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Nitric oxide synthase inhibitors l -NAME and 7-nitroindazole protect rat hippocampus against kainate-induced excitotoxicity
Neuroscience Letters 249 (1998) 75–78
Nitric oxide synthase inhibitors L-NAME and 7-nitroindazole protect rat hippocampus against kainate-induced excitotoxicity P.A. Jones a,, R.A. Smith b, T.W. Stone c ,* a
Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK b Laboratory of Human Anatomy, University of Glasgow, Glasgow G12 8QQ, UK c Division of Neuroscience and Biomedical Systems, West Medical Building, University of Glasgow, Glasgow G12 8QQ, UK Received 9 February 1998; received in revised form 21 April 1998; accepted 27 April 1998
Abstract The role of nitric oxide in cerebral insults remains controversial. While numerous studies have used models of ischaemia and hypoxia, few have examined nitric oxide in the kainate model of excitotoxicity. Kainate (10 mg/kg) was administered to rats via the intraperitoneal (i.p.) route to induce submaximal damage to the CA1, CA2 and CA3a regions of the hippocampus after 7 days. Systemic injections of the nitric oxide synthase (NOS) inhibitors NG-nitro-L-arginine methyl ester (L-NAME) and 7-nitroindazole (7-NI), both at a dose of 5 mg/kg, reduced cell death in all three regions. As 7-NI selectively inhibits the neuronal form of NOS, this study suggests that nitric oxide produced from a neuronal and not epithelial source may contribute to neuronal damage in this model. 1998 Elsevier Science Ireland Ltd. All rights reserved
Keywords: Kainate; Excitotoxicity; Hippocampus; 7-Nitroindazole; NG-nitro-L-arginine methyl ester; Nitric oxide synthase
Nitric oxide (NO) has been viewed as a possible mediator of glutamate-induced neurotoxicity due both to an increase in NO levels after excitotoxicity and ischaemia, and to an apparent upregulation of neuronal nitric oxide synthase (nNOS) and nNOS mRNA during ischaemia. The excitotoxic/ischaemic-associated calcium influx activates both constitutive forms of NOS, elevating the levels of NO. Nitric oxide itself can increase cellular calcium influx by opening the N-methyl-d-aspartate (NMDA)-associated calcium channel [1], or increase the availability of excitatory amino acids by stimulating the release of both glutamate and aspartate and inhibiting glutamate uptake [17]. Nitric oxide has effects on iron metabolism (e.g. activation of iron regulatory factor [9]), which may be significant in the formation of free radicals via the Fenton reaction. A further possible indirect route by which NO may cause damage is by the depletion of the toxic molecule scavenger glutathione, through the formation of intracellular S-nitrosoglu* Corresponding author. 1 Present address: Department of Pharmacology, 1 George Square, University of Edinburgh, Edinburgh EH8 9JZ, UK.
tathione, although Wink et al. reported that NO itself is a free radical scavenger [30]. All this evidence is consistent with studies which indicate that NO itself is neurotoxic [12,18], and that NOS inhibition or deletion can reduce damage produced by ischaemia [8,14,15,20,29]. Some of these data however, have not proved reproducible by others [4]. Against this background the role of NO in excitotoxicity remains controversial. Inhibition of NOS has been claimed to reduce the excitotoxic effect of NMDA receptor activation by NMDA or quinolinic acid [16,26], but not the effects of kainic acid [24]. Conversely there are several reports that NOS inhibition has no effect or can enhance neurotoxicity via NMDA receptors [6,13,19] and can increase kainate-induced seizures. We have now used both a general NOS inhibitor NG-nitro-l-arginine methyl ester (l-NAME) and a competitive selective inhibitor of the neuronal NOS isoform (7nitroindazole; 7-NI) to investigate the role of NO in the kainate model of excitotoxicity. Male Wistar rats weighing 240–310 g, housed under standard conditions, were used in all experiments. All injections were made by the intraperitoneal (i.p.) route in a
0304-3940/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00372- 3
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volume no greater than 3 ml/kg. Kainate and l-NAME were dissolved in saline, 7-NI in a 30% solution of Tween 80 in saline to provide solutions of the correct concentration for injection. The drugs were all injected 10 min prior to the injection of kainate, which was administered at a standard dose of 10 mg/kg. This dose was selected since our previous studies indicated that it produced excitotoxicity which is clearly submaximal in most animals. Following the injection of kainate, the animals were left for 7 days under standard conditions. Rats were anaesthetized with sodium pentobarbitone 60 mg/kg after 7 days. The method of perfusion first described by Brown and Brierley [3] was used, in which 50 ml of physiological medium with 12.5% xylocaine was perfused through a cannula placed into the left ventricle before fixation by 250 ml of FAM (40% formaldehyde/glacial acetic acid/methanol, in the ratio 1:1:8 v/v). The animals were decapitated and the heads stored in fixative for up to a week, at 4°C. The brain was subsequently removed and the left hemisphere marked with Indian ink. The cerebral hemispheres were cut into 2 mm thick slices, dehydrated and infused with paraffin wax throughout before embedding in wax. Sections of 6 mm were cut, mounted on slides and stained with haematoxylin and eosin. Sections were examined under a light microscope by observers who were unaware of the drug treatment received. The left (marked) hippocampus was examined and areas CA1, CA2, CA3a CA3b and CA4 scored for damage. The scale used was a percentage scale, where 0% equated to no damage while 100% indicated a total loss of neurones. The damage was estimated by a cell count of viable (blue stained) cells versus pink stained compromised
Fig. 1. Protection against kainate-induced excitotoxicity by the NOS inhibitor L-NAME in the rat hippocampus. ***P , 0.001 versus kainate alone; + + + P , 0.001 versus saline control.
Fig. 2. The selective nNOS inhibitor 7-NI protected the regions of the hippocampus damaged by kainic acid. ***P , 0.001 versus kainate alone; + + + P , 0.001 versus saline control.
neurones. Account was also taken of decreased neuronal number and general appearance of the hippocampal subareas. Analysis of variance (ANOVA) followed by post-tests was used to determine any statistical significance. Significance refers to results where P , 0.05 was obtained. The administration of 10 mg/kg kainate resulted in neuronal damage to the CA1, CA2 and CA3a regions of the hippocampus at levels significantly different from control values (where no damaged was observed). Damage was greatest in the CA1 (56.7 ± 9.6%) and CA3a regions (51.1 ± 9.0%), with a lower amount in the CA2 region (Fig. 1). No other hippocampal region was significantly damaged. The introduction of 5 mg/g l-NAME 10 min prior to kainate resulted in protection in all three regions damaged by the excitotoxin (Fig. 1). Damage was reduced substantially and significantly, almost reaching zero in all three areas studied. Similarly the i.p. injection of 7-NI significantly reduced the toxicity of kainate to near control levels (Fig. 2) for the CA1, CA2 and CA3a regions. Examples of the histological appearance of the CA1 and CA3 areas are illustrated in Fig. 3. Nitric oxide has been shown both to attenuate or exacerbate excitotoxic damage. Kainate, via an interaction with the kainate receptor, stimulates NO production. This release may be further enhanced by a similar reported action for the NMDA receptor [10,11]. This neuronal NO production can be inhibited by the addition of nitric oxide synthase-inhibitors. In this study two NOS inhibitors were administered systemically 10 min prior to the injection of kainate at a dose known to produce sub-total damage to the hippo-
P.A. Jones et al. / Neuroscience Letters 249 (1998) 75–78
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Fig. 3. Photomicrographs of the hippocampus showing damage and protection. (a,c,e) CA1 region. (b,d,f) CA3 area. (a,b) Control and salinetreated animals, showing a field of clear, well-defined neuronal somata with small nuclei. (c,d) Damage induced by kainic acid, with general disruption, fewer normal cells, and the presence of dead or dying cells with intensely staining, pyknotic nuclei. (e,f) Protection afforded by 7nitroindazole is shown, with cells of apparently normal appearance and no obvious sign of damage. Scale bar, 25 mm.
campus. l-NAME is a general NOS inhibitor, while 7-NI is specific for the neuronal subtype (i.e. does not prevent the actions of epithelial NOS which include cerebral vessel dilatation [11]). In both cases marked protection against kainate-induced excitotoxicity was observed. This suggests that the effects mediated by NO are more harmful than protective in this model, which is in agreement with a number of other studies [7,16,21]. l-NAME at 20 mg/kg inhibits almost 50% of NOS activity. These experiments showed protection at a quarter of this dose, suggesting that even limiting a small proportion of NO production is highly protective. Reports that l-NAME did not protect against kainate used intracerebral injections [2], a route which may not have affected as large a volume of tissue as our systemic application. As free radical-associated cell death is considered important in excitotoxicity, the prevention of the conversion of NO to toxic metabolites (ONOO- and •OH), may represent the most important mechanism by which NOS inhibitors protect against cerebral insults. As well as decreasing free radical production, NOS inhibition by l-NAME has been shown to decrease the extracellular glutamate concentration during seizures after a systemic injection of 10 mg/kg kainate [24]. This was presumably due to the prevention of the presynaptic augmentation of glutamate release by postsy-
naptically released NO [22,17] and the inhibition of glutamate uptake [23]. The dose of NOS inhibitors and the relative amounts of inhibitors and excitotoxin appears to be important in determining the outcome for neurones compromised by NMDA [25]. Low doses of NOS inhibitors exert a protection against cerebral insults, which is not seen when the inhibitors are administered at higher doses [27]. While the dual role of NO on glutamate release may account for this effect, this is unlikely, as low doses of l-NAME enhance NMDA convulsive activity [5]. Low concentrations of the NOS inhibitors have also been shown to increase extracellular glutamate levels in vivo (and enhance NMDA-mediated release), but at a higher concentration this effect was no longer observed [28]. As modulation of glutamate does not appear to correlate with damage, it is unlikely to be a contributory factor to excitotoxic insults. The increase in cellular damage by high doses of NOS inhibitors may be due to inhibition of blood vessel dilatation, which occurs in association with increased neuronal metabolic activity or hypoxia. In conclusion, this study revealed protection against kainate excitotoxicity with a low dose of l-NAME or 7-NI. As both alleviated damage to a similar degree, neuronally-produced nitric oxide may contribute to the brain damage induced by systemically administered kainic acid.
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P.A.J. was funded by a University of Glasgow Postgraduate Scholarship. [1] Akira, T., Henry, D. and Wasterlain, C.G., Nitric oxide mediates the sustained opening of NMDA receptor-gated ionic channels which follows transient excitotoxic exposure in hippocampal slices, Brain Res., 652 (1994) 190–194. [2] Bagetta, G., Ianonne, M., Palma, E., Rodino, P., Granato, T. and Nistico, G., Lack of involvement of nitric oxide in the mechanism of seizures and hippocampal damage produced by kainic acid and ouabain in rats, Neurodegeneration, 4 (1995) 43–49. [3] Brown, A.W. and Brierley, J.B., Anoxic ischaemic cell change in the rat brain. Light microscope and fine structural observations, J. Neurol. Sci., 16 (1972) 15–84. [4] Buchan, A.M., Gertler, S.Z., Huang, Z.-G., Chaundy, K.E. and Xue, D., Failure to prevent selective CA1 neuronal death and reduce cortical infarction following cerebral ischaemia with inhibition of nitric oxide synthase, Neuroscience, 61 (1994) 1–11. [5] Buisson, A., Lakhmeche, N., Verrecchia, C., Plotkine, M. and Boulu, R.G., Nitric oxide: an endogenous anticonvulsant substance, NeuroReport, 4 (1993) 444–446. [6] Connop, B.P., Rolfe, N.G., Boegman, R.J., Jhamandas, K. and Beninger, R.J., Potentiation of NMDA-mediated toxicity on nigrostriatal neurons by a low dose of 7-nitro indazole, Neuropharmacology, 33 (1994) 1439–1445. [7] Dalkara, T., Yoshida, T., Irikura, K. and Moskowitz, M.A., Dual role of nitric oxide in focal ischaemia, Neuropharmacology, 33 (1994) 1447–1452. [8] Dawson, D.A., Graham, D.I., McCulloch, J. and MacRae, I.M., Anti-ischaemic efficacy of a nitric oxide synthase inhibitor and a N-methyl-D-aspartate receptor antagonist in models of transient and permanent focal cerebral ischaemia, Br. J. Pharmacol., 113 (1994) 247–253. [9] Drapier, J.-C., Hirling, H., Wietzerbin, J., Kaldy, P. and Kuhn, L.C., Biosynthesis of nitric oxide activates ion regulatory factor in macrophages, EMBO J., 12 (1994) 3643–3649. [10] East, S.J. and Garthwaite, J., NMDA receptor activation in rat hippocampus induces cyclic GMP formation through the L-arginine-nitric oxide pathway, Neurosci. Lett., 123 (1991) 17–19. [11] Faraci, F.M. and Brian, J.E., 7-Nitroindazole inhibits brain nitric oxide synthase and cerebral vasodilatation in response to Nmethyl-D-aspartate, Stroke, 26 (1995) 2172–2176. [12] Gross, P.M., Weaver, D.F., Bowers, R.J., Nag, S., Ho, L.T., Pang, J.J. and Espinosa, F.J., Neurotoxicity in conscious rats following intraventricular SNAP, a nitric oxide donor, Neuropharmacology, 33 (1994) 915–927. [13] Haberny, K.A., Pou, S. and Eccles, C.U., Potentiation of quinolinate-induced hippocampal lesions by inhibition of NO synthesis, Neurosci. Lett., 146 (1992) 187–190. [14] Izumi, Y., Benz, A.M., Clifford, D.B. and Zorumski, C.F., Nitric oxide inhibitors attenuate ischemic degeneration in the CA1 region of rat hippocampal slices, Neurosci. Lett., 210 (1996) 157–160. [15] Kohno, K., Ohta, S., Furuta, S., Kohno, K., Kumon, Y. and Sakaki, S., Intraventricular administration of nitric oxide
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Nitric oxide synthase inhibitors L-NAME and 7-nitroindazole protect rat hippocampus against kainate-induced excitotoxicity P.A. Jones a,, R.A. Smith b, T.W. Stone c ,* a
Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK b Laboratory of Human Anatomy, University of Glasgow, Glasgow G12 8QQ, UK c Division of Neuroscience and Biomedical Systems, West Medical Building, University of Glasgow, Glasgow G12 8QQ, UK Received 9 February 1998; received in revised form 21 April 1998; accepted 27 April 1998
Abstract The role of nitric oxide in cerebral insults remains controversial. While numerous studies have used models of ischaemia and hypoxia, few have examined nitric oxide in the kainate model of excitotoxicity. Kainate (10 mg/kg) was administered to rats via the intraperitoneal (i.p.) route to induce submaximal damage to the CA1, CA2 and CA3a regions of the hippocampus after 7 days. Systemic injections of the nitric oxide synthase (NOS) inhibitors NG-nitro-L-arginine methyl ester (L-NAME) and 7-nitroindazole (7-NI), both at a dose of 5 mg/kg, reduced cell death in all three regions. As 7-NI selectively inhibits the neuronal form of NOS, this study suggests that nitric oxide produced from a neuronal and not epithelial source may contribute to neuronal damage in this model. 1998 Elsevier Science Ireland Ltd. All rights reserved
Keywords: Kainate; Excitotoxicity; Hippocampus; 7-Nitroindazole; NG-nitro-L-arginine methyl ester; Nitric oxide synthase
Nitric oxide (NO) has been viewed as a possible mediator of glutamate-induced neurotoxicity due both to an increase in NO levels after excitotoxicity and ischaemia, and to an apparent upregulation of neuronal nitric oxide synthase (nNOS) and nNOS mRNA during ischaemia. The excitotoxic/ischaemic-associated calcium influx activates both constitutive forms of NOS, elevating the levels of NO. Nitric oxide itself can increase cellular calcium influx by opening the N-methyl-d-aspartate (NMDA)-associated calcium channel [1], or increase the availability of excitatory amino acids by stimulating the release of both glutamate and aspartate and inhibiting glutamate uptake [17]. Nitric oxide has effects on iron metabolism (e.g. activation of iron regulatory factor [9]), which may be significant in the formation of free radicals via the Fenton reaction. A further possible indirect route by which NO may cause damage is by the depletion of the toxic molecule scavenger glutathione, through the formation of intracellular S-nitrosoglu* Corresponding author. 1 Present address: Department of Pharmacology, 1 George Square, University of Edinburgh, Edinburgh EH8 9JZ, UK.
tathione, although Wink et al. reported that NO itself is a free radical scavenger [30]. All this evidence is consistent with studies which indicate that NO itself is neurotoxic [12,18], and that NOS inhibition or deletion can reduce damage produced by ischaemia [8,14,15,20,29]. Some of these data however, have not proved reproducible by others [4]. Against this background the role of NO in excitotoxicity remains controversial. Inhibition of NOS has been claimed to reduce the excitotoxic effect of NMDA receptor activation by NMDA or quinolinic acid [16,26], but not the effects of kainic acid [24]. Conversely there are several reports that NOS inhibition has no effect or can enhance neurotoxicity via NMDA receptors [6,13,19] and can increase kainate-induced seizures. We have now used both a general NOS inhibitor NG-nitro-l-arginine methyl ester (l-NAME) and a competitive selective inhibitor of the neuronal NOS isoform (7nitroindazole; 7-NI) to investigate the role of NO in the kainate model of excitotoxicity. Male Wistar rats weighing 240–310 g, housed under standard conditions, were used in all experiments. All injections were made by the intraperitoneal (i.p.) route in a
0304-3940/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00372- 3
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volume no greater than 3 ml/kg. Kainate and l-NAME were dissolved in saline, 7-NI in a 30% solution of Tween 80 in saline to provide solutions of the correct concentration for injection. The drugs were all injected 10 min prior to the injection of kainate, which was administered at a standard dose of 10 mg/kg. This dose was selected since our previous studies indicated that it produced excitotoxicity which is clearly submaximal in most animals. Following the injection of kainate, the animals were left for 7 days under standard conditions. Rats were anaesthetized with sodium pentobarbitone 60 mg/kg after 7 days. The method of perfusion first described by Brown and Brierley [3] was used, in which 50 ml of physiological medium with 12.5% xylocaine was perfused through a cannula placed into the left ventricle before fixation by 250 ml of FAM (40% formaldehyde/glacial acetic acid/methanol, in the ratio 1:1:8 v/v). The animals were decapitated and the heads stored in fixative for up to a week, at 4°C. The brain was subsequently removed and the left hemisphere marked with Indian ink. The cerebral hemispheres were cut into 2 mm thick slices, dehydrated and infused with paraffin wax throughout before embedding in wax. Sections of 6 mm were cut, mounted on slides and stained with haematoxylin and eosin. Sections were examined under a light microscope by observers who were unaware of the drug treatment received. The left (marked) hippocampus was examined and areas CA1, CA2, CA3a CA3b and CA4 scored for damage. The scale used was a percentage scale, where 0% equated to no damage while 100% indicated a total loss of neurones. The damage was estimated by a cell count of viable (blue stained) cells versus pink stained compromised
Fig. 1. Protection against kainate-induced excitotoxicity by the NOS inhibitor L-NAME in the rat hippocampus. ***P , 0.001 versus kainate alone; + + + P , 0.001 versus saline control.
Fig. 2. The selective nNOS inhibitor 7-NI protected the regions of the hippocampus damaged by kainic acid. ***P , 0.001 versus kainate alone; + + + P , 0.001 versus saline control.
neurones. Account was also taken of decreased neuronal number and general appearance of the hippocampal subareas. Analysis of variance (ANOVA) followed by post-tests was used to determine any statistical significance. Significance refers to results where P , 0.05 was obtained. The administration of 10 mg/kg kainate resulted in neuronal damage to the CA1, CA2 and CA3a regions of the hippocampus at levels significantly different from control values (where no damaged was observed). Damage was greatest in the CA1 (56.7 ± 9.6%) and CA3a regions (51.1 ± 9.0%), with a lower amount in the CA2 region (Fig. 1). No other hippocampal region was significantly damaged. The introduction of 5 mg/g l-NAME 10 min prior to kainate resulted in protection in all three regions damaged by the excitotoxin (Fig. 1). Damage was reduced substantially and significantly, almost reaching zero in all three areas studied. Similarly the i.p. injection of 7-NI significantly reduced the toxicity of kainate to near control levels (Fig. 2) for the CA1, CA2 and CA3a regions. Examples of the histological appearance of the CA1 and CA3 areas are illustrated in Fig. 3. Nitric oxide has been shown both to attenuate or exacerbate excitotoxic damage. Kainate, via an interaction with the kainate receptor, stimulates NO production. This release may be further enhanced by a similar reported action for the NMDA receptor [10,11]. This neuronal NO production can be inhibited by the addition of nitric oxide synthase-inhibitors. In this study two NOS inhibitors were administered systemically 10 min prior to the injection of kainate at a dose known to produce sub-total damage to the hippo-
P.A. Jones et al. / Neuroscience Letters 249 (1998) 75–78
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Fig. 3. Photomicrographs of the hippocampus showing damage and protection. (a,c,e) CA1 region. (b,d,f) CA3 area. (a,b) Control and salinetreated animals, showing a field of clear, well-defined neuronal somata with small nuclei. (c,d) Damage induced by kainic acid, with general disruption, fewer normal cells, and the presence of dead or dying cells with intensely staining, pyknotic nuclei. (e,f) Protection afforded by 7nitroindazole is shown, with cells of apparently normal appearance and no obvious sign of damage. Scale bar, 25 mm.
campus. l-NAME is a general NOS inhibitor, while 7-NI is specific for the neuronal subtype (i.e. does not prevent the actions of epithelial NOS which include cerebral vessel dilatation [11]). In both cases marked protection against kainate-induced excitotoxicity was observed. This suggests that the effects mediated by NO are more harmful than protective in this model, which is in agreement with a number of other studies [7,16,21]. l-NAME at 20 mg/kg inhibits almost 50% of NOS activity. These experiments showed protection at a quarter of this dose, suggesting that even limiting a small proportion of NO production is highly protective. Reports that l-NAME did not protect against kainate used intracerebral injections [2], a route which may not have affected as large a volume of tissue as our systemic application. As free radical-associated cell death is considered important in excitotoxicity, the prevention of the conversion of NO to toxic metabolites (ONOO- and •OH), may represent the most important mechanism by which NOS inhibitors protect against cerebral insults. As well as decreasing free radical production, NOS inhibition by l-NAME has been shown to decrease the extracellular glutamate concentration during seizures after a systemic injection of 10 mg/kg kainate [24]. This was presumably due to the prevention of the presynaptic augmentation of glutamate release by postsy-
naptically released NO [22,17] and the inhibition of glutamate uptake [23]. The dose of NOS inhibitors and the relative amounts of inhibitors and excitotoxin appears to be important in determining the outcome for neurones compromised by NMDA [25]. Low doses of NOS inhibitors exert a protection against cerebral insults, which is not seen when the inhibitors are administered at higher doses [27]. While the dual role of NO on glutamate release may account for this effect, this is unlikely, as low doses of l-NAME enhance NMDA convulsive activity [5]. Low concentrations of the NOS inhibitors have also been shown to increase extracellular glutamate levels in vivo (and enhance NMDA-mediated release), but at a higher concentration this effect was no longer observed [28]. As modulation of glutamate does not appear to correlate with damage, it is unlikely to be a contributory factor to excitotoxic insults. The increase in cellular damage by high doses of NOS inhibitors may be due to inhibition of blood vessel dilatation, which occurs in association with increased neuronal metabolic activity or hypoxia. In conclusion, this study revealed protection against kainate excitotoxicity with a low dose of l-NAME or 7-NI. As both alleviated damage to a similar degree, neuronally-produced nitric oxide may contribute to the brain damage induced by systemically administered kainic acid.
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