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R. W. Beuerman, V. Barathi, S. R. Weon, J. Chew, L. Tong, S. Ching, D. T. H. Tan; Expression of Transglutaminase 2 in Mouse Sclera in Experimental Myopia. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3718.
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Purpose: Transglutaminase 2 (TG2), a tissue enzyme of the transglutaminase family, plays a central role in wound healing, apoptosis and extra-cellular matrix production, and we have found TG1-5 to be present in mouse and monkey sclera and TG2 in human sclera.
Methods: Contact lenses were placed over the right eyes of 12 Balb/cJ mice by attaching a negative (-10D) contact lens over the eye on post natal day 10. Axial length was measured by AC-Master, OLCI (Carl-Zeiss) and refraction was measured by retinoscopy at 4, 6 and 8 weeks. Mouse sclera was subject to expressions array analysis (Affymetrix) after the induction of myopia. At 8 weeks, sclera from myopic and control eyes without and with atropine treatment (100 uM for 4 weeks) were embedded in OCT for immunohistochemistry or immediately frozen as individual tissues in liquid nitrogen. Total RNA and protein were extracted for real-time PCR and Western blotting. Scleral fibroblasts were cultured from 8 week old naive mice. At P1-P2 they were tested with atropine (0.1, 1.0, 2.0 and 5 uM) for 2, 3 and 5 days. Extracted RNA was used for real-time PCR and protein was used for Western blot analysis.
Microaray studies of mouse sclera showed the significant up-regulation of TG2 comapred to control at 8 weeks after induction. The expression of TG2 was found in choroid, limbus and corneal stroma while TG 1,3 and 5 was found in sclera, limbus, conjunctiva and cornea. Immunohistochemistry showed the presence of TG1,2,3 and 5 in the normal mouse sclera which was confirmed by Western blot. The pattern of expression was slightly different from TG2. After the induction of myopia TG2 increased p<0.05 compared to control sclera. Western blot of TG2 showed the protein to be as well. After treatment of the experimental eye with atropine the expression of TG2 was down regulated five fold after real-time PCR. TG 2 levels in cultured sceral fibroblasts was down regulated after 2 days of atropine treatment which also blocked the progression of experimental myopia
Expression of TG2 in messenger RNA level as well as the protein level was shown to be up-regulated in the mouse myopic sclera as compared to the control sclera. Atropine down-regulates TG2 expression in both cultured scleral fibroblasts and myopic sclera. This result indicates that the TG2 could be involved in scleral growth in myopia.
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