Roles of astrocytes and microglia in seizure-induced aberrant neurogenesis in the hippocampus of adult rats.

Recent evidence showed that epileptic seizures increase hippocampal neurogenesis in the adult rat, but prolonged seizures result in the aberrant hippocampal neurogenesis that often leads to a recurrent excitatory circuitry and thus contributes to epileptogenesis. However, the mechanism underlying the aberrant neurogenesis after prolonged seizures remains largely unclear. In this study, we examined the role of activated astrocytes and microglia in the aberrant hippocampal neurogenesis induced by status epilepticus. Using a lithium-pilocarpine model to mimic human temporal lobe epilepsy, we found that status epilepticus induced a prominent activation of astrocytes and microglia in the dentate gyrus 3, 7, 14, and 20 days after the initial seizures. Then, we injected fluorocitrate stereotaxicly into the dentate hilus to inhibit astrocytic metabolism and found that fluorocitrate failed to prevent the seizure-induced formation of ectopic hilar basal dendrites but instead promoted the degeneration of dentate granule cells after seizures. In contrast, a selective inhibitor of microglia activation, minocycline, inhibited the aberrant migration of newborn neurons at 14 days after status epilepticus. Furthermore, with stereotaxic injection of lipopolysaccharide into the intact dentate hilus to activate local microglia, we found that lipopolysaccharide promoted the development of ectopic hilar basal dendrites in the hippocampus. These results indicate that the activated microglia in the epileptic hilus may guide the aberrant migration of newborn neurons and that minocycline could be a potential drug to impede seizure-induced aberrant migration of newborn neurons.

[Characteristics of continuous spike-and-wave during slow wave sleep syndrome in children].

OBJECTIVE: Continuous spike-and-wave during slow wave sleep (CSWS) syndrome is one of the presentations of electrical status epilepticus during sleep (ESES). The purpose of this study was to investigate the characteristics of CSWS syndrome in children. METHODS: Between 2007 and 2009, a total of 778 nocturnal long-term or 24-hr video-EEG records were included. The EEG, clinical and neuroimaging characteristics were studied in children who met standard criteria for CSWS. RESULTS: Nine children met standard criteria for CSWS in video-EEGs. Their ages ranged 6 to 13 years. Their EEGs were characterized by continuous spike-and-wave (SW) discharges during non-rapid eye movement (NREM) sleep, accounting for 85%-100% of the period of NREM sleep. Clinically, these children had various types of epileptic seizures and exhibited different degrees of neuropsychiatric impairments, language dysfunction, and/or behavioral disturbances. Neuroimaging abnormalities were found in 6 cases, including atelencephalia or atrophy, gray matter heterotopia and leucomalacia. CONCLUSIONS: This study indicates the characteristics of CSWS syndrome in clinical manifestations, EEG and neuroimaging examinations. This will be helpful in understanding CSWS syndrome.

Up-regulation of D-serine might induce GABAergic neuronal degeneration in the cerebral cortex and hippocampus in the mouse pilocarpine model of epilepsy.

Epilepsy is a serious neurological disorder with neuronal loss and spontaneous recurrent seizures, but the neurochemical basis remains largely unclear. We hypothesize that D-serine, a newly identified endogenous co-agonist of N-methyl-D-aspartate (NMDA) receptor, may trigger excitotoxicity and neuronal damage in epileptogenesis. By using a mouse pilocarpine model, immunohistochemistry, Fluoro-Jade staining and double-labeling, the present study revealed up-regulation of D-serine expression in a proportion (41%) of neurons in the cerebral cortex and hippocampus. The D-serine-positive neurons occurred at 4 h, reached peak levels at 12-24 h, and gradually went down at 3-14 days. Moreover, most of D-serine-positive neurons were GABAergic (98%), underwent degenerating death (93%), and were accompanied enhancing phosphorylation of NMDA receptor subunit 1. This study has provided new evidence that up-regulation of D-serine production might induce GABAergic neuronal degeneration through excitotoxic mechanism in the pilocarpine model and may be involved in early pathogenesis and recurrent seizure of chronic epilepsy.

Activation of cerebral peroxisome proliferator-activated receptors gamma exerts neuroprotection by inhibiting oxidative stress following pilocarpine-induced status epilepticus.

Status epilepticus (SE) can cause severe neuronal loss and oxidative damage. As peroxisome proliferator-activated receptor gamma (PPARgamma) agonists possess antioxidative activity, we hypothesize that rosiglitazone, a PPARgamma agonist, might protect the central nervous system (CNS) from oxidative damage in epileptic rats. Using a lithium-pilocarpine-induced SE model, we found that rosiglitazone significantly reduced hippocampal neuronal loss 1 week after SE, potently suppressed the production of reactive oxygen species (ROS) and lipid peroxidation. We also found that treatment with rosiglitazone enhanced antioxidative activity of superoxide dismutase (SOD) and glutathione hormone (GSH), together with decreased expression of heme oxygenase-1 (HO-1) in the hippocampus. The above effects of rosiglitazone can be blocked by co-treatment with PPARgamma antagonist T0070907. The current data suggest that rosiglitazone exerts a neuroprotective effect on oxidative stress-mediated neuronal damage followed by SE. Our data also support the idea that PPARgamma agonist might be a potential neuroprotective agent for epilepsy.

Immunohistochemical demonstration of the calcium channel alpha2 subunit in the chicken dorsal root ganglion and spinal cord: a special reference to colocalization with calbindin-D28k in dorsal root ganglion neurons.

We used immunohistochemical methods to examine the distribution of the calcium channel alpha2 (CCalpha2) subunit in the chicken spinal cord and dorsal root ganglion (DRG) neurons and determine its relationship with calbindin-D28k (CB) in the DRG neurons. In the spinal cord, CCalpha2 subunit was detected in nerve terminals, which were observed as dot-like structures, and in laminae I, II, III and Lissauer's tract in the dorsal horn. In the DRG neurons, approximately 65% of the total neurons were CCalpha2 subunit positive, and most (86%) of these neurons were small to medium sized, suggesting that the CCalpha2 subunit and/or a complex of the CCalpha2 and delta subunits is possibly localized in a number of nociceptive neurons. A majority (77%) of the positive neurons showed CB immunoreactivity and most (88%) of these neurons were small to medium sized. This may indicate a close correlation between the CCalpha2 subunit and CB in the nociceptive neurons. Thus, it is postulated that the mode of nociceptive transmission may involve a cellular Ca(2+)-regulating system that consists of both Ca(2+) entry via calcium channels with the alpha2delta subunit and intracellular Ca(2+)-binding activity of CB in the nociceptive neurons of the DRG.

Topiramate reduced sweat secretion and aquaporin-5 expression in sweat glands of mice.

Decreased sweat secretion is a primary side effect of topiramate in pediatric patients, but the mechanism underlying this effect remains unclear. This study aimed to better understand how topiramate decreases sweat secretion by examining its effect on the expression of carbonic anhydrase (CA) II and aquaporin-5 (AQP5), total CA activity, as well as on tissue morphology of sweat glands in mice. Both developing and mature mice were treated with a low (20 mg/kg/day) and high dose (80 mg/kg/day) of topiramate for 4 weeks. Sweat secretion was investigated by an established technique of examining mold impressions of hind paws. CA II and AQP5 expression levels were determined by immunofluorescence and immunoblotting and CA activity by a colorimetric assay. In mature mice, topiramate treatment decreased the number of pilocarpine reactive sweat glands from baseline in both the low and high dose groups by 83% and 75%, respectively. A similar decrease was seen in developing mice. Mature mice with reactive sweat glands that declined more than 25% compared to baseline were defined as anhidrotic mice. These mice did not differ from controls in average secretory coil diameter, CA II expression and CA activity. In contrast, anhidrotic mice did show a reduction in membrane AQP5 expression in sweat glands after topiramate delivery. Thus, sweat secretion and membrane AQP5 expression in mouse sweat glands decreased following topiramate administration. These results suggest dysregulation of AQP5 may be involved in topiramate-induced hypohidrosis and topiramate may serve as a novel therapy for hyperhidrosis.

[Correlation between hippocampal mossy fiber sprouting and synaptic reorganization and mechanisms of temporal lobe epilepsy].

OBJECTIVE: To explore the effects of the ultrastructural features of sprouted mossy fiber synapses in the mechanism of temporal lobe epilepsy. To explore the correlation between axon guidance molecule-netrin-1 gene expression and mossy fiber synaptic reorganization. METHODS: Sixty-one SD rats underwent intraperitoneal injection of lithium chloride and pilocarpine to establish models of status epilepticus characterized with temporal lobe epilepsy. Nineteen rats were used as controls. One, 2, and 4 weeks after the injection, a certain numbers of rat were killed with their brains taken out. The sprouted mossy fiber synaptic terminals were labeled by Timm histochemistry and the ultrastructure of new synapses were observed by electron microscopy. By in situ hybridization, the mRNA expression of netrin-1 gene was observed. RESULTS: The sprouted mossy fiber synapses in epileptic rats most commonly formed asymmetric synapses with dendritic spines and occasionally with granule cell somata. Seven days after the injection, up-regulation of netrin-1 mRNA expression was seen in the dentate granule cell layers of hippocampus and continued to 4 weeks after the injection. The time course of the increase of netrin-1 mRNA in the dentate granule cell layers was correlated with the time course of mossy fiber sprouting and synaptic reorganization in hippocampus. CONCLUSION: The ultrastructural features of sprouted mossy fiber synapses support the viewpoint that the reorganization of synapses prominently involves the formation of recurrent excitatory circuits. The axon guidance molecule- netrin-1 plays an important role in the process of mossy fiber axonal outgrowth and synaptogenesis in the hippocampal dentate gyrus.

Ionotropic glutamate receptor antagonists inhibit the proliferation of granule cell precursors in the adult brain after seizures induced by pentylenetrazol.

Seizures have been shown to promote the proliferation of granule cell precursors in the adult brain, but the underlying mechanisms remain largely unknown. Using systemic bromodeoxyuridine (BrdU) to label dividing cells, we examined the effects of selective ionotropic glutamate receptor antagonists on granule cell precursor proliferation in adult rats after pentylenetrazol (PTZ)-induced generalized clonic seizures. We found that the NMDA receptor antagonist MK-801 significantly inhibited behavioral and EEG seizures and completely blocked seizure-induced increase in the number of BrdU-labeled cells in the dentate gyrus. Although the AMPA/KA receptor antagonist DNQX was not observed to affect seizures, it significantly suppressed the number of BrdU-labeled cells in the dentate gyrus. Double immunohistochemical staining showed that both the mature granule cells and the majority of BrdU-labeled, mitotically active cells expressed the NMDA receptor subunit NR1 and the AMPA/KA receptor subunit GluR2. Because accumulated evidence showed that mild seizures are sufficient to promote precursor cell proliferation, the present findings that MK-801 inhibited seizures and completely blocked seizure-induced increase in precursor cell proliferation suggest that the direct blockade action of MK-801 on NMDA receptors on the granule cell precursors may play an important role in blocking seizure-induced precursor cell proliferation. The suppression of seizure-induced proliferation of granule cell precursors by DNQX may be achieved by the direct action of DNQX on AMPA/KA receptors on the granule cell precursors. Thus, our findings indicate that seizures may promote cell proliferation in the adult rat dentate gyrus through glutamatergic mechanisms acting on both NMDA and AMPA/KA receptors.

Dentate granule cell neurogenesis after seizures induced by pentylenetrazol in rats.

Epileptic seizures originating from the limbic system have been shown to stimulate the proliferation rate of granule cell precursors in the adult brain, but it is not clear if other type(s) of seizures have the similar effects. This study examined the effects of pentylenetrazol (PTZ)-induced generalized clonic seizures on dentate granule cell neurogenesis in adult rats. Using systemic bromodeoxyuridine (BrdU) to label dividing cells, we studied the proliferation rate of neural precursor cells in the dentate gyrus at various time points after PTZ-induced seizures. The double-label immunofluorescence with confocal microscopy was used to determine the newborn cell phenotypes. Quantitative analysis of BrdU labeling revealed a significant increase in the proliferation rate of neural precursor cells in the dentate gyrus 3, 7, and 14 days after seizures. The number of BrdU-labeled cells in the dentate gyrus returned to baseline levels by 28 days after the initial seizures. Most of newborn cells migrated into the granule cell layer from the subgranular zone, displayed the neuronal phenotype, and developed morphological characteristics of differentiated dentate granule cells. These results indicated that neuron proliferation in the dentate gyrus was enhanced during a time window (3-14 days) after PTZ-induced seizures. Its underlying mechanism is discussed.

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.

[Assessment of autonomic nervous function during orthostatic stress in pilots with history of syncope].

OBJECTIVE: To assess the role of autonomic nervous function during orthostatic tolerance tests (OTT) in pilots with history of G-induced loss of consciousness (G-LOC) or vasovagal syncope (VVS). METHOD: The relation between heart rate variability (HRV) and outcome of OTT result were compared among 3 groups of cases: 1) 30 patients with history of syncope and positive OTT as patient group (PG); 2) 20 cases with history of syncope but negative OTT result as negative group (NG); 3) 15 age-, sex-, flight plane model-, flight time-matched healthy pilots as healthy control (HC). OTT results and HRV, heart rate (HR), systolic blood pressure (SBP) and diastolic blood pressure (DBP) before and after OTT and their time and frequency domain were compared and analyzed among the three groups. RESULT: HR, SBP, and DBP were not significantly different (P>0.05) among the three groups before OTT. But during OTT, HR in PG was higher than those in NG and HC (P<0.01), SBP and DBP in PG were lower than those in NG and HC (P<0.01). 24 h continuous RR interval standard difference (SDNN), HRV triangle index (HRVI), and interval average value in PG were significantly lower than those in NG and HC; Power spectrum analysis of mean 1 h value for low frequency part (LF) and high frequency part (HF) were not significantly different among three groups, but LF/HF ratio in PG was higher than that in NG or HC (P<0.05). CONCLUSION: It is suggested that G-LOC and VVS may be due to increase in sympathetic tone and decrease in parasympathetic tone during orthostatic stimulation, as resulted from autonomic nervous dysfunction.

Thirteen-year follow-up of patients with tri-ortho-cresyl phosphate poisoning in northern suburbs of Xi'an in China.

Based on reports published so far, organophosphorus (OP) compounds do remarkable harm to human health. In 1995 there was an outbreak of organophosphorus-ester induced delayed neuropathy (OPIDN) due to tri-ortho-cresyl phosphate (TOCP) poisoning in northern suburbs of Xi'an in China. The 74 affected patients were treated and followed up after definite diagnosis. 13 years later, all the epidemiological data obtained from 61 survivors were evaluated, and 15 patients underwent clinical, laboratory, neuroimaging and electrophysiological examinations. In addition, a review of the literature about the possible mechanism of OPIDN was made. According to our investigation, of 61 survivors, 35 patients almost regained normal function of limbs and work outside; 23 patients walked with bilateral support and could perform housework; and 3 patients could not self-care. The patients undergoing examinations presented spasticity and minor lower leg muscle atrophy without sensory impairment. Laboratory investigations and brain and spinal cord magnetic resonance imaging examinations were normal. Neurophysiological investigations also showed normal electroencephalogram and visual, brainstem auditory and somatosensory evoked potentials. Motor evoked potential (MEP) obtained from the upper limbs had normal central motor conduction time (CMCT). However, the CMCT of MEP response recorded from the bilateral lower limbs was delayed, or showed even no MEP responses. Motor and sensory nerve conduction velocity and electromyography studies were normal except for two severely affected patients. TOCP showed long-term effects on the nervous system and influenced the quality of life. OP compounds should be strictly regulated to prevent similar occurrences.

Heterogeneous expression of the voltage-gated calcium channel alpha2 subunit and the voltage-gated sodium channel alpha subunit in chicken spinal motoneurons.

The localization of the voltage-gated calcium channel (VGCC) alpha2 and the voltage-gated sodium channel (VGSC) alpha subunits was immunohistochemically investigated in chicken spinal motoneurons. Approximately 83% and 46% of spinal motoneurons were positive for VGCCalpha2 and VGSCalpha subunits, respectively. Almost all VGSCalpha subunit-positive motoneurons exhibited the VGCCalpha2 subunit immunoreactivity. There were different patterns in occurrence, intensity or nuclear/cytoplasmic stainability of the VGCCalpha2 and VGSCalpha subunits among the motoneurons. This study presents the first cellular morphological evidence for the VGCCalpha2 and VGSCalpha subunits in spinal motoneurons, postulating that the heterogeneous expression of VGCCalpha2 and VGSCalpha subunits in the motoneurons may reflect various motor activities.

Time-course of neuronal death in the mouse pilocarpine model of chronic epilepsy using Fluoro-Jade C staining.

Epilepsy is a serious neurological disorder in human beings and the long-term pathological events remain largely obscure. We are interested in elucidating long-term brain injury that may occur in the temporal lobe epilepsy, and time-course of neuronal death was examined in a mouse pilocarpine model of chronic epilepsy by Fluoro-Jade C (FJC) dye that can specifically stain the degenerative neurons in the central nervous system. The FJC stain combined with immunohistochemistry to neuronal nuclear specific protein revealed that pilocarpine-induced status epilepticus (SE) resulted in massive degenerative death of neuronal cells in brains with their dense distribution in the cerebral cortex and hippocampus. The FJC-positive degenerating neurons, most of them also expressed apoptosis signaling molecules such as caspase-9 and activated caspase-3, occurred at 4h, increased into peak levels at 12h-3d, and then gradually went down at 7d-14d after onset of SE. More interestingly, a large percentage (about 88%) of FJC-positive degenerative neurons were GABAergic as indicated with their immunoreactivity to glutamic acid decarboxylase-67, implying that inhibitory function of GABAergic neural system might by seriously damaged in brains subject to SE attack in this mouse pilocarpine model. Taken together with previous studies, time-course of degenerative neurons in the mouse pilocarpine model by Fluoro-Jade C staining further benefits understanding of long-term brain pathological changes and recurrent seizure mechanism, and may also result in finding the most suitable time-window in therapeutic manipulation of the chronic epilepsy in human beings.

Postnatal expression and denervation induced up-regulation of aquaporin-5 protein in rat sweat gland.

The evolution of aquaporin-5 (AQP5) expression during postnatal development has not been defined in the sweat gland. Previous studies have suggested that AQP isoforms in several peripheral targets are regulated by a neural mechanism. We have examined, in rat sweat glands, the expression of AQP5 during postnatal development and the effects of denervation on AQP5 expression. Both AQP5 mRNA and protein begin to be expressed at postnatal day 10, before sweat-secretory responsiveness first appears; this expression coincides with the occurrence of vasoactive intestinal peptide (VIP) immunoreactivity. Early noradrenergic and later cholinergic interaction between sweat glands and their innervation are disrupted by neonatal chemical sympathectomy or postnatal severance of the sciatic nerve. Examination of such denervated developing rats has shown that secretory responsiveness fails to arise later in the adults, and AQP5 immunostaining increases in the denervated glands, whereas gland morphogenesis and the occurrence of AQP5 expression proceed normally. Immunobloting has revealed an increase of AQP5 abundance after the denervated mature glands lose their secretory ability. These findings suggest that AQP5 protein is necessary for sweat secretion, and that the expression of AQP5 in rat sweat glands is independent of sympathetic innervation. Our data also indicate that factor(s) regulating the normal morphological development of sweat gland might be responsible for controlling AQP5 expression.

Topiramate therapy for paroxysmal kinesigenic choreoathetosis.

We observed the clinical efficacy of topiramate for paroxysmal kinesigenic choreoathetosis (PKC). Topiramate was administered as a monotherapy with titrated dosages to 8 patients with PKC. Target daily dose of topiramate was 100 to 200 mg; the follow-up period ranged from 8 months to 2 years. All of the patients became attack-free, and side effects were mild. The results show that topiramate is effective as a monotherapy for treating patients with PKC. The response to topiramate indicates that the disease may be caused by an ion channel defect.

(-)-Epigallocatechin gallate inhibits lipopolysaccharide-induced microglial activation and protects against inflammation-mediated dopaminergic neuronal injury.

Microglial activation is believed to play a pivotal role in the selective neuronal injury associated with several neurodegenerative disorders, including Parkinson's disease (PD) and Alzheimer's disease. We provide evidence that (-)-epigallocatechin gallate (EGCG), a major monomer of green tea polyphenols, potently inhibits lipopolysaccharide (LPS)-activated microglial secretion of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) through the down-regulation of inducible NO synthase and TNF-alpha expression. In addition, EGCG exerted significant protection against microglial activation-induced neuronal injury both in the human dopaminergic cell line SH-SY5Y and in primary rat mesencephalic cultures. Our study demonstrates that EGCG is a potent inhibitor of microglial activation and thus is a useful candidate for a therapeutic approach to alleviating microglia-mediated dopaminergic neuronal injury in PD.