Figure 8. Absence of backlabeled retinal ganglion cells due to axonal transport impairment and cell loss. A-H: These representative examples show whole-mounts of a control retina (A-D) labeled with 10% hydroxystilbamidine methanesulfonate (OHSt) (A) and its isodensity map (C), as well as the same retina immunolabeled with Brn3a (B) to identify surviving RGCs and the corresponding isodensity map (D). To identify RGCs capable of retrograde axonal transport, OHSt was applied to both superior colliculi 1 week before sacrifice. To identify surviving RGCs, retinas were processed for Brn3a immunohistochemistry. Note the presence of intensely stained RGCs distributed throughout the entire control retina, with a typical high-density region along a naso-temporal streak in the superior retina, which is clearly shown in the isodensity maps of OHSt-labeled RGCs (C) and Brn3a-labeled RGCs (D). E-H: Representative examples of experimental retinas at 8 (E, F) and 35 (G, H) days after lasering, showing the isodensity maps of OHSt-labeled RGCs (E, G) and Brn3a-labeled RGCs (F, H). Both experimental retinas show fewer OHSt- and Brn3a-labeled RGCs than in the control retinas. Moreover, the population of Brn3a-labeled RGCs was greater than that of OHSt-labeled RGCs at 8 but not at 35 days, indicating compromised retrograde axonal transport within the first week after lasering and demonstrating that the lack of retrograde labeling in the retina is due not only to RGC degeneration but also to an impairment of the axoplasmic flow. The diminution in the numbers of Brn3a⁺RGCs observed between the control retina and retinas analyzed 8 and 35 days after lasering also documents the loss of RGCs. Isodensity maps were generated by assigning a color code to each of the subdivisions of each individual frame according to its RGC density value within a 45-step color scale range, from 0 (dark blue) to 5,625 RGCs/mm² or higher (red). For all retinas, the dorsal pole is orientated at 12 o’clock (scale bar=1 mm).