Laminar Distribution of Neurochemically-Identified Interneurons and Cellular Co-expression of Molecular Markers in Epileptic Human Cortex / 神经科学通报·英文版

Inhibitory GABAergic interneurons are fundamental elements of cortical circuits and play critical roles in shaping network activity. Dysfunction of interneurons can lead to various brain disorders, including epilepsy, schizophrenia, and anxiety. Based on the electrophysiological properties, cell morphology, and molecular identity, interneurons could be classified into various subgroups. In this study, we investigated the density and laminar distribution of different interneuron types and the co-expression of molecular markers in epileptic human cortex. We found that parvalbumin (PV) and somatostatin (SST) neurons were distributed in all cortical layers except layer I, while tyrosine hydroxylase (TH) and neuropeptide Y (NPY) were abundant in the deep layers and white matter. Cholecystokinin (CCK) neurons showed a high density in layers IV and VI. Neurons with these markers constituted ~7.2% (PV), 2.6% (SST), 0.5% (TH), 0.5% (NPY), and 4.4% (CCK) of the gray-matter neuron population. Double- and triple-labeling revealed that NPY neurons were also SST-immunoreactive (97.7%), and TH neurons were more likely to express SST (34.2%) than PV (14.6%). A subpopulation of CCK neurons (28.0%) also expressed PV, but none contained SST. Together, these results revealed the density and distribution patterns of different interneuron populations and the overlap between molecular markers in epileptic human cortex.

Functional Investigation of a GRIN2A Variant Associated with Rolandic Epilepsy / 神经科学通报·英文版

N-methyl-D-aspartate receptors (NMDARs), a subtype of glutamate-gated ion channels, play a central role in epileptogenesis. Recent studies have identified an increasing number of GRIN2A (a gene encoding the NMDAR GluN2A subunit) mutations in patients with epilepsy. Phenotypes of GRIN2A mutations include epilepsy-aphasia disorders and other epileptic encephalopathies, which pose challenges in clinical treatment. Here we identified a heterozygous GRIN2A mutation (c.1341T>A, p.N447K) from a boy with Rolandic epilepsy by whole-exome sequencing. The patient became seizure-free with a combination of valproate and lamotrigine. Functional investigation was carried out using recombinant NMDARs containing a GluN2A-N447K mutant that is located in the ligand-binding domain of the GluN2A subunit. Whole-cell current recordings in HEK 293T cells revealed that the N447K mutation increased the NMDAR current density by ~1.2-fold, enhanced the glutamate potency by 2-fold, and reduced the sensitivity to Mg inhibition. These results indicated that N447K is a gain-of-function mutation. Interestingly, alternative substitutions by alanine and glutamic acid at the same residue (N447A and N447E) did not change NMDAR function, suggesting a residual dependence of this mutation in altering NMDAR function. Taken together, this study identified human GluN2A N447K as a novel mutation associated with epilepsy and validated its functional consequences in vitro. Identification of this mutation is also helpful for advancing our understanding of the role of NMDARs in epilepsy and provides new insights for precision therapeutics in epilepsy.

Efficacy and safety of cyclophosphamide as a sequential immunotherapy drug for anti-N-methyl-D-aspartate receptor encephalitis in children / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To evaluate the efficacy and safety of cyclophosphamide as a second-line drug in the treatment of children with anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis.</p><p><b>METHODS</b>Six children with anti-NMDAR encephalitis, who showed poor response to steroids and intravenous immunoglobulin, were given cyclophosphamide as a second-line immunotherapy. Follow-up was performed to evaluate the efficacy and safety of cyclophosphamide.</p><p><b>RESULTS</b>After first-line immunotherapy for 1-4 weeks, the six patients had reduced psychiatric symptoms, seizures, and involuntary movements; three patients had an improved level of consciousness and were able to make simple conversations. However, all the patients still showed slow response, as well as cortical dysfunction symptoms such as aphasia, alexia, agraphia, acalculia, apraxia, and movement disorders. The six patients continued to receive cyclophosphamide as a sequential therapy. They were able to answer simple questions 7 days after treatment. Three school-aged patients were able to make simple calculation, had greatly improved reading and writing ability, and almost recovered self-care ability 2-3 weeks later. The cognitive function of the six patients was almost restored to the level before the onset of disease, and their living ability returned to normal 2-3 months later. During the treatment period, there were no adverse reactions or abnormal results of routine blood test and liver and kidney function tests.</p><p><b>CONCLUSIONS</b>Children with anti-NMDAR encephalitis should be given appropriate immunotherapy as soon as possible. Cyclophosphamide as a sequential therapy has good efficacy and safety.</p>

Calcium transient of CA1 pyramidal neurons induced by potassium blocker 4-aminopyridine in acute hippocampal slices / 中华神经医学杂志

Objective To investigate the calcium transient of CA1 pyramidal neurons induced by potassium blocker 4-aminopyridine (4-AP) in acute hippocampal slices to explore the relation between potassium channel function and calcium transient, and their mechanism. Methods Fluorescent probe was employed to mark the hippocampai neurons in acute brain slices of rats; confocal microscopy was used to perform calcium imaging to observe the influences of different concentrations of 4-AP and perfusate with/without calcium on calcium transient of CA1 pyramidal neurons. Results The response of [Ca2+]I to lower concentration of 4-AP (<15 mmol/L) was in a dose-dependent manner (r2=0.910, P=0.000); the higher the concentration of 4-AP (20-80 mmol/L), the lower the peak level of calcium transient. The latency and amplitude of calcium transient induced by 4-AP were obviously reduced when the extracellular condition was switched to an absence of calcium, which was significantly different as compared with that with calcium (P<0.05). Conclusion Blockade of potassium channels with 4-AP can increase [Ca2+]I in the hippocampal pyramidal neurons of acute slices. The increase of [Ca2+]1 to 4-AP could be ascribe to calcium release from intracellular stores and calcium influx from extracellular matrix.

Intron mutations of GABRG2 gene in patients with epilepsy combined with febrile seizures plus:an in vitro splicing assay / 中华神经医学杂志

Objective To screen the GABRG2 gene in Chinese patients diagnosed as having epilepsy with febrile seizures plus (EFS+) and analyze the in vitro splicing of intron mutations of GABRG2 gene. Methods After collecting the blood samples from patients with EFS+, all 9 coding exons and introns relevant to mRNA splice of GA BRG2 gene were sequenced by PCR. PCR products of exons 7, 8 and 9 and part of the introns of both ends of GABRG2 gene were cloned into the pTARGET vector to construct pTARGET-Exon-7-8-9 minigene vector and its Exon8+45C>T mutation vector.Wild-type and Exon8+45C>T mutation vector were transfected into HEK 293 cells and extracted RNA for RT-PCR. Results We did not detect mutation in GABRG2 gene coding region, but found 1 mutation in intron Exon8+45C>T. After splicing, the size of RT-PCR products of Wild-type and Exon8+45 OT mutation were both 522 bp. Conclusion Mutations in GABRG2 gene coding region are not likely to be substantially involved in the etiology of EFS+. Exon8+45C>T mutation does not affect the splicing of GABRG2 gene.

MRI characteristics of different pathologic types of focal cortical dysplasia / 中华神经医学杂志

Objective To investigate the MRI characteristics of different pathologic types of patients with focal cortical dysplasia (FCD). Methods The postoperative clinical data of 23 patients with FCD were retrospectively analyzed and they were divided into patient groups of 4 types according to Palmini' s classification. The MRI characteristics of different pathologic types of patients with FCD were concluded. Results Among these 23 patients, 2 were diagnosed as having FCD ⅠA, 6 as having FCD IB, 8 as having FCD ⅡA and 7 as having FCD IE. As compared with that in patients with FCD Ⅰ, obviously increased signal of Flair images in patients with FCD Ⅱ was more prevalent (2:11,P=0.039). Increased signal in T2 images and tapering of abnormal white matter signals to ventricles were more prevalent in patients with FCD Ⅱ as compared with those in patients with FCD Ⅰ, but no significant differences were noted (P= 0.074, 0.058). As compared with patients with other 3 types of FCD, blurring of the gray matter-white matter junction, increased signal in T2 images, deep sulcus, thickening of the cortex and tapering of abnormal white matter signals to ventricles in patients with FCD ⅡB were more prevalent (P<0.05). Conclusion Different MRI characteristics in each type of FCD are noted, especially in patients with FCDⅡB. These different MRI characteristics are helpful to make preoperative diagnosis and planning of FCD.

Clinical, genetic and cerebral ultrastructural features of late infantile neuronal ceroid lipofuscinosis:a ease report / 中华神经医学杂志

Objective To study the clinical, histopathological and inheritance features of late infantile neuronal ceroid lipofuscinosis (LINCL). Methods The clinical manifestations and family history of a 4-year-old girl with an established diagnosis of LINCL were investigated and the findings in EEG, magnetic resonance imaging (MRI) and histological examination were analyzed. Results EEG of the patient showed diffuse background slowing with bursts of generalized spike-and-wave discharges or polyspike-and-wave activity. Brain MRI for her and her brother revealed brain atrophy, especially diffuse cerebellar atrophy. Histopathological examination also showed diffuse damages in the gray matter where numerous degenerated and atrophic neurons were found. Some immature neurons occurred in the disrupted cortical lamination. Electron microscopy revealed numerous osmiophilic granular lipofuscin inclusions in the cytoplasm of the neurons. Conclusion This patient presented with typical clinical and cerebellar ultrastructural features of LINCL, but the inheritance characteristics of the patient and the prominent lipofuscin pigments in the neurons suggest a case of new LINCL variant.

Association between cutaneous adverse reactions to antiepileptie drugs and HLA-B*IS02 allele / 中华神经医学杂志

Objective To investigate the association between cutaneous adverse drug reactions (CADRs) caused by antiepileptic drugs and HLA-B*1502 allele. Methods In 31 epileptic patients presented to the Epilepsy Clinic of the Second Affiliated Hospital of Guangzhou Medical College between January 2007 and May 2008, 13 had CADR to carbanazepine (CBZ) including 6 with Stevens-Johnson syndrome (SJS) and 7 with mild maculopapular exanthona (MPE);15 were CBZ-tolerant, and 3 had lamotrigine (LTG)-indueed MPE. All the patients underwent examinations using polymerase chain reaction with sequence specific palmers to analyze HLA -B*1502 allele frequencies, with 30 healthy subjects without a history of using CBZ or LTG as the control. Results HLA-B*IS02 allele frequency was 100% (6/6) in patients with CBZ-SJS, 57% (4/7) in patients with CBZ-induced MPE, and 33% (1/3) in patients with LTG-induced MPE. The frequency was 7% (1/15) in CBZ-tolerant patients and 10% (3/30) in the control subjects. Compared with the CBZ-tolerant patients and the control subjects, the patients with CBZ-induced SJS and MPE had significantly increased HLA -B*1502 allele frequency (P<0.05). Conclusions HLA-B*1502 allele is associated with CADRs to CBZ in epileptic patients.

Clinical features and mutations of voltage-gated sodium channel subunit type 1 gene in myoclonic-astatic epilepsy in infancy / 中华神经医学杂志

Objective To study the clinical features and genetic mechanism of myoclonic-astafic epilepsy (MAE) in infancy. Methods This study was conducted among 10 infants with MAE (including 7 male and 3 female patients) diagnosed between 2006 and 2008 according to the criteria of International League Against Epilepsy (2001). The clinical data including onset age, seizure type, physical signs, EEG, brain maguetic resonance imaging (MRI), effects of anti-epileptic drugs and prognosis were analyzed. The mutations of voltage-gated sodium channel subunit type 1 gene (SCN1A gene) were screened by denaturing high performance liquid chromatography and direct sequencing. Results The 10 MAE cases included 8 sporadic cases and 2 with a family history of febrile seizure and epilepsy. The onset age ranged from 5 months to 39 months, and all the MAE patients had multiple generalized seizure types, including myoclonic-atonic, myoclonic, atonic, tonic-clonic and absence seizures. Two patients had myoclonic status epilepticus, and 7 showed mental retardation. All the patients showed normal findings in MRI. SCN1A gene was screened in 8 of the MAE patients, and no mutation was found. Valproate, clonazepam and levetiracetam were effective in these MAE cases. Conclusion MAE is a rare epilepsy syndrome, whose genetic mechanism is still unclear. Valproate, clonazepam and levetiracetam are effective for MAE, which is associated with poor prognosis.

Mitochondrial myopathy,encephalopathy,lactic acidosis and stroke-like episodes:clinical manifestations and gene mutation analysis / 中华神经医学杂志

Objective To investigate the characteristic clinical manifestations and gene mutations in mitochondrial myopathy,encephalopathy,lactic acidosis and stroke-like episodes(MELAS).Methods The clinical manifestations,imaging data and muscle pathologies of a patient with MELASwere analyzed,and the mutations in the mitochondrial DNA were investigated using polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP)and gene sequencing.Results MELAS was clinically characterized by such symptoms as recurrent headache and vomiting,stroke-like episodes,epilepsy,intolerance to exercise,short stature,nerve deafness and lactic acidosis.CT scan demonstrated bilateral basal ganglia calcification,and magnetic resonance imaging(MRI)showed abnormal signals in the occipital lobe.Proton magnetic resonance spectroscopy revealed a visible peak of lactic acid in the area with T2-weighted abnormal signals,while a low peak of lactic acid was identified in the area with T2-weighted normal signals.Muscle biopsy did not find any mitochondrial anomalies.A heterozygous A 3243G mutation in the mitochondrial DNA was found in this patient.Conclusion The diagnosis of MELAS relies on a comprehensive analysis of the clinical features,imaging findings,pathological results and genetic analysis.Normal pathological results do not rule out the possibility of MELAS.Mitochondrial DNA mutation analysis should be carried out as a routine procedure for identifying MELAS.

Variant distribution of voltage-gated sodium channel type Ⅰ on different types of neurons in rat hippocampus / 中华神经医学杂志

Objective To investigate the variant distribution of voltage-gated sodium channel type Ⅰ (Nav1.1) on the different types of neurons in rat hippocampus. Methods In brain tissue sections from adult SD rats, the expression of Nav1.1 on the different types of neurons in rat hippocampus was detected and observed by immunohistochemistry (DAB staining) and laser confocal microscopy. Results Nav1.1 protein was expressed weakly on the soma of pyramidal cells and granular cells of the dentate gyrus, while strongly expressed on the scattered interneurons.Conclusions In the hippocampus of SD rats, the expression of Nav 1.1 protein on interneurons was strong, but weak on the projection neurons (pyramidal cells and granular cells of the dentate gyms). It can be inferred that on different types of neurons, different subtypes of sodium channels are predominately expressed.

Expression changes of microtubule associated protein 1B in the brain of Fmr1 knockout mice / 神经科学通报·英文版

<p><b>OBJECTIVE</b>To explore the regulatory effect of fragile X mental retardation protein (FMRP) on the translation of microtubule associated protein 1B (MAP1B).</p><p><b>METHODS</b>The expressions of MAP1B protein and MAP1B mRNA in the brains of 1-week and 6-week old fragile X mental retardation-1 (Fmr1) knockout (KO) mice were investigated by immunohistochemistry, Western blot, and in situ hybridization, with the age-matched wild type mice (WT) as controls.</p><p><b>RESULTS</b>The mean optical density (MOD) of MAP1B was significantly decreased in each brain region in KO6W compared with WT6W, whereas in KO1W, this decrease was only found in the hippocampus and cerebellum. MAP1B in 6-week mice was much less than that in 1-week mice of the same genotype. The results of Western blot and in situ hybridization showed that MAP1B protein and MAP1B mRNA were significantly decreased in the hippocampus of both KO1W and KO6W.</p><p><b>CONCLUSION</b>The decreased MAP1B protein and MAP1B mRNA in the Fmr1 knockout mice indicate that FMRP may positively regulate the expression of MAP1B.</p>

Antiepileptic drug trials with greater attention to daily clinical practice and special epilepsy syndromes

Most of the present antiepileptic drug trials focus on efficacy and short-term safety with limited information relevant for daily clinical practice, such as spectrum of efficacy, effective dose, titration method, drug interactions, and long-term outcome. Antiepileptic drug trials more akin to daily clinical practice should also be emphasized in future trials. There are, limited randomized control trials for certain seizure types or epilepsy syndromes, such as Lennox-Gastaut syndrome and severe myoclonic epilepsy in infancy. The author proposed self-controlled “class S” study in patients who has well-defined epilepsy syndrome with homogeneity in evolution of illness and high frequency of seizures.

Immunohistochemical investigation of voltage-gated potassium channel-interacting protein 1 in normal rat brain and Pentylenettrazole-induced seizures / 神经科学通报·英文版

Objective To explore the possible role of voltage-gated potassium channel-interacting protein 1 (KChIP1) in the pathogenesis of epilepsy. Methods Sprague Dawley female adult rats were treated with pentylenettrazole (PTZ) to develop acute and chronic epilepsy models. The approximate coronal sections of normal and epilepsy rat brain were processed for immunohistochemistry. Double-labeling confocal microscopy was used to determine the coexistence of KChIP1 and gamma-aminobutyric acid (GABA). Results KChIP1 was expressed abundantly throughout adult rat brain. KChIP1 is highly co-localize with GABA transmitter in hippocampus and cerebral cortex. In the acute PTZ-induced convulsive rats, the number of KChIP1-postive cells was significantly increased especially in the regions of CA1 and CA3 (P < 0.05); whereas the chronic PTZ-induced convulsive rats were found no changes. The number of GABA-labeled and co-labeled neurons in the hippocampus appeared to have no significant alteration responding to the epilepsy-genesis treatments. Conclusion KChIP1 might be involved in the PTZ-induced epileptogenesis process as a regulator to neuronal excitability through influencing the properties of potassium channels. KChIP1 is preferentially expressed in GABAergic neurons, but its changes did not couple with GABA in the epileptic models.

Comparison of Behavioral and Histological Changes between Pilocarpine-Induced Temporal Epilepsy Model and Pentylenetetrazole Kindling Absence Model in Rats / 中国康复理论与实践

@#ObjectiveTo investigate and compare the behavioral changes, neuron loss of hippocampus and mossy fiber sprouting between pilocarpine-induced status epilepticus (SE) model and pentylenetetrazole (PTZ) kindling model in rats.MethodsAfter two different epilepsy models were made, Vedio was adopted to observe the behavioral changes. Nissl staining and Neo-timms' staining were separately used to observe and compare the neuron loss of hippocampus and mossy fiber sprouting in the dentate gyrus (DG) at different time points during epileptogenisis.ResultsNo recurrent spontaneous seizure, no neuron loss and no mossy fiber sprouting were found in PTZ kindling model; whereas obvious neuron loss was found in CA1, CA3 of hippocampus and hilus of DG, and mossy fiber sprouting were found in pilocarpine model in parallel with recurrent spontaneous seizures. ConclusionPTZ kindling model resembles absence epilepsy in human, while pilocarpine-induced status epilepticus model resembles chronic temporal epilepsy in human. Neuron loss and mossy fiber sprouting may play an important role in epileptogenisis. Pilocarpine-induced epilepsy model can be regarded as an ideal chronic temporal epilepsy model.

Changes of amino acid content in hippocampus of epileptic rats treated with volatile oil of Acorus tatarinowii / 中国中药杂志

<p><b>OBJECTIVE</b>To study the changes of excitatory and inhibitory amino acid content in hippocampus of epileptic rats treated with volatile oil of A. tatarinowii, and explore the possible antiepiletic mechanism.</p><p><b>METHOD</b>The volatile oil was extracted through Supercritical-CO2 Fluid Extraction (SFE-CO2), and epileptic models were built up by kainic acid (KA) lateral ventricle injection. The content of amino acid in hippocampus of epileptic rats treated with volatile oil was calculated.</p><p><b>RESULT</b>The content of GABA increased and Glu decreased prominently (P < 0.05) after volatile oil 35 mg x kg(-1) intraperitoneal injection.</p><p><b>CONCLUSION</b>The volatile oil of A. tatarinowii can modulate the balance of excitatory and inhibitory amino acid in epileptic rats, thereby exerting its antiepileptic effect.</p>