Abstract
Purpose:
Retinal ganglion cell (RGC) death is associated with a number of ocular pathologies including glaucoma. In addition to the intrinsic mechanisms of cell death, intercellular communication via gap junctions plays a major role in secondary cell death seen under various pathological conditions. It is believed that gap junctions act as conduits for the movement of toxic molecules from dying cells to their coupled neighbors, thereby amplifying neuronal loss. Since many RGCs are coupled to amacrine cells (ACs) via gap junctions, we examined whether the primary loss of RGCs associated with excitotoxic and ischemic conditions leads to a secondary loss of ACs.
Methods:
Excitotoxicity was induced in vitro by incubation of retina-eyecups of the mouse in NMDA (300 μM). To induce transient retinal ischemia we employed in vivo elevation of intraocular pressure. Antibodies against AC antigens including calretinin (CR), calbindin (CB), and ChAT were used to identify specific subpopulations in the INL and GCL. Application of Lucifer yellow to the cut optic nerve allowed us to identify RGC somata in the GCL.
Results:
Both excitotoxic and ischemic conditions produced a significant loss (50%; p<0.01) of RGCs in the GCL. In addition there was a significant loss of CR-immunoreactive (IR) (63%; p<0.01) and CB-IR ACs (75%; p<0.01) in both the INL and GCL. Interesting, there was a loss of ChAT-IR ACs in the GCL, but no loss of ChAT cells in the INL (p>0.1). Application of the gap junction blocker meclofenamic acid (MFA) to excitotoxic retinas reduced the loss of RGCs by nearly 80%; p<0.01). It also reduced the of CR-IR ACS by 60%; p<0.01) and CB-IR ACs by 75%; p<0.01). For ischemic retinas, MFA injected intravitreally either 30 minutes before or 1 hour after the primary insult reduced RGC loss by about 75%; p<0.01), CB-IR ACs by 65% (p<0.01), and CR-IR ACs by nearly 100% (p<0.01).
Conclusions:
Our results indicate that primary RGC loss under excitotoxic or ischemic conditions is likely followed by a secondary degeneration of neighboring ACs to which they are coupled via gap junctions. Thus, gap junctions may be novel therapeutic targets to significantly reduce cell loss associated with different ocular pathologies.
Keywords: 691 retina: proximal (bipolar, amacrine, and ganglion cells) •
532 gap junctions/coupling •
695 retinal degenerations: cell biology