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.     

Histamine H2-receptors participate in the formation of brain edema induced by kainic acid in rat thalamus

At 4 h after the intraperitoneal administration of kainic acid in a dose of 12 mg/kg, Evans blue extravasation was observed preferentially in the thalamus, accompanied by increases in the water and sodium contents and by a decrease in the potassium content. Subcutaneous pretreatment with a histamine H2-receptor blocking agent, ranitidine, in a dose of 5 mg/kg given 2 h before and at the time of kainic acid injection, partially decreased the edema formation in the thalamus. It is assumed that repetitive discharges evoked by the kainic acid result in the thalamus in an excessive release of histamine from internal (mast cell and neuronal) sources and that this leads to the activation of H2-receptor-coupled adenylate cyclase in the brain microvessels and to the induction of brain edema.     

Some mechanisms of brain edema studied in a kainic acid model

Kainic acid (KA) is a potent neuroexcitatory drug widely used in the experimental study of seizure activity. Subcutaneous injection of KA into rats (10 mg/kg in saline 10 mg/ml; pH 7.0) induced longlasting status epilepticus followed by damage of CNS tissue in the entorhinal/pyriform cortex and in the hippocampus. The studies covered by this report demonstrated the formation of cytotoxic brain edema characterized by massive swelling of perineuronal and perivascular astroglia with microcirculation disturbance after KA injection, resulting in parenchymal necrosis of the affected region; furthermore perivenous hemorrhages and necroses corresponding to herniation lesions of the brain appear. Tracer studies with Na-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. Treatment of brain edema with dexamethasone did not influence the incidence and severity of edematous brain damage. Treatment with mannitol, however, completely prevented the lesion in 54% of animals injected with KA. The present results indicate that brain edema plays an important role in the pathogenesis of epileptic brain damage following systemic KA intoxication. It is suggested that in this model brain edema develops due to massive ionic imbalance caused by KA induced persistent neuronal excitation. In addition the model demonstrates the possible pathogenetic role of selective astrocytic swelling in the production of local hippocampal ischemia followed by herniation and its sequels. Such pathology originating from astrocytes probably may occur also in closed brain injury.     

STIM1在KA致痫大鼠大脑皮质内的表达上调

目的研究基质相互作用分子1(stromal interaction molecule l,STIM1)在海人酸(Kainic acid,KA)致痫大鼠大脑皮质中的表达变化,探讨其与癫痫发病的关系及意义。方法 48只成年雄性SD大鼠随机分为正常对照组(NS组)24只和海人酸致痫组(KA组)24只,海人酸致痫组给予侧脑室注射KA 2μg/kg(7μl左右)制作KA致痫模型,正常对照组给予侧脑室注射等量生理盐水,观察记录各组大鼠癫痫发作的行为学表现,并于癫痫发作后不同时间点(48h、72h)随机取12只大鼠伤侧皮层脑组织,用半定量RT-PCR、Western blot和免疫组织化学技术检测STIM1的mRNA和蛋白的表达。结果 KA致痫组大鼠于造模后5min左右出现典型痫性发作,达Ⅳ~Ⅴ级,持续数小时;海人酸致痫组STIM1mRNA和蛋白表达水平在各时间点均较对照组显著增高(P0.05)。结论 STIM1在KA致痫大鼠皮层脑组织中过表达可能参与癫痫的发病机制。     

Regionally specific and rapid increases in brain-derived neurotrophic factor messenger RNA in the adult rat brain following seizures induced by systemic administration of kainic acid.

In situ hybridization techniques were used to analyse the spatiotemporal pattern of brain-derived neurotrophic factor messenger RNA elevation associated with kainic acid-induced seizure activity in the rat. Pronounced increases in hippocampal brain-derived neurotrophic factor messenger RNA levels were observed as early as 30 min following the onset of behavioral seizures. The greatest increase (10-fold) occurred in the dentate granule cell layer, while pyramidal layers CA1, CA3, and CA4 exhibited increases of two- to six-fold. Peak elevation of brain-derived neurotrophic factor messenger RNA in CA1 hippocampal region was evident at 4 h in CA3, and in the dentate granule layer at 30 min postseizure. Elevations persisted in the dentate and hilar regions to four days, while the increases in CA1 and CA3 returned to control levels by 16 h following seizure. Significant increases in brain-derived neurotrophic factor messenger RNA were also observed in the superficial layers of cortex (II and III) and in the piriform cortex which reached peak elevations by 8 h. No detectable changes were observed in the dorsomedial thalamus. Although histologically defined pyramidal and granule cell layers displayed relatively uniform increases in brain-derived neurotrophic factor messenger RNA in response to kainate, a closer examination of the labeling patterns using emulsion autoradiography revealed discrete areas of high grain densities overlapping uniform, moderate hybridization densities in the dentate granule cell layer and CA3, suggesting that the capacity to upregulate brain-derived neurotrophic factor messenger RNA in these regions may differ among individual neurons. In conclusion, our studies revealed that brain-derived neurotrophic factor messenger RNA induction in response to systemic kainate administration differs in hippocampal and cortical areas, in magnitude, time of onset and duration. The observed temperospatial pattern does not correspond in a simple way to increases in metabolic or electrical activity associated with seizures or neuronal vulnerability coincident with the seizures.     

Kynurenic acid blocks neurotoxicity and seizures induced in rats by the related brain metabolite quinolinic acid

Kynurenic acid (KYNA) was tested as an antagonist of the neurotoxic and epileptogenic effects of the metabolically related brain constituent quinolinic acid (QUIN). In the rat striatum, KYNA blocked the neurotoxic effects of QUIN in preference to those of other excitotoxins. In the hippocampus, KYNA antagonized both the neurodegeneration and seizures caused by the local application of QUIN. These properties of KYNA raise the possibility of a functional link between KYNA and QUIN in the brain which may be of relevance for an understanding of human neurodegenerative disorders.     

Effects of D-(-)-aminophosphonovalerate on behavioral and histological changes induced by systemic kainic acid

In rats, the seizures induced by systemic kainic acid (KA) are followed by extensive neuronal damage, notably in the hippocampal region. We report that the specific N-methyl-D-aspartate (NMDA) receptor antagonist, D-(-)-aminophosphonovalerate (D(-)APV), given i.c.v. prior to or 2 h after i.p. KA injection markedly protected CA1 but not other hippocampal neurons against degeneration. In contrast, D(-)APV had no effect on KA-induced wet dog shakes or on behavioral seizures. We conclude that NMDA receptors participate in the neurotoxic but not in the behavioral effects of systemic KA.     

油酸引起肺水肿的机制探讨

作者从三个不同层次(整体肺、离体肺及肺血管内皮细胞)的肺损伤模型证实了油酸引起的急性肺水肿中有氧自由基参与,来自嘌呤氧化酶的氧自由基起着原发始动作用,而来自嗜中性白细胞的氧自由基起着继发和扩大损伤的作用。     

红藻氨酸诱导PC12细胞凋亡及阿魏酸对神经元的保护作用

 目的： 探讨阿魏酸(ferulic acid, FA)对红藻氨酸(kainic acid, KA)诱导的PC12细胞凋亡的作用及其机制。方法：采用50 μmol/L KA诱导PC12细胞凋亡建立阿尔茨海默病神经细胞模型,然后将处理后的PC12细胞分为KA模型组和KA＋FA (25、50和100 μmol/L)处理的低、中、高剂量组,同时设立正常对照组。采用MTT比色法检测PC12细胞的存活率;采用免疫细胞化学法观察PC12细胞中凋亡蛋白Bcl-2、Bax和细胞色素C (Cyt C)的表达;annexin Ⅴ+PI双染流式细胞术检测PC12的细胞凋亡率;蛋白免疫印记技术检测PC12细胞中Bcl-2、Bax和Cyt C的表达水平。结果：MTT法和免疫细胞化学检测显示,与正常组相比,模型组PC12细胞的存活率明显下降,且细胞中Bcl-2表达减少(P<0.01),而Bax和Cyt C表达升高,Bcl-2/Bax比值下降(P<0.01),流式细胞术检测细胞的凋亡率显示,模型组细胞的凋亡率显著上升(P<0.01)。蛋白印迹术检测显示,模型组细胞中Bcl-2表达量减少,Bax和Cyt C表达量升高,与正常组比较差异显著(均P<0.01)。当采用FA干预后,与模型组相比,25、50和100 μmol/L组细胞的存活率明显上升,细胞凋亡率减少,而且能增加Bcl-2阳性百分率和表达水平,明显减少Bax和Cyt C阳性百分率和表达水平,使Bcl-2/Bax比值增加 (P<0.05或P<0.01)。结论：KA在50 μmol/L时可明显诱导PC12发生凋亡,FA在25～100 μmol/L时能显著抑制KA诱导的PC12细胞凋亡,其神经保护机制可能是通过抑制Bax和Cyt C的表达,升高Bcl-2表达和Bcl-2/Bax比值,从而阻断内源性细胞凋亡通路而提高神经细胞的存活率。     

Effect of myo-inositol on convulsions induced by pentylenetetrazole and kainic acid in rats

We studied the effect of myo-inositol on pentylenetetrazole and kainic acid-induced seizures in rats. Myo-inositol significantly reduced seizure activity. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 143, No. 1, pp. 64–66, January, 2007     

Effects of gamma-decanolactone on seizures induced by PTZ-kindling in mice

Gamma-decanolactone is a monoterpene compound, and its psychopharmacological evaluation in mice revealed that it has a dose-dependent effect on the central nervous system, with hypnotic, anticonvulsant, and hypothermic activity. The aim of the present study was to investigate the effect of gamma-decanolactone on pentylenetetrazole (PTZ)-kindling in mice. Phenobarbital, an antiepileptic drug, was also tested for the purpose of comparison. After the behavioral procedures had been undertaken, the animals were killed and brain tissue was sampled to evaluate DNA damage in the brain using comet assay. The data reported here suggest that the administration of phenobarbital (10 mg/kg) and gamma-decanolactone at 0.3 g/kg, but not at 0.1 g/kg, impairs both the severity and the progression of seizures in the PTZ-kindling model. DNA damage to brain tissue decreased in gamma-decanolactone-treated kindling animals (similar to phenobarbital) as compared to nontreated animals. The results suggest that gamma-decanolactone has dose-dependent anticonvulsant properties, and may also have antiepileptogenic and neuroprotective effects in the PTZ-kindling model.     

地塞米松对海人酸致（癇）大鼠脑P-糖蛋白表达的影响

目的：探讨地塞米松对海人酸致痫大鼠脑P-糖蛋白（P-gp）表达的影响.方法：将SD大鼠随机分为假手术组（Sham组,n=8）、癫痫组（EP组,n=12）、地塞米松干预癫痫组（DEX组,n=12）.后两组采用海马注射海人酸方法制作癫痫模型,DEX组癫痫造模前30 min给予腹腔注射地塞米松0.4 mg/kg.分别记录各组大鼠达到Ⅲ级和Ⅴ级发作时所需的时间（潜伏期）,初次至第6次≥Ⅳ级发作的间隔时间作为评价癫痫发作严重程度的指标;大鼠术后24 h处死,使用HE染色和免疫组织化学方法,比较各组海马CA3区、齿状回、杏仁核复合体区P-gp表达及脑损伤情况.结果：①Sham 组未见癫痫发作;DEX组与EP组达到Ⅲ级发作的潜伏期分别为（87.92±45.80）min和（67.50±22.91）min,达到Ⅴ级发作的潜伏期分别为（103.33±51.27 ）min和（75.60±22.10）min,差异均无统计学意义（P〉0.05）;与EP组相比,DEX组样发作严重程度降低（P=0.004）;②与EP组相比,DEX组于所观察的脑区损伤均减轻,以海马CA3区和杏仁核复合体区较为显著;③与Sham组比较,EP组各观察脑区P-gp表达均明显升高（P〈0.01）;与EP组相比,DEX组海马CA3区和杏仁核复合体区P-gp表达显著减少（P〈0.05）,而在齿状回表达量差异无统计学意义（P=0.078）.结论：地塞米松可降低海人酸致痫大鼠发作严重程度和脑损伤,抑制P-gp表达上调,其中以海马CA3区和杏仁核区较为显著.     

克罗米酚的抗癫癎作用

目的:了解雌激素受体调节剂克罗米酚(clomiphene citrate,CC)对海人藻酸(kainic acid, KA)致癎大鼠癫癎发作行为的影响。方法:健康雌性SD大鼠40只,均行双侧卵巢切除术,术后第八天将动物随机分4组:茶油对照组(OIL组)、KA组、雌二醇(E2) KA组、E2 CC KA组,每组10只。 OIL组、KA组连续5天腹腔注射茶油;E2 KA组连续5天腹腔注射E2(20mg／kg,10mg／ml);E2 CC KA组连续5天腹腔注射E2和CC(2mg／kg,2mg／ml),最后一次打药结束1 h后,致癎各组大鼠(KA 组、E2 KA组、E2 CC KA组)经腹腔注射KA(10mg／kg,2mg／ml),OIL组腹腔注射生理盐水后,连续观察大鼠2 h的行为改变。记录癎样发作的潜伏期、出现重型发作的时间以及发作程度。结果:E2 CC KA组的癫癎发作潜伏期为61．75±19．04 min,较E2 KA组的潜伏期23．8±6．03 min延长,且出现重型癫癎发作的时间晚,为50．20±20．37 min,而E2 KA组出现重型癫癎发作时间平均为30．70 ±13．58 min,两组比较差异有显著意义(P<0．05)。致癎2 h后,致癎各组大鼠的癫癎发作Racine分级比较差异显著意义(P>0．05)。结论:CC有拮抗雌激素的致癎作用,延缓癫癎的发作。     

Increased brain levels of cholecystokinin octapeptide after kainic acid-induced seizures in the rat

Pronounced changes in the content of cholecystokinin octapeptide (CCK-8) have been found after limbic seizures induced by i.p. injection of kainic acid. Three hours after injection of the toxin a significant decrease in CCK-8 was observed in the frontal cortex and amygdala/pyriform cortex reflecting an increased release during acute seizures. A persistent decrease in the content of the peptide in the amygdala/pyriform cortex suggests destruction of the respective neurons. In the substantia nigra and in the striatum and, more moderately, in the hippocampus and frontal cortex increases in CCK-8 were observed 10 days after injection of kainic acid suggesting an increased synthesis or decreased release of the peptide in these brain areas subsequently to the acute seizures.     

Phosphorylated and non-phosphorylated neurofilament proteins: distribution in the rat hippocampus and early changes after kainic acid induced seizures

The regional distribution of neurofilament proteins in the rat hippocampus and their early changes after kainic acid induced seizures were investigated immunocytochemically with antibodies against light weight neurofilament, phosphorylated and non-phosphorylated heavy weight neurofilament. The light weight and non-phosphorylated heavy weight neurofilaments were distributed more unevenly than the phosphorylated neurofilament. The perikarya and processes of pyramidal cells in the CA3 field contained the highest light weight and non-phosphorylated heavy weight neurofilaments, while the perikarya of granule cells contained only few light weight neurofilament and the perikarya of CAI pyramidal cells were even devoid of immunoreactivity of both light and heavy weight neurofilaments. The fiber staining of the light weight and non-phosphorylated heavy weight neurofilaments, especially the former, was less in the CAI field and molecular layer of dentate gyrus. The phosphorylated neurofilament immunoreactivity was identified only in axons. Mossy fibers, the axons of granule cells, contained the light weight and phosphorylated heavy weight neurofilaments, but not the non-phosphorylated neurofilament. Seven days after the kainic acid induced seizures, the phosphorylated neurofilament staining was greatly reduced in the CAI and inner molecular layer of the dentate gyrus, probably resulting from the axonal degeneration of the Schaffer collaterals and the commissural/associational fibers. Furthermore, the non-phosphorylated neurofilament appeared in the mossy fibers of the CA3 stratum lucidum, which normally do not express such immunoreactivity. The results indicate that the neurofilaments are altered following the neuronal degeneration and post lesional plasticity caused by the kainic acid administration. Therefore, the examination of various phosphorylated neurofilaments may offer a comprehensive understanding of major hippocampul pathways, axonal plasticity and the possible roles of neurofilaments in the hippocampus following excitotoxic insults.     

Effect of LiCl pretreatment on cholinomimetic-induced seizures and seizure-induced brain edema in rats

Male Sprague-Dawley rats received LiCl (5 mEq/kg; sc) or saline 24 h prior to injection of cholinomimetics. Physostigmine (PHY, 0.54-0.80 mg/kg), diisopropylfluorophosphate (DFP, 1.3-2.5 mg/kg), pilocarpine (PIL, 23-30 mg/kg), or saline was injected subcutaneously at time 0. Rats were observed for seizure activity for 2 h, survivors were killed 24 h later and edema was measured in samples from parietal and piriform cortices, dorsal thalmus, and hippocampus. None of the rats pretreated with saline had seizures when given doses of cholinomimetics alone. However, rats pretreated with LiCl had the following incidence of seizures: PHY 68%, DFP 71% and PIL 100%. Rats given cholinomimetic agents alone did not have brain edema. In contrast, all LiCl-pretreated rats that seized had pronounced brain edema which was greatest in the piriform cortex. Thus, these studies demonstrate that LiCl pretreatment potentiates cholinomimetic-induced seizures. Further, cholinomimetic-induced seizures produce brain changes resulting in edema.     

Effect of astrocyte-derived factors on ischemic brain edema induced by rat MCA occlusion

To clarify whether astrocyte-derived factors may protect cerebral tissue from ischemia, we examined brain edema, demyelination and astrocyte proliferation in brains with focal ischemia and treated with astrocyte-cultured medium. We occluded the left middle cerebral artery of rats and implanted the Osmotic Minipump, which continuously infused the glial-cultured medium or control medium into the left lateral ventricle. Animals were sacrificed at 3 days or 7 days after occlusion. Brains of both groups were compared by several markers, i.e. extravasation of Evans blue, demyelination by Woelcke's staining and glial proliferation by GFAP staining. We found the astrocyte-cultured medium reduced leakage of Evans blue-plasma protein complex from ischemic lesions and reduced the size of demyelinated lesions. However, the degree of astrocyte proliferation was similar in both groups. From these data, we speculate that humoral factors derived from cultured astrocytes lessened the brain edema by modifying the blood-brain barrier. These factors might also induce proliferation of the microglia, and may protect the neurons from secondary injury by oxygen-free radicals.     

Abnormal distribution of phosphorylated neurofilaments in neuronal degeneration induced by kainic acid.

An abnormal distribution of phosphorylated neurofilaments is present in some human neurodegenerative disorders including Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis. This study reports the changes of phosphorylated neurofilaments observed in rat spinal cord after intrathecal injection of kainic acid. This receptor agonist of excitatory amino acid produces abnormal phosphorylation of neurofilaments in the cell body and proximal neurites of degenerating neurons. These immunocytochemical modifications observed 2 and 10 days after injections are predominantly located in ventral horn neurons. This study indicates that one of the neuronal responses to excitatory amino acid toxicity is the pathological distribution of phosphorylated neurofilaments in affected neurons. Pathological findings are comparable to those observed in neurodegenerative diseases such as amyotrophic lateral sclerosis.     

Effects of nitric oxide on dentate gyrus cell proliferation after seizures induced by pentylenetrazol in the adult rat brain

Epileptic seizures have been shown to increase the proliferation of granule cell precursors in the adult brain, but the underlying mechanisms remain largely unknown. This study examined the effect of nitric oxide (NO) on the proliferation of granule cell precursors in adult rats after pentylenetrazol (PTZ)-induced generalized clonic seizures. Using systemic bromodeoxyuridine (BrdU) to label dividing cells, we found that injection of the neuronal nitric oxide synthase (nNOS) inhibitor 7-nitroindazole (50 mg/kg i.p.) 10 min before PTZ significantly reduced the number of BrdU labeled cells in the dentate gyrus 3, 7, and 14 days after seizures (P < 0.05). Administration of the inducible NOS (iNOS) inhibitor aminoguanidine (100 mg/kg i.p.) also significantly inhibited the proliferation rate of neural precursor cells in the dentate gyrus at various time points after PTZ-induced seizures. Our findings suggest that epileptic seizures lead to increased cell proliferation in the adult rat dentate gyrus through NO-dependent mechanisms. Both the NO originating from nNOS and iNOS may be involved in brain repair after seizures.