Abstract
Purpose :
To investigate scleral structure and permeability using second-harmonic generation (SHG) imaging and fluorescence recovery after photobleaching (FRAP) before and after chemical cross-linking.
Methods :
Scleral tissues were freshly harvested from 6-week old female SD rats, soaked in cross-linking chemicals of two concentrations: paraformaldehyde (PFA, 0.5%, 4%), glutaraldehyde (GLU, 0.5%, 2.5%) and ethanol (EtOH, 20%, 70%) for half an hour each. Untreated fresh rat scleral tissues were used as controls. Changes in collagen structure and organization at the equator region of the sclera were studied by two-photon excited fluorescence microscopy. For permeability assessment, FRAP was utilized to measure the diffusion of 40 kDa fluorescein isothiocyanate-dextran (FITC-dextran) into a photobleached zone at the equator of the sclera. Image J software was used to analyze fluorescence intensity.
Results :
SHG study demonstrated that cross-linking occurred at the higher concentrations with all chemicals used (4% PFA, 2.5% GLU, 70% EtOH). In normal rat sclera, SHG imaging revealed that collagen fibers were irregularly arranged with fibrils of various degrees of waviness and no preferential orientation. In cross-linked tissues, the collagen fibers were more disorientated in larger bundles, and the fibrils more curvy. The diffusion rates of dextran in the sclera were determined by measurement of the 1/2 fluorescence recovery time (t1/2). The mean t1/2 of the high concentration groups were significantly higher compared to control (n=6, control: 11.8s, 4% PFA:14.41s, P = 0.0013, 2.5% GLU: 17.37s, P = 0.00001, 70% EtOH: 16.35s P = 0.00003 ). There was no significant difference between the low concentration groups and control(n=6, 0.5% PFA: 11.63s, P = 0.814, 0.5% GLU: 12.88s, P = 0.195, 20% EtOH: 12.21s P = 0.473).
Conclusions :
SHG imaging showed alteration of the density and waviness of collagen fiber bundles after scleral tissue cross-linking using 4% PFA, 2.5% GLU and 70% EtOH. The measurement of the diffusion rates of dextran in the sclera showed that all 3 chemicals at higher concentration led to decreased scleral permeability. These changes provide insights into the macromolecular changes that are induced by chemical cross-linkers and a valuable tool to study cornea, sclera and other connective tissue collagen structures after cross-linking treatment.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.