Flow Cytometry Approach to Study NGF and p75 in Retinal Cells from Reeler mice

Abstract Purpose:Reeler mice appear to be a good model for exploring the cross-talk between NGF and Reelin in development/maintenance of a physiological retinal function. Therefore, we sought to characterize NGF and trkANGFR/p75NTR expression in retinal cells from Reeler-L7-EGFPreln-/- transgenic mice (E-Reeler, n=2) and C57BL/6J-L7-EGFPreln+/+ transgenic mice (E-control, n=7), both expressing EGFP positivity in Rod Bipolar Cells (RBC). Methods:Retinas were dissected from not pooled whole eyes. Single cell were obtained by DispaseII and/or Trypsin digestion, in the presence of DNAseI, equilibrated in HBSS-EDTA and fixed in 1% PFA. Cells were probed with specific antibodies (NGF, p75NTR and trkANGFR) and at least 10000 cells were acquired/analysed by flow cytometry, according to the MACSquant technology. Apoptosis was also estimated by AnnexinV. Results:Both treatments were successful to obtain single cells from dissected retinas, albeit trypsin allowed a better side-scatter definition/resolution of population. Three main cell populations were separated according to physical parameters: RBC/retinal ganglion cell (RGC), accessory cells (Muller, Amacrine, Horizontal) and photoreceptor. A slight expression of Annexin V was detected (2.9%). EGFP was detected only in RBC (9%). NGF and p75NTR were expressed mainly in RBC/RGC and faintly in accessories cells, while trkANGFR was weakly expressed by RBC and RGC. Both NGF and p75NTR were increased in GFP-positive RBC and RGC, from E-Reeler as compared to E-control. Conclusions:From our studies, single cells can be enzymatic dissociated from unpooled retinas. The isolation method and the selective NGF/p75NTR increase in GFP-bearing RBC and RGC may provide a good experimental system for studying NGF-reelin cross-talk in these cells.