Acetylcholinesterase Inhibition Promotes Retinal Vasoprotection and Increases Ocular Blood Flow in Experimental Glaucoma

Abstract

Purpose.: A clear correlation between vascular deficits and retinal ganglion cell (RGC) loss in glaucoma has not yet been established. The question arose as to whether there is loss of inner retinal vessels following intraocular pressure (IOP) increase and, if so, whether it occurs prior to, concomitantly with, or after RGC death. We also sought to establish whether galantamine, an acetylcholinesterase inhibitor that promotes RGC survival, can protect the retinal microvasculature and enhance blood flow in experimental glaucoma.

Methods.: Ocular hypertension was induced in Brown Norway rats by injection of hypertonic saline into an episcleral vein. Retinas were processed for simultaneous visualization of the retinal microvasculature and RGCs in glaucomatous and control eyes. Retinal blood flow was examined by quantitative autoradiography using N-isopropyl-p-[¹⁴C]-iodoamphetamine. Vascular reactivity was further assessed using an in vitro retinal microvasculature preparation.

Results.: Substantial loss of retinal capillaries was observed after induction of ocular hypertension. The onset of both microvasculature and RGC loss occurred early and proceeded at a similar rate for at least 5 weeks after the initial damage. Systemic administration of galantamine preserved microvasculature density and improved retinal blood flow in glaucomatous retinas. The vasoactive effects of galantamine on retinal microvessels occurred through activation of muscarinic acetylcholine receptors both in vitro and in vivo.

Conclusions.: The onset and progression of microvessel and RGC loss are concomitant in experimental glaucoma, suggesting a tight codependence between these cellular compartments. Early interventions aimed to protect the retinal microvasculature and improve blood supply are likely to be beneficial for the treatment of glaucoma.

Introduction

Glaucoma, a group of diseases characterized by progressive optic nerve degeneration, is the leading cause of irreversible blindness worldwide. ¹ A crucial element in the pathophysiology of all forms of glaucoma is the death of retinal ganglion cells (RGCs), a population of central nervous system neurons that cannot be replaced once they are lost. At present, the only modifiable risk factor in glaucoma is high intraocular pressure (IOP) and standard therapies rely solely on lowering IOP by medication or surgery. A significant number of glaucoma patients, however, continue to lose vision despite responding well to treatments that reduce eye pressure. ² Therefore, there is a need to identify underlying mechanisms that contribute to RGC death, and to develop novel therapies that prevent or delay vision loss in glaucoma. 

The retina is among the most metabolically active tissues in the body and, as such, requires a precise regulation of blood supply to meet its high oxygen demand. It has long been proposed that vascular deficits and ensuing ischemia contribute to glaucoma progression. ^3–7 Indeed, reduced blood flow in the retina and optic nerve head (ONH) of glaucoma patients has been reported by a number of investigators. ^8–13 A reduction in the caliber of retinal vessels ^14,15 as well as atrophy of peripapillary capillaries supplying the retinal fiber layer in this disease have been described. ^6,16 Vascular autoregulation, defined as the capacity of a vascular bed to maintain a constant blood flow despite variations in perfusion pressure, is compromised in glaucoma. ^17,18 Flicker-induced vasodilation of retinal and ONH vessels is appreciably reduced in glaucoma patients, indicating important neurovascular coupling defects in this disease. ^19,20 A relationship between glaucoma and endothelin, a potent vasoactive peptide, has also been established. Elevated plasma and aqueous humor levels of endothelin have been detected in normal tension and primary open-angle glaucoma patients, ^21–24 and endothelin receptor inhibition increases ocular blood flow in individuals with glaucoma. ²⁵ Collectively, these studies support a vascular component in the pathophysiology of glaucomatous optic neuropathies. 

RGCs are in close contact with an elaborate capillary network and critically rely on the retinal microvasculature for metabolic support and nutrition. Conversely, RGCs provide growth factors that stimulate vessel growth and endothelial cell survival. ²⁶ A logical extension of this tight neurovascular cross-talk is that the loss of RGCs in glaucoma will likely have important repercussions on surrounding retinal capillaries and vice versa. However, a clear correlation between RGC death and microvasculature loss in glaucoma has not yet been established. In this study, the question arose as to whether there is loss of inner retinal capillaries in experimental glaucoma and, if so, whether it occurs prior to, concomitantly with, or after RGC death. Moreover, we sought to determine whether galantamine, an acetylcholinesterase inhibitor that promotes RGC survival, can induce vascular protection and blood flow restoration in a rat model of ocular hypertension. 

Materials and Methods

Experimental Animals

All procedures were performed in male Brown Norway rats (300–400 g; Charles River Laboratories International, Inc., St-Constant, QC, Canada) in compliance with the guideline of the Canadian Council on Animal Care for the Use of Experimental Animals (www.ccac.ca) and the Statement for the Use of Animals in Ophthalmic and Visual Research from the Association for Research in Vision and Ophthalmology (ARVO). The number of animals used in each experiment is indicated in the corresponding figure legend or table. 

Ocular Hypertension Surgery

Unilateral elevation of IOP was induced by a single injection of hypertonic saline (1.85 M NaCl) into an episcleral vein, as previously described. ²⁷ Animals were kept in a room with constant low-fluorescent light (40–100 lux) to stabilize circadian IOP variations. IOP from glaucomatous and normal eyes was measured in awake animals using a calibrated tonometer (TonoPen XL; Medtronic Solan, Jacksonville, FL) after corneal application of one drop of proparacaine hydrochloride (0.5%; Alcon Laboratories, Inc., Fort Worth, TX). The tonometer was held exactly perpendicular to the corneal surface and approximately 10 consecutive readings per eye were taken and averaged to obtain an IOP measurement. IOP was measured every other day, at the same time, for the entire duration of the experiment. The mean and peak (maximum) IOP values for each eye were calculated and these values were used to estimate the mean and peak IOPs for experimental and control groups. 

In Vivo Drug Delivery

Galantamine hydrobromide (Gal, 3.5 mg/kg; Tocris Bioscience, Ellisville, MO) was delivered by daily intraperitoneal (IP) injection. Drug delivery started at 1 week after OHT surgery and continued for the entire duration of the experiment. Control animals received daily IP injections of vehicle (phosphate buffered saline [PBS]). Scopolamine (SCO, 100 μM; Tocris Bioscience) or mecamylamine hydrochloride (MMA, 100 μM; Tocris Bioscience), which exhibit poor retinal–blood barrier permeability, ^28,29 were injected intravitreally in a total volume of 5 μL using a 10-μL syringe (Hamilton Medical AG, Bonaduz, Switzerland) adapted with a 32-gauge glass microneedle. The tip of the needle was inserted into the superior hemisphere of the eye at an approximately 45° angle through the sclera into the vitreous body to avoid retinal detachment or injury to eye structures. Surgical glue (Indermill; Tyco Health Care, Mansfield, MA) was used to seal the injection site. 

Quantification of Retinal Microvasculature

Rats were deeply anesthetized and perfused transcardially with 4% paraformaldehyde (PFA). Eyes were enucleated and the dissected retinas were placed in a 24-well plate, washed twice with PBS, and then permeabilized with ice-cold methanol for 10 minutes. TRITC-labeled isolectin ( Griffonia simplicifolia lectin, 4 μg/mL; Sigma-Aldrich, St. Louis, MO) was prepared in PBS containing 1% Triton X-100 and incubated at room temperature overnight. Retinas were flat-mounted on a glass slide with the ganglion cell layer side up and the vessels in the nerve fiber layer were visualized using a fluorescent microscope (Zeiss Axio Vert.A1; Carl Zeiss Microscopy GmbH, Jena, Germany). Retinal vessel density was measured as the total length of retinal capillaries and arterioles in three square areas at 1, 2, and 3 mm from the ONH in each retinal quadrant, for a total of 12 retinal areas, encompassing a total area of 1 mm², using image analysis software (Northern Eclipse; Empix Imaging, Toronto, ON, Canada). 

Quantification of RGC Soma

RGCs were retrogradely labeled with DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate; Molecular Probes, Junction City, OR), prepared by dissolving DiI crystals (3%) in 0.9% NaCl solution containing 10% dimethyl sulfoxide (DMSO) and 0.5% Triton X-100. The superior colliculus was exposed and a small piece of gelfoam (Pharmacia and Upjohn, Inc., Mississauga, ON, Canada), soaked in tracer, was applied to the surface as previously described. ³⁰ Seven days after tracer application, animals were subjected to OHT surgery. For RGC density counts, rats were deeply anesthetized and perfused transcardially with 4% PFA, the eyes were enucleated, and the retinas were dissected out and flat-mounted on a glass slide with the ganglion cell layer side up. DiI-labeled RGCs were counted in three square areas at 1, 2, and 3 mm from the optic disc in each retinal quadrant, for a total of 12 retinal areas encompassing a total area of 1 mm². Macrophages and microglia that incorporated fluorescent tracer after phagocytosis of dying RGCs were excluded from our quantitative analysis based on cell-specific markers and morphology. ³¹  

Quantitative Blood Flow Autoradiography

Retinal blood flow was measured by quantitative autoradiography using the diffusible blood flow tracer N-isopropyl-p-[¹⁴C]-iodoamphetamine ([¹⁴C]-IMP) as previously described. ³² Briefly, [¹⁴C]-IMP (100 μCi/kg; PerkinElmer, Boston, MA) was infused through a femoral vein catheter over a 30-second period at a constant rate of 1.2 mL/min. Arterial blood samples were collected at a continuous rate until euthanasia (2 minutes after [¹⁴C]-IMP infusion onset). The eyes were removed and postfixed in 4% PFA. The retinas were dissected out, flat-mounted on a glass slide with the ganglion cell layer side up, and exposed to x-ray film for 4 days alongside [¹⁴C] standards (ARC, St. Louis, MO). Retinal blood flow was calculated by implementing the principle of indicator-fractionation technique using the equation F = [C _IMP(T) × 10⁻¹/∫₀ ^T Ca(t)] for autoradiographic analysis of flat-mounted retinas. F represents the blood flow (mL/100 g/min), C _IMP(T) is the radioactivity measured from the autoradiogram (μCi/g) at the time (min) of sampling, and Ca(t) is the arterial blood sample radioactivity (μCi/mL). To obtain C _IMP(T) values, a computerized image analysis system (MCID Basic Software, v7.0; Interfocus Imaging, Linton, UK) was used to collect readings from a 0.8 mm² circular area at 1, 2, and 3 mm from the ONH in each eye quadrant. ³² The Ca(t) value was obtained by measuring radioactivity in the collected blood samples using a scintillation counter (LS6500; Beckman Coulter, Mississauga, ON, Canada). 

In Vitro Retinal Microvasculature Preparation

Retinal microvasculature was prepared as previously described. ³² Briefly, animals were euthanized with an overdose of sodium pentobarbital (240 mg/kg) and the eyes were enucleated. Retinas were rapidly dissected out and placed in high Mg²⁺/low Ca²⁺ dissociation buffer (145 mM NaCl, 5 mM KCl, 10 mM HEPES, 5 mM d-glucose, 0.1 mM CaCl₂, 8 mM MgCl₂, pH 7.3), and cut into several pieces, followed by trituration with a fire-polished glass pipette (0.3 mm). Aliquots of the retinal dissociate containing vessel segments (>30 μm length) were plated onto laminin-coated dishes and allowed to adhere before superfusion with bath solution (145 mM NaCl, 5 mM KCl, 1 mM HEPES, 5 mM d-glucose, 2 mM CaCl₂, 1 mM MgCl₂, pH 7.3). Isolated retinal arterioles (10- to 50-μm diameter) were identified under an inverted light microscope. Vessel diameter was measured with video edge detector at 120 Hz (Crescent Electronics, Sandy, UT) and analyzed using commercial software (AxoScope software; MDS Analytical Technologies, Mississauga, ON, Canada). For endothelial denudation, animals received heparin (IP, 1000 USP/mL), to prevent blood coagulation, 30 minutes prior to euthanasia with sodium pentobarbital overdose. Rats were then transcardially perfused with 0.3% CHAPS, which effectively removes endothelial cells, and retinal vessels were isolated as described above. Vessels (endothelium-intact or endothelium-denuded) were superfused with bath solution for 10 minutes, followed by application of human endothelin-1 (10 nM, ET-1; Peptide Institute, Inc., Osaka, Japan) using a rapid switching device for approximately 1 minute or until a stable contraction was obtained, then vessel diameter was recorded for an additional 10 minutes. The following drugs were bath applied in the superfusate immediately prior to, during, and for at least 10 minutes after exposure to ET-1: galantamine hydrobromide (5–50 μM), scopolamine hydrobromide (SCO, 10 μM), mecamylamine hydrochloride (MMA, 10 μM), pirenzepine dihydrochloride (PRZ, 1 μM; Tocris Bioscience), diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP, 1 μM; Tocris Bioscience), 11-([2-[(diethylamino)methyl]-1-piperdinyl]acetyl)-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiaze pine-6-one (DX116, 1–10 μM; Tocris Bioscience), or tropicamide (1–10 μM; Tocris Bioscience). All experiments were carried out at 37°C. Data were generated from six to eight separate vessels isolated from three different animals per group. 

Statistical Analyses

Data analysis and statistics were performed using commercial software (GraphPad Prism software; GraphPad Software, Inc., San Diego, CA). All data are presented as the mean ± SEM and were analyzed by one-way ANOVA followed by Bonferroni or Newman–Keuls post hoc tests. 

Results

Loss of the Retinal Microvasculature Begins Early and Occurs Concomitantly With RGC Death in Experimental Glaucoma

The retinal microvasculature plays a crucial physiologic role by supplying oxygen and nutrients to RGCs and their axons. We sought to determine whether retinal microvessels are lost during OHT and, if so, to establish the time course of vascular damage with respect to RGC loss. For this purpose, a temporal analysis was carried out using flat-mounted retinas processed to simultaneously visualize retinal vessels using TRITC-labeled isolectin, a marker of functional endothelial cells, and RGCs were identified with the retrograde tracer DiI (Fig. 1A). Analysis of glaucomatous retinas demonstrated that early vascular changes, characterized by the loss of capillaries (4- to 6-μm diameter ^33,34 ), started 3 to 7 days after induction of OHT, a time when IOP increase was small (∼3 mm Hg above basal, Table 1) (Figs. 1B, 1C). The loss of capillaries progressed steadily thereafter (Figs. 1D, 1E) and was prominent at 3 and 5 weeks after induction of OHT (Figs. 1E, 1F). Loss of some small arterioles, including precapillary tertiary arterioles (7- to 12-μm diameter) and secondary arterioles (12- to 18-μm diameter), was observed only at 5 weeks after the initial damage (Fig. 1F). 

Figure 1

 

Loss of the retinal microvasculature begins early and occurs concomitantly with RGC death in experimental glaucoma. Flat-mounted retinas were processed to simultaneously visualize retinal capillaries using TRITC-labeled isolectin, a marker of functional endothelial cells; and RGCs, identified with the retrograde tracer DiI. (A, B) Loss of fine capillaries and RGCs, started as early as 3 days after induction of OHT. (C–F) The time course of microvasculature and RGC loss occurred concomitantly and steadily thereafter (n = 6 rats/group). c, capillary; ta, tertiary arteriole; sa, secondary arteriole. Scale bars: 100 μm.

Table 1. 

 

Intraocular Pressure Elevation in Glaucomatous and Control Eyes

Time After OHT Surgery	Group		Mean IOP, mm Hg Glaucoma Control Difference			Peak IOP, mm Hg Glaucoma Control
3 d	No treatment	6	24.82 ± 0.6	23.31 ± 0.2	1.51 ± 0.2	26.70 ± 1.3	25.14 ± 0.6
1 wk	No treatment	6	26.24 ± 1.3	23.05 ± 0.4	3.19 ± 0.3	29.21 ± 1.7	26.15 ± 0.2
2 wk	No treatment	6	30.09 ± 1.2	24.31 ± 2.1	5.78 ± 1.6	33.52 ± 1.7	25.30 ± 1.4
3 wk	Galantamine Vehicle Gal + SCO Gal + MMA No treatment	6 6 6 6 6	34.51 ± 0.3 35.73 ± 1.6 34.20 ± 1.4 34.66 ± 0.5 34.82 ± 0.6	24.13 ± 1.1 24.04 ± 0.5 23.17 ± 0.2 24.08 ± 1.2 23.24 ± 1.3	10.38 ± 0.5 11.69 ± 0.2 11.03 ± 0.7 10.58 ± 1.2 11.58 ± 1.6	37.11 ± 0.7 36.30 ± 0.5 36.33 ± 1.5 37.21 ± 2.1 38.21 ± 1.2	26.55 ± 1.4 26.23 ± 0.9 25.44 ± 1.2 26.71 ± 1.4 26.01 ± 0.4
5 wk	Galantamine Vehicle No treatment	6 6 6	42.12 ± 0.4 41.23 ± 2.1 42.55 ± 1.4	23.22 ± 1.5 23.40 ± 2.0 24.09 ± 0.5	18.90 ± 1.1 17.83 ± 1.8 18.46 ± 1.7	48.77 ± 1.3 49.08 ± 1.4 48.13 ± 1.9	26.35 ± 1.1 25.90 ± 0.3 26.55 ± 1.6

To establish the time course of microvasculature loss with respect to RGC death, we quantified the density of RGCs and retinal vessels in the same retinas (Fig. 2). RGCs were visualized with the fluorescent tracer DiI, applied to the superior colliculus 1 week before OHT surgery, to ensure retrograde labeling prior to any changes in optic nerve function caused by experimental glaucoma. Unlike other retrograde markers that leak from the cell body after several weeks, DiI has been shown to persist in RGCs in vivo for periods of up to 9 months without fading or leakage. ³⁵ Consistent with this, the RGC population detected by DiI in intact, noninjured Brown Norway rat retinas did not significantly change at the following survival times (from tracer application): 7 days: 1841 ± 15 RGCs/mm² (mean ± SEM, n = 9); 10 days: 1835 ± 63 RGCs/mm² (n = 10); 2 weeks: 1899 ± 42 (n = 12); 3 weeks: 1901 ± 51 (n = 9); 4 weeks: 1848 ± 51 (n = 10); and 6 weeks: 1878 ± 57 (n = 12). At 3 and 7 days after induction of OHT (10 and 14 days from DiI application, respectively), there was a small but significant decrease in capillary density (∼10%, Fig. 2A) that correlated with a similar reduction in the density of RGC soma (Fig. 2B). Weekly analysis of retinal vessel density up to 5 weeks after OHT surgery (6 weeks after tracer application) demonstrated a steady loss of the microvasculature that occurred concomitantly with RGC death (Fig. 2C), indicating that degenerative changes begin early and affect both neuronal and vascular compartments. These data support a tight neurovascular relationship between RGCs and retinal vessels, and demonstrate that both neurons and microvasculature are remarkably affected in experimental glaucoma. 

Figure 2

 

Quantitative analysis of microvasculature and retinal ganglion cell density in glaucomatous and control eyes. (A) Microvasculature density was analyzed in isolectin-stained flat-mounted retinas at 3 days, 1 week, 2 weeks, 3 weeks, and 5 weeks after induction of OHT surgery (n = 6 rats/group, ANOVA, *P < 0.05, **P < 0.01). (B) Analysis of RGC survival was carried out by quantification of DiI-labeled neurons in the same retinas and at the same time points as for retinal microvessels (n = 6 rats/group, ANOVA, *P < 0.05, **P < 0.01). (C) Side-by-side comparison of the time course of microvasculature and RGC loss. The average vessel density (black bars) is shown as a percentage of noninjured, nontreated retinas (100% = 19,710 ± 594 μm/mm², mean ± SEM, n = 6). Similarly, the average RGC density is shown as a percentage of noninjured, nontreated retinas (100% = 1830 ± 24 RGCs/mm², mean ± SEM, n = 6). Microvasculature density was not significantly different from RGC density at all time points studied (ANOVA, P > 0.05).

Galantamine Protects the Microvasculature From Glaucomatous Damage

The observation that RGC death occurs together with microvasculature loss led us to put forward the idea that protective therapies for glaucoma should ideally target both neuronal and vascular compartments. We recently demonstrated that galantamine, a small-molecule acetylcholinesterase (AChE) inhibitor, promotes robust protection of RGC soma and axons in experimental glaucoma and its neuroprotective effect is superior to that of other AChE inhibitors such as donepezil. ³⁰ To establish whether galantamine had a vasoprotective effect, we examined retinal microvessel density in animals subjected to experimental glaucoma and daily IP injection of galantamine. Galantamine treatment was initiated at 1 week after induction of OHT and continued thereafter for the entire duration of the experiment. By the time most glaucoma patients seek medical attention there is already some damage and/or visual field defects. Therefore, we chose to initiate galantamine treatment 1 week after induction of OHT, based on our finding that RGC and microvasculature loss were already apparent at that time (Figs. 1, 2). Analysis of isolectin-stained retinas showed that galantamine treatment yielded higher microvasculature density compared with that of PBS-treated control animals (Figs. 3A–D). Specifically, galantamine preserved microvessel density at 3 weeks (78.2%: 15,423 ± 290 μm/mm²) and 5 weeks (71%: 13,986 ± 602 μm/mm²) after OHT surgery, compared with control groups (3 weeks, 67.8%: 13,421 ± 685 μm/mm²; 5 weeks, 39.7%: 7901 ± 618 μm/mm²) (Fig. 3E). The vasoprotective effect of galantamine was most apparent at 5 weeks of glaucoma induction when considerable loss of retinal capillaries and some small arterioles was observed in vehicle-treated control eyes. Importantly, galantamine significantly delayed further microvasculature loss between 3 and 5 weeks after OHT induction (Fig. 3E, black bars). This effect was not due to a reduction of IOP because galantamine treatment did not change eye pressure (Table 1). These data demonstrate that galantamine promotes microvasculature protection in experimental glaucoma. 

Figure 3

 

Galantamine protects the microvasculature from glaucomatous damage. Isolectin-stained flat-mounted retinas demonstrate that galantamine preserves microvasculature density (A, C) compared with PBS-treated control animals (B, D) at both 3 and 5 weeks after induction of OHT (n = 6–8 rats/group). (E) Galantamine (black bars) substantially delayed OHT-induced loss of the retinal microvasculature network relative to PBS-treated control groups (hatched bars). Data are expressed as μm/mm² ± SEM (ANOVA, **P < 0.01, n = 6–8/group). Scale bars (A–D): 100 μm.

Ocular Hypertension–Induced Blood Flow Impairment Is Attenuated by Galantamine

To assess the functional status of the microvascular network in glaucomatous eyes in either the presence or the absence of galantamine, we investigated regional blood flow using a previously described quantitative autoradiographic method. ³² A significant reduction of retinal blood flow was observed in glaucomatous eyes compared with noninjured control eyes, measured from autoradiograms, at 5 weeks after induction of OHT (Figs. 4A, 4B). Galantamine treatment distinctly sustained retinal blood flow in experimental glaucoma relative to PBS-treated control animals (Figs. 4C, 4D). To establish whether galantamine increased blood flow locally or throughout the retina, regional blood flow was analyzed in three consecutive areas at 1, 2, and 3 mm from the ONH in each retinal quadrant. In noninjured, control retinas, higher blood perfusion was measured in central regions compared with the periphery (Fig. 4E, Table 2). Although the central retina underwent the most dramatic reduction in blood supply with IOP elevation, galantamine-induced blood flow increase was proportional in central and peripheral retinal regions (Fig. 4E, Table 2). Quantitative analysis of global blood flow demonstrated that, overall, galantamine preserved 84% of total blood flow compared with only 52% in vehicle-treated animals (Fig. 4F). Collectively, these data indicate that OHT-induced decrease in blood flow is attenuated by galantamine. 

Figure 4

 

OHT-induced blood flow impairment is attenuated by galantamine. Pseudocolored autoradiograms show a significant reduction of retinal blood flow in glaucomatous eyes at 5 weeks after OHT surgery compared with noninjured control eyes ([A, B], n = 4 rats/group). Galantamine treatment preserved retinal blood flow in experimental glaucoma compared with PBS-treated control animals ([C, D], n = 4 rats/group). Both regional (E) and global (F) retinal blood flow were exceptionally improved with galantamine treatment (black bars) compared with PBS-treated controls (hatched bars). Data are expressed as mL/100 g/min ± SEM (ANOVA, *P < 0.05, n = 4 rats/group).

Table 2. 

 

Retinal Blood Flow

Retinal Quadrants, Distance From ONH	Control, mL/100 g/ min	Galantamine, mL/100 g/ min	Vehicle, mL/100 g/ min
Nasal
1 mm	129 ± 17	109 ± 10	67 ± 23
2 mm	111 ± 16	100 ± 18	64 ± 22
3 mm	97 ± 6	86 ± 11	54 ± 20
Temporal
1 mm	132 ± 13	116 ± 15	67 ± 16
2 mm	110 ± 15	97 ± 9	59 ± 21
3 mm	94 ± 11	86 ± 13	49 ± 11
Superior
1 mm	121 ± 20	108 ± 7	58 ± 9
2 mm	114 ± 18	89 ± 12	55 ± 11
3 mm	90 ± 15	81 ± 9	47 ± 6
Inferior
1 mm	119 ± 9	106 ± 11	68 ± 8
2 mm	113 ± 13	99 ± 12	53 ± 6
3 mm	93 ± 20	86 ± 15	44 ± 7

 Values are means ± SEM (n = 4/group).

Galantamine Induces Vasodilation of Isolated Retinal Arterioles

To establish whether the vasoactive effect of galantamine occurred through a direct action on the microvasculature, we performed studies on isolated retinal arterioles (10- to 25-μm diameter) exposed to endothelin-1 (ET-1, 10 nM), a potent vasoconstrictor. ET-1 alone led to a steady reduction in vessel diameter (32% contraction), whereas application of ET-1 in the presence of galantamine (50 μM) resulted in a significant reduction of ET-1–mediated vasoconstriction (Figs. 5A, 5B). A concentration of galantamine as low as 5 μM was sufficient to reduce vessel contraction (not shown); however, the optimal vasodilation-promoting dose of galantamine was 50 μM, which led to maximum relaxation and increased vessel diameter by 50% (17% contraction, Fig. 5C). In isolated endothelium-denuded retinal arterioles, application of ET-1 resulted in increased vasoconstriction (45% contraction) compared with arterioles with intact endothelium (Fig. 5C). Furthermore, in contrast to endothelium-intact arterioles, exposure of endothelium-denuded vessels to galantamine failed to reduce ET-1–mediated contraction (Fig. 5C). Collectively, these data demonstrated that: (1) galantamine acts directly on retinal vessels, (2) galantamine reduces ET-1–mediated vasoconstriction of retinal microvasculature in vitro, and (3) galantamine-mediated vasorelaxation requires the presence of viable endothelial cells. 

Figure 5

 

Galantamine induces vasodilation of isolated retinal arterioles through mAChR activation. (A–C) Application of galantamine reduces ET-1–mediated vasoconstriction of small retinal arterioles. The presence of endothelial cells was necessary for the vasorelaxant effect of galantamine as denudation of endothelium by CHAPS eliminated galantamine-induced relaxation of ET-1–constricted vessels (C). Combined application of galantamine and SCO, a nonselective antagonist of mAChR, arrested the vasorelaxant effect of galantamine on ET-1–constricted vessels, whereas the nAChR antagonist MMA had no effect (D). Galantamine-mediated vasorelaxation was inhibited by blockers of M1 (pirenzepine) and M3 (4-DAMP) mAChR subtypes (E). Data were generated from six to eight separate vessels isolated from three rats/group, and are presented as a percentage of vessel diameter before application of ET-1 (ANOVA, *P < 0.05, **P < 0.01).

Muscarinic Acetylcholine Receptors Mediate the Vasoactive Effects of Galantamine In Vitro and In Vivo

Galantamine increases the availability of acetylcholine (ACh) through its inhibition of AChE, the enzyme responsible for ACh breakdown, thus activating nicotinic receptors (nAChRs) and metabotropic muscarinic receptors (mAChRs). ³⁶ In addition, galantamine is an allosteric modulator of nAChR, enhancing receptor sensitivity to ACh. ³⁷ We previously demonstrated that galantamine-mediated RGC protection in experimental glaucoma occurs through activation of mAChR. ³⁰ To establish whether this mechanism played a role in vasoprotection, we carried out experiments using isolated precontracted vessels exposed to galantamine alone (50 μM) or in combination with pharmacologic inhibitors of AChR. Application of scopolamine (SCO), a nonselective antagonist of all mAChR subtypes, arrested galantamine-induced vasodilation of ET-1–contracted vessels (Fig. 5D). In contrast, application of mecamylamine (MMA), a nonselective antagonist of all nAChR subtypes, did not significantly alter galantamine-induced vasodilation (Fig. 5D). These results indicate that galantamine promotes retinal microvessel relaxation through mAChR activation. 

To establish which mAChR subtypes were involved in this response, we used selective mAChR antagonists in combination with galantamine. Application of pirenzepine (PRZ), a selective blocker of the M1 mAChR subtype, or 4-DAMP, an inhibitor of the M3 mAChR subtype, abolished galantamine-mediated relaxation of ET-1–contracted vessels (Fig. 5E). In contrast, tropicamide or DX116, blockers of M4 or M2 mAChR, respectively, did not have a significant effect on galantamine-induced vasodilation (not shown). These data indicate that M1 and M3 mAChRs mediate the vasodilatory effect of galantamine on isolated retinal microvasculature. 

To assess whether mAChR also played a role in galantamine-induced vasoprotection in vivo, we tested the effect of galantamine in combination with SCO or MMA in experimental glaucoma (Fig. 6). A single intravitreal injection of SCO or MMA was administered at 1 week after OHT surgery, coinciding with the initiation of galantamine treatment, and analysis of microvasculature density was performed 2 weeks later. Analysis of isolectin-stained retinas demonstrated that coadministration of galantamine and SCO inhibited the vasoprotective effect of galantamine, whereas MMA had no effect (Figs. 6A–D). Quantitative analysis of retinal microvasculature density confirmed that SCO blocked galantamine-induced vasoprotection, leading to significant loss of vessels similar to PBS-injected controls (Fig. 6E). Of interest, SCO blocks not only the vasoprotective effect of galantamine (this study), but also galantamine-induced RGC neuroprotection, as previously shown. ³⁰ Although the combination of galantamine and MMA led to a slight reduction in microvasculature density, this effect was not statistically different from that of galantamine alone (Fig. 6E, ANOVA, P > 0.05). A range of MMA concentrations were tested (10 μM–10 mM) with similar outcome, indicating that the lack of effect was not the result of suboptimal doses of this drug. Therefore, we conclude that the vasoprotective effect of galantamine in vivo is mediated through activation of mAChR. 

Figure 6

 

Vasoprotection occurs through galantamine-mediated activation of mAChR in experimental glaucoma. (A–D) Analysis of isolectin-stained retinas demonstrated that coadministration of galantamine and SCO inhibited the vasoprotective effect of galantamine in experimental glaucoma, whereas MMA had no effect (n = 6–8 rats/group). (E) Quantitative analysis of capillary density confirmed that SCO blocked galantamine-mediated vasoprotection, leading to significant loss of vessels similar to PBS-injected controls (n = 6–8 rats/group). Data are expressed as μm/mm² ± SEM (ANOVA, *P < 0.05, n = 6–8 rats/group). Scale bars (A–D): 100 μm.

Discussion

This study reports a number of important findings. First, we detected substantial loss of capillaries in an OHT rat glaucoma model. Second, we found that loss of retinal microvessels occurred concomitantly with the death of RGCs following induction of high IOP. The onset of microvasculature and RGC loss occurred early and proceeded at approximately the same rate for at least 5 weeks after the initial insult. Third, systemic administration of galantamine preserved microvasculature density and improved retinal blood flow in experimental glaucoma. Finally, the vasoactive effects of galantamine on retinal microvessels occurred through activation of mAChR. 

The ONH is considered a primary site of glaucomatous injury and strong experimental evidence indicates that it is an initial point of RGC axonal damage. ^38,39 However, it is increasingly being recognized that changes at the level of the ONH do not occur in isolation but involve cross-talk with other RGC compartments (e.g., soma, dendrites, synapses) as well as other retinal cell types (e.g., astrocytes, microglia, endothelial cells), which respond early and might contribute to disease progression. ^40–44 It is unlikely that therapeutic strategies that target a single cellular compartment in glaucoma will have a long-lasting beneficial effect in disease outcome. Importantly, many of the vascular deficits observed in the ONH in glaucoma have also been reported in the retina, ^8–13 but the correlation between changes in the retinal microvasculature and RGC loss had not been previously studied and is, therefore, a focus of our study. Our data demonstrate a steady and simultaneous loss of RGCs and retinal capillaries, indicating that degenerative changes in glaucoma affect both neuronal and vascular compartments. Based on the high vulnerability of RGCs to glaucomatous damage, we predicted that RGCs would die first and, as the local need for oxygen and nutrient supply decreased, the microvasculature would undergo rapid, but slightly delayed, involution. The surprising observation that microvessels and RGCs disappear at the same rate suggests a tighter codependence between these cellular compartments than previously expected. Furthermore, our data suggest that both RGCs and microvessels are similarly affected by IOP-induced degenerative changes; therefore the symbiotic cross-talk between these cell types is likely to be impaired in glaucoma. 

Microvasculature and RGC loss started early after induction of OHT and continued to progress thereafter. At 3 and 7 days after glaucoma induction, a small but significant loss of RGCs and capillaries (∼10%) was detected. The IOP increase at these time points was also small (∼3 mm Hg above basal), suggesting that a slight increase in IOP might be sufficient to trigger the earliest neurodegenerative changes. Previous studies using acute retinal ischemia rat models, with or without IOP increase, demonstrated that substantial periods of ischemia are needed to induce RGC death. ^45,46 However, when RGCs were quantified at 5 days after a 30-minute period of transient ischemia a significant loss of RGCs (∼14%) ⁴⁵ was found, supporting the idea that the first neurodegenerative changes are likely to take place soon after the initial injury. Nonetheless, it is important to emphasize that the bulk of the cell loss we report here occurred between 2 and 5 weeks of glaucoma induction when mean IOP values in glaucomatous eyes were 30 or 42 mm Hg, respectively, compared with 23 mm Hg in intact eyes (Table 1) and RGC loss was proportional to IOP increase. Our findings are in agreement with previous work showing that the amount of cell death depends on the magnitude of IOP elevation and the duration of the insult. ^45,46  

We show that capillaries are dramatically affected early in the pathology, whereas small arterioles are affected only at later stages (5 weeks after glaucoma induction). Retinal and brain capillaries do not undergo classical vasoconstriction because they lack smooth muscle and their blood flow is regulated primarily by precapillary arterioles. ^47–49 Recent studies show that the diameter of retinal capillaries does not change during functional hyperemia in vivo or acute hydrostatic pressure increase ex vivo. ^49,50 Based on this, it is unlikely that the capillary loss we report here results from capillary vasoconstriction. We propose two plausible scenarios to explain our findings. First, since RGCs also disappear early on, it is possible that the levels of RGC-derived factor(s) essential for capillary maintenance decrease, thus leading to capillary loss. Indeed, RGCs produce vascular endothelial growth factor and angiopoietins 1 and 2, which are required for capillary stability, growth, and survival. ⁵¹ RGCs play an essential role in microvasculature development, and recent studies showed that diphtheria toxin A ablation of RGCs or genetic deletion of Math5 in these neurons resulted in mice completely devoid of retinal vascular plexus. ^51,52 Another possibility is that high IOP stimulates production of toxic factors, including proinflammatory mediators from reactive glia, which threaten the survival of capillaries. Candidates that might potentially contribute to this response include nitric oxide (NO), ET-1, reactive oxygen species, tumor necrosis factor alpha, and glutamate, some of which have the potential to trigger both RGC and endothelial cell dysfunction or apoptosis. ^44,53 The elucidation of the precise mechanism leading to loss of retinal capillaries in glaucoma should be a research priority. 

The branches of retinal arteries expand to form deeper capillary networks, which are most prominent between the inner nuclear and outer plexiform layers. ⁵⁴ These deep capillaries are in close interaction with neuronal and glial elements to meet the metabolic demands of the inner retina. Interestingly, chronic OHT has been shown to induce cone photoreceptor death ⁵⁵ that correlated with important deficits in the electroretinogram response, including a reduction in the amplitude of a- and b-waves. ⁵⁶ Given the extent of retinal microvasculature loss in our OHT model, one would expect amacrine cells to undergo deficits in nutrient and oxygen supply. However, with the exception of one study reporting a decrease in cholinergic and GABAergic amacrine cells, ⁵⁷ most groups have failed to detect a significant loss of these neurons in experimental glaucoma. ^58–61 Nonetheless, a decrease in neurotransmitter levels in amacrine cells has been observed in experimental glaucoma, ^58,61 suggesting that they might lose the ability to function properly. Consistent with this, a decrease in the amplitude of oscillatory potentials, presumed to be generated by amacrine cells, has been reported in human glaucomatous eyes. ⁶² Future studies should address the functional status of amacrine cells following microvasculature loss in glaucoma. 

Given the tight functional relationship between RGCs and the retinal microvasculature during experimental glaucoma onset and progression, we asked whether strategies that promote RGC neuroprotection can also maintain the integrity of the capillary network and enhance blood flow. We previously demonstrated that galantamine promotes structural and functional RGC protection in experimental rat glaucoma. ³⁰ In the present study, we show that galantamine treatment resulted in substantial preservation of microvascular density and increased retinal blood flow in both the central and peripheral retina. RGCs stimulate vessel growth and endothelial cell survival ²⁶ ; therefore, our findings raise the question: is galantamine-induced vasoprotection a consequence of increased RGC survival? Our experiments using isolated retinal arterioles indicate that galantamine can act directly on the vasculature to inhibit vasoconstriction in the absence of RGCs, and that this response depends on the presence of viable endothelial cells. Although these studies do not establish a role for vasoconstriction per se, they identify a direct effect of galantamine on isolated retinal arterioles, suggesting a possible mechanism of action that involves arteriole vasodilation and enhanced blood flow to the glaucomatous retina. Therefore, it is likely that galantamine can boost endothelial cell survival and blood flow independently of its neuroprotective effect on RGCs. This is consistent with a previous study showing that galantamine can enhance the survival of isolated brain endothelial cells in culture. ⁶³ Because galantamine was administered systemically by IP injections, it readily encounters endothelial cells when crossing the blood–retinal barrier, thus acting first on the retinal vasculature. Indeed, recent studies indicate that expression of the organic cation/carnitine transporter-2 by endothelial cells is necessary for the transport of galantamine through the blood–brain barrier, ^64,65 implying that endothelial cells are the first line of action for galantamine in the retina. Therefore, we cannot rule out that the prosurvival effect of galantamine on RGCs, previously reported, ³⁰ is partly due to enhanced endothelial cell survival leading to improved capillary density and blood flow. 

To elucidate whether the mechanisms by which galantamine-induced vasoprotection were different from those leading to RGC survival, we investigated the role of ACh receptors in this response. Using selective pharmacologic inhibitors of mAChR or nAChR, we previously demonstrated that galantamine-induced RGC survival occurred via activation of mAChR, whereas nAChRs were not involved. ³⁰ Here we show that, similar to RGCs, galantamine-mediated vasodilation in vitro and vasoprotection in vivo occurred through activation of mAChR, but not nAChR. An important difference, however, is that although galantamine-induced RGC survival relied on M1 and M4 mAChR activation, the effect of galantamine on retinal microvessels depended on M1 and M3 mAChR subtypes. Retinal and brain endothelial cells abundantly express M1 and M3 mAChR subtypes, ^66,67 which have been implicated in cholinergic-mediated vasomodulation in the brain, ⁶⁸ and ACh-dependent vascular relaxation is lost in M3 mAchR knockout mice. ⁶⁹ Intriguingly, adult RGCs do not express mAChR and muscarine does not elicit membrane currents measured in whole-RGC patch-clamp preparations. ^70,71 Collectively, these findings support a model in which a galantamine-induced increase in ACh leads to mAChR activation on endothelial cells, thus maintaining capillary networks and improving retinal blood flow. Activation of M1 and M4 mAChR on neighboring Müller glia and amacrine cells may also contribute to stimulation of signaling pathways and production of prosurvival factors for both injured microvessels and RGCs. Furthermore, activation of retinal M1 and M3 mAChR subtypes stimulates neuronal NO synthase activity leading to vasodilation ⁷² ; thus, activation of mAChR in cells other than endothelial cells might contribute to vasoprotection and blood flow restoration in glaucoma. 

In summary, this study demonstrates early and progressive loss of the retinal microvasculature intimately associated with RGC death in experimental glaucoma. As new imaging tools become available, it might be relevant to closely monitor microvasculature changes in patients with high risk to develop glaucoma as well as during disease progression. We show that a clinically approved drug, galantamine, promotes effective vasoprotection and restores retinal blood flow in an OHT rat model. These findings are particularly important for the treatment of glaucomatous neuropathies because, although RGC death is irreversible, the retinal microvasculature maintains the capacity to regenerate. ²⁶ Early interventions to protect the microvasculature and improve blood flow are likely to have a beneficial effect on RGC survival and function. Given the dual role of galantamine in neuronal and vascular protection, this drug is a promising therapeutic candidate for glaucoma and other neurodegenerative diseases involving ischemia. A better understating of early microvascular changes in glaucomatous neuropathies as well as the molecular pathways involved in endothelial cell death might reveal new therapeutic targets for the treatment of this disease. 

Acknowledgments

The authors thank Dara O'Connor and Alex Dong for excellent technical assistance. 

Supported by Canadian Institutes of Health Research Grants MOP-165155 (ADP) and MOP-97768 (MEMK). ADP is a Chercheur National of Fonds de recherche en santé du Québec (FRSQ). 

Disclosure: M. Almasieh, None; J.N. MacIntyre, None; M. Pouliot, None; C. Casanova, None; E. Vaucher, None; M.E.M. Kelly, None; A. Di Polo, None