Abstract
Purpose: :
Glaucoma is a leading cause of blindness worldwide. Many groups have shown severe RGC loss in glaucoma. We have previously used NeuN to quantify neurons in the ganglion cell layer (GCL) of the DBA/2J mouse and these data suggested that RGCs persist much longer in the DBA than previously expected. Based on these data, we sought to perform a functional analysis of axon retrograde transport and axon pathology to explore the possibility that axon deficits appear much sooner than cell death. To accomplish this, we compared axon counts and counts of RGCs labeled with two different retrograde tracers, each requiring a different level of axon integrity, to counts of RGCs labeled with NeuN.
Methods: :
Using unbiased stereology, we compared NeuN–labeled RGCs to counts using FluoroGold (FG) and the carbocyanine tracer fast–DiI. Optic nerve axons were counted at the electron microscope level using an automated system from all mice that received FG injections only. A subset of mice received dual injections of FG and DiI to directly compare active transport to passive, membranous transport.
Results: :
There was no change in NeuN–labeled RGCs from 3 to 18–months. Axon counts decreased by 14% from 3 to 13–months. FG–labeled RGCs in retinas corresponding to the optic nerve axon counts decreased by 48% from 3 to 13–months. DiI labeled 18% fewer RGCs than NeuN and 18% more than FG in the dual–injected animals.
Conclusions: :
These data suggest that axon integrity is compromised primary to RGC loss in this model. FG requires active transport and DiI requires an intact membrane, and here we have shown that NeuN labels more RGCs than both FG and DiI. Also, axon loss at 13–months is approximately one–third that of the loss of FG–labeled RGCs. These observation combined with the data that shows DiI labels more RGCs than FG but fewer than NeuN suggests that some RGCs have axons with compromised transport while others have lost intact axons altogether. These data support the hypothesis that RGCs may persist in this model and that glaucomatous damage is evident in axon function prior to RGC death.
Keywords: cell survival • ganglion cells • apoptosis/cell death