7-NITROINDAZOLE , A SELECTIVE NEURONAL NITRIC OXIDE SYNTHASE INHIBITOR IN VIVO , PREVENTS KAINATE-INDUCED INTRAHIPPOCAMPAL NEUROTOXICITY

We investigated the effects of 7-nitroindazole (7-NI), a selective neuronal nitric oxide synthase inhibitor in vivo, on nitrite concentration after kainic acid injection unilaterally into the CA3 region of the rat hippocampus. The accumulation of nitrite, the stable metabolite of NO, was measured by the Griess reaction at different times in hippocampus, forebrain cortex, striatum, and cerebellum homogenates. 7-nitroindazole can effectively inhibit NO synthesis in rat brain after kainate-induced neurotoxicity and suppressed nitrite accumulation. The present results suggest that neuronal NO synthase inhibitors may be useful in the treatment of neurological diseases in which excitotoxic mechanisms play a role. UDC 612.815:599.323.4.


INTRODUCTION
Kainate (KA), a pyrrolidine excitotoxin isolated from the seaweed Digenea simplex, is a potent neuroexcitatory drug acting on glutamate receptors that after intracerebral or systematic injection leads to generalized limbic seizures in rats (Albin et al. 1992;Lipton et al. 1994).Glutamate receptors are the primary excitatory neurotransmitter receptors in the vertebrate brain and are of critical importance to a wide variety of neurological processes (Wo et al. 1994).Kainate-induced seizures are accompanied by severe neuronal damage predominantly in the hippocampus and amygdala/pyriform cortex (Hollmann et al. 1994).Current models of KA toxicity support the hypothesis that the main cause of neurotoxicity is the activation of presynaptic KA receptors and the release of endogenous glutamate.The overstimulation of glutamate receptors has been implicated in the mediation of injury caused by neurotoxins and ischemiarelated insults.Further, KA is also thought to mediate damage partly through an indirect mechanism, which may involve the overproduction of reactive forms of oxygen.The generation of free radicals appears to be pivotal in KA neurotoxicity (Gratacos et al. 2001).
Stimulation of glutamate KA receptors induces neuronal nitric oxide (NO) release, which in turn modulates glutamate transmission (Alabadi et al. 1999;Nakaki et al. 2000).Nitric oxide is a highly reactive signal molecule in the CNS.The agent is a gaseous chemical messenger that acts on interneuronal communications, synaptic plasticity, memory formation, receptor function, intracellular signal transmission, and mediator release (Brown,1999;Heales et al. 1999;Lei et al. 1999).However, pathological conditions may occur when higher fluxes of these mediators are generated, such as during the process referred to as excitotoxicity, i.e., the excessive activation of glutamate KA receptors.This is a condition common to both acute and chronic neurological diseases (Sengpiel et al. 1998;Brorson et al. 1999;Ciriolo et al. 2001).
In view of the above, the present study was undertaken to examine whether the production of NO after intracerebral KA injections can be modulated by pretreatment by 7-nitroindazole (7-NI), a selective neuronal nitric oxi-

Animals
Adult rats of the Wistar strain (Rattus norvegicus) of both sexes, with body weight 200 ± 30 g, were used for experiments.Groups of two or three rats per cage (Erath, FRG), were housed in an air-conditioned room at temperature of 23 ± 2 0 C with 55 ±10% humidity and with lights on 12 h/day (07.00-19.00).The animals were given a commercial rat diet and tap water ad libitum.These animals were anesthetized by giving intraperitoneal injections of pentobarbital sodium (0.0405 g/kg b.w.) and were placed in a stereotaxic frame.

Experimental procedure and intracerebral injection of drugs
The rats were divided into three basic groups (according to drug treatment), each basic group consisting of five different subgroups (according to survival times) and each subgroup consisting of eight animals.The first group received a unilateral KA injection (Sigma Chemical Co., U.S.A., 0.5 mg/ml, dissolved in 0.1 M saline, pH 7.2; 1 µL total volume) into the CA3 region of the hippocampus (coordinates from bregma: anteroposterior: -3.3 mm, dorsoventral: 3.2 mm, and lateral: 3.0 mm) using a Hamilton microsyringe with a beveled tip.The second group received KA and 7-NI (Sigma Chemical Co., U.S.A., 0.5 mg/ml, dissolved in purified olive oil, pH 7.2; 1 µL).Finally, the third group received the same volume (1 µ L), but only saline solution, and served as a control (sham-operated).The animals were allowed to survive from 5 min to seven days (5 min, 15 min, 2 h, 48 h, and 7 days).All animals were anesthetized, decapitated, and the brains immediately removed.The ipsi-and contralateral hippocampus, forebrain cortex, striatum, and cerebellum from individual animals were quickly isolated and homogenized in ice-cold buffer containing 0.25 M sucrose, 0.1 mM EDTA, and 50 mM K-Na phosphate buffer, pH 7.2.Homogenates were centrifuged twice at 1580g for 15 min at 4 0 C. The supernatant obtained by this procedure was then frozen and stored at -70 0 C.

Nitrite measurement
Nitrite and nitrate in biological material are increas-ingly being used as markers of nitric oxide production.We detected nitrite in the rat brain homogenates by the Griess method (Guevara et al. 1998).Nitric oxide production was quantified by measuring nitrite, a stable oxidation end product of NO (Green et al. 1982).Briefly, nitrite production was determined by mixing 50 µL of the assay buffer with 50 µL of Griess reagent (1.5 % sulfanilamide in 1M HCl plus 0.15 % N-(1naphthyl)ethylenediamine dihydrochloride in distilled water, v:v).After 10 min of incubation at room temperature, the absorbance at 540 nm was determined and nitrite concentrations were calculated from the sodium nitrite (Sigma) standard curve.All measurements were performed in triplicate.

Protein measurement
The content of protein in the rat brain homogenates (hippocampus, striatum, forebrain cortex, and cerebellum, ipsilateral and contralateral) was measured by the method of Lowry et al. (1951) using bovine serum albumin (Sigma) as standard.All measurements were performed in triplicate.

Data presentation and analysis
All experiments were done with n = 8.Each assay was performed at least twice under identical conditions.Data are expressed as means ± SD.The statistical significance of differences between groups was assessed by Student's t-test (paired and unpaired) for individual comparisons and regression analysis for overall significance (with p < 0.05 as significant and p < 0.01 as very significant).

Materials
Chemicals were purchased from Sigma (St. Louis, MO, U.S.A.).Other chemicals were of analytical grade.All drug solutions were prepared on the day of experiment.Animals used for procedures were treated in strict accordance with the NIH Guide for Care and Use of Laboratory Animals (1985).

Nitrite levels in the rat hippocampus
The results presented in Fig. 1 show the nitrite levels (mM/mg proteins) in ipsilateral and contralateral hippo-PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com

R E T R A C T E D
campal homogenates, respectively.Injection of KA resulted in generally higher levels of nitrite production at all tested times with statistically significant difference (according to the Student t-test; p<0.05).There was no statistically significant difference between mean nitrite levels obtained from each hemisphere, although the injection site was in the ipsilateral hippocampus.Treatment with 7-NI followed by KA, very clearly inhibited nitrite production in this brain structure.The early tested times (at 5 min, 15 min, and 2 h) showed statistically significant differences (according to the Student t-test; p<0.01) compared with the equivalent control groups (Fig. 1A).The results obtained for the contralateral hippocampus were similar.Measurements at 15 min and 2 h showed statistically significant differences (p<0.01)compared with the equivalent control groups (Fig. 1B).

Nitrite levels in the rat forebrain cortex
Intrahippocampal KA injection resulted in generally higher levels of nitrite production with statistically significant difference, p<0.05 at all tested times (Fig. 2).There was a significant reduction in nitrite levels after KA + 7-NI treatment at all tested times, especially at 5 min, 15 min, and 2 h in the ipsilateral and in the contralateral side of the brain.There was no statistically significant difference between mean nitrite levels obtained from each hemisphere.

Nitrite levels in the rat striatum
The striatum, the main component of the basal ganglia, receives glutamatergic inputs from the cortex and thalamus and considerable attention has therefore been given to the role of excitotoxicity in striatal disorders.The effect of intrahippocampal drug injection on nitrite production in the striatum is shown in Fig. 3.The effect of KA injection on nitrite levels measured at 5 min, 15 min, and 48 h and after 7 days for the ipsilateral and for the contralateral side was a significant increase, p<0.05.Treatment with KA + 7-NI at all tested times showed a significant reduction in nitrite levels, especially at 5 min and 2 h for the ipsi-and contralateral sides, respectively  (Fig. 3).There was no statistically significant difference between mean nitrite levels obtained from each hemisphere.

Nitrite levels in the rat cerebellum
The results obtained for this brain structure were very similar to those for the striatum (Fig. 4).The effect of KA injection on nitrite levels measured for the ipsilateral and the contralateral side at almost all tested times showed a significant increase, p<0.05.There was a significant reduction in nitrite levels after KA + 7-NI treatment at all tested times, especially at 5 min and 2 h ipsilaterally, and at 5 min, 15 min, 2 h, and 48 h contralaterally (Fig. 4B).There was no statistically significant difference between mean nitrite levels obtained from each hemisphere.

The behavioral changes after kainate injection
The purpose of this study was to investigate fine changes in NO levels during the process of excitotoxicity in various brain parts.Our aim was to inject KA (appropriate dose) but to avoid any behavioral changes ("wet dog shake", focal seizure of the limbs and neck, hypersalivation, or generalized convulsion) and typical limbic seizures evolving into status epilepticus, since during status epilepticus hippocampal blood flow, oxygen supply, and body temperatures are modified.These effects are accompanied by severe damage to all subfields of the hippocampal formation.It is a condition of intense metabolic activation and could interfere with our results and measurements.We did not measure epileptic activity by electroencephalogram.Only normal behavioral animals took place in experiments.

DISCUSSION
The role of NO in cerebral insult remains controversial.While numerous studies have used models of ischemia, hypoxia, and status epilepticus, few have examined NO in the KA model of excitotoxicity.Anim-  PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com

R E T R A C T E D
als exposed to KA-induced status epilepticus display a striking pattern of selective neuronal vulnerability in the hippocampus.Neurons in the hilus/CA3 and CA1 subfields appear particularly sensitive, whereas dendate gyrus granule cells are resistant (Becer et al. 1999;Lere etal. 2002), which is likely due to the high concentration of KA receptors on their membranes.Regional distribution of KA receptors of the rat brain was found to be highest in deep layers (layer 5) of the forebrain cortex, cerebellar granule cell layer, and caudate putamen (Carroll et al.1998;Bailey et al. 2001), which is why we tested these particular brain regions: hippocampus, forebrain cortex, striatum, and cerebellum.
It was previously known (Montecot et al. 1998) that during status epilepticus 7-NI significantly reduced the increase in hippocampal blood flow and prevented an increase in the tissue partial pressure of oxygen.Also, seven days later, hippocampal damage in the CA1 and CA3 layers was significantly less in 7-NI-treated rats than in vehicle-treated rats.The authors concluded that the inhibition of nNOS by 7-NI protects neurons from seizure-induced toxicity despite reducing blood flow and oxygen supply to the hippocampus.Kainate enhances hippocampal NO generation (Kashihara et al. 1998) and KA injection results in differential regulation of nNOS mRNA and NO formation in the rat hippocampus (Kashihara et al. 2000).The literature results implicate neuronal NO generation in the pathogenesis of both direct and secondary excitotoxic neuronal injuries in vivo.As such they suggest that nNOS inhibitors may be useful in the treatment of neurological diseases in which excitotoxic mechanisms play a role.Type nNOS has been detected in the cerebellum, the hypothalamus, the striatum, the hippocampus, and the medula oblongata (Torreilles et al. 1999).
In the present study, an appropriate dose of KA (0.5 mg/ml) was used to cause small brain damage in the ipsilateral, but not contralateral, hippocampus, with no behavioral or epileptic effects.It has been previously shown that NO formation was determined in different regions of the rat brain during KA-induced seizures (Mulsch et al. 1994;Yasuda et al. 2001).In our experiments, at various times following intrahippocampal KA injection, nitrite levels were measured in the four rat brain structures.Cortical areas are known to contain the highest packing densities of nNOS-positive interneurones such as the pyriform and entorhinal cortices (Bid-mon et al. 1999), indicating that, in normal animals, neurotransmission and probably cognitive information processing would be affected by the pharmacological modulation of NO production.
We have shown that NO end-product levels in the rat brain increased immediately after KA injection and continued to increase gradually throughout the experiments.Under conditions of normal behavior in the rat, the damage was localized mainly in the CA3 region of hippocampus, where neuronal loss occurred.7-nitroindazole, a selective nNOS inhibitor in vivo, at any dose used did not affect basal nitrite levels before intracerebral KA injections.
In addition, pretreatment with 7-NI was effective in modulating the production of NO following intrahippocampal KA injection.Under the conditions of this experiment, 7-NI produced a rapid (within 2 h) decrease in nitrite levels in all four brain regions.Previously, it was demonstrated that 7-NI can produce designated changes in brain NO content, facilitating the use of 7-NI to probe the pharmacological implications of NO in the CNS (Bush et al. 2001).It was suggested that excessive production of NO is involved in the mechanisms of KAtriggering seizures and neurodegeneration (Bagetta et al. 1995).The results of Takei et al. (1999;2001) suggest that NO is of major importance in the neurodestructive process in spite of its roles in maintaining both the cerebral blood flow and cerebral oxygenation during KAinduced seizures in the neonatal rabbit brain.They suggested that both 7-NI and L-NAME inhibited NO production and EEG abnormalities during the seizures that led to less neuronal damage to the hippocampus.
The study presented here shows that after intrahippocampal injection 7-NI produced an approximately 50% decrease in nitrite levels in all four brain regions, which was sustained.Correlation of the inhibitory effect was demonstrated in all tested brain structures, of which the striatum was shown to be most sensitive and the forebrain cortex most resistant to 7-NI activity.Decreased NO activity in selected areas of the brain suggests that treatment with 7-NI leads to protection of brain neurons against neuronal injuries by impairment of cellular energy metabolism and oxidative stress (Strorch et al. 2000).
The present data indicate that inhibition of nNOS by 7-NI aseptically decreased NO production, at the early PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com

R E T R A C
T E D tested times (from 5 min up to 2 h) in the rat brain after intracerebral KA application.These findings help to explain the equally efficient effect of 7-NI in all tested brain structures in suppressing nitrite accumulation.Also, the results suggest that extremely fine regulation of NO levels in the different neural cell types can modulate excitotoxicity.The NOS inhibitory effect of 7-NI following intracerebral injection should be taken into account when using this drug to evaluate NO central effects.
Finally, increased NO production in distinct brain regions, functionally connected via afferents and efferents suggests that these regions are affected by the injury.It suggests that 7-NI inhibition of nNOS protects the cells in these regions from KA-induced damage and thereforemay limit the retrograde and anterograde spread of neurotoxicity.Mulsch, A., Busse, R., Mordvintcev, P. I., Vanin, A. F., Nielsen, E. O., Scheel-Kruger, J., Olesen, S. F. (1994).Nitric oxide promotes seizure activity in kainate-treated rats.Neuroreport 5, 2325-2328.