Abstract
Abstract: :
Purpose: The anatomical properties of the rat eye, including the large relative size of the lens and the small relative volume of vitreous, can exacerbate vitreous backflow during injection and interfere with the quantification of material in the eye. We have devised a new, efficient injection technique allowing us to deliver a measured amount of material into the vitreous of the rat eye in a reproducible and reliable manner. Methods: In two separate studies, rats were anesthetized and the eyes were gently proptosed and held in place using angled forceps for intravitreal injection. A 33 gauge needle was attached to an Exmire® Micro Syringe and inserted 1 mm posterior to the corneal limbus, maintaining a sharp angle towards the back of the eye. The eye was released from proptosis and 3ul 3H–labeled compound was injected. The material was allowed to settle in the vitreous while holding the syringe in place for 1 minute. After one minute, the conjunctiva around the needle was grasped at the injection site with serrated forceps in order to prevent backflow. In one study, whole eyes were collected 30 minutes post–injection and kept at –20ºC for further processing. In the second study, eyes were enucleated 30 minutes post–injection, and the vitreous was collected by a standardized procedure. Tissues were treated with enzymatic digestion and the amount of radioactivity was measured using a liquid scintillation counter. To estimate the percentage recovery, treated eyes were compared to non–treated control eyes spiked with the same amount of 3H–labeled compound. Results: Due to the inconsistency of material recovered in whole eyes using conventional injecting techniques (average recovery = 66.7% ± 21.8 n=15), a new method was developed to prevent the back flow and loss of material. The grasping technique proved to be efficient and consistent. In whole eyes (n = 8) the average recovery of material using the grasping method was 99.84% ± 10.86. Conclusions: Several methods (e.g., intravitreal and subconjuntival injections) are currently used to deliver therapeutic agents into the eye. We have developed a reproducible and quantifiable method of vitreal collection in the rat. This method is instrumental in the evaluation of compound biodistribution in the eye.
Keywords: vitreous • injection