Effect of ocular hypertension on retinal GABAergic activity

doi:10.1016/j.neuint.2007.08.014

Neurochemistry International

Volume 52, Issues 4–5, March–April 2008, Pages 675-682

https://doi.org/10.1016/j.neuint.2007.08.014 Get rights and content

Abstract

Glutamate and γ-aminobutyric acid (GABA) are major excitatory and inhibitory retinal neurotransmitters. The balance between these signals is a key principle of organization at retinal level. Although glutamate-induced excitotoxicity could mediate retinal ganglion cell death in glaucoma, the GABAergic system was not previously examined in this disease. The aim of this work was to study the retinal GABAergic activity in eyes with ocular hypertension induced by hyaluronic acid (HA). For this purpose, weekly injections of HA were performed unilaterally in the rat anterior chamber, whereas the contralateral eye was injected with saline solution. At 3 weeks of treatment with HA, GABA turnover rate, glutamic acid decarboxylase activity, and both glutamate- and high K⁺-induced GABA release significantly decreased, whereas GABA uptake increased in HA-treated eyes. The binding of t-butylbicyclophosphorothionate (TBPS) to GABA_A/benzodiazepine Cl⁻ channels significantly increased in eyes injected with HA as compared with vehicle-injected eyes. Changes in GABA uptake and TBPS binding persisted at 6 weeks of treatment with HA. These results indicate a dysfunction of the retinal GABAergic activity in hypertensive eyes, which could suggest the involvement of GABA in glaucomatous neuropathy.

Section snippets

Introductory statement

Glaucoma, a chronic disease characterized by visual field loss, cupping of the optic nerve head, and irreversible loss of retinal ganglion cells (RGCs), is a leading cause of blindness worldwide. It is estimated that half of those affected may not be aware of their condition because symptoms may not occur during the early stages of the disease. When vision loss appears, considerable permanent damage has already occurred. Medications and surgery can help slow the progression of some forms of the

Reagents and drugs

GABA, l-glutamic acid, 3-mercaptopropionic acid, GABAse, pyridoxal phosphate and picrotoxin were obtained from Sigma Chemical Co. (St. Louis, MO, USA). [³H]-GABA, 1-[¹⁴C]-l-glutamic acid, [³H]-muscimol, and [³⁵S]-t-butylbicyclophosphorothionate (TBPS) were purchased from New England Nuclear Corp. (Boston, MA, USA).

Animals and tissues

Male Wistar rats (average weight, 200 ± 40 g) were housed in a standard animal room with food and water ad libitum under controlled conditions of humidity and temperature (21 ± 2 °C), under

Effect of HA on IOP

Fig. 1 shows the average IOP of rats injected weekly with HA (in one eye) or vehicle in the other for 3 or 6 weeks. A significant increase of IOP was observed in eyes injected with HA as compared with the respective controls. No differences in IOP of vehicle-injected eyes were detected between these time-points or between non-injected and vehicle-injected eyes.

Effect of high IOP on the retinal GABAergic system

The effect of ocular hypertension on GABA steady state concentrations and GABA turnover rate is shown in Fig. 2. At 3 weeks of treatment

Discussion

For the first time, the foregoing results support a significant dysfunction of the retinal GABAergic system in rats exposed to experimentally elevated IOP. In particular, these results indicate that GABA steady state concentrations, GABA turnover rate, GAD activity, GABA transporters, and GABA receptors were susceptible to HA-induced ocular hypertension. In contrast to these results, Kielczewski et al. (2005) have recently shown that amacrine cells identified by GABA labeling were not affected

Acknowledgments

This research was supported by grants from The University of Buenos Aires, CONICET, and The Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT), Argentina.

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Gamma-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter of the central nervous system (CNS) (Han et al., 2021). A number of studies have determined that alterations in GABA levels are closely related to neurological disorders such as anxiety, panic syndrome, depression, epilepsy, schizophrenia, retinal degeneration, and glaucoma (Liu et al., 2007, Hasler et al., 2009, Akyuz et al., 2021, Moreno et al., 2008, Coghlan et al., 2012, de Jonge et al., 2017 and Assad et al., 2020). As GABA is not metabolized outside the cell, extracellular GABA levels are finely regulated by the action of proteins known as GABA transporters (GATs) (Ishibashi et al., 2019).
The GATs are the membrane proteins responsible for the uptake of GABA in the central nervous system. Alterations in GAT activity are implicated in several neurological diseases, including retinopathies. The present study describes an alternative method to determine GAT activity in tissue preparations of the central nervous system, using high performance liquid chromatography (HPLC) with fluorescence detection. The GABA concentration in the medium was determined using the o-phthaldehyde (OPA)-derivation protocol validated by the Brazilian Health Regulatory Agency (ANVISA) and the United States Food and Drug Administration (US-FDA). The GAT activity in the retinal preparations was determined through the evaluation of the GABA uptake, which was measured by assessing the difference between the initial and final concentrations of GABA in the incubation medium. The evaluation of the GAT kinetics returned values of K_m = 382.5 ± 32.2 μM and V_max = 34 nmol/mg of protein. The data also demonstrated that the GABA uptake was predominantly Na⁺- and temperature-dependent, and was also inhibited by incubation with nipecotic acid, a substrate of GABA transporters. Taken together, these findings confirm that our approach provided a specific measure of GAT activity in retinal tissue. The data presented here thus validate, for the first time, an alternative, simple and sensitive method for the evaluation of GAT activity using high performance chromatography on preparations of the central nervous system.
TSPO activation modulates the effects of high pressure in a rat ex vivo glaucoma model
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Although data indicate a role for glutamate in glaucoma (Naskar et al., 2000; Martin et al., 2002; Moreno et al., 2005; Nucci et al., 2005), the role of GABA has been less thoroughly investigated (Bailey et al., 2014), and prior work indicates that the balance between glutamate and GABA is important for maintaining retinal function (Kishida and Naka, 1968). However, there are only a few reports describing significant dysfunction of the GABAergic system in glaucomatous retinas (Bailey et al., 2014; Kishida and Naka, 1968; Moreno et al., 2008). Allopregnanolone (AlloP) is a neurosteroid that can be locally synthesized in the central nervous system in response to stressful events.
We previously reported that elevated pressure induces axonal swelling and facilitates the synthesis of the neurosteroid, allopregnanolone (AlloP), in the ex vivo rat retina. Exogenously applied AlloP attenuates the axonal swelling, suggesting that the neurosteroid plays a neuroprotective role against glaucomatous pressure-induced injuries, although mechanisms underlying neurosteroidogenesis have not been clarified. The aim of this study was to determine whether AlloP synthesis involves activation of translocator protein 18 kD (TSPO) and whether TSPO modulates pressure-induced retinal injury. Ex vivo rat retinas were exposed to various pressures (10, 35, or 75 mmHg) for 24 h. Expression of TSPO, 5α-reductase (5aRD), and AlloP was examined by quantitative real-time RT-PCR, ELISA, immunohistochemistry, and LC-MS/MS. We also examined the effects of TSPO ligands on AlloP synthesis and retinal damage. In this acute model, quantitative real-time RT-PCR and ELISA analyses revealed that elevated pressure facilitated TSPO expression. Similarly, these methods also detected enhanced 5aRD (mostly type II), which was observed in retinal ganglion cells (RGC) and the inner nuclear layer (INL). Atriol, a TSPO antagonist, suppressed pressure mediated AlloP synthesis and induced more severe histological changes in the inner retina when combined with elevated pressure. PK11195, a TSPO ligand that facilitates AlloP synthesis by itself, remarkably diminished pressure-mediated retinal degeneration. These results suggest that AlloP synthesis is induced by sequential activation of TSPO and 5aRD in an ex vivo glaucoma model, and that TSPO agonists may serve as potential therapeutic agents for the prevention of pressure-induced retinal damage.
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The needle was moved in a clockwise direction, and contact of the needle tip with the iris and the lens was avoided [47]. Induction of OHT was repeated at 1-week intervals for 3 weeks [50,51]. Before and after each injection, IOP was measured with a TonoPen XL applanation tonometer (Mentor) under light anaesthesia, which was induced by 10 mg/kg ketamine hydrochloride and a drop of topically applied proparacaine HCl (Alcaine, Alcon).
The purpose of this study was to investigate the neuroprotective effects of bone marrow bone marrow–derived and adipose tissue–derived mesenchymal stromal cells (MSCs) that were intravitreally transplanted in an experimental ocular hypertension (OHT) model.
An OHT rat model was generated by means of intracameral injection of hyaluronic acid into the anterior chamber. MSCs labeled with green fluorescence protein were transplanted intravitreally 1 week after OHT induction. At the end of the second and fourth weeks, retinal ganglion cells were visualized with the use of a flat-mount retina method and were evaluated by means of immunofluorescence staining against green fluorescence protein, vimentin, CD105, and cytokines (interleukin [IL]-1Ra, prostaglandin E2 receptor, IL-6, transforming growth factor-β1, interferon-γ and tumor necrosis factor-α).
The retinal ganglion cell numbers per area were significantly improved in stem cell–treated OHT groups compared with that in the non-treated OHT group (P < 0.05). The results of immunohistochemical analyses indicated that a limited number of stem cells had integrated into the ganglion cell layer and the inner nuclear layer. The number of cells expressing proinflammatory cytokines (interferon-γ and tumor necrosis factor-α) decreased in the MSC-transferred group compared with that in the OHT group after 4 weeks (P < 0.01). On the other hand, IL-1Ra and prostaglandin E2 receptor expressions were increased in the rat bone marrow–derived MSC group but were more significant in the rat adipose tissue–derived MSC group (P < 0.01).
After intravitreal transplantation, MSCs showed a neuroprotective effect in the rat OHT model. Therefore, MSCs promise an alternative therapy approach for functional recovery in the treatment of glaucoma.
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View full text

Effect of ocular hypertension on retinal GABAergic activity

Abstract

Section snippets

Introductory statement

Reagents and drugs

Animals and tissues

Effect of HA on IOP

Effect of high IOP on the retinal GABAergic system

Discussion

Acknowledgments

Brain Res.

Eur. J. Pharmacol.

Brain Res.

Brain Res. Mol. Brain Res.

Brain Res.

Prog. Retin. Eye Res.

Free Radic. Biol. Med.

J. Biol. Chem.

Free Radic. Biol. Med.

Exp. Eye Res.

Neurotoxicology

Prog. Retin. Eye Res.

Prog. Neurobiol.

Brain Res.

Effect of hyaluronic acid on intraocular pressure in rats

Invest. Ophthalmol. Vis. Sci.

Determination of GABA levels by a [3H]muscimol radioreceptor assay

J. Neurochem.

Application of principles of steady-state kinetics to the estimation of gamma-aminobutyric acid turnover rate in nuclei of rat brain

J. Pharmacol. Exp. Ther.

Glial transporters for glutamate, glycine, and GABA. II. GABA transporters

J. Neurosci. Res.