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J. Kramme, K. Berger, R. Beuerman, C. Sheppard, J. Bille; Structural Characteristics of the Rabbit Sclera Are Depth Related. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5673.
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The collagen structure of the sclera was examined with Second Harmonic Generation (SHG) for eventual application to diseases such as glaucoma. In this study, the collagen structure of the rabbit sclera and its change with depth was measured using SHG imaging which has an advantage of being able to visualize structure in an opaque tissue.
Shock frozen cryostat sections parallel to the surface of the sclera and cross sections both 20 in thickness were imaged with SHG in an inverted microscope with circular polarized light at 830 nm in backward and transmission mode. The power at the sample is between 10-20 mW. The images are averaged over 15 scans.
The cross sections and surface cuts in transmission mode were linked to the known tissue structure. Whereas the signal in the backward direction showed an increased intensity for the inner scleral collagen bundles but with no clear detail information. In our case the normally 170 thick rabbit sclera was swollen to 640 due to storage in PBS before cutting. All images showed a wavy collagen structure. The inner part contains fine, highly criss-crossing bundles (20 deep) changing to a lamellar arrangement (100) towards the scleral stroma. In the inner stroma the lamellae are short, small with no preferred direction. In the middle stroma (360) they are more ordered and oriented mostly parallel to the surface with a preferred direction, but at the outer part (500) a preferred orientation could not be found and the lamellar structure was not as clear.
A change in collagen structure was found correlated with depth. For wound healing after glaucoma surgery it is very important to know more about the scleral structure and its material properties. Obtaining visual knowledge of the substructure is an extra advantage as the strain distribution can be calculated to understand how it is affected by disease. For in vivo applications the backward signal in SHG microscopy has to be improved but some early results are encouraging.
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