Prevention of Glaucoma-Induced Retinal Ganglion Cell Loss Using Alpha7 nAChR Agonists

Special Article - Retinal Diseases

J Ophthalmol & Vis Sci. 2016; 1(1): 1003.

Prevention of Glaucoma-Induced Retinal Ganglion Cell Loss Using Alpha7 nAChR Agonists

Birkholz PJ¹, Gossman CA¹, Webster MK¹, Linn DM² and Linn CL¹*

¹Department of Biological Sciences, Western Michigan University, USA

²Department of Biomedical Sciences, Grand Valley State University, USA

*Corresponding author: Cindy Linn, Department of Biological Sciences, Western Michigan University, USA

Received: February 20, 2016; Accepted: March 28, 2016; Published: March 30, 2016


In this study, the neuroprotective effect of various nicotinic alpha7 acetylcholine receptor agonists in an in-vivo model of glaucoma using adult Long Evans rats was analyzed. Glaucoma-like conditions were induced in the eyes of Long Evans rats after injection of hypertonic saline into episcleral veins to create scar tissue and increase the animal’s intraocular pressure. This procedure produced significant loss of retinal ganglion cells within one month and was associated with an increase of intraocular pressure. Using this model system, various alpha7 nicotinic acetylcholine receptor (a7 nAChR) agonists were applied at different doses as eye drops to the right eye of adult Long Evans rats while the left eye was left as an internal control. The a7 nAChR agonists used in this study prevented loss of RGCs in a dose dependent manner after the procedure to induce glaucoma-like conditions. PHA-543613 and PNU- 282987 provided the largest degree of RGC survival after inducing glaucomalike conditions, followed by nicotine, SEN 12333, tropisetron, 3-Bromocytisine and DMAB. To provide evidence that neuroprotection of RGCs was mediated through activation of a7 nAChR, in some studies different concentrations of the a7 nAChR antagonist, MLA, was intravitreally injected into experimentally treated eyes before initiation of eye drops and the procedure to induce glaucoma-like conditions. In the presence of MLA, RGC neuroprotection was blocked. Results from these studies suggest that selective a7 nAChR agonists may be used in future therapeutic treatments for glaucoma or other CNS diseases associated with a7 nAChRs.

Keywords: Alpha7 nicotinic acetylcholine receptors; Glaucoma; Retina; Retinal ganglion cells; Acetylcholine; Nicotinic agonists


ACh: Acetylcholine; a7 nAChRs: Alpha7 Nicotinic Acetylcholine Receptors; CO2: Carbon Dioxide; DMAB: 4-[(5,6-Dihydro[2,3’- bipyridin]-3(4H)-ylidene)methyl]-N,N-dimethylbenzenamine dihydrochloride; DMSO: Dimethyl Sulfoxide; hERG: human Ether-a-go-go Related Gene; IACUC: Institutional Animal Care and Use Committee; IOP: Intraocular Pressure; KAX: Ketamine, Acepromazine and Xylaxine cocktail; LC MS/MS: Liquid Chromatography, Mass Spectroscopy, Mass Spectroscopy; ONH: Optic Nerve Head; PBS: Phosphate Buffer; RGCs: Retinal Ganglion Cells; NaCl: Sodium Chloride


Glaucoma is one of the leading causes of blindness worldwide [1- 3]. It is a neurodegenerative disorder characterized by a progressive optic neuropathy, cupping of the optic disk, the death of RGCs and degeneration of axons in the optic nerve [4,5]. The primary risk factor associated with glaucoma is an increase in intraocular pressure, which has been linked to apoptosis of Retinal Ganglion Cells (RGCs) [6-8]. Recently, a great deal of research has explored the agents and mechanisms that provide neuroprotection against neurodegenerative conditions. Activation of alpha7 nicotinic acetylcholine receptors (a7 nAChRs) in the brain have been linked to neuroprotection against several neurodegenerative diseases [9,10]. There is strong evidence that a7 nAChRs are neuroprotective, reducing β-amyloid induced toxicity in Alzheimer’s disease [11,12] and that the a7 nAChRs plays a role in the pathophysiology of schizophrenia [13,14]. In the retina, RGCs contain a7 nAChRs [15-17] and receive cholinergic input from a well-described population of starburst amacrine cells [18,19]. They are the only source of ACh in the vertebrate retina. Previous studies have demonstrated that intravitreal injections or eye drop application of the a7 nAChR agonist, PNU-282987, in an in vivo rat model, prevented the loss of RGCs typically associated with a procedure to induce glaucoma-like conditions [20,21].

Binding studies in rat chimera cells using PNU-282987 have demonstrated that PNU-282987 is a potent specific agonist for a7 nAChRs [22]. These studies demonstrated that Methyllycaconitine (MLA), a specific a7 nAChR antagonist, preferentially bound to a7 nAChRs when both PNU-282987 and MLA were present. In electrophysiology studies using rat hippocampal neurons, PNU- 282987 evoked a rapidly desensitizing inward whole-cell current associated with the opening of the a7 nAChR channel. This current was eliminated if MLA was introduced before PNU-282987 [22].

Although PNU-282987 has been used in animal schizophrenia models, it has not been found to be suitable for systemic use in humans because of excessive inhibition of a hERG anti target in the heart [23]. As a result, other a7 nAChR agonists were investigated to determine their neuroprotective effect following topical delivery in a rat glaucoma model. Comparisons between the neuroprotective effect of PNU-282987 and the other a7 nAChR agents are discussed. The results from these studies support the hypothesis that a7 nAChR agonists may have a role in the future as a therapeutic intervention for glaucoma.

Materials and Methods


Both male and female adult Long Evans rats were used in this study. Rats were used at 3 months of age. Out bred Long Evans rats were chosen because of their docile nature, pigmented eyes, the lack of genetic defects that are associated with other inbred rat species and for consistency with studies already completed [20,21]. All animals were cared for in accordance with the approved guidelines of the Institutional Animal Care and Use Committee (IACUC) at Western Michigan University.


To anesthetize Long Evans rats, animals were injected intraperitoneally with 1.0 ml/kg KAX standard rat cocktail; consisting of a solution of 5 ml of ketamine (100 mg/ml), 2.5 ml of xylazine (20 mg/ml), and 1 ml of acepromazine (10 mg/ml) in 3 ml sterile water. KAX was injected prior to hypertonic saline injections to induce glaucoma and prior to intravitreal injections (if utilized in the experiment). To ensure complete anesthesia, a lack of tail and toe pinch reflex were needed before proceeding with procedures. After the procedures were completed, rats were placed on a warm circulating water pad and watched until they were fully awake and functioning normally before being returned to the animal colony.

Procedure to induce glaucoma

To induce glaucoma, rats were anesthetized with an intraperitoneal injection of KAX and two drops of pilocarpine were added to the right eye of each rat to prevent the blinking reflex. After applying betadine solution around the eye, a hemostat was used to pinch the skin surrounding the eye to bulge the eye from the eye socket and reveal the episcleral vein for hypertonic saline injection. The right eye in each animal was used for procedural manipulation while the left eye served as an untreated internal control for each experiment unless noted otherwise. Under a dissection microscope, the episcleral vein of the right eye was injected with 50 μL of 2M NaCl using a micro needle assembly [20]. Injection of 2M NaCl caused blanching of the vein, which ensured a successful injection. Observation of a blanched vein correlated to a significant loss of RGCs one month following the procedure [20]. Following the injection, a small amount of antibiotic ointment was applied over the injection site and where the hemostat pinched the skin around the eye. This procedure was modified from the procedure originally developed by Morrison et al. [24]. In sham procedure studies, injections were made with PBS instead of ACh agonists.

IOP measurements using Tonopen

IOP measurements were obtained before hypertonic injections to obtain a baseline and after hypertonic injections into the episcleral veins to measure any increase of IOP. IOP measurements were obtained from awake behaving rats using the Tono-Pen XL tonometer (Mentor, Norwell MA) according to instructions outlined by Morrison et al. [24]. Rat eyes were anesthetized with 1 drop of 0.5% proparacaine hydrochloride before using the Tono-Pen. Rats were loosely held in the experimenter’s hand during this procedure. IOP measurements were obtained each day for 1 week before hypertonic injections and 5 times each week after hypertonic injections until the rats were sacrificed [20].

Eye treatments

Previous in vivo studies on adult Long Evans rats have shown that the a7 nAChR specific agonist, PNU-282987, prevented the loss of RGCs typically associated with inducing glaucoma-like conditions when applied as eye drops [21]. Other studies have also demonstrated that pharmacological agents can reach the retina if the agents are dissolved in appropriate vehicles [25-27]. In this study, the neuroprotective effect of the following commercially available a7 nAChR agonists were examined, including PHA-543613, PNU- 282987, nicotine, SEN 12333, tropisetron, 3-bromocytisine and DMAB. Three different concentrations of each agent were used for dose-response studies. The concentrations of agents used were based on previously obtained preliminary results. ACh agonists were dissolved in Dimethyl Sulfoxide (DMSO) to make a stock solution and then diluted in PBS and sterilized using syringe filters. 30 μl eye drops were applied for three days to the bulbar conjunctiva of the eye before the injection of hypertonic solution to induce glaucomalike conditions and for one month following the procedure [20,21]. Eye drops were applied twice a day based on previous LC MS/MS studies that demonstrated evidence of PNU-282987 in the retina up to 12 hours after treatment [20]. Between 4 and 20 animals were used for each experiment. The left eye in each animal was untreated and served as an internal control to compare against the experimentally treated right eye. After one month, animals were sacrificed by C02 asphyxiation and retinas were removed for RGC labeling. Vehicle control experiments were performed concurrently using only PBS eye drops (N=4), or eye drops containing up to 1% DMSO (N=4).

To support the hypothesis that the ACh agonists act through a7 nAChRs, experiments were performed using a specific a7 nAChR antagonist, MLA. In these experiments, 5 μL of various concentrations of the a7 nAChR specific antagonist, MLA (0, 1, 10, 100nM) (N=4 for each concentration),was injected directly into the vitreal chamber of the right eye using a Hamilton syringe 1 hour before initiation of eye drop treatments and before the procedure to induce glaucoma-like conditions.

Tissue preparation and analysis

One month following the hypertonic saline injection, rats were euthanized using CO2 asphyxiation. Both eyes were subsequently removed and processed [20]. After removal of the cornea, lens and vitreous humor, the whole retina was peeled away from the back of the eye cup. Care was taken to remove the retina in one piece to maintain geographical orientation and landmarks. Once the whole retinas were removed, four short evenly spaced slits were made around the retina’s periphery that allowed the retinas to be flattened. The retina was then pinned out flat onto sylgard plates using cactus needles and fixed in 4% paraformaldehyde for 24 hours at 4oC. The next day, retinas were rinsed, blocked and permeabilized with 2% bovine serum and 1% triton X-100 (Sigma) for two hours at room temperature. Retinas were then immunostained with antibodies against Thy 1.1 or double-labeled with antibodies against Thy 1.1 and Brn3a. Anti Thy 1.1 (mouse anti-rat, BD Biosciences) is a monoclonal antibody against glycoproteins found exclusively on the plasma membrane of RGCs in the retina [28]. Anti-Brn3a is an antibody that labels the transcription factor found in RGCs [29]. For single labeling of RGCs, the primary antibody, mouse anti-Thy1.1 (1:300, BD Pharmingen) was added to the flat-mounted retina and incubated overnight on a rocker, in a humidified chamber at room temperature. 24 hours later, retinas were rinsed twice in 0.1% Triton X-100 and three times in PBS. The secondary antibody, Alexa Fluor 594 goat anti-mouse, was then applied in PBS (1:300, Life technologies) and the retinas were incubated for 24 hours on a rocker, in a darkened humidified chamber at room temperature. After incubation in secondary antibody, the cactus needles were removed and the retinas were transferred to glass slides and mounted using 50% glycerol and 50% PBS. When double-labeling RGCs, anti-Thy 1.1 and rabbit anti-Brn3a (1:300) were used in PBS containing 2% bovine serum. The secondary antibodies used to visualize double labeled RGCs were Alexa Fluor 594 goat anti-mouse and Alexa Fluor 488 donkey anti-rabbit (1:300, Life technologies) [20].

Once stained, the number of single or double-labeled RGCs throughout the RGC layer at specific regions of the retina was counted from images obtained 4 mm from the Optic Nerve Head (ONH) using the Z-stack capabilities of a Nikon confocal microscope. In each retina, 200 μm2 images were obtained from the dorsal retina containing the highest density of RGCs in the visual streak, the ventral, nasal and temporal regions of the retina [20]. All images were obtained 4 mm from the ONH as a previous study demonstrated that the greatest loss of RGCs occurred in the peripheral retina under glaucoma conditions [20]. The number of RGCs counted from each experimentally treated retina were averaged and compared directly to the number of RGCs obtained from internal control retinas and normalized by calculating the percent change from each internal control. Four to twenty different rats were used for each experimental condition and in control studies to determine if the delivery method or vehicle had any effect on RGC counts. In other control studies, experiments were conducted to display specificity of the antibodies used. In four negative control experiments, retinas were processed with the primary antibodies omitted, while another four experiments substituted non-immune mouse immunoglobulin (dilution: 0.1 - 1.0 μg/ml) for the antibodies. In other experiments, pre absorption controls were performed where the primary antibody and antigens were added together before applying to tissue (N=4). No significant epifluorescence was observed under any of these conditions.

Statistical analysis

For normalized data, statistical analysis was performed using Kruskall-Wallis non parametric analysis of variance with post hoc comparisons (Dunn’s test). P-values of < 0.05 represented significance. Graphs were plotted with Prism GraphPad version 4.0 software (GraphPad Software, Inc., San Diego, CA). All data values are reported as the mean ± Standard Error (S.E.). Errors bars are presented one-sided to prevent error bar clutter in dose-response studies and for consistency.


Glaucoma effects on RGCs

The primary risk factor associated with glaucoma is an increase in Intraocular Pressure (IOP) [30-32]. Earlier studies from this lab using the Tono-Pen XL tonometer (Mentor, Norwell MA) have demonstrated that injection of hypertonic saline into Long Evans rat’s episcleral veins significantly increased intraocular pressure from an average of 13 mm Hg before the procedure to induce glaucoma to an average of 21 mm Hg within one month afterward [20]. Corresponding with this increase of intraocular pressure is a significant loss of RGCs. (Figure 1) illustrates the typical loss of RGCs that result from 50 μl injection of 2M NaCl into the episcleral vein of adult anesthetized Long Evans rats. (Figure 1A) is an image obtained from the left control untreated eye of a flat-mounted retina doublelabeled with antibodies against the glycoprotein, Thy 1.1 and the RGC nuclear marker, Brn3a. Images were obtained from the nerve fiber layer and the RGC layer using a Nikon confocal microscope. The antibody against Thy 1.1clearly labeled the plasma membrane of RGC bodies (arrows) when secondarily labeled with Alexa Fluor 594. Anti-Brn3a also labeled RGCs and was found to be co-localized in cell bodies of the RGC layer that labeled with Thy 1.1. The anti-Brn3a antibody was secondarily labeled with Alexa Fluor 488. Co-labeled RGCs occurred 95.5% (+/-3.5; N=5) of the time.