Abstract:
Purpose: Atropine is widely used to inhibit the axial elongation that underlies human myopia progression, but its site of action remains unclear. This thesis describes a series of experiments aimed at localising the action of atropine in the chick model of myopia using an anti-atropine antibody for the first time. Method: After the anti-atropine antibody was validated, it was used in conjunction with enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC) to localise atropine in chick ocular tissues. Crucial to this project was the use of tissues from eyes in which myopia had been inhibited by atropine in vivo, as atropine does not affect ocular growth in normal eyes. Thus, 6 days of continuous unilateral diffuser wear was used to induce unilateral form-deprivation myopia (FDM) and intravitreally injected atropine was delivered to inhibit FDM. IHC protocols were developed and trialled to address the expected difficulties in accurately localising atropine, which is prone to diffusion given its small molecular weight and lipophilic nature. Results: Chick rearing experiments successfully induced FDM, and successfully inhibited FDM using atropine. ELISA showed that 1 hour after a single, unilateral intravitreal injection of atropine, approximately half of the injected atropine was still in the injected eye. Most of the atropine was located in the vitreous, with lesser amounts in the aqueous-iris, retina-RPE-choroid, sclera, lens and cornea. The distribution of atropine was the same in myopic and emmetropic chick eyes. After a series of intravitreal atropine injections to inhibit FDM, IHC localised atropine in the inner nuclear layer of the retina. Atropine was identified in this layer 1.5 hours after the final injection was given. IHC at 4 hours and 24 hours after the final injection did not detect atropine in the eye. However, the immunolocalisation of atropine in the retina after 1.5 hours was not replicated in a follow-up experiment. Conclusions: New approaches were used to localise atropine within chick eyes at both the tissue and cellular level. Immunohistochemistry results were equivocal, but suggest that atropine may act on the inner retina when inhibiting myopia, an interaction which appears to be short-lived.