An investigation into the potential mechanisms underlying the neuroprotective effect of clonidine in the retina

Brain Research 877 (2000) 47–57 www.elsevier.com / locate / bres

Research report

An investigation into the potential mechanisms underlying the neuroprotective effect of clonidine in the retina H.M. Chao, G. Chidlow, J. Melena, J.P.M. Wood, N.N. Osborne* Nuffield Laboratory of Ophthalmology, University of Oxford, Walton Street, Oxford OX2 6 AW, UK Accepted 9 May 2000

Abstract a 2 -Adrenoceptor agonists, such as clonidine, attenuate hypoxia-induced damage to brain and retinal neurones by a mechanism of action which likely involves stimulation of a 2 -adrenoceptors. In addition, the neuroprotective effect of a 2 -adrenoceptor agonists in the retina may involve stimulation of bFGF production. The purpose of this study was to examine more thoroughly the neuroprotective properties of clonidine. In particular, studies were designed to ascertain whether clonidine acts as a free radical scavenger. It is thought that betaxolol, a b 1 -adrenoceptor antagonist, acts as a neuroprotective agent by interacting with sodium and L-type calcium channels to reduce the influx of these ions into stressed neurones. Studies were therefore undertaken to determine whether clonidine has similar properties. In addition, studies were undertaken to determine whether i.p. injections of clonidine or betaxolol affect retinal bFGF mRNA levels. In vitro data were generally in agreement that clonidine and bFGF counteracted the effect of NMDA as would occur in hypoxia. No evidence could be found that clonidine interacts with sodium or L-type calcium channels, reduces calcium influx into neurones or acts as a free radical scavenger at concentrations below 100 mM. Moreover, i.p. injection of clonidine, but not betaxolol, elevated bFGF mRNA levels in the retina. The conclusion from this study is that the neuroprotective properties of a 2 -adrenoceptor agonists, like clonidine, are very different from betaxolol. The fact that both betaxolol and clonidine blunt hypoxia-induced death to retinal ganglion cells suggests that combining the two drugs may be a way forward to producing more effective neuroprotection.  2000 Elsevier Science B.V. All rights reserved. Keywords: Clonidine; a 2 -Adrenoceptor agonist; Betaxolol; Neuroprotection; Calcium channel; Sodium channel; Reactive oxygen species; Lipid peroxidation; Basic fibroblast growth factor

1. Introduction a 2 -Adrenoceptor agonists have been reported to be neuroprotective in both brain and retinal tissues. For example, they blunt the effect of focal or global cerebral ischaemia [23,17,1], pressure-induced retinal hypoxia and mechanical injury to the retinal ganglion cells [55,57]. In most experiments, co-administration of a 2 -adrenoceptor antagonists was found to counteract the protective effects of the agonists. There are a variety of possible ways in which neuroprotection can be induced through activation of a 2 -adrenoceptors. Hypoxia / ischaemia is considered to result in the excessive release of neurotransmitters from affected cells, leading to overstimulation of neurones and unwanted depolarisation [35]. Neurones that are most at risk in this *Corresponding author. Tel.: 144-186-5248-996; fax: 144-186-5794508. E-mail address: [email protected] (N.N. Osborne).

situation are those which possess a high density of receptors for excitatory transmitters such as glutamate [35,34]. a 2 -Adrenoceptors are often situated presynaptically and / or somatodendritically and their activation leads to a decrease in the release of the neuronal transmitter [18,3]. Thus, if a 2 -adrenoceptors are present on glutamatergic neurones, then administration of a 2 -adrenoceptor agonists should theoretically decrease the release of glutamate from these neurones during hypoxia. Since ‘released’ glutamate is the major cause of neuronal death in hypoxia, it is quite feasible that a 2 -adrenoceptor agonists may function in this way. Moreover, neurones which contain inhibitory a 2 adrenoceptors and excitatory glutamate receptors will probably be less overexcited than neurones which contains the same number and type of glutamate receptors [35], since activation of a 2 -adrenoceptors may help alleviate the excessive depolarisation occurring during hypoxia. Administration of a 2 -adrenoceptor agonists induces basic fibroblast growth factor (bFGF) expression in the photoreceptors of the retina but not in the brain [54].

0006-8993 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )02592-0

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Moreover, such treatment protects against light-induced photoreceptor damage [54]. bFGF itself is known to protect retinal neurones from injury induced by both ischaemia [58] and excitotoxicity [13]. Thus, at least in the retina, it may be argued that a 2 -adrenoceptor agonists act as neuroprotectants by stimulating the formation of bFGF. Peng et al. [43] have also found that mitogen-activated protein kinase, MAPK, is up-regulated by a 2 -adrenoceptors agonists in the retina, which may contribute to the neuroprotective effect of a 2 -adrenoceptor agonists. The possibility that a 2 -adrenoceptor agonists have other characteristics that may contribute to their neuroprotective action needs consideration. Very effective ways of protecting a neurone from injury include substances which prevent an elevation of internal levels of sodium (Na 1 ), calcium (Ca 21 ) or free radicals [35,34]. To our knowledge no investigation has been conducted to examine whether a 2 -adrenoceptor agonists can have an effect on these parameters. The presence of a 2 -adrenoceptors in the mammalian retina was first indicated by radioligand binding studies [5,31] and subsequent work showed that the receptors are negatively linked to adenylate cyclase [32]. Cloning techniques have revealed the existence of multiple subtypes of the a 2 -adrenoceptor and the a 2A , a 2C and a 2D isoforms have all been found in the retina [2,56,6]. The precise localisation of these receptors remains uncertain although recent data have shown that bovine photoreceptors express the a 2D -adrenoceptor subtype [53]. It is interesting to note that bFGF receptors are also associated with photoreceptors [54,10]. Over the past 5 years a variety of experimental studies have shown that many different substances can counteract the effect of ischaemia / hypoxia to the retina [34]. In the context of glaucoma, it has been hypothesised that hypoxia plays a part in the death of the ganglion cells [35,34,36] and interestingly, two substances presently used to lower IOP, betaxolol (b 1 -adrenoceptor antagonist) and brimonidine (a 2 -adrenoceptor agonist) also protect ganglion cells from injury in animals [55,57,37,38]. The neuroprotective properties of betaxolol are believed to result from the ability of the compound to reduce Ca 21 and Na 1 entry into neurones. Betaxolol displays considerable affinity for both L-type Ca 21 channels [24] and Na 1 channels [8] and reduces the influx of these ions through direct, competitive, allosteric binding to the channels. These actions of betaxolol are likely to become important in conditions of stress when there is an excessive influx of these ions into injured neurones. The present studies were designed to explore in more detail than previously the potential mechanisms underlying the neuroprotective effect of clonidine in the retina. Since betaxolol and clonidine are thought to possess entirely different ways of protecting neurones, involving blockade of Ca 21 and Na 1 influx and stimulation of bFGF, respectively, the effect of clonidine was compared with that of betaxolol.

2. Materials and methods All investigations involving animals conformed to the ARVO Statement for the Use of Animals in Ophthalmology and Vision Science Research. Albino rabbits and Wistar rats, bred in our laboratory, were kept on a 12-h light / dark cycle and provided with food and water ad libitum.

2.1. In vitro studies on rabbit retinal tissues Rabbits were killed by injection of sodium pentobarbital into the ear vein and retinas were rapidly dissected and placed in Locke’s solution (Mg 21 -free) containing: 154 mM NaCl, 5.6 mM KCl, 2.3 mM CaCl 2 , 3.6 mM NaHCO 3 , 5 mM HEPES and 5.6 mM D-glucose. The Locke’s solution was briefly equilibrated before use with O 2 / CO 2 (95% / 5%), pH 7.2 at 378C. Pieces of retina in Locke’s solution were incubated alone, or with NMDA (100 or 200 mM) at 378C. Parallel experiments were carried out where the samples contained 100 mM clonidine or bFGF (1 mg / 500 ml). In certain cases the a 2 -antagonist yohimbine (1 mM)) was added 15 min (preincubation) before the addition of clonidine. Then, 45 min after the addition of clonidine the tissues were fixed in 2% paraformaldehyde. Frozen sections of similar eccentricity were then cut and processed for the localization of GABA as described elsewhere [39].

2.2. ROS assay The assay is based on that described by LeBel and Bondy [20,19]. This involves the use of DCFH-DA (29,79dichloro-fluorescein diacetate) which is a stable nonfluorescent molecule that readily crosses cell membranes and is hydrolysed by intracellular esterases to non-fluorescent 29,79-dichloro-fluorescein (DCFH). DCFH is then rapidly oxidised in the presence of oxygen reactive species to highly fluorescent DCF (29,79-dichloro-fluorescein). The retinas from 14-day chicken embryos were dissociated by an enzymatic procedure which consisted of incubating the retinas in L-15 medium containing 4 mg / ml dispase (two retinas / 2 ml) for 10 min at 378C. Repetitive pipetting with Pasteur pipette was carried out to dissociate the tissue mechanically. Cellular debris and undissociated tissues were removed until a homogenous suspension was obtained. The cells were washed with HBSS medium and centrifuged. Cells were then suspended in fresh HBSS (10 ml for 14–18 retinas) containing DCFH-DA (20 mM) for 30 min at 308C. After loading with DCFH-DA, the cellular suspension was centrifuged at 30003g for 3 min at 48C. The pellet was resuspended in fresh HBSS and divided in 2-ml aliquots (two retinas / 2 ml) in cuvettes and incubated for 60 min at 308C. Oxygen reactive species formation was stimulated by ascorbate at 308C and the fluorescence (with excitation and emission wavelengths 488 and 525 nm, respectively, and band widths 5 nm) measured at intervals

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of 0 (prior to stimulant addition), 15, 30 and 60 min after stimulation. The ROS formed was quantified by constructing a standard curve using various amounts of pure DCF (0.005–1 nmol). The results are expressed in nmol DCF / h / mg.

2.3. Lipid peroxidation assay The retinas from 14-day chicken embryos were isolated and incubated in Locke’s solution (one retina / 2 ml, pH 7.4) for 15 min at 378C. Then, 5 mM ascorbate and 100 mM iron sulphate were used to induce lipid peroxidation. Tested drugs (clonidine, deferoxamine, flupirtine, melatonin) were added 5 min before stimulation. Retinas were then transferred from the Locke’s solution to 1.15% KCl (one retina / 200 ml) and sonicated. Finally, 5 ml of tissue homogenates were assayed for protein content using bicinchoninic acid [49]. The reaction mixture contained 100 ml retinal homogenate, 100 ml 8.1% SDS, 750 ml of 20% acetic acid (adjusted to pH 3.5 with NaOH), 750 ml of 0.8% aqueous solution of thiobarbituric acid and 300 ml of distilled water. The reaction mixture was heated at 958C for 60 min using a glass bowl as a condenser. After cooling with tap water, 0.5 ml of distilled water and 2.5 ml of the mixture of n-butanol and pyridine (15:1, v / v) were added and the mixture was shaken vigorously. After the centrifugation at 4000 rpm for 10 min, the absorbance of the organic layer (upper) was measured on a Perkin-Elmer LS50B spectrofluorimeter (with excitation and emission wavelengths 515 and 553 nm, respectively, and band widths 5 nm). The lipid peroxide level was quantified by constructing a standard curve using various amounts of pure malondialdehyde (MDA: 1–8 nmol). The results are expressed in nmol MDA / h / mg.

2.4. Assay for bFGF mRNA by RT-PCR Levels of bFGF mRNA present in retinas were determined using a semi-quantitative reverse transcriptase polymerase chain reaction technique (RT-PCR). These were compared with determined mRNA levels for the house-keeping gene cyclophilin, in each case, to correct for experimental variations. Wistar rats (10–12 weeks old) were initially treated as follows: One group of rats was injected i.p. with 200 ml 0.9% sterile sodium chloride solution, a further group with clonidine (1 mg / kg in saline) and another group with betaxolol (2 mg / kg in saline). After 6 h, rats were killed and the retinas removed and sonicated in TriReagent (as per manufacturer’s instructions). Total retinal RNA was isolated and first strand cDNA synthesis was performed on 2 mg DNase-treated RNA essentially as described previously [27]. Individual cDNAs were amplified in a 10-ml reaction volume with PCR buffer (10 mM Tris–HCl, pH 8.3, plus 50 mM KCl), 4 mM MgCl 2 , 200 mM each dNTP, 4 ng / ml

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of both sense and anti-sense primers and 2.5 U AmpliTaq Gold. The PCR reaction was initiated by an incubation at 948C for 10 min and this was followed by the required number of PCR cycles that ensured saturation had not been reached, but were suitable for comparison of cDNA levels in each sample (cycle: 948C, 45 s; 528C, 30 s; 728C, 60 s; cyclophilin: 28 cycles, bFGF: 30 cycles). Cycling was performed using a Hybaid PCR Sprint temperature cycling system (Ashford, UK). After the final cycle was completed, there followed a final incubation of samples at 728C for 3 min. PCR reaction products were separated on 1.5% agarose gels using ethidium bromide for visualisation. The relative abundance of each PCR product was determined by analysing photographs of gels with SigmaScan (Jandel Scientific, Erkrath, Germany). Data were analysed using the Student’s unpaired t-test and a P value less than 0.05 was considered significant.

2.5. Radioligand binding to L-type calcium channels Radioligand binding studies with [ 3 H]nitrendipine were performed as described elsewhere [24]. Rat cortices from Wistar rats (250–350 g) were homogenised in ice-cold 50 mM Tris–HCl buffer (pH 7.4 at 258C). The homogenate was washed by means of three consecutive centrifugation cycles (48 0003g, 10 min, 48C). The final pellet was resuspended in 50 mM Tris–HCl buffer (pH 7.4) to yield an original tissue concentration of 200 mg / 3 ml and stored at 2708C until use. Aliquots (200 ml) of 4-fold diluted tissue homogenate (50 mg / 3 ml) were incubated with 0.1 nM [ 3 H]nitrendipine and various concentrations of the drugs tested for 90 min at 258C in 0.5 ml 50 mM Tris–HCl buffer (pH 7.4). At the end of incubation, samples were diluted with 5 ml of ice-cold buffer, filtered through Whatman GF / B filters and washed twice with 5 ml of ice-cold buffer. Radioactivity on the filters was measured by liquid scintillation spectrometry in 5 ml of Insta-gel Plus. Non-specific binding of [ 3 H]nitrendipine was determined in the presence of 1 mM nifedipine. These experiments were carried out in subdued light to minimise [ 3 H]nitrendipine and nifedipine degradation.

2.6. Calcium influx into rat retinas Wistar rats (250–350 g) were killed and retinas rapidly removed and preincubated (378C for 25 min) in Mg 21 -free Krebs–Ringer bicarbonate buffer (138 mM NaCl, 1 mM CaCl 2 , 11 mM NaH 2 PO 4 , 1 mM Na 2 HPO 4 , 10 mM glucose, 20 mM HEPES, pH 7.2) continuously perfused with oxygen. Antagonists were then added and the retinas incubated for 10 min. Calcium influx was initiated by the addition of 1 mCi 45 CaCl 2 and 100 mM NMDA in a final volume of 2 ml. After 15 min incubation, the reaction was halted by addition of 2 ml ice-cold 10 mM EGTA / 0.9%

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NaCl. Retinas were then washed twice with 2 ml ice-cold 1 mM EGTA / 0.9% NaCl and sonicated in 1 ml distilled water. Radioactivity was determined by liquid scintillation spectrometry and protein concentration using a bicinchoninic acid protein assay kit.

2.7. [ 3 H] BTX-B and [ 3 H] saxitoxin binding assays for sodium channels To prepare cortical membranes from rats (250–350 g Wister rats) animals were killed by decapitation and the cerebral cortex homogenised in 10 volumes of ice-cold 0.32 M sucrose, 5 mM K 2 HPO 4 , pH 7.4 using a motordriven polytetrafluoroethylene-glass homogeniser. The homogenate was centrifuged at 10003g for 10 min at 48C and the resulting supernatant centrifuged at 39 0003g for 20 min at 48C. The pellet was resuspended in Na 1 free buffer (130 mM choline chloride, 5.4 mM KCl, 0.8 mM MgSO 4 , 5.5 mM D-glucose, 50 mM HEPES–Tris, pH 7.4) and recentrifuged at 39 0003g for 20 min at 48C. The pellet was resuspended in Na 1 free buffer at an approximate protein concentration of 3 mg / ml, snap frozen in liquid N 2 and stored at 2808C until required. [ 3 H]Batrachotoxinin-A 20-a-benzoate ([ 3 H]BTX-B) binding was determined as described elsewhere [47]. Briefly, aliquots of cortical membranes (200–400 mg protein) were incubated for 60 min at 378C in Na 1 free buffer containing 10 nM [ 3 H]BTX-B, 1 mM tetrodotoxin, 30 mg scorpion venom (Leiurus quinquestriatus) and 1 mg / ml BSA, with or without clonidine. Nonspecific binding was measured in the presence of 300 mM veratridine. The binding reactions were terminated by the addition of 3 ml of ice-cold washing buffer (163 mM choline chloride, 0.8 mM MgCl 2 , 1.8 mM CaCl 2 , 5 mM HEPES–Tris, pH 7.4). Following rapid vacuum filtration through Whatman GF / B glass fibre filters, the samples were washed a further three times with the same buffer. Bound radioactivity was measured by liquid scintillation spectrometry in 5 ml of Insta-gel Plus. In [ 3 H]saxitoxin binding assays, 100 ml of membrane suspension (200–400 mg protein) was incubated with 50 3 ml of [ H]STX (2.5 nM final concentration) and 50 ml of 1 Na free buffer or clonidine at 378C for 30 min. The binding reactions were terminated as described for [ 3 H]BTX-B assays. Specific binding was defined as the difference between total binding and binding obtained in the presence of 1 mM tetrodotoxin.

2.8. Materials [ 3 H]Batrachotoxinin-A 20-a-benzoate (34 Ci / mmol), NaCl (1 mCi / ml) and [ 3 H]nitrendipine (87 Ci / mmol) were obtained from NEN Research Products (Stevenage, UK) and [11- 3 H]saxitoxin diacetate (14.9 Ci / mmol) and 45 CaCl 2 (2 mCi / ml) from Amersham (Amersham, UK). 22

29,79-dichloro-fluorescein diacetate was from Molecular Probes (Holland). Oligo(dT) 30 was a generous gift from Dr S.L. Eastwood (Oxford University, Oxford, UK). Moloney murine leukaemia virus (M-MLV) reverse transcriptase, RQ1 RNase-free DNase, RNase inhibitor (RNAsin  ) and stock deoxynucleotide triphosphates (each 100 mM) were from Promega UK Ltd. (Southampton, UK). Amplitaq Gold (Taq) with GeneAMP  (103) PCR buffer and MgCl 2 (25 mM) were from Perkin-Elmer (Warrington, UK). All other drugs and reagents were purchased from Sigma (Poole, UK) except for tetrodotoxin and Insta-gel Plus which were from Semat Technical Ltd. (St. Albans, UK) and Packard (Groningen, The Netherlands), respectively. L-15 and HBSS media and PCR oligonucleotide primers were obtained from Gibco Life Technologies (Paisley, Scotland, UK). These were as follows: cyclophilin sense, 59-TGGTCAACCCCACCGTGTTCTTCG-39; cyclophilin antisense, 59GTCCAGCATTTGCCATGGACAAGA-39; bFGF sense, 59-GCCTTCCCACCCGGCCACTTCAAGG-39; bFGF antisense, 59-GCACACACTCCCTTGATGGACACAA-39.

3. Results

3.1. Immunohistochemistry The whole of the inner plexiform layer ‘stains’ positively for GABA immunoreactivity in the rabbit retina (Fig. 1a). A number of perikarya on either side of the inner plexiform layer also ‘stain’ positively for GABA (Fig. 1a). The GABA immunoreactivity was unaffected in retinas incubated in physiological saline for 45 min (Fig. 1b). However, when NMDA (100 or 200 mM) was included in the medium GABA ‘staining’ in the inner plexiform layer was no longer homogenous in appearance but appeared as four bands of immunoreactivity (Fig. 1c and d). This effect of NMDA on the GABA immunoreactivity was almost completely blunted by the inclusion of clonidine (Fig. 1e) or bFGF (Fig. 1f). Moreover, the nullifying effect of clonidine on NMDA was ineffective when the a 2 -adrenoreceptor antagonist, yohimbine, was present (Fig. 1g). Yohimbine (Fig. 1h) and clonidine (Fig. 1i) had no effect individually on the GABA immunoreactivity.

3.2. Effect of clonidine on reactive oxygen species ( ROS) ROS formation was determined using DCFH-DA which is oxidised to the fluorescent substance DCF. As shown in Fig. 2, ROS is generated in the presence of ascorbate / FeSO 4 but this is not the case for the iron chelator, deferoxamine (results not shown), clonidine or flupirtine. However, while flupirtine and deferoxamine completely attenuate the effect of ascorbate / FeSO 4 this was not the

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Fig. 1. (a) Shows the distribution of GABA immunoreactivity in the rabbit retina fixed with 2% paraformaldehyde. GABA is associated with a number of amcrine cell bodies (thick arrows) and their processes in the inner plexiform layer, giving a homogenous appearance. Incubation of the rabbit retina in physiological saline for 45 min has little or no effect on the GABA immunoreactivity (b). Exposure of the retina in vitro to 100 mM (c) or 200 mM (d) NMDA caused a change in the nature of the GABA immunoreactivity. Particularly clear to see is that the GABA immunoreactivity in the inner plexiform layer appears as three to four bands (thin arrows). The NMDA effect on the changes seen in the GABA immunoreactivity is significantly blunted when clonidine (e) or bFGF (f) is present. The blunting influence of clonidine on the NMDA-induced changes to the GABA immunoreactivity is counteracted by the presence of yohimbine (g). Yohimbine (h) and clonidine (i) have no effect on the GABA ‘staining’.

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Fig. 2. Production of reactive oxygen species (ROS) quantified as the amount of DCF formed in dissociated chick retinal cells (see Materials and methods). Results are mean values 6S.E.M. from 8 to 16 different cell preparations. It can be seen that clonidine (100 or 500 mM) and flupirtine (100 mM) do not stimulate the production of DCF. In contrast, ascorbate (AA) together with iron (FeSO 4 ) stimulates a huge increase in DCF. This effect is not significantly counteracted by inclusion of clonidine although it is significantly blunted by bFGF, deferoxamine and flupirtine. Statistics by Student’s t-test. * P,0.05.

Fig. 3. This figure shows that ascorbate and FeSO 4 stimulate lipid peroxidation indicated by the formation of MDA. The effect is significantly blunted by clonidine (500 mM), deferoxamine, flupirtine and melatonin. Clonidine (100 or 500 mM) has no effect on lipid peroxidation. Results are mean6S.E.M., where n57–10. Statistics by Student’s t-test. * P,0.05.

bFGF mRNA in the retina 6 h later (Fig. 4). However, betaxolol, when injected, had no significant effect on bFGF mRNA. case for clonidine. Indeed high concentrations of clonidine (500 mM) tended to exacerbate the effect of ascorbate / FeSO 4 although the effect was insignificant. Interestingly, bFGF had a weak but significant inhibition on the stimulation of ROS by ascorbate / FeSO 4 .

3.3. Effect of clonidine on lipid peroxidation In this procedure lipid peroxidation is determined by measuring malondialdehyde (MDA) as an index for thiobarbituric-reactive substances (TBARS) formed between decomposed lipid peroxides and thiobarbituric acid. Fig. 3 shows that ascorbate / FeSO 4 stimulate the formation of MDA and that this effect is significantly blunted by melatonin (10 mM), flupirtine (100 mM), deferoxamine (200 mM) and clonidine (500 mM). Clonidine (100 mM) did not significantly attenuate the effect of ascorbate / FeSO 4 , nor did it (up to 500 mM) stimulate MDA formation by itself.

3.4. Effect of clonidine and betaxolol on bFGF mRNA Injection of clonidine i.p. caused an upregulation of

Fig. 4. The effect of clonidine and betaxolol on the levels of bFGF mRNA in rat retina 6 h after treatment. The data represent the ratio of cyclophilin:bFGF mRNA and are expressed as mean6S.E.M. of 12 experiments. * P,0.05 by Student’s unpaired t-test vs. saline.

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Fig. 5. The effect of clonidine (s) on the specific binding of [ 3 H]nitrendipine (0.1 nM) to rat cortical membranes. The effect of nifedipine (d) is also shown for comparison. Each point represents the mean6S.E.M. of four experiments performed in duplicate.

3.5. Effect of clonidine on calcium influx and interaction with L-type channels Fig. 5 shows the effect of nifedipine and clonidine on the specific binding of [ 3 H]nitrendipine to rat cortical membranes. Nifedipine competitively inhibited [ 3 H]nitrendipine binding with an IC 50 of 2.9 nM (2log IC 50 58.5460.06, slope factor50.760.1, n54). Clonidine did not significantly displace [ 3 H]nitrendipine binding to rat cortical membranes at any of the concentrations tested. NMDA (100 mM) produced a 4-fold increase in the basal influx of 45 Ca 21 into rat retinas from 25356394 to 100846980 cpm / mg protein (n56, P,0.001 by unpaired Student’s t-test) (Fig. 6). While MK-801 (5 mM) completely inhibited the NMDA-stimulated 45 Ca 21 influx into rat retinas, clonidine did not produce any significant (by one-way ANOVA followed by Bonferroni’s post-test) effect at the concentrations tested (10 and 100 mM). MK-801 (5 mM) or clonidine (100 mM) alone showed no significant effect on the basal influx of 45 Ca 21 into rat retinas (Fig. 6).

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Fig. 6. The effects of clonidine and MK-801 on the basal and 100 mM NMDA-stimulated 45 Ca 21 influx into the rat retina. Left side of the dashed line shows un-stimulated values, whereas values in presence of 100 mM NMDA are presented on the right. Each value represents the mean6S.E.M. of four to six experiments. * P,0.001 compared with 100 mM NMDA-stimulated 45 Ca 21 influx by one-way ANOVA (Bonferroni’s post-test).

an IC 50 value of 37 nM (Fig. 7). Fig. 8 shows the effect of veratridine and clonidine on the specific binding of [ 3 H]BTX-B to rat cortical membranes. Veratridine competitively inhibited [ 3 H]BTX-B binding with an IC 50 of 4.4 mM (2log IC 50 55.3660.03, slope factor50.97, n53). At concentrations up to 100 mM clonidine did not signifi-

3.6. Effect of clonidine on sodium channels Clonidine displayed practically no affinity for [ 3 H]saxitoxin binding sites on cortical membranes even at concentrations as high as 250 mM (Fig. 7). In contrast, tetrodotoxin potently displaced [ 3 H]saxitoxin binding with

Fig. 7. The effect of clonidine (h) on the specific binding of [ 3 H]saxitoxin (2.5 nM) to rat cortical membranes. The effect of tetrodotoxin (j) is also shown for comparison. Each point represents the mean6S.E.M. of three experiments performed in duplicate.

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Fig. 8. The effect of clonidine (h) on the specific binding of [ 3 H]BTX-B (10 nM) to rat cortical membranes. The effect of veratridine (j) is also shown for comparison. Each point represents the mean6S.E.M. of four experiments performed in duplicate.

cantly inhibit the binding of [ 3 H]BTX-B. However, very high concentrations of clonidine (1 mM) did displace [ 3 H]BTX-B binding by about 50%.

4. Discussion The present results support previous data showing that a 2 -adrenoceptor agonists such as clonidine can attenuate the detrimental effects of glutamate mimetics [4,51,15]. It is known that excessive stimulation of ionotropic glutamate receptors leads to an influx of Na 1 and Ca 1 into neurones. This results in sustained neuronal depolarisation and ultimately to death of these cells [21,30]. We have shown in previous experiments that exposure of retinal tissues to the ionotropic glutamate receptor agonist, NMDA, causes a change in the nature of GABA immunostaining [33]. These changes have been interpreted as showing that some GABA-containing neurones express NMDA receptors and upon excessive stimulation of these, cellular stores of this neurotransmitter are released and cells eventually die. Thus, a change in the nature of GABA immunolabelling can be used to index the initial stages of cell death. Furthermore, substances which blunt the effect of NMDA receptor stimulation act as neuroprotectants, under these circumstances, when used in vivo. An example of this is flupirtine, a non-opioid centrally acting analgesic, which blunts both NMDA-induced changes to GABA immunoreactivity in vitro [39] and also retinal neuronal death, in vivo [40]. In the present study, clonidine and bFGF attenuate

NMDA-induced changes to the retina, in vitro, suggesting that both substances would protect against hypoxia / ischaemia-induced insults to the retina in situ. This has indeed been found to be the case [57,13,52]. Moreover, the clonidine effect is shown to be receptor-mediated,since its effect on NMDA-induced changes to the GABA immunoreactivity, in vitro, is nullified by yohimbine. This is similar to that which has been found, in vivo, where the neuroprotective effect of a 2 -adrenoceptor agonists have been shown to be receptor mediated [57]. It is generally accepted that the initial processes involved in neuronal death brought about by hypoxia, ischaemia and oxidative stress are excessive influxes of calcium, sodium and chloride [34,48,9]. The subsequent biochemical events that take place generate free radicals / ROS and the excessive production of these leads to cell damage [34,9,11]. Indeed there is very good evidence that free radical scavengers such as melatonin [7], vitamin E [44], flupirtine [40,26], Ginkgo biloba extract [50] or nitric oxide inhibitors [28] can counteract hypoxia-induced damage to retinal neurones. In this study experiments were carried out, in vitro, to determine whether any evidence could be amassed to support the notion that clonidine could act as a free radical scavenger. Ascorbate / iron is known to act on cells to generate ROS, which in turn can induce lipid peroxidation [12]. It was found that ascorbate / iron induced formation of ROS in chick retinal neurones and that this was blunted by both flupirtine and melatonin, as expected, but not by clonidine (see Fig. 2). There was, indeed, a suggestion that clonidine was potentiating the production of ROS, although this was only seen at very high concentrations (i.e. 500 mM). Paradoxically, 500 mM clonidine blunted the ascorbate / iron induced formation of MDA (index for lipid peroxidation) whereas 100 mM did not. As expected, flupirtine or melatonin nullified ascorbate / iron-induced production of MDA. From the combined data it may, therefore, be concluded that little evidence was found to support the idea that clonidine acts as a free radical scavenger at fairly high concentrations (100 mM). Moreover, since the data obtained in the ROS-formation and lipid peroxidation experiments were conflicting, it is probable that these high concentrations simply give rise to non-physiological effects. The finding that intraperitoneal injections of clonidine increase levels of bFGF in the retina (but not the brain; [54]) and that bFGF is known to attenuate hypoxia-induced destruction to the retina [58,13,52] suggest a unique neuroprotective action for clonidine in the retina. The idea of a neuroprotective action for bFGF is supported by the fact that this factor can attenuate the ascorbate / iron-induced formation of ROS and is also known to attenuate injury to neurones [52]. It remains unknown how bFGF protects neurones from insults such as hypoxia. The present studies suggest that it may do so, at least in part, by acting as a free radical scavenger. Further detailed

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studies are needed to resolve this issue. Another factor that should be considered in the context of neuroprotection is the known action of clonidine on body temperature and blood pressure [46,22,45]. In is generally agreed that raising the body temperature would potentiate an ischaemic insult so any rise in body temperature due to clonidine administration should not contribute to neuroprotection. However, this may not be the case for blood pressure because a case can be made out that either an increase or decrease can potentiate the effects of ischaemia / reperfusion. In addition changes in blood pressure and body temperature may initiate a number of indirect actions on an ischaemic insult. Overactivation of ionotropic glutamate receptors results in Ca 21 and Na 1 entering the cell, which in turn activates Ca 21 and Na 1 channels to allow further entry of these ions [21,16,14]. During hypoxia, this process is perpetuated such that the internal Ca 21 and Na 1 levels become abnormally high and these set in motion biochemical events which eventually lead to cell death. Na 1 channels contain a number of neurotoxin binding sites, which have proved very useful in determining the function of the channel. The most important of these binding sites are receptor sites 1 and 2, which bind specifically to saxitoxin and batrachotoxin, respectively. The saxitoxin site is located in the vestibule of the channel and binding of water soluble toxins including saxitoxin and tetrodotoxin directly leads to inhibition of ion conductance. The batrachotoxin site is found in the transmembrane region and is involved in the gating of the channel. A number of lipophilic pharmacological agents have an indirect negative allosteric interaction with this site in the Na 1 channel and reduce Na 1 entry in situations where the channel is overactivated. Overactivation of Na 1 channels occurs in ischaemia and compounds which reduce Na 1 influx have been found to be neuroprotective in models of ischaemia [47,25,29]. Unlike betaxolol, which binds to the batrachotoxin site and reduces Na 1 entry [8] clonidine has no affinity for the sodium channel and thus blockade of Na 1 entry is unlikely to contribute to its action as a neuroprotectant Pharmacological agents which interact with voltagedependent Ca 21 channels to reduce Ca 21 influx into a stressed cell will generally have neuroprotectant properties [49,53]. Examples of agents which decrease calcium entry are betaxolol [37,38,24,41], that directly inhibits L-type calcium channels, and flupirtine [26,42]. In the present study, no evidence could be found that clonidine acts in this way, i.e. interacts with L-type calcium channels and / or reduces NMDA-induced influx of calcium. It would appear, therefore, that the characteristics which make betaxolol a neuroprotectant (interacting with calcium and sodium channels to reduce influx of these ions into stressed neurones) are not associated with clonidine. This conclusion is further supported by the RT-PCR studies which show that i.p. injection of clonidine, but not betaxolol, stimulates bFGF. Thus, a 2 -adrenoceptor agon-

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ists and betaxolol act as neuroprotectants in fundamentally different ways. Whether this means that combining the two substances for neuroprotection will be more efficacious than simply increasing the concentration of one remains to be established.

Acknowledgements JM is grateful to the European Commission for a Marie Curie fellowship. NNO thanks the The Glaucoma Foundation (New York, USA) for providing consumable support.

References [1] M.Z. Berkman, T.A. Zirh, K. Berkman, M.N. Pamir, Tizanidine is an effective agent in the prevention of focal cerebral ischaemia in rats: an experimental study, Surg. Neurol. 50 (1998) 264–270. [2] J.R. Berlie, L.J. Iversen, H.S. Blaxall, M.E. Cooley, D.M. Chacko, D.B. Bylund, Alpha-2 adrenergic receptors in the bovine retina: Presence of only the alpha-2D subtype, Invest. Ophthalmol. Vis. Sci. 36 (1995) 1885–1892. [3] M. Bertolino, S. Vicini, R. Gillis, A. Travagli, Presynaptic alpha2adrenoceptors inhibit excitatory synaptic transmission in rat brain stem, Am. J. Physiol. 272 (1997) G654–661. [4] P.E. Bickler, B.M. Hansen, Alpha 2-adrenergic agonists reduce glutamate release and glutamate receptor-mediated calcium changes in hippocampal slices during hypoxia, Neuropharmacology 35 (1996) 679–687. [5] H. Bittiger, J. Heid, N. Wigger, Are only alpha-2-adrenergic receptors present in bovine retina?, Nature 287 (1980) 645–647. [6] D.B. Bylund, D.M. Chacko, Characterization of alpha-2 adrenergic receptor subtypes in human ocular tissue homogenates, Invest. Ophthalmol. Vis. Sci. 40 (1999) 2299–2306. [7] C. Cazevieille, N.N. Osborne, Retinal neurones containing kainate receptors are influenced by exogenous kainate and ischaemia while neurones lacking these receptors are not — melatonin counteracts the effects of ischaemia and kainate, Brain Res. 755 (1997) 91–100. [8] Chidlow, G., Melena, J. and Osborne, N.N., Betaxolol, a b 1 adrenoceptor antagonist, reduces Na 1 influx into cortical neurones by direct interaction with Na 1 channels: comparison with other b-adrenoceptor antagonists, Br. J. Pharmacol. 2000 (in press). [9] J.T. Coyle, P. Puttfarcken, Oxidative stress, glutamate, and neurodegenerative disorders, Science 262 (1993) 689–695. [10] H. Gao, J.G. Hollyfield, J. Jolkkonen, K. Puurunen, J. Koistinaho, R. Kauppinen, A. Haapalinna, L. Nieminen, J. Sivenius, Basic fibroblast growth factor: increased gene expression in inherited and light-induced photoreceptor degeneration, Exp. Eye Res. 62 (1996) 181–189. [11] M.E. Gotz, G. Kunig, P. Riederer, M.B. Youdim, Oxidative stress: free radical production in neural degeneration, Pharmacol. Ther. 63 (1994) 37–122. [12] B. Halliwell, Vitamin C: antioxidant or pro-oxidant in vivo?, Free Radic. Res. 25 (1996) 439–454. [13] D. Hicks, V. Heidinger, S. Mohand Said, J. Sahel, H. Dreyfus, Growth factors and gangliosides as neuroprotective agents in excitotoxicity and ischaemia, Gen. Pharmacol. 30 (1998) 265–273. [14] A.J. Hunter, Calcium antagonists: their role in neuroprotection, Int. Rev. Neurobiol. 40 (1997) 95–108. [15] J. Jolkkonen, K. Puurunen, J. Koistinaho, R. Kauppinen, A. Haapalinna, L. Nieminen, J. Sivenius, Neuroprotection by the

56

[16] [17]

[18]

[19]

[20]

[21] [22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30] [31]

[32] [33]

[34]

[35]

[36]

H.M. Chao et al. / Brain Research 877 (2000) 47 – 57 alpha2-adrenoceptor agonist, dexmedetomidine, in rat focal cerebral ischaemia, Eur. J. Pharmacol. 372 (1999) 31–36. T. Kobayashi, Y. Mori, Ca 21 channel antagonists and neuroprotection from cerebral ischaemia, Eur. J. Pharmacol. 363 (1998) 1–15. J. Kuhmonen, J. Pokorny, R. Miettinen, A. Haapalinna, J. Jolkkonen, P. Riekkinen Sr., J. Sivenius, Neuroprotective effects of dexmedetomidine in the gerbil hippocampus after transient global ischaemia, Anesthesiology 87 (1997) 371–377. S.Z. Langer, P.K. Smith, R.I. Krohn, G.T. Hermanson, A.K. Mallia, F.H. Gartner, M.D. Provenzano, E.K. Fujimoto, N.M. Goeke, B.J. Olson, D.C. Klenk, Presynaptic regulation of the release of catecholamines, Pharmacol. Rev. 32 (1980) 337–362. C.P. LeBel, S.C. Bondy, Persistent protein damage despite reduced oxygen radical formation in the aging rat brain, Int. J. Dev. Neurosci. 9 (1991) 139–146. C.P. LeBel, S.F. Ali, M. McKee, S.C. Bondy, Organometal-induced increases in oxygen reactive species: the potential of 29,79-dichlorofluorescin diacetate as an index of neurotoxic damage, Toxicol. Appl. Pharmacol. 104 (1990) 17–24. J.M. Lee, G.J. Zipfel, D.W. Choi, The changing landscape of ischaemic brain injury mechanisms, Nature 399 (1999) A7–14. A. Livingston, J. Low, B. Morris, Effects of clonidine and xylazine on body temperature in the rat, Br. J. Pharmacol. 81 (1984) 189–193. C. Maier, G.K. Steinberg, G.H. Sun, G.T. Zhi, M. Maze, Neuroprotection by the alpha 2-adrenoreceptor agonist dexmedetomidine in a focal model of cerebral ischaemia, Anesthesiology 79 (1993) 306–312. J. Melena, J.P.M. Wood, N.N. Osborne, Betaxolol, a beta-1-adrenoceptor antagonist, has an affinity for L-type Ca(21) channels, Eur. J. Pharmacol. 378 (1999) 317–322. T. Narahashi, C.S. Huang, J.H. Song, J.Z. Yeh, Ion channels as targets for neuroprotective agents, Ann. NY Acad. Sci. 825 (1997) 380–388. M.S. Nash, J.P.M. Wood, J. Melena and N.N. Osborne, Flupirtine ameliorates ischaemic-like death of rat retinal ganglion cells by preventing calcium influx, Brain Res. 2000 (in press). M.S. Nash, N.N. Osborne, Assessment of Thy-1 mRNA levels as an index of retinal ganglion cell damage, Invest. Ophthalmol. Vis. Sci. 40 (1999) 1293–1298. A.H. Neufeld, A. Sawada, B. Becker, Inhibition of nitric-oxide synthase 2 by aminoguanidine provides neuroprotection of retinal ganglion cells in a rat model of chronic glaucoma, Proc. Natl. Acad. Sci. USA 96 (1999) 9944–9948. T.P. Obrenovitch, Neuroprotective strategies: voltage-gated Na 1 channel down-modulation versus presynaptic glutamate release inhibition, Rev. Neurosci. 9 (1998) 203–211. J.W. Olney, Excitotoxic amino acids and neuropsychiatric disorders, Annu. Rev. Pharmacol. Toxicol. 30 (1990) 47–71. N.N. Osborne, Binding of (2)[(3)H]noradrenaline to bovine membrane of the retina. Evidence for the existence of alpha-2-receptors, Vis. Res. 22 (1982) 1401–1407. N.N. Osborne, Inhibition of cAMP production by alpha-2-adrenoceptor stimulation in rabbit retina, Brain Res. 553 (1991) 84–88. N.N. Osborne, A.J. Herrera, The effect of experimental ischaemia and excitatory amino acid agonists on the GABA and serotonin immunoreactivities in the rabbit retina, Neuroscience 59 (1994) 1071–1081. N.N. Osborne, M. Ugarte, M. Chao, G. Chidlow, J.H. Bae, J.P.M. Wood, M.S. Nash, Neuroprotection in relation to retinal ischaemia and relevance to glaucoma, Surv. Ophthalmol. 43 (1999) S102– S128. N.N. Osborne, J.P.M. Wood, G. Chidlow, J.H. Bae, J. Melena, M.S. Nash, Ganglion cell death in glaucoma: What do we really know?, Br. J. Ophthalmol. 83 (1999) 980–986. N.N. Osborne, G. Chidlow, M.S. Nash, J.P.M. Wood, The potential of neuroprotection in glaucoma treatment, Curr. Opin. Ophthalmol. 10 (1999) 82–92.

[37] N.N. Osborne, C. Cazevieille, A.L. Carvalho, A.K. Larsen, L. DeSantis, In vivo and in vitro experiments show that betaxolol is a retinal neuroprotective agent, Brain Res. 751 (1997) 113–123. [38] N.N. Osborne, L. DeSantis, J.H. Bae, M. Ugarte, J.P.M. Wood, M.S. Nash, G. Chidlow, Topically applied betaxolol attenuates NMDAinduced toxicity to ganglion cells and the effects of ischaemia to the retina, Exp. Eye Res. 69 (1999) 331–342. [39] N.N. Osborne, G. Pergande, F. Block, M. Schwarz, Immunohistochemical evidence for flupirtine acting as an antagonist on the Nmethyl-D-aspartate and homocysteic acid-induced release of GABA in the rabbit retina, Brain Res. 667 (1994) 291–294. [40] N.N. Osborne, M. Schwarz, G. Pergande, Protection of rabbit retina from ischemic injury by flupirtine, Invest. Ophthalmol. Vis. Sci. 37 (1996) 274–280. [41] N.N. Osborne, L. DeSantis, J. Melena, G. Chidlow, J.P.M. Wood, Attenuation of ganglion cell dysfunction: A goal for glaucoma treatment in the new millennium, Ophthalmol. Clin. North Am. 13 (2000) p123–30. [42] N.N. Osborne, C. Cazevieille, J.P. Wood, M.S. Nash, G. Pergande, F. Block, C. Kosinski, M. Schwarz, Flupirtine, a nonopioid centrally acting analgesic, acts as an NMDA antagonist, Gen. Pharmacol. 30 (1998) 255–263. [43] M. Peng, Y. Li, Z. Luo, C. Liu, A.M. Laties, R. Wen, Alpha2adrenergic agonists selectively activate extracellular signal-regulated kinases in Muller cells in vivo, Invest. Ophthalmol. Vis. Sci. 39 (1998) 1721–1726. [44] A.C. Rego, M.S. Santos, C.R. Oliveira, Influence of the antioxidants vitamin E and idebenone on retinal cell injury mediated by chemical ischaemia, hypoglycemia, or oxidative stress, Free Radic. Biol. Med. 26 (1999) 1405–1417. [45] D.J. Reis, S. Regunathan, M.P. Meeley, Imidazole receptors and clonidine-displacing substance in relationship to control of blood pressure, neuroprotection, and adrenomedullary secretion, Am. J. Hypertens. 5 (1992) 51s–57s. [46] A.A. Romanovsky, O. Shido, A.L. Ungar, C.M. Blatteis, Genesis of biphasic thermal response to intrapreoptically microinjected clonidine, Brain Res. Bull. 31 (1993) 509–513. [47] T. Shimidzu, Y. Itoh, S. Tatsumi, S. Hayashi, Y. Ukai, Y. Yoshikuni, K. Kimura, Blockade of voltage-sensitive sodium channels by NS-7, a novel neuroprotective compound, in the rat brain, Naunyn Schmiedeberg’s Arch. Pharmacol. 355 (1997) 601–608. [48] B.K. Siesjo, Pathophysiology and treatment of focal cerebral ischaemia. Part I. Pathophysiology, J. Neurosurg. 77 (1992) 169– 184. [49] P.K. Smith, R.I. Krohn, G.T. Hermanson, A.K. Mallia, F.H. Gartner, M.D. Provenzano, E.K. Fujimoto, N.M. Goeke, B.J. Olson, D.C. Klenk, Measurement of protein using bicinchoninic acid [published erratum appears in Anal Biochem 1987 May 15;163(1):279], Anal. Biochem. 150 (1985) 76–85. [50] M.E. Szabo, M.T. Droy Lefaix, M. Doly, P. Braquet, Ischaemia- and reperfusion-induced Na1, K1, Ca21 and Mg21 shifts in rat retina: effects of two free radical scavengers, SOD and EGB 761, Exp. Eye Res. 55 (1992) 39–45. [51] P. Talke, P.E. Bickler, J.M. Lee, G.J. Zipfel, D.W. Choi, Effects of dexmedetomidine on hypoxia-evoked glutamate release and glutamate receptor activity in hippocampal slices, Anesthesiology 85 (1996) 551–557. [52] K. Unoki, M.M. LaVail, Protection of the rat retina from ischemic injury by brain-derived neurotrophic factor, ciliary neurotrophic factor, and basic fibroblast growth factor, Invest. Ophthalmol. Vis. Sci. 35 (1994) 907–915. [53] V. Venkataraman, T. Duda, K. Galoian, R.K. Sharma, Molecular and pharmacological identity of the a(2D)-adrenergic receptor subtype in bovine retina and its photoreceptors, Mol. Cell. Biochem. 159 (1996) 129–138. [54] R. Wen, T. Cheng, Y. Li, W. Cao, R.H. Steinberg, Alpha-2-adrenergic agonists induce basic fibroblast growth factor expression in

H.M. Chao et al. / Brain Research 877 (2000) 47 – 57 photoreceptors in vivo and ameliorate light damage, J. Neurosci. 16 (1996) 5986–5992. [55] L.A. Wheeler, R. Lai, E. Woldemussie, From the lab to the clinic: activation of an alpha-2 agonist pathway is neuroprotective in models of retinal and optic nerve injury, Eur. J. Ophthalmol. 9 (Suppl. 1) (1999) S17–21. [56] A. Wikberg Matsson, J.E.S. Wikberg, S. Uhlen, Characterization of alpha-2-adrenoceptor subtypes in the porcine eye: Identification of alpha (2A)-adrenoceptors in the choroid, ciliary body and iris, and

57

alpha (2A)- and alpha (2C)-adrenoceptors in the retina, Exp. Eye Res. 63 (1996) 57–66. [57] E. Yoles, L.A. Wheeler, M. Schwartz, Alpha2-adrenoreceptor agonists are neuroprotective in a rat model of optic nerve degeneration, Invest. Ophthalmol. Vis. Sci. 40 (1999) 65–73. [58] C. Zhang, K. Takahashi, T.T. Lam, M.O.M. Tso, Effects of basic fibroblast growth factor in retinal ischaemia, Invest. Ophthalmol. Vis. Sci. 35 (1994) 3163–3168.