David M. Linn

Calcium preconditioning triggers neuroprotection in retinal ganglion cells

In the mammalian retina, excitotoxicity has been shown to be involved in apoptotic retinal ganglion cell (RGC) death and is associated with certain retinal disease states including glaucoma, diabetic retinopathy and retinal ischemia. Previous studies from this lab [Wehrwein E, Thompson SA, Coulibaly SF, Linn DM, Linn CL (2004) Invest Ophthalmol Vis Sci 45:1531-1543] have demonstrated that acetylcholine (ACh) and nicotine protects against glutamate-induced excitotoxicity in isolated adult pig RGCs through nicotinic acetylcholine receptors (nAChRs). Activation of nAChRs in these RGCs triggers cell survival signaling pathways and inhibits apoptotic enzymes [Asomugha CO, Linn DM, Linn CL (2010) J Neurochem 112:214-226]. However, the link between binding of nAChRs and activation of neuroprotective pathways is unknown. In this study, we examine the hypothesis that calcium permeation through nAChR channels is required for ACh-induced neuroprotection against glutamate-induced excitotoxicity in isolated pig RGCs. RGCs were isolated from other retinal tissue using a two step panning technique and cultured for 3 days under different conditions. In some studies, calcium imaging experiments were performed using the fluorescent calcium indicator, fluo-4, and demonstrated that calcium permeates the nAChR channels located on pig RGCs. In other studies, the extracellular calcium concentration was altered to determine the effect on nicotine-induced neuroprotection. Results support the hypothesis that calcium is required for nicotine-induced neuroprotection in isolated pig RGCs. Lastly, studies were performed to analyze the effects of preconditioning on glutamate-induced excitotoxicity and neuroprotection. In these studies, a preconditioning dose of calcium was introduced to cells using a variety of mechanisms before a large glutamate insult was applied to cells. Results from these studies support the hypothesis that preconditioning cells with a relatively low level of calcium before an excitotoxic insult leads to neuroprotection. In the future, these results could provide important information concerning therapeutic agents developed to combat various diseases involved with glutamate-induced excitotoxicity.

ACh Receptors Link Two Signaling Pathways to Neuroprotection against Glutamate-Induced Excitotoxicity in Isolated RGCs

J. Neurochem. (2010) 112, 214–226.

Neuroprotection of rat retinal ganglion cells mediated through alpha7 nicotinic acetylcholine receptors.

Glutamate-induced excitotoxicity is thought to play an important role in several neurodegenerative diseases in the central nervous system (CNS). In this study, neuroprotection against glutamate-induced excitotoxicity was analyzed using acetylcholine (ACh), nicotine and the α7 specific nicotinic acetylcholine receptor (α7 nAChR) agonist, N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride (PNU-282987), in cultured adult rat retinal neurons. Adult Long Evans rat retinas were dissociated and retinal ganglion cells (RGCs) were isolated from all other retinal tissue using a two-step panning technique. Once isolated, RGCs were cultured under various pharmacological conditions to demonstrate excitotoxicity and neuroprotection against excitotoxicity. After 3 days, RGCs were immunostained with antibodies against the glycoprotein, Thy 1.1, counted and cell survival was assessed relative to control untreated conditions. 500 μM glutamate induced excitotoxicity in large and small RGCs in an adult rat dissociated culture. After 3 days in culture with glutamate, the cell survival of large RGCs decreased by an average of 48.16% while the cell survival of small RGCs decreased by an average of 42.03%. Using specific glutamate receptor agonists and antagonists, we provide evidence that the excitotoxic response was mediated through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainic acid (KA) and N-methyl-d-aspartate (NMDA) glutamate receptors through an apoptotic mechanism. However, the excitotoxic effect of glutamate on all RGCs was eliminated if cells were cultured for an hour with 10 μM ACh, 100 μM nicotine or 100 nM of the α7 nAChR agonist, PNU-282987, before the glutamate insult. Inhibition studies using 10nM methyllycaconitine (MLA) or α-bungarotoxin (α-Bgt) supported the hypothesis that neuroprotection against glutamate-induced excitotoxicity on rat RGCs was mediated through α7 nAChRs. In immunocytochemical studies, double-labeled experiments using antibodies against Thy 1.1 and α7 nAChR subunits demonstrated that both large and small RGCs contained α7 nAChR subunits. The data presented in this study support the hypothesis that ACh and nicotinic acetylcholine receptor (nAChR) agonists provide neuroprotection against glutamate-induced excitotoxicity in adult rat RGCs through activation of α7 nAChR subunits. These studies lay the groundwork required for analyzing the effect of specific α7 nAChR agonists using in vivo models of excitotoxicity. Understanding the type of ACh receptors involved in neuroprotection in the rat retina could ultimately lead to therapeutic treatment for any CNS disease that involves excitotoxicity.

A Nicotinic Acetylcholine Receptor Agonist Prevents Loss of Retinal Ganglion Cells in a Glaucoma Model

PURPOSE The purpose of this study was to analyze the neuroprotective effect of an α7 nAChR agonist, PNU-282987, using an in vivo model of glaucoma in Long Evans rats. METHODS One eye in each animal was surgically manipulated to induce glaucoma in control untreated animals and in animals that were treated with intravitreal injections of PNU-282987. To induce glaucoma-like conditions, 0.05 mL of 2 M NaCl was injected into the episcleral veins of right eyes in each rat to create scar tissue and increase intraocular pressure. The left eye in each rat acted as an internal control. One month following NaCl injection, rats were euthanized, retinas were removed, flatmounted, fixed, and nuclei were stained with cresyl violet or RGCs were immunostained with an antibody against Thy 1.1 or against Brn3a. Stained nuclei in the RGC layer and labeled RGCs in NaCl-injected retinas were counted and compared with cell counts from untreated retinas in the same animal. RESULTS NaCl injections into the episcleral veins caused a significant loss of cells by an average of 27.35% (± 2.12 SEM) in the RGC layer within 1 month after NaCl injection, which corresponded to a significant loss of RGCs. This loss of RGCs was eliminated if 5 μL of 100 μM PNU-282987 was injected into the right eye an hour before NaCl injection. CONCLUSIONS The results from this study support the hypothesis that the α7 agonist, PNU-282987, has a neuroprotective effect in the rat retina. PNU-282987 may be a viable candidate for future therapeutic treatments of glaucoma.

Tropisetron as a neuroprotective agent against glutamate-induced excitotoxicity and mechanisms of action

The objective of this study was to determine the neuroprotective role of tropisetron on retinal ganglion cells (RGCs) as well as to explore the possible mechanisms associated with alpha7 nAChR-induced neuroprotection. Adult pig RGCs were isolated from all other retinal tissue using a two-step panning technique. Once isolated, RGCs were cultured for 3 days under control untreated conditions, in the presence of 500 μM glutamate to induce excitotoxicity, and when tropisetron was applied before glutamate to induce neuroprotection. 500 μM glutamate decreased RGC survival by an average of 62% compared to control conditions. However, RGCs pretreated with 100 nM tropisetron before glutamate increased cell survival to an average of 105% compared to controls. Inhibition studies using the alpha7 nAChR antagonist, MLA (10 nM), support the hypothesis that tropisetron is an effective neuroprotective agent against glutamate-induced excitotoxicity; mediated by α7 nAChR activation. ELISA studies were performed to determine if signaling cascades normally associated with excitotoxicity and neuroprotection were up- or down-regulated after tropisetron treatment. Tropisetron had no discernible effects on pAkt levels but significantly decreased p38 MAPK levels associated with excitotoxicity from an average of 15 ng/ml to 6 ng/ml. Another mechanism shown to be associated with neuroprotection involves internalization of NMDA receptors. Double-labeled immunocytochemistry and electrophysiology studies provided further evidence that tropisetron caused internalization of NMDA receptor subunits. The findings of this study suggest that tropisetron could be an effective therapeutic agent for the treatment of degenerative disorders of the central nervous system that involves excitotoxicity.

Neuroprotective Strategies in Glaucoma.

BACKGROUND Glaucoma is characterized as a neuropathic disease that causes progressive degeneration of retinal ganglion cells (RGCs) in the retina, resulting in irreversible loss of vision. All conventional treatments for glaucoma are focused on reducing intraocular pressure (IOP) in the anterior chamber of the eye. However, these treatments alone are insufficient to halt the progression of the disease. As a result, neuroprotective strategies have been developed that prevent retinal neuron loss and disease progression. METHODS The goal of this review is to summarize and discuss neuroprotective strategies in glaucoma at the level of the retina and the ganglion cell layer instead of treatments targeting IOP. Recent and past neuroprotective therapies used to prevent the loss of retinal ganglion cells, the loss of axons in the optic nerve and the loss of vision and function associated with glaucoma are presented. RESULTS Pharmacological approaches have targeted specific receptors, signaling cascades and neurotrophic factors to induce neuroprotection in the retina, while others have focused on the mechanism of cellular loss associated with glaucoma, including excitotoxicity, oxidative stress and apoptotic processes. In addition to neuroprotective pharmacological treatments, stem cell, gene therapy and viral research have demonstrated neuroprotection against the loss of RGCs in glaucomatous conditions. CONCLUSION It is likely that future development for glaucoma treatment will include a combination of these treatments to prevent the pathophysiology of glaucoma.

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Glaucoma is a disease of the central nervous system affecting retinal ganglion cells (RGCs). RGC axons making up the optic nerve carry visual input to the brain for visual perception. Damage to RGCs and their axons leads to vision loss and/or blindness. Although the specific cause of glaucoma is unknown, the primary risk factor for the disease is an elevated intraocular pressure. Glaucoma-inducing procedures in animal models are a valuable tool to researchers studying the mechanism of RGC death. Such information can lead to the development of effective neuroprotective treatments that could aid in the prevention of vision loss. The protocol in this paper describes a method of inducing glaucoma - like conditions in an in vivo rat model where 50 µl of 2 M hypertonic saline is injected into the episcleral venous plexus. Blanching of the vessels indicates successful injection. This procedure causes loss of RGCs to simulate glaucoma. One month following injection, animals are sacrificed and eyes are removed. Next, the cornea, lens, and vitreous are removed to make an eyecup. The retina is then peeled from the back of the eye and pinned onto sylgard dishes using cactus needles. At this point, neurons in the retina can be stained for analysis. Results from this lab show that approximately 25% of RGCs are lost within one month of the procedure when compared to internal controls. This procedure allows for quantitative analysis of retinal ganglion cell death in an in vivo rat glaucoma model.