In vivo removal of KS GAGs from murine cornea by intrastromal injection of keratanase II enzyme.

The composition and microstructure of corneal extracellular matrix are important for its transparency and mechanical properties. Collagen fibrils, proteoglycans, and glycosaminoglycans (GAGs) are the primary constituents of corneal extracellular matrix. The primary objective of the present study was to investigate the effect of GAG removal on in vivo thickness and biomechanical properties of cornea. To this end, the enzyme keratanase II from Bacillus circulans, which hydrolyzes keratan-sulfate (KS), was used to remove KS GAGs from mouse corneas in vivo. This was accomplished by intrastromal injections of keratanase II enzyme solution (treatment group) and buffer (control group) into the right and left corneas, respectively. The thickness was measured in vivo by anterior segment OCT up to twenty-eight days after which the animals were euthanized and their corneas were subjected to mechanical testing by performing uniaxial tensile tests. It was found that GAG depletion caused a significant reduction in the in vivo corneal thickness. In particular, the thickness of enzyme group decreased significantly from 90.6 ± 11.2 μm to 70.9 ± 7.1 μm after 7 days and to 78.1 ± 8.3 μm after 28 days (P < 0.05). The thickness variation of control samples because of buffer solution injection was insignificant. The enzymatic treatment significantly reduced corneal tensile properties, evidenced by substantially lower peak tangent modulus (1.02 ± 0.31 MPa) in the treatment group compared to the control samples (2.46 ± 0.40 MPa), (P < 0.05). This study validated the use of intrastromal injection of keratanase II enzyme for manipulating the composition of corneal extracellular matrix. It also highlighted the role of GAGs in defining corneal thickness and mechanical properties.