Enhancement of progenitor cell division in the dentate gyrus triggered by initial limbic seizures in rat models of epilepsy

PURPOSE: Mitogenic effects of seizures on granule cell progenitors in the dentate gyrus were studied in two rat models of epilepsy. We investigated which stage of epileptogenesis is critical for eliciting progenitor cell division and whether seizure-induced neuronal degeneration is responsible for the enhancement of progenitor cell division. METHODS: Seizures were induced by either kainic acid (KA) administration or electrical kindling. Neurogenesis of dentate granule cells was evaluated using the bromodeoxyuridine (BrdU) labeling method, and neuronal degeneration was assessed by in situ DNA fragmentation analysis. RESULTS: After injection of KA, the number of BrdU-positive granule cells began to increase at day 3 after the treatment, peaked at day 5, and returned to baseline at day 10. By day 13, the values were lower than control. After kindling, the number of BrdU-positive cells began to increase after five consecutive experiences of stage I seizures. The increase occurred from day 1 to day 3 after the last electrical stimulation, but returned to baseline by day 7. After generalized seizures were well established, repeated stimulation did not facilitate division of granule cell progenitors. DNA fragmentation was noted in pyramidal neurons in the CA1, CA3, and hilus regions at 18 h after KA injection, but not in the kindling model. CONCLUSIONS: These observations indicate that a mechanism in epileptogenesis boosts dentate progenitor cell division, but progenitor cells may become unreactive to prolonged generalized seizures. Pyramidal neuronal degeneration is not necessary for triggering the upregulation. It is suggested that newly born granule cells may play a role in the network reorganization that occurs during epileptogenesis.     

红藻氨酸致癫痫持续状态大鼠海马neuropilin-2 mRNA及其蛋白表达的研究

目的研究轴索导向分子NPN-2mRNA及其蛋白对癫痫持续状态(SE)后大鼠海马内神经纤维外向性生长和突触重建中的调控作用。方法采用侧脑室内注射红藻氨酸(KA)制作TLE大鼠模型,用Nissl染色、原位杂交和免疫组织化学的方法,分别检测致SE后1d、1w、2w、3w、4w大鼠海马齿状回(DG)、CA1区、CA3区、门区神经元丢失程度以及NPN-2mRNA及其蛋白的表达。结果 KA致SE后1d开始出现神经元丢失,至4w神经元丢失明显增多。KA致SE后1d,NPN-2mRNA及其蛋白在DG和CA1区表达明显下降,持续至3w(P0.01),4w恢复至正常(P0.05);NPN-2mRNA及其蛋白在门区、CA3区表达实验组与对照组无明显差别(P0.05)。结论 KA致SE后,海马DG及CA1区神经元下调NPN-2mRNA及其蛋白的表达,促进DG及CA1区神经纤维外向性生长和突触的重建。     

Suppression of limbic motor seizures by electrical stimulation in thalamic reticular nucleus

Kindling is a model of temporal lobe epilepsy in which repeated electrical stimulations in limbic areas lead to progressive increase of seizure susceptibility, culminating in generalized convulsions and the establishment of a permanent epileptic syndrome. We studied here the effect of stimulations in the thalamic reticular nucleus (TRN) on the development of seizures and hippocampal hyperexcitability in kindling elicited from the ventral hippocampus in rats. Animals given 12 kindling stimulations per day with 30-min intervals for 4 consecutive days developed generalized convulsions on day 4. Stimulations in TRN delivered simultaneously with those in the hippocampus induced marked suppression of seizure generalization. Similarly, the number of generalized seizures and the duration of behavioral convulsions were reduced when rats subjected to 40 kindling stimulations with 5-min intervals during about 3 h were costimulated in the TRN. The anticonvulsant effect of TRN costimulation was detected also when rats were test-stimulated in the hippocampus at 24 h and 2 and 4 weeks after the initial 40 hippocampal stimulations. Our data provide the first evidence that TRN stimulations can act to suppress limbic motor seizures in hippocampal kindling and suggest a new approach for seizure control in temporal lobe epilepsy.     

Spatio-temporal profile of DNA fragmentation and its relationship to patterns of epileptiform activity following focally evoked limbic seizures

The specific electrographic activity responsible for seizure-induced DNA damage remains little explored. We therefore examined the regional and temporal appearance of DNA fragmentation and cell death and its relationship to specific electrographic seizure patterns in a rat model of focally evoked limbic epilepsy. Animals received intra-amygdaloid injection of kainic acid (KA) to induce seizures for 45 min during continuous electroencephalographic (EEG) monitoring, after which diazepam (30 mg/kg) was administered. DNA polymerase I-mediated biotin-dATP nick translation (PANT) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) were used to detect single- and double-stranded DNA breaks, respectively. Injection of 0.01 microg KA induced seizures characterized by ictal fast activity but without consequent brain injury. By contrast, 0.1 microg KA induced an additional pattern of seizure activity characterized by bursts of high frequency polyspike paroxysmal discharges. In these animals, there was a significant reduction in numbers of pyramidal neurons within the ipsilateral and contralateral CA3 subfield of the hippocampus, detectable as little as 4 h following seizures. PANT- and TUNEL-positive cells appeared in similar numbers 16 h following seizure cessation within the CA3, declining after 72-96 h. Varying the duration of polyspike paroxysmal discharges determined that as little as 30 s elicited maximal injury. These data suggest single- and double-stranded DNA breaks are generated during the cell death process and are consequent on a specific component of seizure activity electrographically determined.     

红藻氨酸致痫后大鼠海马ERK、P38 MAPK和JNK的活性变化

目的研究红藻氨酸(KA)诱导大鼠癫痫发作后海马组织细胞外调节蛋白激酶(ERK)、p38MAPK和c-jun氨基末端激酶(JNK)的活性(磷酸化状态)的变化情况。方法立体定向大鼠侧脑室内注射KA引起大鼠癫痫发作,采用Western-blot方法观察KA致痫后大鼠海马中活性ERK、p38MAPK和JNK的变化。结果KA诱导大鼠癫痫发作后,海马组织ERK、p38MAPK和JNK的磷酸化水平开始增高,分别于30min、1h和30min后达高峰,呈对照组的4.76倍、2.16倍和3.95倍,两组比较差异具有显著性(P<0.01),之后逐渐下降。结论KA致痫大鼠癫痫发作后,海马组织MAPKs的活性产生变化,其信号通路可能参与癫痫发作后海马组织的病理生理反应过程。     

Activation of Rho after traumatic brain injury and seizure in rats

Traumatic brain injury (TBI) is characterized by a progressive cell loss and a lack of axonal regeneration. In the central nervous system (CNS), the Rho signaling pathway regulates the neuronal response to growth inhibitory proteins and regeneration of damaged axons, and Rho activation is also correlated with an increased susceptibility to apoptosis. To evaluate whether traumatic brain injury (TBI) results in changes in Rho activation in vulnerable regions of the brain, GTP-RhoA pull down assays were performed on rat cortical and hippocampal tissue homogenates obtained from 24 h to 3 days following lateral fluid percussion brain injury (FPI). Following FPI, a significantly increased RhoA activation was observed from 24 h to 3 days post-injury in the cortex and by 3 days in the hippocampus ipsilateral to the injury. We also detected activated RhoA in the cortex and hippocampus contralateral to the injury, without concomitant changes in total RhoA levels. To determine if immediate post-traumatic events such as seizures may activate Rho, we examined RhoA activation in the brains of rats with kainic acid-induced seizures. Severe seizures resulted in bilateral RhoA activation in the cortex and hippocampus. Together, these results indicate that RhoA is activated in vulnerable brain regions following traumatic and epileptic insults to the CNS.     

Ganglioside GT1B and melatonin inhibit brain mitochondrial DNA damage and seizures induced by kainic acid in mice

The effects of ganglioside GT1b or melatonin on damage to brain mitochondrial DNA (mtDNA) and seizures induced by kainic acid were investigated both in vivo and in vitro. An intraperitoneal (i.p.) injection of kainic acid (45 mg/kg) produced broad-spectrum limbic and severe sustained seizures in all of the treated mice. These seizures were completely abolished by an intracerebroventricular (i.c.v.) injection of ganglioside GT1b (90 nmol/brain), a potent inhibitor of glutamate receptor mediated activation and translocation of protein kinase C and lipid peroxidation, or an i.p. injection of melatonin (20 mg/kg), a potent scavenger of hydroxyl radicals (*OH). The administration of kainic acid caused damage to mtDNA in brain frontal and central portion of cortex in mice. The damage to mtDNA was abolished by pre-injection of ganglioside GT1b (90 nmol/brain, i.c.v.) or melatonin (20 mg/kg, i.p.). In vitro exposure of kainic acid (0.25, 0.5 or 1.0 mM) inflicted damage to mtDNA in a concentration-dependent manner. The damage to mtDNA induced by 1.0 mM kainic acid was attenuated by the co-treatment with 60 microM ganglioside GT1b or 1.5 mM melatonin. Furthermore, kainic acid (0.5 or 1.0 mM) increased lipid peroxidation in a concentration-dependent manner when incubated with a homogenate prepared from mice brain at 37 degrees C for 20 or 60 min. However, the increased lipid peroxidation was completely abolished by the co-treatment with ganglioside GT1b (60 microM) or melatonin (1.5 mM). These results suggest that reactive oxygen species including hydroxyl radical (*OH) may play a role in the damage to brain mtDNA and seizures induced by kainic acid. We conclude that the preventive effect of melatonin or ganglioside GT1b against kainic acid-induced mtDNA damage or seizures may be due to its scavenging of reactive oxygen species including the *OH.     

Protective effects of cortistatin (CST-14) against kainate-induced neurotoxicity in rat brain

Cortistatin (CST-14) is a recently discovered endogenous peptide which shares similarity to somatostatin and binds to somatostatin receptors. In this study, we show that CST-14 exhibits anticonvulsive and neuroprotective effects in rats. Injection of rats with kainic acid (KA; 10 mg/kg; i.p.) generated a strong seizure activity which was attenuated by the i.c.v. application of 1 and 10 nmol CST-14 when given 10 min before KA. Moreover, 3 days after KA injection, a marked loss of neurons in cortex and hippocampus of rats was observed which was inhibited by pretreatment with CST-14. An immunohistochemical analysis using specific antibodies revealed that KA reduced immunoactive sst_2A and sst₃ somatostatin receptors in the hippocampus—an effect which was largely prevented by pretreatment with CST-14. Superfusion of hippocampal slices with CST-14 also reduced the stimulated release of ³H- -aspartate. We conclude that CST-14 exerts neuroprotective effects by binding to somatostatin receptors which in turn leads to a reduced release of excitotoxic neurotransmitters.     

An early decrease in cell proliferation after pentylenetetrazole-induced seizures

There are increasing data on the influence of seizures on neurogenesis in the adult brain. However, data on cell proliferation and differentiation during the early stages of kindling are scarce. We have used pentylenetetrazole (PTZ)-induced kindling to investigate the temporal profile of cytogenesis in the germinative zones of adult rat brain. For comparison, we also used a single PTZ-induced generalized tonic–clonic seizure. During kindling development, the density of 5-bromo-2'-deoxyuridine-positive cells demonstrated similar changes in all germinative zones: a dramatic decrease after the first subthreshold PTZ injection, and a gradual increase to the control level following repeated PTZ administration. On the contrary, a single PTZ-induced generalized tonic–clonic seizure was followed by an increase in the number of proliferating cells in both the dentate gyrus and the subventricular zone. These results may indicate the existence of global mechanisms affecting cellular proliferation in adult brain during seizures. Different temporal profiles of neuronal damage and proliferation changes suggest that neurodegeneration is unlikely to be a global proliferation-regulating factor. The data may contribute to better understanding of the initial phase of kindling development and epileptogenesis.     

红藻氨酸诱导性癫痫发作大鼠海马cNOS及iNOS的变化及作用

目的研究组成型一氧化氮合酶（ｃＮＯＳ）及诱导型一氧化氮合酶（ｉＮＯＳ）在红藻氨酸（ＫＡ）诱导癫痫发作中的变化及作用。方法采用免疫组织化学方法显示ｃＮＯＳ及ｉＮＯＳ的变化;Ｎｉｓｓｌ染色显示神经元的损害。结果ＫＡ３０分钟ｃＮＯＳ较对照组明显增加（Ｐ＜０．０５）,随后下降至正常水平;ＫＡ诱导２小时ｉＮＯＳ明显升高,以ＣＡ１区为著,至ＫＡ６小时达高峰,然后在高水平缓慢下降;Ｎｉｓｓｌ染色神经元变性坏死ＣＡ３＝齿状回＞ＣＡ１。结论ＫＡ诱导癫痫发作导致海马ｃＮＯＳ及ｉＮＯＳ表达增多,神经元的坏死与ＮＯＳ表达增多无明显关系。     

Effect of nifedipine and anticonvulsants of kainic acid-induced seizures in mice

The calcium-channel inhibitor nifedipine and several anticonvulsant drugs were evaluated for effects on seizures induced by intracerebroventricular injection of 0.14 μg of kainic acid. These seizures were markedly exacerbated by valproic acid and moderately inhibited by diazepam. Nifedipine decreased the duration of each individual seizure episode, but did not block the development of seizures. It is concluded that nifedipine prevents the maintenance or propagation of kainate-induced seizures.     

Theophylline reduces cerebral hyperaemia and enhances brain damage induced by seizures

Hippocampal and neocortical blood flows and tissue pO2 were investigated by mass spectrometry in unanaesthetized spontaneously breathing rats during kainic acid-induced seizures to determine whether adenosine is involved in the coupling of cerebral blood flow to metabolism during enhanced metabolic demand. The possible involvement of adenosine in the neuronal damage induced by seizures was also analyzed. The intrinsic effects of theophylline and the duration of the adenosine receptor blockade by this xanthine were first tested in 8 rats. Two groups of rats were then compared: one (n = 6) received kainic acid, and the other (n = 10) theophylline 15 min prior to kainic acid administration. An additional group of 10 rats was taken for classical histology 48 h after kainic acid treatment. Theophylline significantly reduced the hyperaemia observed during seizures, prevented any tissue hyperoxia and enhanced brain damage. This strongly suggests that adenosine is partly responsible for the increase in cerebral blood flow during kainic acid-induced seizures and has neuroprotective properties.     

Seizures in the developing brain cause adverse long-term effects on spatial learning and anxiety

PURPOSE: Seizures in the developing brain cause less macroscopic structural damage than do seizures in adulthood, but accumulating evidence shows that seizures early in life can be associated with persistent behavioral and cognitive impairments. We previously showed that long-term spatial memory in the eight-arm radial-arm maze was impaired in rats that experienced a single episode of kainic acid (KA)-induced status epilepticus during early development (postnatal days (P) 1-14). Here we extend those findings by using a set of behavioral paradigms that are sensitive to additional aspects of learning and behavior. METHODS: On P1, P7, P14, or P24, rats underwent status epilepticus induced by intraperitoneal injections of age-specific doses of KA. In adulthood (P90-P100), the behavioral performance of these rats was compared with that of control rats that did not receive KA. A modified version of the radial-arm maze was used to assess short-term spatial memory; the Morris water maze was used to evaluate long-term spatial memory and retrieval; and the elevated plus maze was used to determine anxiety. RESULTS: Compared with controls, rats with KA seizures at each tested age had impaired short-term spatial memory in the radial-arm maze (longer latency to criterion and more reference errors), deficient long-term spatial learning and retrieval in the water maze (longer escape latencies and memory for platform location), and a greater degree of anxiety in the elevated plus maze (greater time spent in open arms). CONCLUSIONS: These findings provide additional support for the concept that seizures early in life may be followed by life-long impairment of certain cognitive and behavioral functions. These results may have clinical implications, favoring early and aggressive control of seizures during development.     

Age-dependent cyclooxygenase-2 induction and neuronal damage after status epilepticus in the postnatal rat hippocampus

PURPOSE: Epileptic seizures lead to age-dependent neuronal damage in the developing brain, particularly in the hippocampus, but the mechanisms involved have remained poorly elucidated. In this study, we investigated the contribution of apoptosis and inflammatory processes to neuronal damage after status epilepticus (SE) in postnatal rats. METHODS: SE was induced by an intraperitoneal injection of kainic acid (KA) in 21- and 9-day-old (P21 and P9) rats. The expression of Bax, Bcl-2 and caspase-3, markers for apoptosis, and cyclooxygenase-2 (COX-2), an indicator for activation of inflammatory processes, were studied from 6 h up to 1 week after SE by Western blotting and immunocytochemistry. Neuronal damage was verified by Fluoro-Jade B staining. RESULTS: In P21 rats, SE resulted in neuronal damage in the CA1 neurons of the hippocampus. COX-2 expression was extensively, but transiently, increased and its immunoreactivity pronouncedly enhanced in several hippocampal subregions, amygdala, and piriform cortex by 24 h after SE. The expression of Bax and caspase-3 remained unchanged, whereas the antiapoptotic factor Bcl-2 transiently decreased by 24 h. Single caspase-3 positive neurons appeared in the CA1 region of both control and KA-treated rats. In P9 rats, no neuronal death was detected, and COX-2 expression and immunoreactivity remained at the control level. DISCUSSION: Our results suggest that SE provokes age-specific effects on COX-2 expression. This together with the activation of putative inflammatory processes may contribute to neuronal cell death in the hippocampus of postnatal rats, whereas caspase-dependent apoptosis seems not to be involved in the death process.     

Neuraminidase activity in different regions of the seizing epileptic and non-epileptic brain

The sialic acid in the brain is split from sialoglucoconjugates by sialidases (neuraminidases, EC 3.2.1.18), and is postulated to act as an inhibitor of cellular adhesion and to play a role in various membrane functions. Since epilepsy alters cellular interactions and connectivity, it is reasonable to propose that sialidases can be affected by this pathological state or, alternately, by seizures. Therefore, we studied the activity of total, soluble, and membranal sialidases in various brain regions in normal, kindled epileptic and non-epileptic seizing rats. The results showed that in kindled rats, the total activity of the sialidases significantly decreased in cerebral cortex (11.38%) and cerebellum (28.58%), whereas it increased in brainstem (35.51%), hypothalamus (2.88%) and hippocampus (9.37%). The activity of the membranous sialidases in kindled rats followed the same pattern as the total activity, whereas the activity of soluble sialidase was significantly lower than membranous activity. Interestingly, the activity of total and membranal sialidases in non-epileptic seizing rats paralleled that observed in kindled rats. We suggest that the seizure-induced decrease of sialidasic activity may not modify the number of sialic acid molecules bound to gangliosides in cell membranes, as compared to areas of increased activity, that may decrease them. These changes in sialidases' activity may reflect functional disturbances of membrane polysialylated gangliosides related to the functional and anatomical plastic changes associated to seizures. Our data indicate that these changes are related to the presence of seizures rather than to an established epileptic state.     

Mapping of c-Fos expression in the rat brain during the evolution of pentylenetetrazol-kindled seizures

c-Fos protein immunocytochemistry was used to map the brain structures recruited during the evolution of seizures that follows repeated administration of a subconvulsive dose (35 mg/kg, ip) of pentylenetetrazol in rats. c-Fos appeared earliest in nucleus accumbens shell, piriform cortex, prefrontal cortex, and striatum (stages 1 and 2 of kindling in comparison to control, saline-treated animals). At the third stage of kindling, central amygdala nuclei, entorhinal cortex, and lateral septal nuclei had enhanced concentrations of c-Fos. At the fourth stage of kindling, c-Fos expression was increased in basolateral amygdala and CA1 area of the hippocampus. Finally, c-Fos labeling was enhanced in the dentate gyrus of the hippocampus only when tonic–clonic convulsions were fully developed. The most potent changes in c-Fos were observed in dentate gyrus, piriform cortex, CA1, lateral septal nuclei, basolateral amygdala, central amygdala nuclei, and prefrontal cortex. Piriform cortex, entorhinal cortex, prefrontal cortex, lateral septal nuclei, and CA3 area of the hippocampus appeared to be the brain structures selectively involved in the process of chemically induced kindling of seizures.     

Changes in microtubule-associated protein-2 (MAP2) expression during development and after status epilepticus in the immature rat hippocampus

In this study, we analyzed the spatiotemporal expression patterns of the high-molecular weight (MAP2a and b) and low-molecular weight (MAP2c and d) cytoskeletal microtubule-associated protein-2 (MAP2) isoforms with Western blotting, and the cellular localization of the high-molecular weight MAP2 isoforms with immunocytochemistry in the hippocampi of 1- to 21-day-old rats. Moreover, the temporal profile (from 30 min to 1 week) of MAP2 isoform reactivity to kainic acid-induced status epilepticus was studied in P9 rats. During development, the expression of the high-molecular weight MAP2 isoforms significantly increased, while the low-molecular weight isoforms decreased, the most prominent changes occurring during the second postnatal week. This developmental increase in the high-molecular weight MAP2 expression was also confirmed with immunocytochemistry, which showed increased immunoreactivity, particularly in the molecular layers of the dentate gyrus, and in CA1 and CA3 stratum radiatum. In 9-day-old rats, status epilepticus resulted in a rapid transient increase (about 210%) in the high-molecular weight MAP2 expression, without any effect on the low-molecular weight MAP2. Moreover, disturbed dendritic structure in the CA1 and CA3 stratum radiatum was manifested as formation of varicosities 3h after the kainic acid treatment. The strictly developmentally regulated MAP2 isoform expression suggests different functional roles for these proteins during the postnatal development in the rat hippocampus. Moreover, high-molecular weight MAP2s may play a role in nerve cell survival during cell stress.     

Influence of the plasma glucose level on brain damage after systemic kainic acid injection in the rat

Summary Systemic administration of kainic acid (KA), 11 mg/kg body weight, to hyperglycemic rats induced lethal seizures in all animals, while 40% of normoglycemic rats survived the KA treatment and all hypoglycemic rats survived. An inverse correlation (P<0.01) between the plasma glucose level and survival during KA-induced seizures was demonstrated (Chi-square-test). Histopathological observations on the surviving rats clearly divided them into a group with severe hippocampal CA-1 damage and a group with mild hippocampal CA-1 damage. Hippocampal pyramidal cells and CA-1 interneurons were counted 3 weeks after the insult. The pyramidal cell loss in the CA-1 region was significant within mildly, as well as severely, affected rats with normo- and with hypoglycemia. CA-1 interneurons and CA-4 interneurons were only lost in the severely affected group. Hypoglycemia seemed to protect those CA-1 interneurons situated close to the alveus and within the stratum radiatum in these animals. The increased mortality in the hyperglycemic rats could be due to increased brain lactate accumulation, but extracerebral damage of hyperglycemia in association with KA is also a possibility. The study indicated a correlation between loss of interneurons and pronounced CA-1 pyramidal cell death and furthermore that hypoglycemia possibly protected some interneurons against KA.