-induced.