Methods of identifying neuroprotective compounds for retinal ganglion cells
Neuroprotective compounds for treating optic neuropathies and screening methods for identifying neuroprotective compounds.
1. A high throughput method of identifying a neuroprotective compound, comprising:
(1) contacting dissociated retinal cells with a magnetic bead coupled to an anti-cd11 b/c antibody:
(2) separating cells bound to the anti-cd11 b/c antibody from other retinal cells to obtain a macrophage-depleted cell population;
(3) contacting the macrophage-depleted cell population with a magnetic bead coupled to an anti-Thy1 antibody;
(4) retaining cells bound to the anti-Thy1 antibody as a test enriched population of retinal ganglion cells;
(5) culturing the test enriched population of retinal ganglion cells in the absence of growth factors;
(6) contacting the test enriched population of retinal ganglion cells with a test compound previously unknown to have any neuroprotective pharmacological activity;
(7) staining the test enriched population of retinal ganglion cells with at least a dye capable of staining living cells and their neurites and a dye capable of staining the nuclei of dead cells:
(8) imaging retinal ganglion cells in the test enriched population to quantify the number of live cells and the extent of neurite outgrowth present in the test enriched population;
(9) comparing the number of live cells and the extent of neurite outgrowth present in the test enriched population of ganglion cells contacted with the test compound to a control number of live cells and extent of neurite outgrowth present in a control enriched population of retinal ganglion cells cultured in the presence of BDNF and forskolin and the absence of the test compound; and
(10) identifying the test compound as a neuroprotective compound if the number of live cells and/or the extent of neurite outgrowth in the compound treated population of retinal ganglion cells is statistically greater than the control number of live cells and/or extent of neurite outgrowth in the control enriched population of retinal ganglion cells grown in the presence of BDNF and forskolin and the absence of the test compound;
wherein steps (1) to (10) are performed as a high throughput screen in a multi-well plate.
2. The method of claim 1 wherein test compound is a small molecule.
3. The method of claim 1 wherein the test compound is a cDNA expression product.
4. The method of claim 1 wherein the multi-well plate comprises a multi-well microtiter plate.