Glaucoma, a chronic eye disease that is responsible for 9%-12% of the blindness in the USA, is caused by damage to the optic nerve (“Learn”). An increased intra-ocular pressure, caused by the build-up of aqueous humors (which supply the retina and cornea with nutrients), has been shown to be a major risk factor for damage to the optic nerve. Intra-ocular pressure can be controlled either through the production of the aqueous humor, which takes place in the ciliary epithelium, or through the regulation of aqueous humor outflow, through the trabelcular meshwork and Schelmm’s canal (Figure 1). Recent studies have shown that tetrahydracannabinol, the active ingredient in marijuana, may decrease intraocular pressure by targeting receptor sites in ocular tissue.
The objective of this project is to investigate the expression of cannabinoid receptors at different sites in ocular tissue, including ciliary epithelium, and to investigate the ability of cannabinoids to activate intracellular signalling molecules that are coupled to CB1 receptors. It is hypothesized that cannabinoid action is controlled by CB1 cannabinoid receptors, at different sites in the eye, particularly the ciliary body epithelium, and that further investigation could show that THC and cannabinoid agonists will act at multiple sites in the eye to decrease intraocular pressure, either by decreasing the rate of aqueous humor production or by increasing the rate of aqueous humor outflow.
This experiment will be carried out using three techniques to identify the prescence of cannabinoid receptors (particularly CB1 receptors) within the ciliary epithelium. The Immunohistochemistry technique was used to identify and visualize the location of cannabinoid receptors in the CBE. The Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) technique was used to examine mRNA for CB1 receptors in the tissue of the ciliary epithelium. The final technique used was Western Blot Analysis with the purpose of determining the presence of CB1 receptor proteins. Immunohistochemistry revealed the presence of CB1 receptors in the ciliary body epithelium and retina of the rat eye. The CB1 receptors were then localized to the non-pigmented epithelium tissue. RT-PCR showed that mRNA, which contains the code for the protein receptors for CB1, was present in the rat’s ciliary epithelium and retinal tissues. The oligonucleotide sequence was then found to be identical to that of hCB1 receptor cDNA reported in GenBank (GenBank#: XM 004350). The Western Blotting procedure indicated that the proteins that make up the CB1 receptors are present in the ciliary epithelium and retina, however results remained inconclusive as the technique depends upon the use of a specific antibody in the correct concentration.
CB1 receptors were discovered in the ciliary body epithelium, and were localized to non-pigmented ciliary epithelial cells. They were also revealed to be expressed on radial muscle tissue. This suggests that cannabinoid compounds may exert their intraocular pressure lowering effects through the reduction of aqueous humor production as well as through an increase in the aqueous humor outflow.