Rat Model Of Chronic Glaucoma Research Articles

Differential Modulation of the Excitatory and Inhibitory Synaptic Circuits of Retinal Ganglion Cells via Asiatic Acid in a Chronic Glaucoma Rat Model.

To investigate whether asiatic acid (AA) can improve the quantity and function of retinal ganglion cells (RGCs), as well as how AA regulates synaptic pathways in rat models with chronic glaucoma. In our study, a rat model of chronic glaucoma was prepared via the electrocoagulation of the episcleral veins. The numbers of surviving RGCs were counted via retrograde Fluorogold labeling, and a whole-cell patch clamp was used to clamp RGCs in normal retinal sections and in retinal sections 4 weeks after glaucoma induction. Retrograde-Fluorogold-labeled RGC loss caused by persistent glaucoma was decreased by AA. Additionally, AA reduced the postsynaptic current produced by N-methyl-D-aspartate (NMDA) and diminished miniature glutamatergic excitatory neurotransmission to RGCs. On the other hand, AA increased miniature gamma-aminobutyric acid (GABA)-ergic inhibitory neurotransmission to RGCs and enhanced the GABA-induced postsynaptic current. The excitability of the RGC itself was also decreased by AA. RGCs in glaucomatous slices were less excitable because AA decreased their spontaneous action potential frequency and membrane potential, which led to a hyperpolarized condition. AA directly protected RGCs in a chronic glaucoma rat model by lowering their hyperexcitability. To enhance RGCs' survival and function in glaucoma, AA may be a viable therapeutic drug.

Sodium Hyaluronate-Induced Ocular Hypertension in Rats Damages the Direction-Selective Circuit and Inner/Outer Retinal Plexiform Layers.

PurposeTo assess the changes in retinal morphology in a rat model of chronic glaucoma induced by ocular hypertension.MethodsIntraocular pressure (IOP) was surgically increased through weekly injections of sodium hyaluronate (HYA) in the anterior eye chamber of the left eye of male Wistar rats, whereas the right eyes were sham operated (salt solution). During the 10-week experimental period, IOP was measured weekly with a rebound tonometer. Retinal cryosections were prepared for histological/immunohistochemical analysis and morphometry.ResultsIOP was higher in HYA-treated eyes than in sham-operated eyes along the 10-week period, which was significant from the fourth to the nineth week. Ocular hypertension in HYA-treated eyes was associated with morphologic and morphometric changes in bipolar cells, ON-OFF direction-selective ganglion cells, ON/OFF starburst amacrine cells, and inner plexiform layer sublamina.ConclusionsSerial HYA treatment in the rat anterior eye chamber results in mild-to-moderate elevated and sustained IOP and ganglion cell death, which mimics most human open-angle glaucoma hallmarks. The reduced number of direction-selective ganglion cells and starburst amacrine cells accompanied by a deteriorated ON/OFF plexus in this glaucoma model could lend insight to the abnormalities in motion perception observed in patients with glaucoma.

Human Umbilical Cord-Mesenchymal Stem Cells Survive and Migrate within the Vitreous Cavity and Ameliorate Retinal Damage in a Novel Rat Model of Chronic Glaucoma.

Glaucoma is the leading cause of irreversible blindness worldwide, and pathologically elevated intraocular pressure (IOP) is the major risk factor. Neuroprotection is one of the potential therapies for glaucomatous retinal damage. Intravitreal mesenchymal stem cell (MSC) transplantation provides a viable therapeutic option, and human umbilical cord- (hUC-) MSCs are attractive candidates for cell-based neuroprotection. Here, we investigated the ability of transplanted hUC-MSCs to survive and migrate within the vitreous cavity and their neuroprotective effects on chronic glaucomatous retina. For this, we developed a chronic ocular hypertension (COH) rat model through the intracameral injection of allogeneic Tenon's fibroblasts. Green fluorescent protein-transduced hUC-MSCs were then injected into the vitreous cavity one week after COH induction. Results showed that a moderate IOP elevation lasted for two months. Transplanted hUC-MSCs migrated toward the area of damaged retina, but did not penetrate into the retina. The hUC-MSCs survived for at least eight weeks in the vitreous cavity. Moreover, the hUC-MSCs were efficient at decreasing the loss of retinal ganglion cells; retinal damage was attenuated through the inhibition of apoptosis. In this study, we have developed a novel COH rat model and demonstrated the prolonged neuroprotective potential of intravitreal hUC-MSCs in chronic glaucoma.

Silencing of GAS5 Alleviates Glaucoma in Rat Models by Reducing Retinal Ganglion Cell Apoptosis.

Retinal ganglion cells (RGCs) play a key role in the pathogenesis and development of glaucoma. The present study aims to investigate the underlying mechanism of long noncoding RNA growth arrest-specific transcript 5 (GAS5) in glaucoma development through regulating the apoptosis of RGCs. Rat models of chronic glaucoma were successfully established by translimbal laser photocoagulation. Retinal tissues were collected to determine the density of RGCs through Toluidine blue staining. The overexpression vector or short hairpin RNA for GAS5 or enhancer of zeste homolog 2 (EZH2) was transfected into RGCs after in vitro pressurization culture to examine the function of GAS5 in RGC apoptosis. The involvement of EZH2 and ATP-binding cassette transporter A1 (ABCA1) was further identified. Cell apoptosis after laser treatment and transfection was assessed by flow cytometry. We found abundant GAS5 expression and a reduction in RGC density in the retinal tissues of glaucoma rats. Silencing of GAS5 led to increased EZH2 expression and decreased ABCA1 expression in RGCs. In addition, upregulation of EZH2 promoted trimethylation of lysine 27 on histone H3, thereby suppressing ABCA1 expression and eventually leading to the inhibition of RGC apoptosis. These findings provide further understanding of the function of GAS5 in RGC apoptosis. We conclude that downregulation of GAS5 could help relieve glaucoma symptoms. GAS5 is therefore a promising target for developing novel therapeutic approaches for treating patients with glaucoma.

Brimonidine enhances inhibitory postsynaptic activity of OFF- and ON-type retinal ganglion cells in a Wistar rat chronic glaucoma model

Glaucoma is a multifactorial disease in which retinal ganglion cells (RGCs) undergo excitotoxic damage, leading to their degeneration. The α2-adrenoceptor (α2-AR) agonist brimonidine exerts a neuroprotective effect by regulating postsynaptic excitatory N-methyl-D-aspartate (NMDA) receptor activity in RGCs. However, researchers have not clearly determined whether or how brimonidine regulates inhibitory synaptic transmission in rat models of chronic glaucoma. Whole-cell voltage-clamp and current-clamp recordings were performed in ON- and OFF-type RGCs in retinal slices. Brimonidine directly and acutely enhanced γ-aminobutyric acidergic (GABAergic) transmission mediated by ionotropic GABAA receptors in ON- and OFF-type RGCs in rat retinal slices; this effect occurred at the synaptic terminals and was independent of action potentials and multi-synaptic connections. The highly selective α2-AR antagonist yohimbine blocked the effects of brimonidine. Regarding the postsynaptic GABA receptor sensitivity, brimonidine also increased the amplitude of the GABA-induced current. Additionally, compared to RGCs from the control group, the frequencies and amplitudes of spontaneous excitatory postsynaptic currents (sEPSCs) and miniature excitatory postsynaptic currents (mEPSCs) did not change after brimonidine gravity perfusion. Brimonidine significantly decreased the spontaneous firing frequency of rat RGCs with intact synaptic inputs and decreased the resting membrane potential of RGCs, changes that were blocked by the highly selective GABAA receptor antagonist SR95531. SR95531 alone increased spontaneous action potentials and the resting membrane potential. Based on these findings, an α2-AR agonist facilitated the frequency of the GABAergic inhibitory postsynaptic currents (IPSCs), directly increased the amplitude of the postsynaptic GABA-induced current (GABA receptor reactivity/sensitivity), suppressed the firing frequency of spontaneous action in RGCs with intact synaptic inputs and decreased the resting membrane potential of RGCs, thus deactivating RGCs from the neural network level and reducing the excitotoxic damage occurring during the pathological process of chronic glaucoma.

Quercetin Enhances Inhibitory Synaptic Inputs and Reduces Excitatory Synaptic Inputs to OFF- and ON-Type Retinal Ganglion Cells in a Chronic Glaucoma Rat Model.

BackgroundGlaucoma is a neurodegenerative disease caused by excitotoxic injury of retinal ganglion cells (RGCs). In our previous model of high intraocular pressure, prepared by injecting magnetic beads into the anterior chamber, we demonstrated that an important natural dietary flavonoid compound (quercetin) can improve RGC function. However, it is unclear whether quercetin can improve the synaptic function of RGCs and how quercetin regulates synaptic transmission in rat models of chronic glaucoma.MethodsA rat model of chronic glaucoma was prepared by electrocoagulation of the superior scleral vein. Electrophysiological electroretinography was used to detect the photopic negative response (PhNR). The whole-cell patch-clamp technique was used to clamp ON- and OFF- type RGCs in sections from normal retinas and from retinas that had been subjected to glaucoma for 4 weeks.ResultsQuercetin can reverse the decrease in PhNR amplitude caused by chronic glaucoma. The baseline frequency of miniature GABAergic inhibitory postsynaptic currents (mIPSCs) in the RGCs of glaucomatous retinal slices was lower than that of the control group. The frequencies of miniature excitatory postsynaptic currents (mEPSCs) were not significantly different between control and glaucomatous RGCs. The baseline frequencies of GABAergic mIPSCs and mEPSCs in OFF-type glaucomatous RGCs were greater than those in ON-type glaucomatous RGCs. Quercetin increased miniature GABAergic inhibitory neurotransmission to RGCs and decreased miniature glutamatergic excitatory neurotransmission, reducing the excitability of the RGCs themselves, thus alleviating the excitability of RGCs in glaucomatous slices.ConclusionQuercetin may be a promising therapeutic agent for improving RGC survival and function in glaucomatous neurodegeneration. Quercetin exerted direct protective effects on RGCs by increasing inhibitory neurotransmission and decreasing excitatory neurotransmission to RGCs, thus reducing excitotoxic damage to those cells in glaucoma.

Morphological and Functional Changes of Locus Coeruleus in Patients with Primary Open-Angle Glaucoma and Animal Models

Primary open-angle glaucoma (POAG) is a leading cause of irreversible blindness. Magnetic resonance imaging (MRI) studies report an association between POAG and nonvisual pathway alterations. Locus coeruleus (LC) is a major source of norepinephrine released in the brain, and norepinephrine can reduce intraocular pressure via increasing aqueous outflow. This study aimed to investigate the relationship between glaucoma and the LC in patients with POAG and animal models. Resting-state functional MRI was performed using a 3-Tesla MR scanner with an eight-channel phased-array head coil, and MRI data were analyzed. A rat model of chronic glaucoma was generated by episcleral vein ligation and cauterization. DBA/2J mice that develop glaucoma with age were also acquired. Immunohistochemistry and immunofluorescence staining were used to investigate LC tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DβH) expression, as well as cell apoptosis by terminal deoxynucleotidyl transferase dUTP Nick-End Labeling staining. Patients with POAG showed significantly increased amplitude of low-frequency fluctuation (ALFF) in the LC compared with controls. LC ALFF values showed significant correlations with cup-to-disk ratio, retinal nerve fiber layer thickness, and visual function (P < 0.05). Functional connectivity (FC) between the LC and frontal and insular lobes was reduced, but elevated between the LC and parahippocampal gyrus, compared with controls. Glaucoma animal models revealed reduced expression of TH and DβH in the LC, and increased cell apoptosis. In this study, we provide novel evidence for the relationship between the LC and glaucoma. The LC may act as a network in POAG pathogenesis and intraocular pressure regulation.

Protective effects of triptolide on retinal ganglion cells in a rat model of chronic glaucoma.

PurposeTo study the effects of triptolide, a Chinese herb extract, on retinal ganglion cells (RGCs) in a rat model of chronic glaucoma.MethodsEighty Wistar rats were randomly divided into triptolide group (n=40) and normal saline (NS) group (n=40). Angle photocoagulation was used to establish the model of glaucoma, with right eye as laser treated eye and left eye as control eye. Triptolide group received triptolide intraperitoneally daily, while NS group received NS. Intraocular pressure (IOP), anti-CD11b immunofluorescent stain in retina and optic nerve, RGCs count with Nissel stain and microglia count with anti-CD11b immunofluorescence stain in retina flat mounts, retinal tumor necrosis factor (TNF)-α mRNA detection by reverse transcription–polymerase chain reaction, and double immunofluorescent labeling with anti-TNF-α and anti-CD11b in retinal frozen section were performed.ResultsMean IOP of the laser treated eyes significantly increased 3 weeks after photocoagulation (P<0.05), with no statistical difference between the two groups (P>0.05). RGCs survival in the laser treated eyes was significantly improved in the triptolide group than the NS group (P<0.05). Microglia count in superficial retina of the laser treated eyes was significantly less in the triptolide group (30.40±4.90) than the NS group (35.06±7.59) (P<0.05). TNF-α mRNA expression in the retina of the laser treated eyes in the triptolide group decreased by 60% compared with that in the NS group (P<0.01). The double immunofluorescent labeling showed that TNF-α was mainly distributed around the microglia.ConclusionTriptolide improved RGCs survival in this rat model of chronic glaucoma, which did not depend on IOP decrease but might be exerted by inhibiting microglia activities and reducing TNF-α secretion.

The expression of dynein light chain DYNLL1 (LC8-1) is persistently downregulated in glaucomatous rat retinal ganglion cells

High intraocular pressure induces glaucomatous degeneration of retinal ganglion cells (RGCs). The cellular mechanisms leading to activation of the apoptosis cascade are multidimensional and only partially understood. A small dynein subunit, the light chain DYNLL1 (synonym LC8-1, PIN) has recently been shown to be an important regulator of neuron proteins known to be involved in glaucomatous RGC death including NO synthases, the pro-apoptotic protein Bim and the dynein intermediate chain. Also, DYNLL1 is a regulator of mitochondria anchorage in axons, which is impaired in glaucoma. We investigated expression of DYNLL1 and 2 and its dynein binding partner dynein intermediate chain in a rat model of chronic glaucoma. Laser capture microdissection (LCM) allowed us to collect distinct cell layers and cell bodies from the retina to gain data highly specific for retinal ganglion cells. Glaucoma was induced in 23 rats by laser treatment to the aqueous outflow tract. RNA was extracted from LCM dissected ganglion cell layers (GCL) and 100 pooled RGCs per retina. Expression levels for 1, 2 and 4 week timepoints were analysed by quantitative PCR for DYNLL1 and 2, dynein intermediate chain and GFAP. DYNLL protein abundance in RGCs was quantified in immunostained retina sections. DYNLL gene 1 but not 2 was expressed in RGCs. In the glaucoma model DYNLL1 was strongly and persistently downregulated at all timepoints. DYNLL protein was significantly less abundant at the 4 week timepoint. In contrast, the motorprotein binding partner dynein intermediate chain 1 was more stably expressed. DYNLL2 was upregulated in glia cells at 2 weeks. Expression of DYNLL1, the only form of the dynein light chain expressed in RGCs, is downregulated persistently in glaucoma, while its binding partner dynein IC-1 is unchanged. The specific lack of DYNLL1 could have an impact on the function of their regulatory binding partners and contribute in several ways to neuron dysfunction and apoptosis.

Proton magnetic resonance spectroscopy revealed choline reduction in the visual cortex in an experimental model of chronic glaucoma

Glaucoma is a neurodegenerative disease of the visual system. While elevated intraocular pressure is considered to be a major risk factor, the primary cause and pathogenesis of this disease are still unclear. This study aims to employ in vivo proton magnetic resonance spectroscopy ( 1H MRS) to evaluate the metabolic changes in the visual cortex in a rat model of chronic glaucoma. Five Sprague–Dawley female rats were prepared to induce ocular hypertension unilaterally in the right eye by photocoagulating the episcleral and limbal veins using an argon laser. Single voxel 1H MRS was performed on each side of the visual cortex 6 weeks after laser treatment. Relative to the creatine level, the choline level was found to be significantly lower in the left glaucomatous visual cortex than the right control visual cortex in all animals. In addition, a marginally significant increase in glutamate level was observed in the glaucomatous visual cortex. No apparent difference was observed between contralateral sides of the visual cortex in T1-weighted or T2-weighted imaging. The results of this study showed that glaucoma is accompanied with alterations in the metabolism of choline-containing compounds in the visual cortex contralateral to the glaucomatous rat eye. These potentially associated the pathophysiological mechanisms of glaucoma with the dysfunction of the cholinergic system in the visual pathway. 1H MRS is a potential tool for studying the metabolic changes in glaucoma in vivo in normally appearing brain structures, and may possess direct clinical applications for humans. Measurement of the Cho:Cr reduction in the visual cortex may be a noninvasive biomarker for this disease.

GD-DTPA enhanced MRI of ocular transport in a rat model of chronic glaucoma

Glaucoma is a neurodegenerative disease of the visual system characterized by the elevation of intraocular pressure. While this elevated pressure is related to an increased resistance to the outflow of aqueous humor from the eye, their impacts to the etiology and pathogenesis of the disease are not fully understood. This study aims to employ in vivo Gd-DTPA enhanced magnetic resonance imaging to evaluate the ocular transport following an induction of ocular hypertension in a rat model of chronic glaucoma. An experimental ocular hypertension model was induced in adult rats using an argon laser to photocoagulate the episcleral and limbal veins on the surface of the eyeball. The enhancements of the MRI signal intensity in the anterior chamber and vitreous body were measured as a function of time following systemic administration of Gd-DTPA solution at 3 mmol/kg. Results showed a progressive T1-weighted signal increase in the vitreous body of the glaucomatous eye but not the control eye. This increase occurred earlier in the anterior vitreous body than the preretinal vitreous. Further, there was an earlier Gd-DTPA transport into the anterior chamber in the majority of glaucomatous eyes. Our findings revealed the leakage of Gd-DTPA at the aqueous–vitreous interface, which was likely resulted from increased permeability of blood–aqueous or aqueous–vitreous barrier. These may explain the sources of changing biochemical compositions in the glaucomatous chamber components, which may implicate the cascades of neurodegenerative processes in the retina and the optic nerve.

Evaluation of the retina and optic nerve in a rat model of chronic glaucoma using in vivo manganese-enhanced magnetic resonance imaging

Glaucoma is a neurodegenerative disease of the visual system. While elevated intraocular pressure is considered to be a major risk factor, the primary cause and pathogenesis of the disease are still unclear. This study aims to employ in vivo manganese-enhanced magnetic resonance imaging (MEMRI) to evaluate dynamically the Mn2+ enhancements in the visual components following an induction of ocular hypertension in a rat model of chronic glaucoma. The episcleral and limbal veins were photocoagulated unilaterally in the right eye using an argon laser to maintain a consistent elevation of intraocular pressure by about 1.6 times above the normal level. Two and six weeks after glaucoma induction, MnCl2 solution (50 mM, 3 μL) was injected intravitreally into both eyes, and MEMRI was performed 2 to 5 h after injection. Results showed a delayed increase in T1-weighted signal intensity in the glaucomatous optic nerve at 6 weeks but not 2 weeks after glaucoma induction. In addition, there was an accumulation of Mn2+ ions in the vitreous humour of the glaucomatous eye, with a high concentration of Mn2+ ions at the optic nerve head and the retina. These MEMRI findings may help understand the disease mechanisms, monitor the effect of drug interventions in glaucoma models and complement the conventional techniques in examining the glaucomatous visual components.

Expression of neuronal nitric oxide synthase in the retina of a rat model of chronic glaucoma

We investigated the expression of neuronal nitric oxide synthase (nNOS) in a rat retina model of chronic glaucoma, which was produced by electrocauterization of the episcleral vessels. Western-blot analysis showed that nNOS expression was significantly increased in cauterized retinas. nNOS immunoreactivity was observed in the cells of both the inner nuclear layer and the ganglion cell layer. Double labeling of retinal ganglion cells (RGCs) revealed that RGCs in the retina of cauterized rat was nNOS-immunopositive. Systemic administration of l-NAME ( N G-nitro- l-arginine-methyl-ester), a non-specific NOS inhibitor, reduced RGC loss in cauterized rat retina, but there was no statistical significance ( P = .06). These results suggest that the cytotoxicity of excessive NO plays a role in selective RGC loss in glaucoma.

The Ginkgo biloba extract (EGb 761) provides a neuroprotective effect on retinal ganglion cells in a rat model of chronic glaucoma

Purpose. To investigate the effect of Ginkgo biloba extract (EGb 761) against neurotoxicity of retinal ganglion cells of rats with chronic moderately elevated intraocular pressure (IOP). Methods. Unilateral chronic moderately elevated IOP was produced in rats by cautery of three episcleral vessels. Secondary degeneration was measured with and without EGb 761 for 5 months. At 5 months, retinal ganglion cells were labeled with a fast blue tracer applied to both superior colliculi. Densities of surviving retinal ganglion cells were estimated by counting fast blue labeled cells in whole mounted retinas. Results. When compared with their contralateral control eyes with normal IOP, in the peripheral retina, retinal ganglion cell loss in eyes with chronic, moderately elevated IOP was 29.8 ± 1.5% (n = 5) at 5 months in untreated animals and 4.6 ± 4.5% (n = 5) at 5 months in treated animals with EGb 761. Conclusions. Pretreatment and early posttreatment with EGb 761 is an effective neuroprotectant in a rat model of chronic glaucoma.

Inhibition of nitric-oxide synthase 2 by aminoguanidine provides neuroprotection of retinal ganglion cells in a rat model of chronic glaucoma.

Glaucoma is an optic neuropathy with cupping of the optic disk, degeneration of retinal ganglion cells, and characteristic visual field loss. Because elevated intraocular pressure (IOP) is a major risk factor for progression of glaucoma, treatment has been based on lowering IOP. We previously demonstrated inducible nitric-oxide synthase (NOS-2) in the optic nerve heads from human glaucomatous eyes and from rat eyes with chronic, moderately elevated IOP. Using this rat model of unilateral glaucoma, we treated a group of animals for 6 months with aminoguanidine, a relatively specific inhibitor of NOS-2, and compared them with an untreated group. At 6 months, untreated animals had pallor and cupping of the optic disks in the eyes with elevated IOP. Eyes of aminoguanidine-treated animals with similar elevations of IOP appeared normal. We quantitated retinal ganglion cell loss by retrograde labeling with Fluoro-Gold. When compared with their contralateral control eyes with normal IOP, eyes with elevated IOP in the untreated group lost 36% of their retinal ganglion cells; the eyes with similarly elevated IOP in the aminoguanidine-treated group lost less than 10% of their retinal ganglion cells. Pharmacological neuroprotection by inhibition of NOS-2 may prove useful for the treatment of patients with glaucoma.