Caspase-3-dependent neuronal death in the hippocampus following kainic acid treatment.

In this study, we examined the levels of activated caspase-3 in the kainic acid (KA) model of hippocampal degeneration in both sensitive (FVB/N) and resistant (129/SvEMS) strains of mice. At 30 h, 2 and 4 days following KA administration, animals were sacrificed and brains examined for pyknosis, TUNEL labeling, and activated caspase-3 immunoreactivity. Catalytically active caspase-3 was first detected 30 h following KA treatment in the sensitive, FVB/N strain. This was 18 h before the appearance of pyknosis or TUNEL labeling. The expression of activated caspase-3 continues up to 4 days post-injection. No activated caspase-3 immunoreactivity was detected in the resistant, 129/SvEMS strain, neither was there evidence of pyknosis or TUNEL staining. This suggests that activation of caspase-3 is a necessary component of KA-induced cell death.     

Volumetric structural magnetic resonance imaging (MRI) of the rat hippocampus following kainic acid (KA) treatment

An in vivo MRI study employing a high field (7T) magnet and a T1- and T2-weighted imaging sequence with subsequent histopathological evaluations was undertaken to develop and evaluate MRI-based volumetric measurements in the rat. The brain structures considered were the hippocampus, the cingulate cortex, the retrosplenial granular cortex and the ventricles. Control (n=3) and kainic acid (KA; n=4) treated rats were scanned 10 days following the manifestation of stage four seizures. The MRI images exhibited anatomical details (125 microm in-plane resolution) that enabled volumetric analysis with high intra-rater reliability. Volumetric analysis revealed that KA-treated rats had significantly smaller hippocampi, and a significant increase in ventricular size. The cingulate cortex and the retrosplenial granular cortex did not differ in volume between the two groups. The histological observations supported the MRI data showing neuronal loss and neuronal degeneration in CA1 and CA3 of the hippocampus, which was accompanied by strong microglia activation. These data demonstrate a reliable and valid method for the measurement of the rat hippocampus in vivo using MRI with a high field magnet, thereby providing a useful tool for future studies of rodent models of neuro-degenerative diseases.     

Amino acids in rat neostriatum: Alteration by kainic acid lesion

Unilateral stereotaxic injection of 10 nmol of the glutamomimetic substance, kainic acid, into the rat striatum caused permanent, significant decreases in the levels of glutamate (40--50%), aspartate (35--40%), taurine (20--30%) and GABA (60--70%). There were initial, transient decreases in serine, glycine and alanine which returned to normal values within 16--32 days after injection. Glutamine levels were not altered in lesioned striatum. This coincided with a 55% increase in glutamine synthetase activity in the lesioned striatum compared either to the non-injected striatum or controls injected with saline. The high affinity uptake of choline by synaptosomal preparations of lesioned striatum was decreased by 70% compared to controls whereas that of glutamate/aspartate was either unchanged or somewhat on a per mg protein basis. This latter point may be illusory in that, because of widespread neuronal destruction, the total 'synaptosomal' protein obtained from the lesioned striata was only about 50% that from control tissue. The biochemical data are consistent with the histological and behavioral effects of kainic acid administration. The unchaning glutamine levels and increase in glutamine synthetase activity are consistent with the widespread gliosis and the lack of change in glutamate/aspartate high affinity uptake is consistent with a sparing of afferent terminals. The large decrease in glutamate and aspartate is consistent with hypotheses concerning the intraneuronal localization of a major pool of these amino acids, especially in GABAergic neurons. The decrease in taurine suggests that a portion of this amino acid in striatum is probably associated with neurons destroyed by kainic acid. The bulk of the taurine, however, is therefore associated either with glial cells or the afferents to the striatum.     

Cognitive effects of long-term treatment with phenobarbital and valproic acid in school children

The Wechsler Intelligence Scales for Children was applied to 64 epileptic children and 60 healthy subjects; patients followed chronic treatment with valproic acid (n = 32) or phenobarbital (n = 32). None of the children suffered mental retardation or neurological abnormalities. The test was repeated after a 9-12 month interval: 26 of the valproate treated children and 23 of the phenobarbital-treated children performed the second evaluation. At baseline, total, verbal and performance IQ scores of children receiving phenobarbital were lower than those of controls. When the results of the first and the second tests were compared, a significant increase in IQ scores was detected among controls and patients treated with valproic acid, but not among phenobarbital-treated patients. It is concluded that long-term phenobarbital therapy induces a significant impairment in learning ability whereas long-term valproate therapy does not exert a noticeable noxious effect at this respect.     

Hypoxia prevents seizures and neuronal damages of the hippocampus induced by kainic acid in rats

The effects of hypoxia on the epileptic seizures and neuronal damages induced by kainic acid were studied in rats using hypoxic chamber equipment. Rats treated with kainic acid and placed in atmospheric pressure showed typical limbic seizures and regressive neuronal changes in CA3 and CA4 of the hippocampus, while those kept in a hypoxic chamber with 8.5% O2 and 91.5% N2 showed moderate hypoxia and a slight decline of mean arterial blood pressure. In these hypoxic rats, seizures were completely prevented and there was remarkably less regressive neuronal injury of the hippocampus. Thus hypoxia has a rather ameliorative effect on the occurrence of seizures and excitotoxic neuronal injuries induced by kainic acid. The contribution of oxygen radicals and endogenous adenosine to preventing excitotoxic neuronal damages by kainic acid was discussed.     

Accumulation of calcium in the rat hippocampus during kainic acid seizures

The change in the total calcium content of the rat hippocampus was investigated quantitatively and the sites of the accumulated calcium in the CA₁ region were demonstrated cytochemically by the oxalate-pyroantimonate technique 4 h following intraperitoneal kainic acid administration. Kainic acid was found to induce a significant increase in the total calcium content of the hippocampus (from 4.75 ± 0.39to6.64 ± 0.60 μmol/g dry weight). Calcium accumulated mainly in the mitochondria of the neuronal somata, the dendrites and the synaptic terminals. Consequently, the degenerative alterations caused by kainic acid are associated with intracellular calcium accumulation, which can overload the capacity of the mitochondria for calcium sequestration, and may have a role, besides other factors, in the development of neuronal damage.     

Induction and activation of protein kinase C delta in hippocampus and cortex after kainic acid treatment

Various isoforms of protein kinase C (PKC), especially the novel PKC subtypes delta, epsilon, and the atypical subtype PKC zeta, are involved in delayed cell death. We studied the expression and late activation of the latter PKC isoforms in comparison with classic PKC alpha, beta, and gamma in the brains of rats exposed to systemic kainate injection. The expression of PKC delta mRNA was strikingly upregulated (13-fold) in the cortex and the CA1 and CA3 hippocampal regions on 1 day after kainate administration, whereas PKC zeta mRNA was only moderately increased (about 100%) in these three brain regions on day 2 following the drug. PKC epsilon mRNA was slightly increased only in the cortex on days 2 and 6, while the mRNA levels of the classic PKC subtypes (alpha, beta, and gamma) remained unchanged or decreased after the treatment. Immunoblotting analyses revealed that the level of PKC delta protein started to increase on day 1 after kainate and was significantly elevated on day 2 in both the membrane and cytosol fractions of cortex and hippocampus. PKC epsilon protein only showed a marginal increase and the level of PKC zeta protein remained unaltered in response to the treatment. Cortical and CA1-3 pyramidal neurons displayed strong immunoreactivity for PKC delta on days 1 and 2, and microglia on days 1, 2, and 4 after the drug. The results indicate that the expression of apoptosis-associated isoforms of PKC, most notably that of delta, but to lesser extent also that of epsilon and zeta, is increased during kainate-induced neuronal death. The predominant induction of PKC delta in neurons and microglia suggests that PKC delta could be the major mediator or modulator of apoptotic and inflammatory responses to excitotoxic insults.     

Selective kainic acid lesions in cultured explants of rat hippocampus

Summary The influence of the excitotoxin kainic acid (KA) on cultivated explants of rat hippocampus was investigated. Addition of 3 M KA to the culture medium over 24–48 h induced a destruction of the pyramidal cells in the CA3 region, whereas the CA1 pyramidal cells and the granule cells were left undamaged. Higher concentrations (10–100 M) of KA destroyed also the latter cell groups. The selectivity of the KA lesion at 3 M was further indicated by the fact that the acetylcholinesterase-positive neurons in the hippocampus were not destroyed through KA administration and that the stereoisomer dihydrokainic acid was ineffective in inducing lesions. Application of tetrodotoxin did not protect the CA3 pyramidal cells from KA lesion, whereas -glutamylaminomethylsulphonic acid (GAMS) only offered a very small, statistically not significant, protection. Baclofen protected the cultures slightly from KA lesions but not when added together with GAMS. Possible mechanisms responsible for the KA lesions in these cultures are discussed.Supported in part by a grant from the Swiss National Foundation for Scientific Research (No. 3.528.-0.83)     

Modifications in recognition sites for neurotransmitters in rat hippocampus by kainic acid lesion

The specific binding of the tritiated radioligands of dexetimide, serotonin, clonidine, prazosin, WB-4101 and dihydroalprenolol to hippocampal membranes was determined two weeks after producing a virtual complete degeneration of perikarya by the local application of 0.5 μg of kainic acid in the dorsal and ventral parts of the hippocampus. Afferent terminals were unaffected by the neurotoxin since the contents of noradrenaline, serotonin and acetylcholine, as well as the activity of choline acetyltransferase, were not modified.Scatchard analysis is revealed that the kainic acid lesion produced a 60% decrease in the density of both cholinergic muscarinic binding sites and serotonin binding sites. A significant portion ofα₁-andα₂-adrenoceptor binding sites are also associated with intrinsic neurons of the hippocampus, as shown by the approximately 30% reduction in the densities of tritiated WB-4101, prazosin and clonidine produced by the action of kainic acid. By contrast, the affinity and density of β-adrenoceptor binding sites were unaffected by the lesion. It is suggested that the recognition sites of the different receptor populations surviving the lesion most likely reside on homologous and/or heterologous nerve terminals.     

Ketogenic diet prevents clusterin accumulation induced by kainic acid in the hippocampus of male ICR mice

We investigated the effect of ketogenic diet (KD) on clusterin accumulation in the kainic acid (KA)-induced seizure model. Two days after KA administration, strong clusterin-like immunoreactivity (IR) was detected in the hippocampus in the normal diet (ND)-fed mice. But in the KD-fed mice, few clusterin-like IR was detected. These results indicate that KD has neuroprotective effects throughout diminishing nuclear clusterin accumulation that is involved in caspase-3 independent cell death mechanism.     

Cyclooxygenase-2 selective inhibitors aggravate kainic acid induced seizure and neuronal cell death in the hippocampus.

Cyclooxygenase-2 (COX-2) in the brain is expressed constitutively and also increased in pathological conditions such as seizure, cerebral ischemia, and inflammation. This study examined the role of COX-2 in kainic acid-induced seizure and in the following neuronal death by using selective inhibitors. Systemic kainate injection (50 mg/kg; i.p.) in mice evoked seizure within 15 min and led to 29% mortality within 2 h. TUNEL-positive neuronal death peaked at 3 days after injection and was prominent in CA(3a) regions of the hippocampus. NS-398 or celecoxib (10 mg/kg, COX-2 selective inhibitor) and indomethacin (5 mg/kg, nonselective inhibitor) exaggerated kainic acid-induced seizure activity and mortality. COX-2 selective inhibitors induced the seizure at earlier onset and more severe mortality within the first hour than indomethacin and aspirin. NS-398 also aggravated kainic acid-induced TUNEL positive neuronal death and decreased Cresyl violet stained viable neurons, and extended lesions to CA(1) and CA(3b). Kainic acid increased the levels of PGD(2), PGF(2a) and PG E(2) in the hippocampus immediately after injection. Indomethacin attenuated the production of basal and kainic acid-induced prostaglandins. In contrast, NS-398 failed to reduce until the first 30 min after kainic acid injection, during which the animals were severely seizured. It has been challenged the endogenous PGs might have anticonvulsant properties. Thus, COX-2 selective inhibitor, including nonselective inhibitor such as indomethacin, aggravated kainic acid-induced seizure activity and the following hippocampal neuronal death even with variable prostaglandin levels.     

Transient and long-lasting beneficial behavioral effects of grafts in the damaged hippocampus of rat

One week after receiving bilateral neurotoxic lesions of their dorsal hippocampi, adult Wistar rats were bilaterally grafted with fetal hippocampal tissue suspensions. The behavior of the animals was tested during a period of 5 months after grafting to determine changes in lesion-induced deficits. The transplants caused various behavioral effects with different time courses. Grafted animals showed an early, however, transient amelioration of behavioral deficits in a T-maze alternation task and they performed with a long-lasting improvement in the alcove-test. Transplant histology demonstrated high levels of AChE-activity in patches correlating with clusters or rudimentary layers of pyramidal neurons.     

Changes in inhibitory processes in the hippocampus following recurrent seizures induced by systemic administration of kainic acid

Rats were chronically prepared with stimulation electrodes in the angular bundle and recording electrodes in the dentate gyrus under electrophysiological guidance. Following testing of dentate gyrus field potentials, the animals were given a single injection of kainic acid which caused repeated seizures and led to status epilepticus. The seizures were stopped by administration of a barbiturate anesthetic after 60 min. Changes in inhibition during seizure development were monitored by administering pulse pairs at regular intervals. The results revealed a progressive kainic acid-induced loss in inhibition that preceded the occurrence of seizures. This breakdown of inhibition was transient, and generally disappeared within 24 h. Over subsequent testing, recurrent inhibition, as measured by the double pulse test, increased beyond baseline levels. This increase persisted for at least one month and was restricted to the early phase of inhibition with a conditioning/test pulse interval of less than 50 ms. A later phase of inhibition, measured at interpulse intervals between 200 and 300 ms, showed a transient decrease which lasted about a week. These results contrast with previous reports of a long-term period of hyperexcitability following recurrent seizures. Procedural differences which might account for such discrepancies are discussed.     

Molecular events involved in neuronal death induced in the mouse hippocampus by in-vivo injection of kainic acid

Apoptosis results from the activation of a programmed cellular cascade involving several mechanisms. In the present study, we have investigated the implication of three molecules of this cascade, p53, Bax and caspase-3, in neuronal death induced by kainic acid (KA) administration in mouse hippocampus. Using immunocytochemistry, western blot and quantification of enzyme activity, we observed in p53+/+ and p53-/- animals that KA induced neuronal death by both p53-dependent and independent pathways. Moreover, apoptosis (labeled by TUNEL) and the increase of bax and caspase-3 protein expression after the neurotoxic insult appeared to clearly depend on p53 expression.     

Pregnancy decreases the frequency of spontaneous recurrent seizures in rats with kainic acid lesions of the hippocampus

Spontaneous recurrent seizures (SRSs) were observed in female rats following the injection of kainic acid into the dorsal hippocampus. Pregnancy and nursing decreased the frequency of SRSs in such animals. The finding of a protective effect of pregnancy and lactation in this animal model of temporal lobe epilepsy stresses the usefulness of this model.     

神经颗粒素在海人酸诱发癫(疒间)大鼠皮质及海马中表达的研究

目的探讨海人酸(KA)诱发癫疒间后不同时间大鼠皮质及海马各区神经颗粒素的表达及意义。方法将52只SD大鼠随机分为癫疒间诱发组(KA组)和正常对照组。运用免疫荧光染色共聚焦显微镜观察大鼠制模成功后6 h、12 h、18 h、24 h、48 h 5个时点皮质及海马各区神经颗粒素表达的变化;蛋白质免疫印记(W estern B lot)技术对皮质及海马神经颗粒素作选择性半定量分析,并进行比较。结果与正常对照组相比,KA组制模18 h大鼠皮质神经颗粒素表达开始减少(P<0.05),24 h为最低(P<0.01),48 h恢复正常;制模12 h海马齿状回神经颗粒素明显下降(P<0.05),18 h最低(P<0.01),48 h恢复正常;海马CA1区制模后神经颗粒素持续下降,48 h时与正常对照组比较差异有显著性(P<0.05);CA3区未见显著变化。W estern B lot实验印证了这一结果。结论KA诱发的复杂部分性癫疒间发作急性期能引起大鼠皮质及海马神经颗粒素表达减少,在各脑区的变化不同,且有可恢复的趋势,可能与癫疒间发作后引起大脑神经元突触可塑性改变有关。