Arterial spin labeling demonstrates that focal amygdalar glutamatergic agonist infusion leads to rapid diffuse cerebral activation

Munasinghe JP, Banerjee M, Acosta MT, Banks M, Heffer A, Silva AC, Koretsky A, Theodore WH. Arterial spin labeling demonstrates that focal amygdalar glutamatergic agonist infusion leads to rapid diffuse cerebral activation.
Acta Neurol Scand: 2010: 121: 209–216.
© 2009 The Authors Journal compilation © 2009 Blackwell Munksgaard. Objectives – To investigate acute effects of intra‐amygdalar excitatory amino acid administration on blood flow, relaxation time and apparent diffusion coefficient in rat brain. Materials and methods – Several days after MR‐compatible cannula placement in right basolateral amygdala, anesthetized rats were imaged at 7 T. Relative cerebral blood flow (CBF) was measured before and 60 min after infusion of 10 nmol KA, cAMPA, ATPA, or normal saline using arterial spin labeling. Quantitative T₂ and diffusion‐weighted images were acquired. rCBF, T₂ and ADC values were evaluated in bilateral basolateral amygdala, hippocampus, basal ganglia, frontal and parietal regions. Results – KA led to the highest, and ATPA lowest bilateral rCBF increases. Time courses varied among drugs. T₂ for KA and AMPA was higher while ADC was lower for KA. Conclusions – Intra‐amygdalar injection of GluR agonists evoked bilateral seizure activity and increased rCBF, greater for KA and AMPA than selective ATPA GluR5 activation.     

Seizures in the intrahippocampal kainic acid epilepsy model: characterization using long‐term video‐EEG monitoring in the rat

Objective – Intrahippocampal injection of kainic acid (KA) in rats evokes a status epilepticus (SE) and leads to spontaneous seizures. However to date, precise electroencephalographic (EEG) and clinical characterization of spontaneous seizures in this epilepsy model using long‐term video‐EEG monitoring has not been performed. Materials and Methods – Rats were implanted with bipolar hippocampal depth electrodes and a cannula for the injection of KA (0.4 μg/0.2 μl) in the right hippocampus. Video‐EEG monitoring was used to determine habitual parameters of spontaneous seizures such as seizure frequency, severity, progression and day–night rhythms. Results – Spontaneous seizures were detected in all rats with 13 out of 15 animals displaying seizures during the first eight weeks after SE. A considerable fraction (35%) of the spontaneous seizures did not generalize secondarily. Seizure frequency was quite variable and the majority of the KA‐treated animals had less than one seizure per day. A circadian rhythm was observed in all rats that showed sufficient seizures per day. Conclusions – This study shows that the characteristics of spontaneous seizures in the intrahippocampal KA model display many similarities to other SE models and human temporal lobe epilepsy.     

AIDA,a class I metabotropic glutamate-receptor antagonist limits kainate-induced hippocampal dysfunction

PURPOSE: In the developing animal, intraperitoneal injections of kainic acid (KA) lead to a prolonged initial seizure followed by chronic recurrent seizures and long-term hippocampal dysfunction. We investigated whether the class I metabotropic glutamate receptor (mGluR) antagonist 1-aminoindan-1,5-dicarboxylic acid (AIDA) is neuroprotective in the KA model of epilepsy. METHODS: Immature rats aged postnatal day 20 (P20) and P30 were injected with fixed volumes of KA, KA + AIDA, AIDA, or saline. We monitored recurrent seizures. Thirty days later, we tested hippocampal function with the Morris water-maze test or prepared hippocampal slices to record extracellularly evoked and spontaneous potentials from the CA1 area. In a third group, we performed neuronal counts. RESULTS: In both age groups, acute seizures were similar in KA and KA + AIDA groups. Rare spontaneous recurrent seizures occurred only in KA-injected rats. The KA P20 group performed significantly worse than controls in the water-maze test. The KA + AIDA group showed impaired performance on day 1, but learning improved substantially, reaching control values in the remaining 3 days. The P30 KA rats performed worse than controls on all trial days, whereas the KA + AIDA rats improved by day 3, but did not reach control values. Electrophysiologic recordings showed small but consistent differences between KA and control animals, suggestive of an adaptive modification in the gamma-aminobutyric acid (GABA)ergic system, reversed by AIDA. On histology, we observed a loss of CA1 interneurons in both ages. Cell loss was reversed by the use of AIDA. CONCLUSIONS: Blockade of the class I mGluR during KA-induced seizures in the developing brain limits seizure-induced hippocampal dysfunction.     

Comparison of excitotoxic profiles of ATPA, AMPA, KA and NMDA in organotypic hippocampal slice cultures

The excitotoxic profiles of (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)propionic acid (ATPA), (RS)-2-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), kainic acid (KA) and N-methyl-D-aspartate (NMDA) were evaluated using cellular uptake of propidium iodide (PI) as a measure for induced, concentration-dependent neuronal damage in hippocampal slice cultures. ATPA is in low concentrations a new selective agonist of the glutamate receptor subunit GluR5 confined to KA receptors and also in high concentrations an AMPA receptor agonist. The following rank order of estimated EC(50) values was found after 2 days of exposure: AMPA (3.7 mM)>NMDA (11 mM)=KA (13 mM)>ATPA (33 mM). Exposed to 30 microM ATPA, 3 microM AMPA and 10 microM NMDA, CA1 was the most susceptible subfield followed by fascia dentata and CA3. Using 8 microM KA, CA3 was the most susceptible subfield, followed by fascia dentata and CA1. In 100 microM concentrations, all four agonists induced the same, maximal PI uptake in all hippocampal subfields, corresponding to total neuronal degeneration. Using glutamate receptor antagonists, like GYKI 52466, NBQX and MK-801, inhibition data revealed that AMPA excitotoxicity was mediated primarily via AMPA receptors. Similar results were found for a high concentration of ATPA (30 microM). In low GluR5 selective concentrations (0.3-3 microM), ATPA did not induce an increase in PI uptake or a reduction in glutamic acid decarboxylase (GAD) activity of hippocampal interneurons. For KA, the excitotoxicity appeared to be mediated via both KA and AMPA receptors. NMDA receptors were not involved in AMPA-, ATPA- and KA-induced excitotoxicity, nor did NMDA-induced excitotoxicity require activation of AMPA and KA receptors. We conclude that hippocampal slice cultures constitute a feasible test system for evaluation of excitotoxic effects and mechanisms of new (ATPA) and classic (AMPA, KA and NMDA) glutamate receptor agonists. Comparison of concentration-response curves with calculation of EC(50) values for glutamate receptor agonists are possible, as well as comparison of inhibition data for glutamate receptor antagonists. The observation that the slice cultures respond with more in vivo-like patterns of excitotoxicity than primary neuronal cultures, suggests that slice cultures are the best model of choice for a number of glutamate agonist and antagonist studies.     

Simultaneous BOLD fMRI and local field potential measurements during kainic acid-induced seizures

Purpose: To investigate how kainic acid–induced epileptiform activity is related to hemodynamic changes probed by blood oxygenation level–dependent functional magnetic resonance imaging (BOLD fMRI). Methods: Epileptiform activity was induced with kainic acid (KA) (10 mg/kg, i.p.), and simultaneous fMRI at 7 Tesla, and deep electrode local field potential (LFP) recordings were performed from the right hippocampus in awake and medetomidine‐sedated adult Wistar rats. Key Findings: Recurrent seizure activity induced by KA was detected in LFP both in medetomidine‐sedated and awake rats, even though medetomidine sedation reduced the mean duration of individual seizures as compared to awake rats (33 ± 24 and 46 ± 34 s, respectively, mean ± SD p < 0.01). KA administration also triggered robust positive BOLD responses bilaterally in the hippocampus both in awake and medetomidine‐sedated rats; however, in both animal groups some of the seizures detected in LFP recording did not cause detectable BOLD signal change. Significance: Our data suggest that medetomidine sedation can be used for simultaneous fMRI and electrophysiologic studies of normal and epileptic brain function, even though seizure duration after medetomidine administration was shorter than that in awake animals. The results also indicate that neuronal activity and BOLD response can become decoupled during recurrent kainic acid–induced seizures, which may have implications to interpretation of fMRI data obtained during prolonged epileptiform activity.     

Gradation of Kainic Acid-induced Rat Limbic Seizures and Expression of Hippocampal Heat Shock Protein-70

Systemic injection of kainic acid (KA) induces limbic seizures in rats, which resemble human temporal lobe epilepsy, the most common form of adult human epilepsy. In this study, we have investigated KA-elicited limbic seizures in the rats by correlating the severity of the seizure attacks with the expression of hippocampal heat shock protein-70 (HSP70) which has been suggested to be a marker for neuronal injury/death in this model of seizures. After a systemic injection of KA, six stages of limbic seizures have been classified, namely, staring (stage 1), wet dog shake (stage 2), hyperactivity (stage 3), rearing (stage 4), rearing and falling (stage 5), and jumping (stage 6). Stages 4, 5 and 6 were further divided into mild and severe sub-stages. HSP70 expression was not detected in animals with stages 1 and 2 seizures. At stage 3 a small amount of HSP70 immunoreactive neurons was detected in the CA3 field and the dentate hilus. From stage 4 to stage 5 the degree of HSP70 immunoreactivity increased in the CA1 field from a few positive cells in stage 4 mild to large numbers of immunoreactive neurons in stage 5 severe. HSP70 became detectable in pyramidal cells in the CA2 field from stage 5 severe and higher. In animals with stage 6 seizures, the majority of HSP70 expression became located in glial cells throughout the whole hippocampus. We concluded that HSP70 expression in the hippocampus positively correlates with the severity of KA-elicited limbic seizures.     

Talampanel suppresses the acute and chronic effects of seizures in a rodent neonatal seizure model

Purpose: To test the efficacy of the novel candidate anticonvulsant talampanel (GYKI 53773) in a rodent model of hypoxic neonatal seizures. Talampanel is a noncompetitive antagonist of the alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐propionic acid subtype of the glutamate receptor (AMPAR). We have previously shown that AMPARs play a critical role in the generation of acute seizures and later‐life seizure susceptibility in this model of neonatal seizures. Methods: Seizures were induced in postnatal day (P) 10 Long‐Evans rat pups by a 15 min exposure to global hypoxia. Acute seizure activity at P10 and subsequent susceptibility to seizure‐induced neuronal injury with a “second‐hit” kainate‐induced seizure at P30–31 were compared between animals receiving talampanel (1, 5, 7.5, or 10 mg/kg) intraperitoneally (i.p.) versus saline vehicle treatment. Results: Talampanel treatment suppressed seizures in a dose‐dependent manner, with maximal effect at 7.5 and 10 mg/kg. In addition, talampanel treatment 30 min before hypoxia prevented later‐life increases in seizure‐induced neuronal injury as assessed by in situ DNA nick end‐labeling (ISEL). Discussion: We have previously demonstrated efficacy of other AMPAR antagonists such as NBQX and topiramate in this model. The present finding shows that the novel agent talampanel, under evaluation as an antiepileptic drug in children and adults, may have clinical potential in the treatment of neonatal seizures, particularly those occurring in the context of hypoxic encephalopathy.     

Anticonvulsant activity of ginseng on seizures induced by chemical convulsants

PURPOSE: To test the anticonvulsant activity of three preparations of American ginseng: whole root extract, whole leaves/stems extract, and a partially purified extract that concentrates the Rb ginsenosides (Rb extract). METHODS: One hour after treatment with normal saline, or one of the three ginseng preparations, seizures were induced in adult, male, Sprague-Dawley rats with kainic acid (KA; 10 mg/kg), pilocarpine (300 mg/kg, preceded by methylscopolamine, 1 mg/kg, s.c.), or pentylenetetrazol (PTZ, 50 mg/kg). Time to onset of seizure activity, duration of seizure activity for PTZ, seizure severity, and weight change for KA and pilocarpine were determined for each animal. The brains from animals who had received KA or pilocarpine were examined for severe neuronal stress, by using immunoreactivity for heat-shock protein (HSP)72. RESULTS: The Rb extract had a dose-dependent anticonvulsant effect in all three models of chemically induced seizures: increasing the latency to the seizures; decreasing the seizure score, weight loss, and subsequent neuronal damage after pilocarpine; and shortening the seizure duration and reducing mortality after PTZ. The Rb extract also significantly reduced the effects of KA, including completely blocking behavioral seizures. The root preparation increased the mortality rate after administration of pilocarpine, but had no other significant effects. The leaves/stems preparation, at 120 mg/kg, reduced the weight loss after pilocarpine, but had no other significant effects. CONCLUSIONS: Ginseng extract made from either the root or leaves/stems is ineffective against chemically induced seizures. A partial purification of the whole extract that concentrates the Rb1 and Rb3 ginsenosides has significant anticonvulsant properties.     

米诺环素对红藻氨酸诱导的癫痫大鼠海马组织凋亡相关蛋白表达的影响

目的:观察癫发作后不同时间海马组织凋亡相关蛋白表达水平的变化,探讨米诺环素(MT)减轻癫所致的海马神经元损伤的机制。方法:杏仁核立体定向注射红藻氨酸(KA)建立大鼠癫模型,随机分为治疗(MT)组和非治疗(KA)组,以杏仁核注射生理盐水为空白对照(SC)组。用免疫印迹法检测caspase-3裂解片段及Bcl-2和Bcl-xl表达水平的变化。结果:与KA组相比,MT组caspase-3裂解水平明显降低(P<0.05)并接近SC组;Bcl-2在癫发作终止后2h开始上调,24h达高峰并持续到72h。Bcl-xl上调不明显。结论:米诺环素能减少caspase-3的裂解,抑制线粒体凋亡通路激活,从而减轻癫发作对神经元的损伤。这一作用与抗凋亡因子Bcl-2的上调有关,提示Bcl-2可能是癫潜在的治疗靶点。     

Glial activation links early-life seizures and long-term neurologic dysfunction: evidence using a small molecule inhibitor of proinflammatory cytokine upregulation

PURPOSE: Early-life seizures increase vulnerability to subsequent neurologic insult. We tested the hypothesis that early-life seizures increase susceptibility to later neurologic injury by causing chronic glial activation. To determine the mechanisms by which glial activation may modulate neurologic injury, we examined both acute changes in proinflammatory cytokines and long-term changes in astrocyte and microglial activation and astrocyte glutamate transporters in a "two-hit" model of kainic acid (KA)-induced seizures. METHODS: Postnatal day (P) 15 male rats were administered KA or phosphate buffered saline (PBS). On P45 animals either received a second treatment of KA or PBS. On P55, control (PBS-PBS), early-life seizure (KA-PBS), adult seizure (PBS-KA), and "two-hit" (KA-KA) groups were examined for astrocyte and microglial activation, alteration in glutamate transporters, and expression of the glial protein, clusterin. RESULTS: P15 seizures resulted in an acute increase in hippocampal levels of IL-1beta and S100B, followed by behavioral impairment and long-term increases in GFAP and S100B. Animals in the "two-hit" group showed greater microglial activation, neurologic injury, and susceptibility to seizures compared to the adult seizure group. Glutamate transporters increased following seizures but did not differ between these two groups. Treatment with Minozac, a small molecule inhibitor of proinflammatory cytokine upregulation, following early-life seizures prevented both the long-term increase in activated glia and the associated behavioral impairment. CONCLUSIONS: These data suggest that glial activation following early-life seizures results in increased susceptibility to seizures in adulthood, in part through priming microglia and enhanced microglial activation. Glial activation may be a novel therapeutic target in pediatric epilepsy.     

Downregulation of kainate receptors in the hippocampus following repeated seizures in immature rats

There are significant differences in seizure-induced sequelae between the immature and mature brain. We have previously demonstrated that repeated doses of the chemoconvulsant kainic acid is associated with a progressive increase in severity of seizures in adult animals while in immature rats the opposite occurs; seizure intensity decreases with subsequent doses of kainic acid. Likewise, repeated kainic acid seizures causes severe hippocampal damage in mature rats while in the immature brain serial administration of kainic acid causes no demonstrable cell loss. Here we show that recurrent kainic acid seizures in immature rats are associated with a downregulation of kainate receptor binding. No histological damage was noted in any of the rats exposed to recurrent seizures. Furthermore, when tested for visual-spatial memory immature rats with recurrent kainate seizures did not differ from controls. The downregulation of KA receptors following repeated exposure to KA suggests that the decrease in glutamate receptor density might account in part for the observed lack of neuronal loss and decrease in seizure intensity in these animals.     

Electrophysiological Effects of Kainic Acid on Vasopressin‐Enhanced Green Fluorescent Protein and Oxytocin‐Monomeric Red Fluorescent Protein 1 Neurones Isolated from the Supraoptic Nucleus in Transgenic Rats

The supraoptic nucleus (SON) contains two types of magnocellular neurosecretory cells: arginine vasopressin (AVP)‐producing and oxytocin (OXT)‐producing cells. We recently generated and characterised two transgenic rat lines: one expressing an AVP‐enhanced green fluorescent protein (eGFP) and the other expressing an OXT‐monomeric red fluorescent protein 1 (mRFP1). These transgenic rats enable the visualisation of AVP or OXT neurones in the SON. In the present study, we compared the electrophysiological responses of AVP‐eGFP and OXT‐mRFP1 neurones to glutamic acid in SON primary cultures. Glutamate mediates fast synaptic transmission through three classes of ionotrophic receptors: the NMDA, AMPA and kainate receptors. We investigated the contributions of the three classes of ionotrophic receptors in glutamate‐induced currents. Three different antagonists were used, each predominantly selective for one of the classes of ionotrophic receptor. Next, we focused on the kainate receptors (KARs). We examined the electrophysiological effects of kainic acid (KA) on AVP‐eGFP and OXT‐mRFP1 neurones. In current clamp mode, KA induced depolarisation and increased firing rates. These KA‐induced responses were inhibited by the non‐NMDA ionotrophic receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3(1H4H)‐dione in both AVP‐eGFP and OXT‐mRFP1 neurones. In voltage clamp mode, the application of KA evoked inward currents in a dose‐dependent manner. The KA‐induced currents were significantly larger in OXT‐mRFP1 neurones than in AVP‐eGFP neurones. This significant difference in KA‐induced currents was abolished by the GluK1‐containing KAR antagonist UBP302. At high concentrations (250–500 μm ), the specific GluK1‐containing KAR agonist (RS)‐2‐amino‐3‐(3‐hydroxy‐5‐tert‐butylisoxazol‐4‐yl) propanoic acid (ATPA) induced significantly larger currents in OXT‐mRFP1 neurones than in AVP‐eGFP neurones. Furthermore, the difference between the AVP‐eGFP and OXT‐mRFP1 neurones in the ATPA currents was approximately equal to the difference in the KA currents. These findings suggest that the GluK1‐containing KARs may be more highly expressed in OXT neurones than in AVP neurones. These results may provide new insight into the physiology and synaptic plasticity of SON neurones.     

GnRH-immunoreactive fiber changes with unilateral amygdala-kindled seizures

PURPOSE: To assess whether unilateral amygdala seizures are associated with a change in the number and lateral distribution of gonadotropin releasing hormone (GnRH)-staining fibers in the ventromedial hypothalamus of female rats. METHODS: The study compared three groups of female rats: (1) amygdala seizures induced by focal injection of kainic acid (KA); (2) saline injected controls; and (3) nai;ve controls. The animals were sacrificed at 4 weeks in the diestrus phase. GnRH fibers were counted in the ventromedial hypothalamus and compared among groups. RESULTS: GnRH fiber counts were significantly lower in KA than saline and nai;ve animals ipsilaterally but not contralaterally. CONCLUSIONS: This finding may support a potential mechanism by which (1) temporolimbic epilepsy may promote the development of reproductive endocrine disorders and (2) the laterality of the epilepsy may influence the particular nature of the reproductive endocrine disorder.     

Rapamycin has paradoxical effects on S6 phosphorylation in rats with and without seizures

Purpose: Accumulating data have demonstrated that seizures induced by kainate (KA) or pilocarpine activate the mammalian target of rapamycin (mTOR) pathway and that mTOR inhibitor rapamycin can inhibit mTOR activation, which subsequently has potential antiepileptic effects. However, a preliminary study showed a paradoxical exacerbation of increased mTOR pathway activity reflected by S6 phosphorylation when rapamycin was administrated within a short period before KA injection. In the present study, we examined this paradoxical effect of rapamycin in more detail, both in normal rats and KA‐injected animals. Methods: Normal rats or KA‐treated rats pretreated with rapamycin at different time intervals were sacrificed at various time points (1, 3, 6, 10, 15, and 24 h) after rapamycin administration or seizure onset for western blotting analysis. Phosphorylation of mTOR signaling target of Akt, mTOR, Rictor, Raptor, S6K, and S6 were analyzed. Seizure activity was monitored behaviorally and graded according to a modified Racine scale (n = 6 for each time point). Neuronal cell death was detected by Fluoro‐Jade B staining. Key Findings: In normal rats, we found that rapamycin showed the expected dose‐dependent inhibition of S6 phosphorylation 3–24 h after injection, whereas a paradoxical elevation of S6 phosphorylation was observed 1 h after rapamycin. Similarly, pretreatment with rapamycin over 10 h before KA inhibited the KA seizure–induced mTOR activation. In contrast, rapamycin administered 1–6 h before KA caused a paradoxical increase in the KA seizure–induced mTOR activation. Rats pretreated with rapamycin 1 h prior to KA exhibited an increase in severity and duration of seizures and more neuronal cell death as compared to vehicle‐treated groups. In contrast, rapamycin pretreated 10 h prior to KA had no effect on the seizures and decreased neuronal cell death. The paradoxical effect of rapamycin on S6 phosphorylation was correlated with upstream mTOR signaling and was reversed by pretreatment of perifosine, an Akt inhibitor. Significance: These data indicate the complexity of S6 regulation and its effect on epilepsy. Paradoxical effects of rapamycin need to be considered in clinical applications, such as for potential treatment for epilepsy and other neurologic disorders.     

Amygdaloid lesion increases the toxicity of intrahippocampal kainic acid injection and reduces the late occurrence of spontaneous recurrent seizures in rats

Spontaneous recurrent seizures (SRS) following intrahippocampal kainic acid (KA) injection have been described in a previous paper from our laboratory. The SRSs are clinically similar to the seizures induced by kindling the amygdala and we suggested that the amygdala plays a role in initiating the SRSs. Accordingly, the present paper examines the effect of amygdaloid lesions on intrahippocampal KA-treated rats. There were short- and long-term effects. (1) Short-term: the toxicity of KA was increased in lesioned animals. Status epilepticus followed by death of the animals was evoked with half of the dose required to cause the same effect in intact rats. Moreover, a gross haematuria was encountered 6–12 h after KA injection. This was not observed in non-lesioned rats even following the highest KA doses. (2) Long-term: amygdaloid lesions delayed the occurrence of the SRSs, reduced their incidence and modified their expression. In lesioned animals seizures has began with a period of tonic immobility with no sign of the masticatory movements seen in intact animals. Histological examination of the KA-induced lesions did not show any major differences between lesioned and intact animals. It is suggested that the short-term effects are due to an unspecific effect on homeostatic mechanisms, whereas the long-term ones reflect a specific involvement of the amygdala in the late appearing seizures.     

IL-1β Induction and IL-6 Suppression Are Associated with Aggravated Neuronal Damage in a Lipopolysaccharide-Pretreated Kainic Acid-Induced Rat Pup Seizure Model

Objectives: Reportedly, hippocampal neuronal degeneration by kainic acid (KA)-induced seizures in rats <14 days old was enhanced by lipopolysaccharide (LPS). This study was to test the hypothesis that cytokines such as interleukin (IL)-1β, IL-6 and tumor necrosis factor-α are associated with aggravated neuronal damage. Materials and Methods: Sixty male Sprague-Dawley, 14-day-old rats were used. Experiments were conducted in saline, LPS + saline, saline + KA and LPS + KA groups. Intraperitoneal LPS injections (0.04 mg/kg) were administered 3 h prior to KA injection (3 mg/kg). Results: The LPS + KA group showed a tendency toward shorter latency to seizure onset (p = 0.086) and significantly longer seizure duration (p < 0.05) compared with the KA group. Induction of the proconvulsant cytokine IL-1β in rat pup brains was significantly greater in the LPS + KA group compared to the KA group (38.8 ± 5.5 vs. 9.2 ± 1.0 pg/μg; p < 0.05); however, IL-6 levels were higher in the KA group than in the LPS + KA group (108.7 ± 6.8 vs. 60.9 ± 4.7 pg/μg; p < 0.05). The difference in tumor necrosis factor-α between the LPS + KA group and the KA group was insignificant (12.1 ± 0.6 vs. 10.9 ± 2.3 pg/μg; p = 0.64). Conclusions: Our results showed an increase in the proconvulsant cytokine IL-1β and a decrease in a potentially neuroprotective cytokine, IL-6, in rat pups treated with LPS + KA. These results warrant further investigation into the possible role of IL-1β induction and IL-6 suppression in LPS-promoted neuronal damage.     

Kainic acid-induced seizures cause neuronal death in infant rats pretreated with lipopolysaccharide

A major controversy in human epilepsy is whether severe seizures in infants or young children cause brain damage and subsequent epilepsy. Kainic acid (KA) produces severe seizures in infant rats, but hippocampal neuronal death and mossy fibre sprouting have not been previously demonstrated. There are similarities between lipopolysaccharide (LPS) pretreatment and KA-induced seizures in rats and the febrile convulsion of young children, in that both processes are associated with an immune stimulus and seizures. Infant rats, co-treated with LPS and KA, showed hippocampal neuronal death and mossy fibre sprouting. Taken together, our results suggest that severe febrile convulsion of young children may cause hippocampal damage and synaptic reorganization.     

Coactivation of GABA receptors inhibits the JNK3 apoptotic pathway via disassembly of GluR6‐PSD‐95‐MLK3 signaling module in KA‐induced seizure

Purpose: Past work has demonstrated that kainic acid (KA)–induced seizures could cause the enhancement of excitation and lead to neuronal death in rat hippocampus. To counteract such an imbalance between excitation and inhibition, we designed experiments by activating the inhibitory γ‐aminobutyric acid (GABA) receptor to investigate whether such activation suppresses the excitatory glutamate signaling induced by KA and to elucidate the underlying molecular mechanisms. Methods: Muscimol coapplied with baclofen was intraperitoneally administrated to the rats 40 min before KA injection by intracerebroventricular infusion. Subsequently we used a series of methods including immunoprecipitation, immunoblotting, histologic analysis, and immunohistochemistry to analyze the interaction, expression, and phosphorylation of relevant proteins as well as the survival of the CA1/CA3 pyramidal neurons. Results: Coadministration of muscimol and baclofen exerted neuroprotection against neuron death induced by KA; inhibited the increased assembly of the GluR6‐PSD‐95‐MLK3 module induced by KA; and suppressed the activation of MLK3, MKK7, and JNK3. Discussion: Taken together, we demonstrate that coactivation of the inhibitory GABA receptors can attenuate the excitatory JNK3 apoptotic signaling pathway via inhibiting the increased assembly of the GluR6‐PSD‐95‐MLK3 signaling module induced by KA. This provides a new insight into the therapeutic approach to epileptic seizure.     

Prolonged neonatal seizures exacerbate hypoxic-ischemic brain damage: correlation with cerebral energy metabolism and excitatory amino acid release

BACKGROUND: Perinatal hypoxia-ischemia (HI) is the most common precipitant of seizures in the first 24-48 h of a newborn's life. In a previous study, our laboratory developed a model of prolonged, continuous electrographic seizures in 10-day-old rat pups using kainic acid (KA) as a proconvulsant. Groups of animals included those receiving only KA, or HI for 15 or 30 min, followed by KA infusion. Our results showed that prolonged electrographic seizures following 30 min of HI resulted in a marked exacerbation of brain damage. We have undertaken studies to determine alterations in hippocampal high-energy phosphate reserves and the extracellular release of hippocampal amino acids in an attempt to ascertain the underlying mechanisms responsible for the damage promoted by the combination of HI and KA seizures. METHODS: All studies were performed on 10-day-old rats. Five groups were identified: (1) group I--KA alone, (2) group II--15 min of HI plus KA, (3) group III--15 min of HI alone, (4) group IV--30 min of HI plus KA, and (5) group VI--30 min of HI alone. HI was induced by right common carotid artery ligation and exposure to 8% oxygen/balance nitrogen. Glycolytic intermediates and high-energy phosphates were measured. Prior to treatment, at the end of HI (both 15 and 30 min), prior to KA injection, and at 1 (onset of seizures), 3, 5 (end of seizures), 7, 24 and 48 h, blood samples were taken for glucose, lactate and beta-hydroxybutyrate. At the same time points, animals were sacrificed by decapitation and brains were rapidly frozen for subsequent dissection of the hippocampus and measurement of glucose, lactate, beta-hydroxybutyrate, adenosine triphosphate (ATP) and phosphocreatine (PCr). In separate groups of rats as defined above, microdialysis probes (CMA) were stereotactically implanted into the CA2-3 region of the ipsilateral hippocampus for measurement of extracellular amino acid release. Dialysate was collected prior to any treatment, at the end of HI (15 and 30 min), prior to KA injection, and at 1 (onset of seizures), 3, 5 (end of seizures), 7 and 9 h. Determination of glutamate, serine, glutamine, glycine, taurine, alanine, and GABA was accomplished using high-performance liquid chromatography with EC detection. RESULTS: Blood and hippocampal glucose concentrations in all groups receiving KA were significantly lower than control during seizures (p < 0.05). beta-Hydroxybutyrate values displayed the inverse, in that values were significantly higher (p < 0.01) in all KA groups compared with pretreatment controls during seizure activity. Values returned to control by 2 h following the cessation of seizures. Lactate concentrations in brain and blood mimicked those of beta-hydroxybutyrate. ATP values declined to 0.36 mmol/l in both the 15 and 30 min hypoxia groups compared with 1.85 mmol/l for controls (p < 0.01). During seizures, ATP and PCr values declined significantly below their homologous controls. Following seizures, ATP values only for those animals receiving KA plus HI for 30 min remained below their homologous controls for at least 24 h. Determination of amino acid release revealed elevations of glutamate, glycine, taurine, alanine and GABA above pretreatment control during HI, with a return to normal prior to KA injections. During seizures and for the 4 h of recovery monitored, only glutamate in the combined HI and KA group rose significantly above both the 15 min of HI plus KA and the KA alone group (p < 0.05). CONCLUSION: Under circumstances in which there is a protracted depletion of high-energy phosphate reserves, as occurs with a combination of HI- and KA-induced seizures, excess amounts of glutamate become toxic to the brain. The latter may account for the exacerbation of damage to the newborn hippocampus, and serve as a target for future therapeutic intervention.