Sunday, February 22, 2009

TRPV1 and Retinal Ganglion Cell Apoptosis

The publications referencing our TRPV (Vanilloid); TRPM; TRPA and TRPC Antibodies keep on coming.

In this February 2009 publication, Dr. David Calkins and his team at Vanderbilt demontrate that Retinal Ganglian Cells express the TRPV1 channel and that TRPV1 activation contributes to their death with exposure to hydrostatic pressure . We also demonstrated that activation of TRPV1 alone was sufficient to induce apoptosis of RGCs.
Rebecca M. Sappington, Tatiana Sidorova, Daniel J. Long, and David J. Calkins. TRPV1: Contribution to Retinal Ganglion Cell Apoptosis and Increased Intracellular Ca2+ with Exposure to Hydrostatic Pressure. Invest. Ophthalmol. Vis. Sci., Feb 2009; 50: 717 - 728.
...we used rabbit anti-mouse TRPV1 IgG (1:500; catalog number RA14113; Neuromics, Edina, MN) against the absolute C terminus of mouse TRPV1 (EDAEVFKDSMAPGEK)...

TRPV1 localization in RGCs of rat retina. (A) Immunocytochemical labeling for TRPV1 shows strong localization in the outer retina and in RGCs (large cell bodies); there is little or no label in smaller displaced amacrine cells of the GCL. Clear examples of amoeboid-shaped cell bodies of microglia cells are indicated (ovals). Right: Control section preabsorbed using the TRPV1 blocking peptide (+BP). (B) Confocal image stack through GCL and NFL showing labeling for TRPV1 in wholemount preparation counterlabeled with antibodies against heavy-chain neurofilaments that recognize broad-field RGCs (SMI32). Image shows punctate localization to dendrites (arrows) as well as intense label to cell bodies (brackets); smaller cell bodies with TRPV1 label are in the background. (C) Confocal image stack through GCL and NFL of peripheral retina shows TRPV1 in RGC cell bodies (bracket) and in small bundles of RGC axons (arrows). (D) Confocal stack from central retina shows TRPV1 in RGC cell bodies (bracket) and in axon bundles in the NFL as they course toward the optic nerve head. Amoeboid-shaped cell bodies of microglia are apparent (ovals). (E) Confocal image in single plane at GCL/NFL border shows Iba-1–labeled microglia processes colocalizing with TRPV1, as we previously demonstrated.51 TRPV1-label RGC cell bodies (brackets) and axons (arrows) are indicated for reference. (F) Immunocytochemical labeling demonstrates strong perinuclear and dendritic localization of TRPV1 in cultured RGCs counterstained with the nuclear label DAPI. Localization to dendritic processes and neurites (dashed circles) includes node-like clusters; right: these regions are shown at higher magnification. (G, top) Western blot against TRPV1 in brain and whole retina from adult rat shows band at expected molecular weight (arrowheads; 100–113 kDa). Retina demonstrates an additional band with a slightly lower molecular weight that probably corresponds to a different glycosylation state for this antibody.79 Bottom: Control Western blot with preabsorption of TRPV1 antibody using the blocking peptide prevents detection of both bands. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; NFL, nerve fiber layer.

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