A QUANTITATIVE APPROACH TO IDENTIFY AND ISOLATE PURE POPULATIONS OF FLUORESCENTLY LABELED ADULT RETINAL GANGLION-CELLS USING A PRESSURE-DRIVEN MICROASPIRATION TECHNIQUE

We developed a technique to obtain pure subsets of neuronal population s for specific analysis at the molecular level. The fact that most are as of the brain consist of mixed types of neuronal and non-neuronal ce lls was circumvented by retrograde labeling of retinal ganglion cells (RGCs) either from the superior colliculus (SC) or the optic nerve (ON ) using fluorescent dyes (4Di-10ASP or Rhodamine). Subsequent enzymati c dissociation of the retina with papain allowed to identify and colle ct pure populations of RGC by using the pressure-driven microaspiratio n technique. Enzymatic treatment and additional mechanical dissociatio n destroy most of the vulnerable ganglion cells leaving between 1.8% a nd 6% of the labeled cells intact. This low outcome was consistent amo ng the techniques used to apply the dye and the two different dyes use d in the study. However, the total numbers of cells obtained from each retina were sufficient to collect about 200 cells within 1 day and pr ocess these cells for molecular biology. As an example of further proc essing of collected cells, the expression of beta-actin gene was analy zed.