Endothelin B receptors contribute to retinal ganglion cell loss in a rat model of glaucoma

Glaucoma is an optic neuropathy, commonly associated with elevated intraocular pressure (IOP) characterized by optic nerve degeneration, cupping of the optic disc, and loss of retinal ganglion cells which could lead to loss of vision. Endothelin-1 (ET-1) is a 21-amino acid vasoactive peptide that plays a key role in the pathogenesis of glaucoma; however, the receptors mediating these effects have not been defined. In the current study, endothelin B (ET(B)) receptor expression was assessed in vivo, in the Morrison's ocular hypertension model of glaucoma in rats. Elevation of IOP in Brown Norway rats produced increased expression of ET(B) receptors in the retina, mainly in retinal ganglion cells (RGCs), nerve fiber layer (NFL), and also in the inner plexiform layer (IPL) and inner nuclear layer (INL). To determine the role of ET(B) receptors in neurodegeneration, Wistar-Kyoto wild type (WT) and ET(B) receptor-deficient (KO) rats were subjected to retrograde labeling with Fluoro-Gold (FG), following which IOP was elevated in one eye while the contralateral eye served as control. IOP elevation for 4 weeks in WT rats caused an appreciable loss of RGCs, which was significantly attenuated in KO rats. In addition, degenerative changes in the optic nerve were greatly reduced in KO rats compared to those in WT rats. Taken together, elevated intraocular pressure mediated increase in ET(B) receptor expression and its activation may contribute to a decrease in RGC survival as seen in glaucoma. These findings raise the possibility of using endothelin receptor antagonists as neuroprotective agents for the treatment of glaucoma.     

Aldosterone: a mediator of retinal ganglion cell death and the potential role in the pathogenesis in normal-tension glaucoma

Glaucoma is conventionally defined as a chronic optic neuropathy characterized by progressive loss of retinal ganglion cells (RGCs) and optic nerve fibers. Although glaucoma is often associated with elevated intraocular pressure (IOP), significant IOP reduction does not prevent progression of the disease in some glaucoma patients. Thus, exploring IOP-independent mechanisms of RGC loss is important. We describe chronic systemic administration of aldosterone and evaluate its effect on RGCs in rat. Aldosterone was administered via an osmotic minipump that was implanted subcutaneously into the mid-scapular region. Although systemic administration of aldosterone caused RGC loss associated with thinning of the retinal nerve fiber layer without elevated IOP, the other cell layers appeared to be unaffected. After chronic administration of aldosterone, RGC loss was observed at 2 weeks in the peripheral retina and at 4 weeks in the central retina. However, administration of mineralocorticoid receptor blocker prevented RGC loss. These results demonstrate aldosterone is a critical mediator of RGC loss that is independent of IOP. We believe this rat normal-tension glaucoma (NTG) animal model not only offers a powerful system for investigating the mechanism of neurodegeneration in NTG, but can also be used to develop therapies directed at IOP-independent mechanisms of RGC loss.     

Alpha2-macroglobulin is a mediator of retinal ganglion cell death in glaucoma

Glaucoma is defined as a chronic and progressive optic nerve neuropathy, characterized by apoptosis of retinal ganglion cells (RGC) that leads to irreversible blindness. Ocular hypertension is a major risk factor, but in glaucoma RGC death can persist after ocular hypertension is normalized. To understand the mechanism underlying chronic RGC death we identified and characterized a gene product, alpha2-macroglobulin (alpha2M), whose expression is up-regulated early in ocular hypertension and remains up-regulated long after ocular hypertension is normalized. In ocular hypertension retinal glia up-regulate alpha2M, which binds to low-density lipoprotein receptor-related protein-1 receptors in RGCs, and is neurotoxic in a paracrine fashion. Neutralization of alpha2M delayed RGC loss during ocular hypertension; whereas delivery of alpha2M to normal eyes caused progressive apoptosis of RGC mimicking glaucoma without ocular hypertension. This work adds to our understanding of the pathology and molecular mechanisms of glaucoma, and illustrates emerging paradigms for studying chronic neurodegeneration in glaucoma and perhaps other disorders.     

Neurotrophic rationale in glaucoma: a TrkA agonist, but not NGF or a p75 antagonist, protects retinal ganglion cells in vivo

Glaucoma is a major cause of vision impairment, which arises from the sustained and progressive apoptosis of retinal ganglion cells (RGC), with ocular hypertension being a major risk or co-morbidity factor. Because RGC death often continues after normalization of ocular hypertension, growth factor-mediated protection of compromised neurons may be useful. However, the therapeutic use of nerve growth factor (NGF) has not proven effective at delaying RGC death in glaucoma. We postulated that one cause for the failure of NGF may be related to its binding to two receptors, TrkA and p75. These receptors have distinct cellular distribution in the retina and in neurons they induce complex and sometimes opposing activities. Here, we show in an in vivo therapeutic model of glaucoma that a selective agonist of the pro-survival TrkA receptor was effective at preventing RGC death. RGC loss was fully prevented by combining the selective agonist of TrkA with intraocular pressure-lowering drugs. In contrast, neither NGF nor an antagonist of the pro-apoptotic p75 receptor protected RGCs. These results further a neurotrophic rationale for glaucoma.     

Opposing roles for membrane bound and soluble Fas ligand in glaucoma-associated retinal ganglion cell death

Glaucoma, the most frequent optic neuropathy, is a leading cause of blindness worldwide. Death of retinal ganglion cells (RGCs) occurs in all forms of glaucoma and accounts for the loss of vision, however the molecular mechanisms that cause RGC loss remain unclear. The pro-apoptotic molecule, Fas ligand, is a transmembrane protein that can be cleaved from the cell surface by metalloproteinases to release a soluble protein with antagonistic activity. Previous studies documented that constitutive ocular expression of FasL maintained immune privilege and prevented neoangeogenesis. We now show that FasL also plays a major role in retinal neurotoxicity. Importantly, in both TNFα triggered RGC death and a spontaneous model of glaucoma, gene-targeted mice that express only full-length FasL exhibit accelerated RGC death. By contrast, FasL-deficiency, or administration of soluble FasL, protected RGCs from cell death. These data identify membrane-bound FasL as a critical effector molecule and potential therapeutic target in glaucoma.     

Transplantation of Human Neural Progenitor Cells Expressing IGF-1 Enhances Retinal Ganglion Cell Survival

We have previously characterized human neuronal progenitor cells (hNP) that can adopt a retinal ganglion cell (RGC)-like morphology within the RGC and nerve fiber layers of the retina. In an effort to determine whether hNPs could be used a candidate cells for targeted delivery of neurotrophic factors (NTFs), we evaluated whether hNPs transfected with an vector that expresses IGF-1 in the form of a fusion protein with tdTomato (TD), would increase RGC survival in vitro and confer neuroprotective effects in a mouse model of glaucoma. RGCs co-cultured with hNP^IGF-TD cells displayed enhanced survival, and increased neurite extension and branching as compared to hNP^TD or untransfected hNP cells. Application of various IGF-1 signaling blockers or IGF-1 receptor antagonists abrogated these effects. In vivo, using a model of glaucoma we showed that IOP elevation led to reductions in retinal RGC count. In this model, evaluation of retinal flatmounts and optic nerve cross sections indicated that only hNP^IGF-TD cells effectively reduced RGC death and showed a trend to improve optic nerve axonal loss. RT-PCR analysis of retina lysates over time showed that the neurotrophic effects of IGF-1 were also attributed to down-regulation of inflammatory and to some extent, angiogenic pathways. This study shows that neuronal progenitor cells that hone into the RGC and nerve fiber layers may be used as vehicles for local production and delivery of a desired NTF. Transplantation of hNP^IGF-TD cells improves RGC survival in vitro and protects against RGC loss in a rodent model of glaucoma. Our findings have provided experimental evidence and form the basis for applying cell-based strategies for local delivery of NTFs into the retina. Application of cell-based delivery may be extended to other disease conditions beyond glaucoma.     

An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible vision loss. Examples of such diseases in human include traumatic optic neuropathy and optic nerve degeneration in glaucoma. It is characterized by typical changes in the optic nerve head, progressive optic nerve degeneration, and loss of retinal ganglion cells, if uncontrolled, leading to vision loss and blindness.The optic nerve crush (ONC) injury mouse model is an important experimental disease model for traumatic optic neuropathy, glaucoma, etc. In this model, the crush injury to the optic nerve leads to gradual retinal ganglion cells apoptosis. This disease model can be used to study the general processes and mechanisms of neuronal death and survival, which is essential for the development of therapeutic measures. In addition, pharmacological and molecular approaches can be used in this model to identify and test potential therapeutic reagents to treat different types of optic neuropathy.Here, we provide a step by step demonstration of (I) Baseline retrograde labeling of retinal ganglion cells (RGCs) at day 1, (II) Optic nerve crush injury at day 4, (III) Harvest the retinae and analyze RGC survival at day 11, and (IV) Representative result.Download video file.^{(53M, mov)}     

Susceptibility to neurodegeneration in a glaucoma is modified by Bax gene dosage

In glaucoma, harmful intraocular pressure often contributes to retinal ganglion cell death. It is not clear, however, if intraocular pressure directly insults the retinal ganglion cell axon, the soma, or both. The pathways that mediate pressure-induced retinal ganglion cell death are poorly defined, and no molecules are known to be required. DBA/2J mice deficient in the proapoptotic molecule BCL2-associated X protein (BAX) were used to investigate the roles of BAX-mediated cell death pathways in glaucoma. Both Bax+/- and Bax-/- mice were protected from retinal ganglion cell death. In contrast, axonal degeneration was not prevented in either Bax+/- or Bax-/- mice. While BAX deficiency did not prevent axonal degeneration, it did slow axonal loss. Additionally, we compared the effects of BAX deficiency on the glaucoma to its effects on retinal ganglion cell death due to two insults that are proposed to participate in glaucoma. As in the glaucoma, BAX deficiency protected retinal ganglion cells after axon injury by optic nerve crush. However, it did not protect retinal ganglion cells from N-methyl-D-aspartate (NMDA)-induced excitotoxicity. BAX is required for retinal ganglion cell death in an inherited glaucoma; however, it is not required for retinal ganglion cell axon degeneration. This indicates that distinct somal and axonal degeneration pathways are active in this glaucoma. Finally, our data support a role for optic nerve injury but not for NMDA receptor-mediated excitotoxicity in this glaucoma. These findings indicate a need to understand axon-specific degeneration pathways in glaucoma, and they suggest that distinct somal and axonal degeneration pathways may need to be targeted to save vision.     

Cannabinol modulates neuroprotection and intraocular pressure: A potential multi-target therapeutic intervention for glaucoma

ObjectivesGlaucoma is characterized by progressive damage of the retinal ganglion cells (RGCs), resulting in irreversible vision loss. Cannabinoids (CBs) ameliorate several factors that contribute to the progression of glaucoma, including increased intraocular pressure (IOP), degeneration of RGC and optical nerve (ON) damage. However, a direct correlation of specific CBs with the molecular events pertaining to glaucoma pathology is not well established. Therefore, this study aims to evaluate the role of cannabinol (CBN) on RGC protection, modulation of IOP, and its effects on the level of extracellular matrix (ECM) proteins using both in vitro and in vivo models of glaucoma.Methods and resultsWhen exposed to elevated hydrostatic pressure, CBN, in a dose-dependent manner, protected differentiated mouse 661W retinal ganglion precursor-like cells from pressure-induced toxicity. In human trabecular meshwork cells (hTM), CBN attenuated changes in the ECM proteins, including fibronectin and α-smooth muscle actin (α-SMA), as well as mitogen-activated protein kinases (phospho-ERK1/2) in the presence or absence of transforming growth factor-beta 2 (TGF-β₂) induced stress. Ocular pharmacokinetic parameters were evaluated post-intravitreal (IVT) CBN delivery in vivo. Furthermore, we demonstrated that IVT-administered CBN improved pattern electroretinogram (pERG) amplitudes and reduced IOP in a rat episcleral vein laser photocoagulation model of glaucoma.ConclusionCBN promotes neuroprotection, abrogates changes in ECM protein, and normalizes the IOP levels in the eye. Therefore, our observations in the present study indicate a therapeutic potential for CBN in the treatment of glaucoma.     

Protection of Pattern Electroretinogram and Retinal Ganglion Cells by Oncostatin M after Optic Nerve Injury

Injury to retinal ganglion cell (RGC) axons leads to selective loss of RGCs and vision. Previous studies have shown that exogenous neurotrophic factors promote RGC survival. We investigated the neuroprotective effects of oncostatin M (OSM), a member of the IL-6 family of cytokines, on pattern electroretinogram (PERG) and RGC survival after optic nerve crush (ON-crush) in the mouse. BALB/C mice received ON-crush in the left eyes for either 4-second or 1-second duration (4-s or 1-s). Fluoro-gold retrograde labeling was used to identify RGCs. RGC function was assessed by PERG measurement. OSM or CNTF protein was injected intravitreally immediately after ON-crush. OSM responsive cells were identified by localization of increased STAT3 phosphorylation. Significant higher RGC survival (46% of untreated control) was seen in OSM-treated eyes when assessed 2 weeks after 4-s ON-crush as compared to that (14% of untreated control) of the PBS-treated eyes (P<0.001). In addition, PERG amplitude was significantly higher in eyes treated with OSM or CNTF 1 week after 1-s ON-crush (36% of baseline) as compared with the amplitude of PBS-treated eyes (19% of the baseline, P = 0.003). An increase in STAT3 phosphorylation was localized in Müller layer after OSM treatment, suggesting that Müller cells mediate the effect of OSM. Our results demonstrate that one single injection of either OSM or CNTF after ON-crush improves RGC survival together with their electrophysiological activity. These data provide proof-of-concept for using neurotrophic factors OSM and CNTF for RGC degenerative diseases, including glaucoma and acute optic nerve trauma.     

Hydrostatic Pressure Does Not Cause Detectable Changes in Survival of Human Retinal Ganglion Cells

Purpose

Elevated intraocular pressure (IOP) is a major risk factor for glaucoma. One consequence of raised IOP is that ocular tissues are subjected to increased hydrostatic pressure (HP). The effect of raised HP on stress pathway signaling and retinal ganglion cell (RGC) survival in the human retina was investigated.

Methods

A chamber was designed to expose cells to increased HP (constant and fluctuating). Accurate pressure control (10-100mmHg) was achieved using mass flow controllers. Human organotypic retinal cultures (HORCs) from donor eyes (<24h post mortem) were cultured in serum-free DMEM/HamF12. Increased HP was compared to simulated ischemia (oxygen glucose deprivation, OGD). Cell death and apoptosis were measured by LDH and TUNEL assays, RGC marker expression by qRT-PCR (THY-1) and RGC number by immunohistochemistry (NeuN). Activated p38 and JNK were detected by Western blot.

Results

Exposure of HORCs to constant (60mmHg) or fluctuating (10-100mmHg; 1 cycle/min) pressure for 24 or 48h caused no loss of structural integrity, LDH release, decrease in RGC marker expression (THY-1) or loss of RGCs compared with controls. In addition, there was no increase in TUNEL-positive NeuN-labelled cells at either time-point indicating no increase in apoptosis of RGCs. OGD increased apoptosis, reduced RGC marker expression and RGC number and caused elevated LDH release at 24h. p38 and JNK phosphorylation remained unchanged in HORCs exposed to fluctuating pressure (10-100mmHg; 1 cycle/min) for 15, 30, 60 and 90min durations, whereas OGD (3h) increased activation of p38 and JNK, remaining elevated for 90min post-OGD.

Conclusions

Directly applied HP had no detectable impact on RGC survival and stress-signalling in HORCs. Simulated ischemia, however, activated stress pathways and caused RGC death. These results show that direct HP does not cause degeneration of RGCs in the ex vivo human retina.     

Pilocarpine Protects Cobalt Chloride-induced Apoptosis of RGC-5 Cells: Involvement of Muscarinic Receptors and HIF-1α Pathway

The retina is the most metabolically active tissue in the human body and hypoxia-induced retinal ganglion cell (RGC) death has been implicated in glaucomatous optic neuropathy. The aim of this study is to determine whether muscarinic receptor agonist pilocarpine, a classic antiglaucoma drug, possesses neuroprotection against cobalt chloride (CoCl₂)-mimetic hypoxia-induced apoptosis of rat retinal ganglion cells (RGC-5 cells) and its underlying mechanisms. Cell viability was determined by Cell Counting Kit-8 assay and apoptosis was examined by annexin V and mitochondrial membrane potential (MMP) assays. Expressions of hypoxia-induced factor-1α (HIF-1α), p53, and BNIP3 were investigated by quantitative real-time PCR and western blot analysis. After treatment of 200 μM CoCl₂ for 24 h, RGC-5 cells showed a marked decrease of cell viability by approximately 30%, increased apoptosis rate and obvious decline in MMP, which could largely be reversed by the pretreatment of 1 μM pilocarpine mainly via the activation of muscarinic receptors. Meanwhile, pretreatment of 1 μM pilocarpine could significantly prevent CoCl₂-induced HIF-1α translocation from cytoplasm to nucleus and down-regulate the expression of HIF-1α, p53, and BNIP3. These studies demonstrated that pilocarpine had effective protection against hypoxia-induced apoptosis in RGCs via muscarinic receptors and HIF-1α pathway. The findings suggest that HIF-1α pathway as a “master switch” may be used as a therapeutic target in the cholinergic treatment of glaucoma.     

Pigment Epithelium-Derived Factor Released by Müller Glial Cells Exerts Neuroprotective Effects on Retinal Ganglion Cells

Survival of retinal ganglion cells (RGC) is compromised in several vision-threatening disorders such as ischemic and hypertensive retinopathies and glaucoma. Pigment epithelium-derived factor (PEDF) is a naturally occurring pleiotropic secreted factor in the retina. PEDF produced by retinal glial (Müller) cells is suspected to be an essential component of neuron-glial interactions especially for RGC, as it can protect this neuronal type from ischemia-induced cell death. Here we show that PEDF treatment can directly affect RGC survival in vitro. Using Müller cell-RGC-co-cultures we observed that activity of Müller-cell derived soluble mediators can attenuate hypoxia-induced damage and RGC loss. Finally, neutralizing the activity of PEDF in glia-conditioned media partially abolished the neuroprotective effect of glia, leading to an increased neuronal death in hypoxic condition. Altogether our results suggest that PEDF is crucially involved in the neuroprotective process of reactive Müller cells towards RGC.     

Ocular neuroprotection by siRNA targeting caspase-2

Retinal ganglion cell (RGC) loss after optic nerve damage is a hallmark of certain human ophthalmic diseases including ischemic optic neuropathy (ION) and glaucoma. In a rat model of optic nerve transection, in which 80% of RGCs are eliminated within 14 days, caspase-2 was found to be expressed and cleaved (activated) predominantly in RGC. Inhibition of caspase-2 expression by a chemically modified synthetic short interfering ribonucleic acid (siRNA) delivered by intravitreal administration significantly enhanced RGC survival over a period of at least 30 days. This exogenously delivered siRNA could be found in RGC and other types of retinal cells, persisted inside the retina for at least 1 month and mediated sequence-specific RNA interference without inducing an interferon response. Our results indicate that RGC apoptosis induced by optic nerve injury involves activation of caspase-2, and that synthetic siRNAs designed to inhibit expression of caspase-2 represent potential neuroprotective agents for intervention in human diseases involving RGC loss.     

Rescue of Retinal Function by BDNF in a Mouse Model of Glaucoma

Vision loss in glaucoma is caused by progressive dysfunction of retinal ganglion cells (RGCs) and optic nerve atrophy. Here, we investigated the effectiveness of BDNF treatment to preserve vision in a glaucoma experimental model. As an established experimental model, we used the DBA/2J mouse, which develops chronic intraocular pressure (IOP) elevation that mimics primary open-angle glaucoma (POAG). IOP was measured at different ages in DBA/2J mice. Visual function was monitored using the steady-state Pattern Electroretinogram (P-ERG) and visual cortical evoked potentials (VEP). RGC alterations were assessed using Brn3 immunolabeling, and confocal microscope analysis. Human recombinant BDNF was dissolved in physiological solution (0.9% NaCl); the effects of repeated intravitreal injections and topical eye BDNF applications were independently evaluated in DBA/2J mice with ocular hypertension. BDNF level was measured in retinal homogenate by ELISA and western blot. We found a progressive decline of P-ERG and VEP responses in DBA/2J mice between 4 and 7 months of age, in relationship with the development of ocular hypertension and the reduction of Brn3 immunopositive RGCs. Conversely, repeated intravitreal injections (BDNF concentration = 2 µg/µl, volume = 1 µl, for each injection; 1 injection every four days, three injections over two weeks) and topical eye application of BDNF eye-drops (12 µg/µl, 5 µl eye-drop every 48 h for two weeks) were able to rescue visual responses in 7 month DBA/2J mice. In particular, BDNF topical eye treatment recovered P-ERG and VEP impairment increasing the number of Brn3 immunopositive RGCs. We showed that BDNF effects were independent of IOP reduction. Thus, topical eye treatment with BDNF represents a promisingly safe and feasible strategy to preserve visual function and diminish RGC vulnerability to ocular hypertension.     

Axonal protection by Nmnat3 overexpression with involvement of autophagy in optic nerve degeneration

Axonal degeneration often leads to the death of neuronal cell bodies. Previous studies demonstrated the crucial role of nicotinamide mononucleotide adenylyltransferase (Nmnat) 1, 2, and 3 in axonal protection. In this study, Nmnat3 immunoreactivity was observed inside axons in the optic nerve. Overexpression of Nmnat3 exerts axonal protection against tumor necrosis factor-induced and intraocular pressure (IOP) elevation-induced optic nerve degeneration. Immunoblot analysis showed that both p62 and microtubule-associated protein light chain 3 (LC3)-II were upregulated in the optic nerve after IOP elevation. Nmnat3 transfection decreased p62 and increased LC3-II in the optic nerve both with and without experimental glaucoma. Electron microscopy showed the existence of autophagic vacuoles in optic nerve axons in the glaucoma, glaucoma+Nmnat3 transfection, and glaucoma+rapamycin groups, although preserved myelin and microtubule structures were noted in the glaucoma+Nmnat3 transfection and glaucoma+rapamycin groups. The axonal-protective effect of Nmnat3 was inhibited by 3-methyladenine, whereas rapamycin exerted axonal protection after IOP elevation. We found that p62 was present in the mitochondria and confirmed substantial colocalization of mitochondrial Nmnat3 and p62 in starved retinal ganglion cell (RGC)-5 cells. Nmnat3 transfection decreased p62 and increased autophagic flux in RGC-5 cells. These results suggest that the axonal-protective effect of Nmnat3 may be involved in autophagy machinery, and that modulation of Nmnat3 and autophagy may lead to potential strategies against degenerative optic nerve disease.     

Metal chelator combined with permeability enhancer ameliorates oxidative stress-associated neurodegeneration in rat eyes with elevated intraocular pressure

Because as many as half of glaucoma patients on intraocular pressure (IOP)-lowering therapy continue to experience optic nerve toxicity, it is imperative to find other effective therapies. Iron and calcium ions play key roles in oxidative stress, a hallmark of glaucoma. Therefore, we tested metal chelation by means of ethylenediaminetetraacetic acid (EDTA) combined with the permeability enhancer methylsulfonylmethane (MSM) applied topically on the eye to determine if this noninvasive treatment is neuroprotective in rat optic nerve and retinal ganglion cells exposed to oxidative stress induced by elevated IOP. Hyaluronic acid (HA) was injected into the anterior chamber of the rat eye to elevate the IOP. EDTA–MSM was applied topically to the eye for 3 months. Eyeballs and optic nerves were processed for histological assessment of cytoarchitecture. Protein–lipid aldehyde adducts and cyclooxygenase-2 (COX-2) were detected immunohistochemically. HA administration increased IOP and associated oxidative stress and inflammation. Elevated IOP was not affected by EDTA–MSM treatment. However, oxidative damage and inflammation were ameliorated as reflected by a decrease in formation of protein–lipid aldehyde adducts and COX-2 expression, respectively. Furthermore, EDTA–MSM treatment increased retinal ganglion cell survival and decreased demyelination of optic nerve compared with untreated eyes. Chelation treatment with EDTA–MSM ameliorates sequelae of IOP-induced toxicity without affecting IOP. Because most current therapies aim at reducing IOP and damage occurs even in the absence of elevated IOP, EDTA–MSM has the potential to work in conjunction with pressure-reducing therapies to alleviate damage to the optic nerve and retinal ganglion cells.     

Exploring the potential for subtype-selective muscarinic agonists in glaucoma

Pilocarpine has been used to lower intraocular pressure (IOP) in glaucoma patients for more than 100 years. Since the identification of five muscarinic receptor subtypes, there has been an interest in separating the IOP-lowering effects from the ocular side effects of pupil constriction and lens accommodation. However, all these actions seem to be mediated by the M3 receptor. A novel muscarinic receptor agonist, AGN 199170, that has no activity on the M3 subtype was compared to pilocarpine in a monkey glaucoma model. This compound lowered IOP suggesting that muscarinic agonists targeted at muscarinic receptors other than the M3 subtype may be able to selectively lower IOP.