Temporal Changes in Proton MRS Metabolites After Kainic Acid-Induced Seizures in Rat Brain

Summary: Purpose : In situ ¹H-magnetic resonance spectroscopy (MRS) was used to study temporal metabolic changes in a rat model of temporal lobe epilepsy (TLE) by using kainic acid (KA).
Methods: Rat brains were scanned at the level of the hippocampal body for MRS measurements. Relative ratios of N- acetyl groups (NA: N-acetylaspartate and N-acetylaspartyl glutamate), choline, and lactate (Lac) over creatine (Cr) were calculated.
Results: NA/Cr ratios increased significantly during the ictal phase. During the postictal and interictal phases, the NA/Cr ratio decreased. There was a significant and prolonged increase of the lactate/Cr ratio in the hippocampi of rats that started 1 h after the onset of KA-induced seizure activity and persisted up to 24 h after the injection. The prolonged lactate/Cr increase in an area susceptible to neuronal damage (e.g., hippocampus) correlated with the onset of seizure activity but remained elevated thereafter.
Conclusions: The ictal and early postictal increase in lactate ratios may reflect increased cellular activity and metabolism resulting from KA excitotoxicity. Assuming that the changes in NA/Cr ratios are due to NAA increase, we speculate that an activation of the N-acetylaspartylglutamate (NAAG) dipeptidase pathway may explain the ictal increase in NA/Cr ratios. The late postictal decrease in NNCr ratios is a reflection of KA-induced neuronal cell loss.     

Protective effect of topiramate on kainic acid–induced cell death in mice hippocampus

The protective effect of topiramate (TPM) on seizure-induced neuronal injury is well known; however, its molecular basis has yet to be elucidated. We investigated the effect and signaling mediators of TPM on seizure-induced hippocampal cell death in kainic acid (KA)-treated ICR mice. KA-induced hippocampal cell death was identified by terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling. Immunoreactivity (IR) of p-Erk, p-Jnk, p-P38, and caspase-3, and caspase-3 activity were observed in the hippocampal region 3 h after KA (0.1 μg/5 μL, i.c.v.) administration, and/or TPM (100 mg/kg, i.p.) pretreatment. TPM attenuated seizure-induced neuronal cell death and reduced KA-induced p-Erk IR in the CA3 region of the hippocampus, but did not affect p-Jnk and p-P38. In addition, TPM reduced caspase-3 IR and activation by KA. KA-induced seizures were also suppressed by TPM pretreatment. TPM inhibits seizures, and decreases Erk phosphorylation and caspase-3 activation by KA, thereby contributing to protection from neuronal injury.     

Protective effect of topiramate on kainic acid-induced cell death in mice hippocampus.

The protective effect of topiramate (TPM) on seizure-induced neuronal injury is well known; however, its molecular basis has yet to be elucidated. We investigated the effect and signaling mediators of TPM on seizure-induced hippocampal cell death in kainic acid (KA)-treated ICR mice. KA-induced hippocampal cell death was identified by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. Immunoreactivity (IR) of p-Erk, p-Jnk, p-P38, and caspase-3, and caspase-3 activity were observed in the hippocampal region 3 h after KA (0.1 microg/5 microL, i.c.v.) administration, and/or TPM (100 mg/kg, i.p.) pretreatment. TPM attenuated seizure-induced neuronal cell death and reduced KA-induced p-Erk IR in the CA3 region of the hippocampus, but did not affect p-Jnk and p-P38. In addition, TPM reduced caspase-3 IR and activation by KA. KA-induced seizures were also suppressed by TPM pretreatment. TPM inhibits seizures, and decreases Erk phosphorylation and caspase-3 activation by KA, thereby contributing to protection from neuronal injury.     

Cycloheximide inhibits neurotoxic responses induced by kainic acid in mice

In the present study, we examined the effect of cycloheximide on various pharmacological responses induced by kainic acid (KA) administered intracerebroventricularly (i.c.v.) in mice. In a passive avoidance test, a 20-min cycloheximide (200mg/kg, i.p.) pretreatment prevented the memory impairment induced by KA. The morphological damage induced by KA (0.1microg) in the hippocampus was markedly concentrated in the CA3 pyramidal neurons and cycloheximide effectively prevented the KA-induced pyramidal cell death in CA3 hippocampal region. In immunohistochemical study, KA dramatically increased the phosphorylation of extracellular signal-regulated protein kinase (p-ERK), c-Jun N-terminal kinase 1 (p-JNK1), and calcium/calmodulin-dependent protein kinase II (p-CaMK II). Cycloheximide attenuated the increased p-ERK, p-JNK1, and p-CaMK II levels induced by KA. Furthermore, cycloheximide inhibited the increased c-Fos and c-Jun protein expression levels induced by KA in the hippocampus. The activation of microglia was detected in KA-induced CA3 cell death region by immunostaining with a monoclonal antibody against the OX-42. Cycloheximide inhibited KA-induced increase of OX-42 immunoreactivity. Our results suggest that the increased expression of the c-Fos, c-Jun, and phosphorylation of ERK, JNK1, and CaMK II proteins may play important roles in the memory impairment and the cell death in CA3 region of the hippocampus induced by i.c.v. KA administration in mice. Furthermore, the activated microglia may be related to phagocytosis of degenerated neuronal elements induced by KA.     

Resistance of immature hippocampus to morphologic and physiologic alterations following status epilepticus or kindling.

Seizures in adult rats result in long-term deficits in learning and memory, as well as an enhanced susceptibility to further seizures. In contrast, fewer lasting changes have been found following seizures in rats younger than 20 days old. This age-dependency could be due to differing amounts of hippocampal neuronal damage produced by seizures at different ages. To determine if there is an early developmental resistance to seizure-induced hippocampal damage, we compared the effects of kainic acid (KA)-induced status epilepticus and amygdala kindling on hippocampal dentate gyrus anatomy and electrophysiology, in immature (16 day old) and adult rats. In adult rats, KA status epilepticus resulted in numerous silver-stained degenerating dentate hilar neurons, pyramidal cells in fields CA1 and CA3, and marked numerical reductions in CA3c pyramidal neuron counts (-57%) in separate rats. Two weeks following the last kindled seizure, some, but significantly less, CA3c pyramidal cell loss was observed (-26%). Both KA status epilepticus and kindling in duced mossy-fiber sprouting, as evidenced by ectopic Timm staining in supragranular layers of the dentate gyrus. In hippocampal slices from adult rats, paired-pulse stimulation of perforant path axons revealed a persistent enhancement of dentate granule-cell inhibition following KA status epilepticus or kindling. While seizures induced by KA or kindling in 16-day-old rats were typically more severe than in adults, the immature hippocampus exhibited markedly less KA-induced cell loss (-22%), no kindling-induced loss, no detectable synaptic rearrangement, and no change in dentate inhibition. These results demonstrate that, in immature rats, neither severe KA-induced seizures nor repeated kindled seizures produce the kind of hippocampal damage and changes associated with even less severe seizures in adults. The lesser magnitude of seizure-induced hippocampal alterations in immature rats may explain their greater resistance to long-term effects of seizures on neuronal function, as well as future seizure susceptibility. Conversely, hippocampal neuron loss and altered synaptic physiology in adults may contribute to increased sensitivity to epileptogenic stimuli, spontaneous seizures, and behavioral deficits.     

Effect of theophylline and trimethobenzamide when given during kainate-induced status epilepticus: an improved histopathologic rat model of human hippocampal sclerosis

PURPOSE: The most common pathology in temporal lobe epilepsy (TLE) is hippocampal sclerosis. It is controversial whether status epilepticus (SE) or prolonged seizures plus secondary cerebral injuries are pathogenic mechanisms of hippocampal sclerosis. This study addressed this question in rat models of TLE. METHODS: Hippocampal neuron densities and supragranular mossy fiber sprouting were determined in adult rats subjected to systemic kainate-induced SE (KA-only) and KA-induced SE followed 75 minutes later by theophylline (KA/Theo) or trimethobenzamide (KA/Tri). These drugs probably decrease seizure-induced cerebral hyperemia or hypertension. RESULTS: Compared with controls and KA-only rats, KA/Tri and KA/Theo rats showed decreased CA3b and CA1 neuron densities (i.e., greater Sommer's sector injury). In addition, KA/Tri rats showed that increased trimethobenzamide dosages were associated with decreased hilar, CA3c, CA3b, CA1, and subiculum neuron densities. There were no significant differences in supragranular mossy fiber sprouting between KA-only, KA/Tri, and KA/Theo rats. CONCLUSIONS: Pharmacologic manipulations during KA-induced SE are associated with differences in hippocampal pathology, especially in Sommer's sector, and the final pattern of damage and axon sprouting shows histopathologic similarities to that in patients with hippocampal sclerosis. Our findings support the hypothesis that secondary physiologic insults during SE that are likely to decrease seizure-induced cerebral hyperemia and hypertension may generate greater hippocampal neuronal injury compared with SE alone, and this may be a pathogenic mechanism of human hippocampal sclerosis in patients with TLE.     

NBQX or topiramate treatment after perinatal hypoxia-induced seizures prevents later increases in seizure-induced neuronal injury

PURPOSE: To evaluate the efficacy of NBQX (2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f) quinoxaline-2,3-dione) and topiramate (TPM) given after hypoxia-induced seizures in preventing the delayed effect of hypoxia on subsequent susceptibility to seizures and neuronal injury. METHODS: We used "two-hit" rodent seizure model to study the long-term effect of perinatal hypoxia on later kainate (KA) seizure-induced neuronal damage and investigated the therapeutic efficacy of a postseizure treatment protocol in reversing the conditioning effect of early-life seizures. RESULTS: Hypoxia at P10 induces seizures without cell death but causes an increase in susceptibility to second seizures induced by KA as early as 96 h after hypoxia, and this lowered seizure threshold persists to adulthood. Furthermore, perinatal hypoxia increases KA-induced neuronal injury at postnatal day (P)21 and 28/30. Repeated doses of NBQX (20 mg/kg) or TPM (30 mg/kg) given for 48 h after hypoxia-induced seizures prevent the increase in susceptibility to KA seizure-induced hippocampal neuronal injury at P28/30. CONCLUSIONS: Our results suggest that alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor blockade after hypoxia prevents the priming effect of perinatal hypoxia-induced seizures and that this protection occurs independent of its anticonvulsant action.     

Effects of nitric oxide synthase inhibition on the cerebral circulation and brain damage during kainic acid-induced seizures in newborn rabbits.

The nitric oxide (NO) synthase inhibitor, N-omega-nitro-L-arginine methyl ester (L-NAME), was used to investigate the effect of endogenous NO on the cerebral circulation and brain damage during kainic acid (KA)-induced seizures in newborn rabbits. The cerebral blood flow (CBF), by laser doppler flowmetry, cerebral oxygenation (concentrations of oxy-(HbO2), deoxy-(HbR) and total hemoglobin (tHb) in brain tissue), by near-infrared spectroscopy (NIRS), mean arterial blood pressure (MABP), electroencephalography (EEG), and hippocampal neuronal damage were evaluated. Pretreatment with L-NAME caused significant decreases in CBF, HbO2, and tHb, and a significant increase in HbR during KA-induced seizures, compared with pretreatment with saline (P < 0.05), without a significant difference in MABP. Our study also demonstrated that pretreatment with L-NAME reduced the seizure activity and neuronal cell death in the hippocampus elicited by the systemic administration of KA in the neonatal brain. These results suggest that NO is of major importance in the neurodestructive process in spite of its roles in maintaining both the CBF and cerebral oxygenation during KA-induced seizures in the neonatal brain.     

[31P]/[1H] Nuclear Magnetic Resonance Study of Mitigating Effects of GYKI 52466 on Kainate-Induced Metabolic Impairment in Perfused Rat Cerebrocortical Slices

Summary: Purpose: Kainic acid (KA) has long been used in experimental animals to induce status epilepticus (SE). A mechanistic implication of this is the association between ex-citotoxicity and brain damage during or after SE. We evaluated KA-induced metabolic impairment and the potential mitigating effects of GYKI 52466 [1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine] in superfused rat cerebral cortical slices. Methods: Interleaved [³¹P]/[¹H] magnetic resonance spectroscopy (MRS) was used to assess energy metabolism, intra-cellular pH (pH_i), N-acetyl-L-aspartate (NAA) level, and lactate (Lac) formation before, during, and after a 56-min exposure to 4 mM KA in freshly oxygenated artificial cerebrospinal fluid (OXY-ACSF). Results: In the absence of GYKI 52466 and during the KA exposure, NAA, PCr, and ATP levels were decreased to 91.1 ± 0.8, 62.4 ± 3.9, and 59.1 ± 4.3% of the control, respectively; Lac was increased to 118.2 ± 2.1%, and pH, was reduced from 7.27 ± 0.02 to 7.13 2 0.02. During 4-h recovery with KA-free ACSF, pH_i rapidly and Lac gradually recovered, NAA decreased further to 85.5 ± 0.3%, and PCr and AW showed little recovery. Removal of Mg²⁺ from ACSF during KA exposure caused a more profound Lac increase (to 147.1 ± 4.0%) during KA exposure and a further NAA decrease (to 80.4 ± 0.5%) during reperfusion, but did not exacerbate PCr, ATP, and pH, changes. Inclusion of 100 μM GYKI 52466 during KA exposure significantly improved energy metabolism: the PCr and ATP levels were above 76.6 ± 2.1 and 82.0 & 2.9% of the control, respectively, during KA exposure and recovered to 101.4 ± 2.4 and 95.0 ± 2.4%, respectively, during reperfusion. NAA level remained at 99.8 ± 0.6% during exposure and decreased only slightly at a later stage of reperfusion. Conclusions: Our finding supports the notion that KA-induced SE causes metabolic disturbance and neuronal injury mainly by overexcitation through non-N-methy1-D-aspartate (NMDA) receptor functions.     

Proton Magnetic Resonance Spectroscopic Imaging in Patients with Extratemporal Epilepsy

Summary: Purpose: Reduced levels of N-acetylaspartate (NAA) in temporal lobes responsible for temporal lobe epilepsy have been observed consistently in proton magnetic resonance spectroscopy (MRS) studies.
Methods: We investigated the potential of proton MRS to detect low NAA outside of the temporal lobes in patients with non-lesional partial extratemporal epilepsy. Proton MR spectroscopic imaging (MRSI) data of both frontal lobes and central/postcentral regions were obtained in 20 such patients and 16 normal control subjects. The epileptogenic region was determined by an extensive clinical-EEG investigation, including the recording of habitual seizures in each patient, and intracranial EEG recordings in 10 patients.
Results: The relative NAA resonance intensities (i.e., NAN/phosphocreatine plus creatine (CR_t), NAN/choline-containing metabolites (Cho_t) and NAA/Cr_t+ Cho_t), were all significantly reduced throughout the spectroscopic image as compared with that of the controls. Furthermore, reduction of the NAA ratios was greater in the epileptogenic region as compared with the nonepileptogenic regions, on EEG investigation.
Conclusions: In vivo proton MRSI of patients with nonlesional partial extratemporal epilepsy detected evidence of widespread neuronal damage or dysfunction that was greatest in the region of seizure focus.     

Inhibition of delayed induction of p38 mitogen-activated protein kinase attenuates kainic acid-induced neuronal loss in the hippocampus

The activation of p38 mitogen-activated protein kinase (MAPK) has been implicated in the pathological changes accompanying inflammatory and apoptotic processes of various cell types including neurons. In a kainic acid (KA)-induced mouse seizure model, p38 MAPK is induced in reactive astrocytes in the CA3 region of the hippocampus where severe neuronal loss occurs. Here we report the delayed and protracted activation of p38 MAPK in the CA3 region of the hippocampus of mice treated with KA. In this model, the inhibition of p38 MAPK isoforms by SB203580, a specific inhibitor, attenuated neuronal loss in the CA3 and CA1 regions of the hippocampus, which was accompanied by the suppression of the p38 MAPK activation as well as astrogliosis. Thus, the delayed and sustained induction of p38 MAPK plays a crucial role in the neuronal damage of KA-induced brain seizures.     

Hypoxic preconditioning attenuated in kainic acid-induced neurotoxicity in rat hippocampus

The neuroprotective effect of hypoxic preconditioning on kainate (KA)-induced neurotoxicity, including apoptosis and necrosis, was investigated in rat hippocampus. Female Wistar-Kyoto rats were subjected to 380 mm Hg in an altitude chamber for 15 h/day for 28 days. Intrahippocampal infusion of KA was performed in chloral hydrate anesthetized rats, which acutely elevated 2,3-dihydroxybenzoic acid levels in normoxic rats. Seven days after the infusion, KA increased lipid peroxidation in the infused hippocampus and resulted in hippocampal CA3 neuronal loss. A 4-week hypoxic preconditioning attenuated KA-induced elevation in hydroxyl radical formation and lipid peroxidation as well as KA-induced neuronal loss. The effects of hypoxic preconditioning on KA-induced apoptosis and necrosis were investigated further. Two hours after KA infusion, cytosolic cytochrome c content was increased in the infused hippocampus. Twenty-four hours after KA infusion, pyknotic nuclei, cellular shrinkage, and cytoplasmic disintegration, but not TUNEL-positive staining, were observed in the CA3 region of hippocampus. Forty-eight hours after KA infusion, both DNA smear and DNA fragmentation were demonstrated in the infused hippocampus. Furthermore, TUNEL-positive cells, indicative of apoptosis, in the infused hippocampus were detected 72 h after KA infusion. Hypoxic pretreatment significantly reduced necrotic-like events in the KA-infused hippocampus. Moreover, hypoxic preconditioning attenuated apoptosis induced by KA infusion, including elevation in cytosolic cytochrome c content, TUNEL-positive cells, and DNA fragmentation. Our data suggest that hypoxic preconditioning may exert its neuroprotection of KA-induced oxidative injuries via attenuating both apoptosis and necrosis in rat hippocampus.     

Effect of the calcineurin inhibitor FK506 on K+–Cl? cotransporter 2 expression in the mouse hippocampus after kainic acid-induced status epilepticus

Calcineurin (CaN)-mediated excitotoxicity impairs γ-aminobutyric acid (GABA) transmission and induces neuronal apoptosis. Ca(2+)-dependent K(+)-Cl(-) cotransporter 2 (KCC2) participates in GABAergic inhibitory transmission. However, the mechanism by which CaN mediates GABA receptor-mediated KCC2 in seizures is not fully understood. In the present study, we investigated the altered expression of KCC2 and the effects of the CaN inhibitor FK506 on KCC2 expression in the mouse hippocampus following kainic acid (KA) treatment. FK506 was injected twice 24 h and 30 min before KA treatment and then mice were treated with KA and killed 2 days later. FK506 had anticonvulsant effect on KA-induced seizure activities. CaN cleavage was evident in the hippocampus 24 h after KA treatment. FK506 pretreatment blocked the truncation of CaN in the KA-treated hippocampus. Cresyl violet and TUNEL staining showed that FK506 prevented KA-induced hippocampal cell death. In particular, Western blot analysis showed that KCC2 expression was time dependent, with a peak at 6 h and a return to decreased levels at 48 h, whereas FK506 pretreatment inhibited the KA-induced decrease in KCC2 expression in the hippocampus. Immunofluorescence showed that FK506 pretreatment protected the loss of inhibitory GABAergic KCC2-expressing neurons following KA treatment. Taken together, these results provide evidence that altered KCC2 expression may be associated with Ca(2+)-mediated seizure activity and indicate that neuron-specific KCC2 may be involved in neuroprotection after seizures.     

急性热应激与热惊厥幼龄大鼠海马ZnT3 mRNA表达和代谢变化的1H-MRS波谱研究

目的 探讨氢质子磁共振波谱分析(1H-MRS)结合神经病理手段对急性热应激(HS)和热惊厥(FC)后脑神经元代谢和损伤的早期检测价值.方法 采用热水浴诱导21 d龄大鼠FC模型,应用1H-MRS检测HS和FC后脑内N-乙酰天门冬氨酸(NAA)、复合胆碱(Cho)、肌酸(Cr)和乳酸(Lac)含量的变化,原位杂交检测海马锌离子转运体3(ZnT3)mRNA表达.结果 MRS检测结果显示对照组、HS组与FC组NAA/Cr比值分别为1.50±0.42,1.57±0.50和1.61±0.37,Cho/Cr比值分别为0.93±0.27,1.13±0.17和1.28±0.31,各组间差异均无统计学意义(P0.05);Lac/Cr仅见于FC组,为0.64±0.23.FC组海马齿状回检测到ZnT3 mRNA表达.结论 Lac波峰是惊厥性神经元损伤的特征性1H-MRS变化;ZnT3与海马苔藓纤维再生性发芽密切相关;单次短暂的FC发作也能使脑神经元早期发生不同于HS的显著的神经活性物质表达和物质代谢的变化,对脑神经元造成一定的损伤.     

Seizure activity-induced changes in polyamine metabolism and neuronal pathology during the postnatal period in rat brain.

Systemic injection of kainic acid (KA) does not cause neuronal pathology in limbic structures in rat brain prior to postnatal day (PND) 21. The present study tested if the development of the pathogenic response is associated with the maturation of a link between seizure activity and polyamine metabolism. Pathology was assessed with histological techniques and with the binding of [3H]Ro5-4864, a ligand for the peripheral type benzodiazepine binding sites (PTBBS), a marker of glial cell proliferation. In agreement with previous results, peripherally administered kainate at doses sufficient to induce intense behavioral seizures produced a loss of Nissl staining in hippocampus after PND 21 but not at earlier ages. The pattern of neuronal damage observed after PND 21 resembled that found in adult animals: extensive losses of Nissl staining in area CA3 of hippocampus and in piriform cortex, more modest effects in CA1 and sparing of the granule cells of the dentate gyrus. Similarly, no increase in [3H]Ro5-4864 binding as a result of KA administration was observed in hippocampus and piriform cortex until PND 21. Ornithine decarboxylase (ODC) activity and putrescine levels were high in the neonatal brain and decreased to reach adult values by PND 21. KA-induced seizure activity did not significantly alter both variables until PND 21. After PND 21, ODC activity and putrescine levels markedly increased 16 h after KA-induced seizure activity in hippocampus and piriform cortex. The magnitude of the effects increased between PND 21 and PND 30, at which point the changes in both parameters were comparable to those found in adults. Polyamines stimulate the activity of the calcium-dependent proteases calpain in brain fractions and may increase calpain-mediated proteolysis in situ. In accord with this, kainate-induced breakdown of spectrin, a preferred substrate of calpain, measured 16 h after KA injection followed a developmental curve parallel to that for kainate-induced increases in putrescine levels. These results indicate that the onset of vulnerability to seizure activity triggered by kainic acid is correlated with the development of an ODC/polyamine response to the seizures and further support a critical role for the ODC/polyamine pathway in neuronal pathology following a variety of insults.     

Differential NPY mRNA expression in granule cells and interneurons of the rat dentate gyrus after kainic acid injection

Using in situ hybridization histochemistry neuropeptide Y (NPY) mRNA expression was investigated after intraperitoneal injection of kainic acid (KA) and after local application of KA or quinolinic acid into the dentate gyrus of the rat. Enhanced concentrations of NPY mRNA were observed in interneurons of the hilus, including presumptive fusiform neurons and pyramidal-shaped basket cells already 4 hours after initiation of limbic seizures by KA (10 mg/kg, i.p.). IncreaseD NPY expression persisted in neurons resistant to seizure-induced cell death (6–48 h after i.p. KA). Exceptionally high hybridization signals were found in interneurons of the hilus and the CA1 and CA3 sectors 8 months after KA-induced limbic seizures. In the granule cell layer only a transient but pronounced increase in NPY mRNA was observed 12–24 h after injection. Only moderate changes were observed in this cell layer at later intervals. Anticonvulsant treatment with thiopental, after a brief period of generalized seizures, prevented the increase in NPY mRNA in granule cells but not in interneurons. No change in NPY message was found also in granule cells of rats which responded with mild “wet dog shake” behvior but not with motor seizures to KA injection. Local injections of low doses of KA (0.05–0.2 nmol) or quinolinic acid (6.5–100 nmol) into the dentate gyrus of the hippocampus under deep thiopental anesthesia, after 24 h, resulted in increased concentrations of NPY message in interneurons of the ipsilateral, but not of the contralateral hilus and not in granule cells. Higher doses of the excitatory amino acid analogs caused partial neurodegeneration at the injection site, but enhanced NPY expression in interneurons of the contralateral dentate. Only the highest dose of quinolinic acid (100 nmol), resulting in general neuronal cell loss at the injection area, induced enhanced NPY mRNA expression also in granule cells of the contralateral dentate gyrus. The experiments suggest different mechanisms for NPY mRNA expression in interneurons and in granule cells of the dentate gyrus. Whereas in the stratum granulosum NPY mRNA expression was only observed after generalized limbic seizures, in hilar interneurons it was augmented by only moderate neuronal stimulation or directly by KA. © 1994 Wiley-Liss, Inc.     

Ketogenic diet reduces Smac/Diablo and cytochrome c release and attenuates neuronal death in a mouse model of limbic epilepsy

The ketogenic diet (KD) is effective in the treatment of refractory epilepsy, yet the molecular mechanisms underlying its antiepileptic effects have not been determined. There is increasing evidence that neuronal cell death induced by seizures via mitochondrial pathway and seizures can lead to mitochondrial release of cytochrome c, and we have shown previously that translocation of Smac/DIABLO into the cytosol play a role in the brain damage in a model of limbic seizure. In the present study, we explored the neuroprotective effect of KD in C57BL/6 mice with seizures induced by kainic acid (KA). Status epilepticus triggered by intra-amygdaloid microinjection of KA lead to neuronal death in the selective ipsilateral CA3 subfield of the hippocampus and mitochondrial release of Smac/DIABLO and cytochrome c. We found that KD significantly decreased neuronal death in the ipsilateral CA3 at 24h after KA-induced seizures. Furthermore, KD reduced Smac/DIABLO and cytochrome c release from mitochondria, attenuated activation of casepase-9 and caspase-3 following seizures. These results demonstrate that the neuroprotective effect of KD against brain injury induced by limbic seizures, at least partially, is associated with inhibition of mitochondrial release of Smac/DIABLO and cytochrome c.     

Inflammation contributes to seizure-induced hippocampal injury in the neonatal rat brain

Objective –  The extent of neuronal injury in the hippocampus produced by experimental status epilepticus (SE) is age dependent and is not readily demonstrable in many models of neonatal seizures. Neonatal seizures often occur in clinical settings that include an inflammatory component. We examined the potential contributory role of pre-existing inflammation as an important variable in mediating neuronal injury.
Materials and methods –  Postnatal day 7 (P7) and P14 rat pups were injected with lipopolysaccharide (LPS), 2 h prior to SE induced by lithium–pilocarpine (LiPC). Neuronal injury was assessed by well-described histologic methods.
Results –  While LPS by itself did not produce any discernible cell injury at either age, this treatment exacerbated hippocampal damage induced by LiPC–SE. The effect was highly selective for the CA1 subfield.
Conclusions –  Inflammation can contribute substantially to the vulnerability of immature hippocampus to seizure-induced neuronal injury. The combined effects of inflammation and prolonged seizures in early life may impact long-term outcomes of neonatal seizures.     

Mitochondrial dysfunction and ultrastructural damage in the hippocampus during kainic acid-induced status epilepticus in the rat

PURPOSE: Prolonged and continuous epileptic seizure (status epilepticus) results in cellular changes that lead to neuronal damage. We investigated whether these cellular changes entail mitochondrial dysfunction and ultrastructural damage in the hippocampus, by using a kainic acid (KA)-induced experimental status epilepticus model. METHODS: In Sprague-Dawley rats maintained under chloral hydrate anesthesia, KA (0.5 nmol) was microinjected unilaterally into the CA3 subfield of the hippocampus to induce seizure-like hippocampal EEG activity. The activity of key mitochondrial respiratory chain enzymes in the dentate gyrus (DG), or CA1 or CA3 subfield of the hippocampus was measured 30 or 180 min after application of KA. Ultrastructure of mitochondria in those three hippocampal subfields during KA-induced status epilepticus also was examined with electron microscopy. RESULTS: Microinjection of KA into the CA3 subfield of the hippocampus elicited progressive build-up of seizure-like hippocampal EEG activity. Enzyme assay revealed significant depression of the activity of nicotinamide adenine dinucleotide cytochrome c reductase (marker for Complexes I+III) in the DG, or CA1 or CA3 subfields 180 min after KA-elicited temporal lobe status epilepticus. Conversely, the activities of succinate cytochrome c reductase (marker for Complexes II+III) and cytochrome c oxidase (marker for Complex IV) remained unaltered. Discernible mitochondrial ultrastructural damage, varying from swelling to disruption of membrane integrity, also was observed in the hippocampus 180 min after hippocampal application of KA. CONCLUSIONS: Our results demonstrated that dysfunction of Complex I respiratory chain enzyme and mitochondrial ultrastructural damage in the hippocampus are associated with prolonged seizure during experimental temporal lobe status epilepticus.     

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.