Rabbit Anti-Human RBPMS Polyclonal Antibody

Background: A reliable model of ischemia-reperfusion is required to evaluate the efficacy and safety of neuroprotective therapies for stroke. We present a novel reproducible pterygopalatine-ophthalmic artery ligation model of ischemia-reperfusion injury in the retina. Methods: Rats were subjected to ophthalmic artery/meningeal sheath ligation (OAML-standard method) or clamping of the pterygopalatine-ophthalmic artery (OAC-new method) for 30 minutes. Retinal ganglion cell (RGC) survival was assessed by prelabeling with FluoroGold (FG) (Santa Cruz Biotechnology, CA, USA) and RNA-binding protein with multiple splicing (RBPMS) at 14 days after ischemia, and all results were compared with a sham group (n = 7 in each group). Results: RGC density in the normal-uninjured (FG-labeled) group was 2111 +/- 38 cells/mm(2) (mean +/- standard error of mean) and that in the RBPMS-labeled group was 2142 +/- 35 cells/mm(2). The OAML procedure significantly reduced RGC density to 738 +/- 23 cells/mm(2) and 780 +/- 41 cells/mm(2) (P < .001) in the FG-labeled and RBPMS-labeled groups, respectively. Similarly, OAC reduced RGC survival to 782 +/- 19 cells/mm(2) and 813 +/- 22 cells/mm(2) (P < .001) in the FG-labeled and RBPMS-labeled groups, respectively. RGC survival was similar following OAC and OAML models, suggesting that both induce comparable levels of damage. However, RGC survival in the OAC model was found to have less dispersion than OAML-induced ischemia. Conclusions: These results suggest that the OAC procedure is a reliable reproduction of ischemia-reperfusion injury that mimics the effects of ophthalmic artery occlusion in humans and provides a useful research model for testing manipulations directed against pathways involved in RGC ischemic degeneration.

Axonopathy is a hallmark of many neurodegenerative diseases including glaucoma, where elevated intraocular pressure (ocular hypertension, OHT) stresses retinal ganglion cell (RGC) axons as they exit the eye and form the optic nerve. OHT causes early changes in the optic nerve such as axon atrophy, transport inhibition, and gliosis. Importantly, many of these changes appear to occur prior to irreversible neuronal loss, making them promising points for early diagnosis of glaucoma. It is unknown whether OHT has similarly early effects on the function of RGC output to the brain. To test this possibility, we elevated eye pressure in mice by anterior chamber injection of polystyrene microbeads. Five weeks post-injection, bead-injected eyes showed a modest RGC loss in the peripheral retina, as evidenced by RBPMS antibody staining. Additionally, we observed reduced dendritic complexity and lower spontaneous spike rate of Ona RGCs, targeted for patch clamp recording and dye filling using a Opn4-Cre reporter mouse line. To determine the influence of OHT on retinal projections to the brain, we expressed Channelrhodopsin-2 (ChR2) in melanopsin-expressing RGCs by crossing the Opn4-Cre mouse line with a ChR2-reporter mouse line and recorded post-synaptic responses in thalamocortical relay neurons in the dorsal lateral geniculate nucleus (dLGN) of the thalamus evoked by stimulation with 460 nm light. The use of a Opn4-Cre reporter system allowed for expression of ChR2 in a narrow subset of RGCs responsible for image-forming vision in mice. Five weeks following OHT induction, paired pulse and high-frequency stimulus train experiments revealed that presynaptic vesicle release probability at retinogeniculate synapses was elevated. Additionally, miniature synaptic current frequency was slightly reduced in brain slices from OHT mice and proximal dendrites of post-synaptic dLGN relay neurons, assessed using a Sholl analysis, showed a reduced complexity. Strikingly, these changes occurred prior to major loss of RGCs labeled with the Opn4-Cre mouse, as indicated by immunofluorescence staining of ChR2-expressing retinal neurons. Thus, OHT leads to pre-and postsynaptic functional and structural changes at retinogeniculate synapses. Along with RGC dendritic remodeling and optic nerve transport changes, these retinogeniculate synaptic changes are among the earliest signs of glaucoma.