Quantifying the Effects of Enzymatic Vitreolysis Reveals Biomechanical Roles of Structural Macromolecules in Shear and Extension

Abstract

 

Purpose

 

Clinical goals of enzymatic vitreolysis are two-fold: reduce vitreous stiffness and vitreoretinal traction to aid in separation of the vitreous from the retina. The purpose of this study was to develop a quantitative, in vitro method to simultaneously probe the viscoelastic response of the vitreous in shear (to gauge stiffness) and extension (to estimate traction) following enzymatic degradation of structural macromolecules.

 

Methods

 

Vitreous (10 mL total volume) from fresh bovine eyes was injected (200 μL) with PBS, purified collagenase (50-200 U/mL), or hyaluronidase (50-200 U/mL) and stored overnight at 4°C. The vitreous was isolated for analysis on a AR-G2 rheometer (TA Instruments) with cleated parallel plate (20mm) geometry. An oscillatory frequency sweep (Fig. 1A) (to estimate stiffness) at fixed 3% strain was followed by uniaxial extension (250 μm/sec; Fig. 1B) (to estimate traction) until detachment.

 

Results

 

Overnight exposure to hyaluronidase or collagenase significantly decreased the storage modulus (G’; solid-like structure) of the bovine vitreous relative to PBS controls (n > 7 per group) as assessed by oscillatory shear testing (G’_PBS = 8.6 Pa - Fig. 1A'; G’_Hyal = 5.4 Pa; G’_Collag = 4.6 Pa). During extensional testing, the vitreous exhibited bimodal behavior. In the first mode, the vitreous gradually sloughed from the test geometry with little normal (adhesive) force generation (Fig. 1B’, red). The second mode was characterized by significant axial stretch (> 1 cm; 100% strain), an increase in adhesive force, and rapid release (Fig. 1B’, blue). Collagenase treatment always caused the first mode (n = 7/7; F_A = 7.9 mN), which also predominately occurred in controls (n = 5/7; F_A = 11.1 mN). Hyaluronidase exposure more frequently resulted in the second scenario (n = 4/8; F_A = 12.6 mN) as the vitreous generated significantly more tractional force.

 

Conclusions

 

Enzymatic digestion of hyaluronan or collagen should soften the vitreous, whereas collagen (but not hyaluronan) digestion may help prevent vitreoretinal traction. This finding supports the idea that the local increases in collagen density that occur with age (synchesis via vitreous liquefaction) may generate tensile forces that cause retinal detachments and tears. Future testing with other vitreolytic enzymes (e.g. ocriplasmin) is warranted.

 

 

Fig. 1: Vitreous in shear (A, A’) and extension (B, B’).

 

Keywords: 763 vitreous • 692 retinal adhesion • 661 proteoglycans/glycosaminoglycans