Halina Baran

The role of brain edema in epileptic brain damage induced by systemic kainic acid injection.

Edema formation and blood-brain barrier permeability was studied in animals with epileptic seizures induced by subcutaneous injection of kainic acid. Brain edema was most pronounced between 3 and 24 h after kainic acid injection. It was reflected by massive swelling of perineuronal and perivascular astroglia. Three hours after kainic acid perivascular astroglia swelling resulted in disturbance of local microcirculation in the affected brain areas. In addition, compression of drainage veins by the edematous brain induced focal perivenous hemorrhages similar to herniation damage in human brain edema. Tracer studies with sodium fluorescein, Evans blue, albumin and horseradish peroxidase revealed only a mild increase in the permeability of cerebral vessels, topographically unrelated to areas of brain edema. This finding indicates the presence of cytotoxic brain edema in kainic acid-induced epileptic brain damage. Treatment of brain edema with dexamethasone did not influence the incidence and severity of kainic acid-induced epileptic brain damage. However, in 54% of animals injected with kainic acid, lesions were completely prevented by treatment of brain edema with mannitol. The present results indicate that brain edema plays an important role in the pathogenesis of epileptic brain damage following systemic kainic acid intoxication. It is suggested that in this model of limbic epilepsy the brain edema is due to the massive ionic imbalance elicited in the affected brain regions by the kainic acid-induced persistent neuronal excitation.

The cyclooxygenase and lipoxygenase inhibitor BW755C protects rats against kainic acid-induced seizures and neurotoxicity

In this study the effect of the anti-inflammatory drugs indomethacin, ibuprofen, ebselen (PZ 51, 2-phenyl-1,2-benzoisoselenazol-3(2H)-one), and BW755C (3-amino-1-(m-(trifluoromethyl-phenyl)-2-pyrazoline) on kainic acid (KA)-induced behavioral and neurochemical changes in rats was investigated. Rats injected with KA (10 mg/kg s.c.) developed seizure activity with a 20% mortality within the first 4 h and neuronal degeneration in the limbic system after 3 days. Pretreatment with the cyclooxygenase inhibitor indomethacin (10 mg/kg i.p.) augmented KA-induced epileptic activity and increased the mortality in status epilepticus to 80%. Another cyclooxygenase inhibitor, ibuprofen (20 mg/kg i.p.), and the lipoxygenase inhibitor ebselen (20 mg/kg i.p.) showed no effect on KA-induced symptoms and neurochemical changes. Application of the cyclooxygenase/lipoxygenase inhibitor BW755C (40 mg/kg i.p.) reduced the severity of seizures and protected significantly from irreversible brain lesions induced by KA. The marked reduction of glutamate decarboxylase (GAD; 53.3 +/- 12.2% of control) and choline acetyltransferase (ChAT; 60.9 +/- 9.1% of control) activities in amygdala/pyriform cortex and GAD activity in hippocampus (69.4 +/- 5.6% of control) observed 3 days after KA injection was abolished by BW755C treatment. Histopathological analyses of brain tissue showed that treatment with BW755C prevented the KA-induced nerve cell degeneration, edema, hemorrhages, and tissue necrosis in amygdala/pyriform cortex.(ABSTRACT TRUNCATED AT 250 WORDS)

Anticonvulsant effects of the glycine/NMDA receptor ligands D-cycloserine and D-serine but not R-(+)-HA-966 in amygdala-kindled rats

1 The effects of the glycine/NMDA receptor partial agonists, d‐cycloserine and (+)‐HA‐966 and the full agonist, d‐serine, on focal seizure threshold and behaviour have been determined in amygdala‐kindled rats, i.e. a model of focal (partial) epilepsy. The uncompetitive NMDA receptor antagonist, MK‐801, was used for comparison. 2 The high efficacy glycine partial agonist, d‐cycloserine, did not alter the threshold for induction of amygdaloid afterdischarges (ADT) at doses of 20–80 mg kg−1 i.p., but significant ADT increases were determined after application of higher doses (160 and 320 mg kg−1). The ADT increases after these high doses were long‐lasting; significant elevations were still observed 2 days after drug injection. Determination of d‐cycloserine in plasma and brain tissue showed that it was rapidly eliminated from plasma. Compared to peak levels in plasma, only relatively low concentrations of d‐cycloserine were measured in brain tissue. 3 The low efficacy glycine partial agonist, (+)‐HA‐966, 10–40 mg kg−1 i.p., did not alter the ADT or seizure recordings (seizure severity, seizure duration, afterdischarge duration) at ADT currents. However, the drug dose‐dependently increased the duration of postictal behavioural and electroencephalographic depression in kindled rats. At the higher dose tested, postictal immobilization was dramatically increased from 3 min to about 120 min. This might indicate that glutamatergic activity is decreased postictally, which is potentiated or prolonged by (+)‐HA‐966. 4 Like d‐cycloserine, the glycine receptor full agonist, d‐serine, injected bilaterally into the lateral ventricles at a dose of 5 μmol, significantly increased the ADT, while no effect was seen at a lower dose (2.5 μmol). 5 The anticonvulsant effects observed with d‐cycloserine were completely antagonized by combined treatment with (+)‐HA‐966, indicating that the effects of d‐cycloserine were mediated by the glycine/NMDA receptor complex. 6 MK‐801, 0.1 mg kg−1, did not alter the focal seizure threshold or seizure recordings at ADT current, but induced marked phencyclidine(PCP)‐like behavioural alterations, such as hyperlocomotion, stereotypies and motor impairment. No PCP‐like behaviours were observed after d‐cycloserine, d‐serine or (+)‐HA‐966. High doses of (+)‐HA‐966 induced moderate motor impairment in kindled rats. 7 The long lasting increases in seizure threshold observed after the high efficacy glycine partial agonist, d‐cycloserine but not the low efficacy partial agonist, (+)‐HA‐966, may suggest that the effects of d‐cycloserine are mediated by adaptive changes in the NMDA receptor complex in response to glycine receptor stimulation. 8 Pharmacological intervention at the strychnine‐insensitive glycine receptor by high‐efficacy partial agonists with systemic bioavailability may be an effective means of increasing seizure threshold without concomitantly inducing PCP‐like adverse effects.

Effect of mannitol treatment on brain neurotransmitter markers in kainic acid-induced epilepsy.

The effect of mannitol treatment on the behavioural, morphological and neurochemical brain damage induced after subcutaneously applied kainic acid (10 mg/kg) was studied in the rat. Mannitol at a dose of 1.5 g/kg was injected intravenously 10 min, 1.5 h and 3 h respectively after kainic acid administration. A protective effect of mannitol was observed only when mannitol was given 1.5 h after kainic acid application, i.e. within the early phase of kainic acid-induced brain oedema development. At this time period, mannitol prevented the development of kainic acid-induced seizures as well as irreversible brain lesions and neurochemical changes, the latter being reduction of noradrenaline levels in amygdala/pyriform cortex measured 3 h, and reduction of glutamate decarboxylase and choline acetyltransferase activities measured 3 days after kainic acid treatment. Similarly loss of glutamate decarboxylase activity in dorsal hippocampus induced by kainic acid was prevented by mannitol treatment. It is concluded that by washing out brain oedema, mannitol treatment may prevent propagation of seizures and brain damage in the kainic acid model of epilepsy.

Age-Related Increase of Kynurenic Acid in Human Cerebrospinal Fluid – IgG and β2-Microglobulin Changes

Kynurenic acid (KYNA) is an endogenous metabolite in the kynurenine pathway of tryptophan degradation and is an antagonist at the glycine site of the N-methyl-D-aspartate as well as at the alpha 7 nicotinic cholinergic receptors. In the brain tissue KYNA is synthesised from L-kynurenine by kynurenine aminotransferases (KAT) I and II. A host of immune mediators influence tryptophan degradation. In the present study, the levels of KYNA in cerebrospinal fluid (CSF) and serum in a group of human subjects aged between 25 and 74 years were determined by using a high performance liquid chromatography method. In CSF and serum KAT I and II activities were investigated by radioenzymatic assay, and the levels of β2-microglobulin, a marker for cellular immune activation, were determined by ELISA. The correlations between neurochemical and biological parameters were evaluated. Two subject groups with significantly different ages, i.e. <50 years and >50 years, p < 0.001, showed statistically significantly different CSF KYNA levels, i.e. 2.84 ± 0.16 fmol/µl vs. 4.09 ± 0.14 fmol/µl, p < 0.001, respectively; but this difference was not seen in serum samples. Interestingly, KYNA is synthesised in CSF principally by KAT I and not KAT II, however no relationship was found between enzyme activity and ageing. A positive relationship between CSF KYNA levels and age of subjects indicates a 95% probability of elevated CSF KYNA with ageing (R = 0.6639, p = 0.0001). KYNA levels significantly correlated with IgG and β2-microglobulin levels (R = 0.5244, p = 0.0049; R = 0.4253, p = 0.043, respectively). No correlation was found between other biological parameters in CSF or serum. In summary, a positive relationship between the CSF KYNA level and ageing was found, and the data would suggest age-dependent increase of kynurenine metabolism in the CNS. An enhancement of CSF IgG and β2-microglobulin levels would suggest an activation of the immune system during ageing. Increased KYNA metabolism may be involved in the hypofunction of the glutamatergic and/or nicotinic cholinergic neurotransmission in the ageing CNS.

Effect of the glycine/NMDA receptor partial agonist, D-cycloserine, on seizure threshold and some pharmacodynamic effects of MK-801 in mice

Acute treatment of mice with D-cycloserine (a high efficacy, partial agonist at strychnine-insensitive glycine receptors) resulted in dose- and time-dependent increases in the threshold for electrically induced tonic seizures. This anticonvulsant effect was observed at doses which did not induce motor impairment, as determined by the rotarod test. Despite the relatively high intrinsic efficacy of D-cycloserine at glycine receptors, this drug did not produce proconvulsant effects in mice at any of the doses (5-320 mg/kg) or time points examined. Prolonged treatment with D-cycloserine led to a reduction of its anticonvulsant effect. Similar to D-cycloserine, the uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801 (dizocilpine), dose dependently increased the electroconvulsive threshold. Combined treatment with MK-801 and D-cycloserine led to significant anticonvulsant effects, but these effects were simply additive and not synergistic. In contrast to anticonvulsant activity, the motor impairing effect of MK-801 was markedly potentiated by D-cycloserine. The data substantiate that high efficacy glycine/NMDA receptor partial agonists such as D-cycloserine exert anticonvulsant activity at non-toxic doses. The finding that motor impairing but not anticonvulsant effects of MK-801 were potentiated by D-cycloserine suggests that different pharmacodynamic actions of NMDA receptor antagonists are differentially modulated by the glycine receptor, which could be related to the regional heterogeneity of the NMDA receptor complex in the brain.

α2-adrenoceptors modulate kainic acid-induced limbic seizures

We have tested several compounds interfering with the brain monoamine (noradrenaline, dopamine, serotonin) and acetylcholine systems for their effects on limbic seizures produced by systemically (s.c.) injected kainic acid as well as on neurochemical changes in amygdala/pyriform cortex resulting from the kainic acid treatment. The characteristic neurochemical changes induced by s.c. kainic acid were a decrease in noradrenaline and an increase in 5-hydroxyindoleacetic acid in the acute (3 h after kainic acid injection) suggesting strongly increased neurotransmitter turnover in noradrenergic and serotonergic neurons. This was followed by a reduction of glutamic acid decarboxylase and choline acetyltransferase activities during the chronic phase (3 days) of the kainic acid action, indicating destruction of GABAergic and cholinergic neurons. The compounds tested in this model of limbic epilepsy included 1-propranolol, prazosin, clonidine, yohimbine, metergoline, atropine and haloperidol. Among these compounds the alpha 2-adrenergic agonist clonidine (0.1 mg/kg, i.p.) exhibited a powerful protective action on kainic acid-induced limbic seizures as well as on the neurochemical changes in the amygdala and pyriform cortex. In addition, the adrenoceptor antagonists prazosin (alpha 1) and propranolol (beta) as well as the dopamine receptor antagonist haloperidol had significant but less potent - protective actions upon kainic acid-induced seizures and subsequent neurochemical changes. On the other hand, yohimbine (alpha 2-antagonist) and metergoline (serotonin-antagonist) potentiated the limbic seizure syndrome and no effect was found with atropine.(ABSTRACT TRUNCATED AT 250 WORDS)

Systemic administration of kainate induces marked increases of endogenous kynurenic acid in various brain regions and plasma of rats

The endogenous neuroinhibitory and neuroprotective excitatory amino acid receptor antagonist kynurenic acid has been hypothetically linked to the pathogenesis of epilepsy and several other brain disorders. In the present study, alterations in kynurenic acid levels were examined in the kainate model of temporal lobe epilepsy. Kainate was systemically injected in rats at a dose (10 mg/kg s.c.) which induces a characteristic behavioural syndrome with stereotypies and focal (limbic) and generalized seizures, eventually progressing into severe status epilepticus. Kynurenic acid was determined 3 h after kainate injection in various brain regions (olfactory bulb, frontal cortex, piriform cortex, amygdala, hippocampus, nucleus accumbens, caudate/putamen, thalamus, superior and inferior colliculus, pons and medulla, and cerebellar cortex) and in plasma, using a sensitive high-performance liquid chromatographic method. When data were analysed irrespective of individual seizure severity, significant increases in kynurenic acid were determined in all brain regions examined except the hippocampus, nucleus accumbens and pons/medulla. The most marked (200-500%) increases above controls were seen in the piriform cortex, amygdala, and cerebellar cortex. Furthermore, a significant kynurenic acid increase of about 200% above control was determined in plasma. When kynurenic acid levels were determined in subgroups of rats with different behavioural alterations in response to kainate, the most marked kynurenic acid increases were seen in subgroups with status epilepticus. Rats which only developed mild (focal) seizures or stereotyped behaviours (wet dog shakes) also exhibited significantly increased kynurenic acid levels, thus indicating that the increase in kynurenic acid in response to kainate was not solely due to sustained convulsive seizure activity. Whereas it was previously proposed that kynurenic acid is involved only in later stages of seizure disorders, the present data demonstrate that marked increases in central and peripheral kynurenic acid levels occur early after the onset of neuroexcitation, at least in the kainate model.

The glycine/NMDA receptor partial agonist D-cycloserine blocks kainate-induced seizures in rats. Comparison with MK-801 and diazepam

Systemic administration of kainic acid in the rat results in the development of a characteristic excitotoxic syndrome, consisting of automatisms (wet dog shakes, WDS), sustained limbic seizures and brain damage. Since kainate increases the release of excitatory amino acid neurotransmitters such as glutamate, this syndrome is thought to be due, at least in part, to excessive activation of glutamate receptors, particularly of the N-methyl-D-aspartate (NMDA) subtype. We examined the effect of D-cycloserine, a partial agonist for the NMDA receptor-associated glycine binding site, in the kainate model of limbic seizures in rats. For comparison, the uncompetitive NMDA antagonist MK-801 (dizocilpine) and the GABAmimetic anticonvulsant diazepam were used. D-Cycloserine exerted a potent, dose-dependent and long-lasting anticonvulsant effect against kainate-induced seizures. At 160 mg/kg, seizures were almost completely suppressed by D-cycloserine over a 3 h observation period. No adverse effects were observed at anticonvulsant doses of D-cycloserine. In contrast to its potent effect on kainate-induced seizures, D-cycloserine did not significantly alter the number of automatisms (WDS) determined after kainate. MK-801, 0.3 mg/kg, also markedly reduced seizure severity in response to kainate, but this anticonvulsant effect was accompanied by marked motor impairment. Similarly, diazepam, 5 mg/kg, significantly attenuated kainate-induced seizures but marked ataxia was observed at this dosage. In contrast to D-cycloserine, both MK-801 and diazepam reduced WDS behaviour caused by kainate. The data demonstrate that pharmacological manipulation of the strychnine-insensitive glycine site is a powerful means of protecting against kainate-induced seizures.

Kynurenines and the respiratory parameters on rat heart mitochondria

It has been shown recently that the L-kynurenine metabolite kynurenic acid lowers the efficacy of mitochondria ATP synthesis by significantly increasing state IV, and reducing respiratory control index and ADP/oxygen ratio of glutamate/malate-consuming heart mitochondria. In the present study we investigated the effect of L-tryptophan (1.25 microM to 5 mM) and other metabolites of L-kynurenine as 3-hydroxykynurenine (1.25 microM to 2.5 mM), anthranilic acid (1.25 microM to 5 mM) and 3-hydroxyanthranilic acid (1.25 microM to 5 mM) on the heart mitochondria function. Mitochondria were incubated with saturating concentrations of respiratory substrates glutamate/malate (5 mM), succinate (10 mM) or NADH (1 mM) in the presence or absence of L-tryptophan metabolites. Among tested substances, 3-hydroxykynurenine, 3-hydroxyanthranilic acid and anthranilic acid but not tryptophan affected the respiratory parameters dose-dependently, however at a high concentration, of a micro molar range. 3-Hydroxykynurenine and 3-hydroxyanthranilic acid lowered respiratory control index and ADP/oxygen ratio in the presence of glutamate/malate and succinate but not with NADH. While, anthranilic acid reduced state III oxygen consumption rate and lowered the respiratory control index only of glutamate/malate-consuming heart mitochondria. Co-application of anthranilic acid and kynurenic acid (125 or 625 microM each) to glutamate/malate-consuming heart mitochondria caused a non-additive deterioration of the respiratory parameters determined predominantly by kynurenic acid. Accumulated data indicate that within L-tryptophan metabolites kynurenic acid is the most effective, followed by anthranilic acid, 3-hydroxykynurenine, 3-hydroxyanthranilic acid to influence the respiratory parameters of heart mitochondria. Present data allow to speculate that changes of kynurenic acid and/or anthranilic acid formation in heart tissue mitochondria due to fluctuation of L-kynurenine metabolism may be of functional importance for cardiovascular processes. On the other hand, beside the effect of 3-hydroxyanthranilic acid and 3-hydroxykynurenine on respiratory parameters, their oxidative reactivity may contribute to impairment of mitochondria function, too.