Humans ; Infant ; Spasms, Infantile ; therapy

Objective To explore the different influence of antiepileptic drugs(AEDs)at therapeutic levels to the maturation of brain.Methods 180 healthy Sprague-Dawley(SD)rats were divided into infant and adult group.Each age group was administered with PB,CNP,VPA,TPM or normal saline respectively in persistent 5 weeks.The steady-state plasma concentrations of AEDs at the experimental dosage were coincided with the range of clinical therapeutic concentrations.After AEDs withdrawed,the effects of AEDs on cognitive function were assessed by Morris water maze and two-way shuttle box at different time points.Body and brain weight were got immediately when the rats were sacrificed.Histological changes of brain were observed by HE staining,Nissl staining and transmission electron microscopy.Results(1) For immature rats,1 day or 14 days after AEDs withdrawed,there were significant differences between groups exposed to PB or CNP and control group in escape response latency(ERL)in the two-way shuttle box.Even after one month ERLs of immature rats receiving CNP((6.05?2.04)s)or PB((5.81? 1.75)s)were still longer than that of untreated controls((4.75?2.43)s,P

The specific fragment of Pneumococcal surface protein A(PspA)and Pneumococcal Surface Adhesin A(PsaA)gene was amplified by PCR from Streptococcus pneumonia 5 and Streptococcus pneumonia 19.The amplified fragnent of PspA and PsaA gene was ligated into pET-27b(+)vector and transformed into BL 21 E.coli for expression and obtain the expressive production of PspA and PsaA.Induced by IPTG,the expression level was as high as 75 % of the total disolube protein.The result showed that the recombinant plasmid could express a specific 75 kDa and 37 kDa fusion protein in E.coli BL 21,which showed the good immunogenicity and a broadly cross reactivity with the other serotypes.

The study aims to identify the role of cAMP-PKA pathway in the group Ⅱ metabotropic glutamate receptors (mGluRs)-mediated regulation of respiratory rhythm from the brainstem slice. Neonatal (aged 0-3 d) Sprague-Dawley rats of either sex were used. The brainstem slice containing the medial region of the nucleus retrofacialis (mNRF) and the hypoglossal nerve rootlets was prepared, and the surgical procedure was performed in the modified Kreb's solution (MKS) with continuous carbogen (95% O2 and 5% CO2) bubbling, and ended in 3 min. Respiratory rhythmical discharge activity (RRDA) of the hypoglossal nerve rootlets was recorded by suction electrode. Eighteen brainstem slice preparations were divided into 3 groups. In group 1, group Ⅱ mGluRs specific antagonist (2S)-α-ethylglutamic acid (EGLU) was added into the perfusion solution for 10 min. In group 2, after application of Forskolin for 10 min, washout with MKS, the slice was perfused with Rp-cyclic 3', 5'-hydrogen phosphorothioate adenosine triethylammonium salt (Rp-cAMPS) alone for another 10 min. In group 3, after application of Rp-cAMPS for 10 min, additional EGLU was added into the perfusion for another 10 min. The results showed EGLU shortened respiratory cycle (RC), but the changes of integral amplitude (IA) and inspiratory time (TI) were not statistically significant. Forskolin induced significant decreases in RC, and increased TI, IA. Rp-cAMPS could make the opposite effect compared with the changes of RRDA with Forskolin. The effect of EGLU on the RRDA was inhibited after blocking the cAMP-PKA pathway. Taken together, cAMP-PKA pathway may play an important role in the group Ⅱ mGluRs-mediated regulation of RRDA in the brainstem slice of neonatal rats.
Animals ; Animals, Newborn ; Brain Stem ; physiology ; Cyclic AMP-Dependent Protein Kinases ; metabolism ; Female ; In Vitro Techniques ; Male ; Rats ; Rats, Sprague-Dawley ; Receptors, Metabotropic Glutamate ; physiology ; Respiration ; Signal Transduction ; physiology

OBJECTIVETo study the autophagy activity between rat bone marrow stem cells (BMSCs) neural differentiation in order to explore the mechanism involve in this process.

METHODSBMSCs were passed by 3 generation, then was induced with the revulsant 2% (DMSO) + 200 µmol/L (BHA), NSE expression was detected by immunocytochemical stain, the mRNA expression of autophagy associated genes L3B, Beclinl, Atg5, Atg7, Atg10 were detected by RT-PCR, the autophagy protein LC3B was examined by Western blot and flow cytometry analysis.

RESULTSBMSCs were passed by 3 generation, the purity of BMSCs could reach more than 90%, the morphology of cells were like fibroblasts, after the revulsant 2% DMSO + 200 µmol/L BRA induced, cells were extended long neurites, like nerve cells, positive rate of NSE staining was (83±5) %, RT-PCR results showed that the expression of autophagy associated genes LC3B, Beclinl, Atg5, Atg7 Atg0 were rised after BMSCs neural differentiation, Western blot analysis showed that the LC3B-II protein expression was increased after neural differentiation and the MFI of L3B was highten by flow cytometry.

CONCLUSIONAutophagy is increased after rat BMSC neural differentiation.

Animals ; Autophagy ; Cell Differentiation ; Cells, Cultured ; Flow Cytometry ; Mesenchymal Stromal Cells ; cytology ; Neurons ; cytology ; Rats

OBJECTIVETo explore the possibility of brain damage induced by several anti-epileptic drugs (AEDs) at therapeutic level to immature brain of rat.

METHODSTotally 160 healthy Spraque-Dawley (SD) rats selected for the study were divided into infant and adult groups. Each age group was treated with phenobarbital (PB), clonazepam (CZP), valproic acid (VPA), topiramate (TPM) or normal saline respectively for 2 or 5 weeks with 8 rats in each group. The steady-state plasma concentrations of AEDs at the experimental dosage were coincided with the range of clinical therapeutic concentrations. Drug levels in plasma were determined by fluorescence polarization. Body and brain weights were measured when the rats were sacrificed. Histological studies on the tissues of frontal lobes and hippocampus were performed by Nissl staining. And ultrastructural changes of brain were observed by the transmission electron microscopy. Plasma neuron-specific enolase (NSE) was determined by ELISA. Expression of apoptosis-related proteins Bcl-2 and Bax in neurons was detected by immunohistochemistry. Neuronal apoptosis was detected by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL).

RESULTS(1) There were no significant differences in brain weight among all adults groups. While remarkable reduction of brain weight was observed in immature rats exposed to CZP or PB (P < 0.01) for long term. (2) Significant neurodegeneration, neuronal necrosis and decrease in the number of neurons can be observed in the immature rats exposed to CZP or PB for long period. (3) For immature rats, concentration of plasma NSE was increased even after short-term treatment with PB [(8.84 +/- 2.10) nmol/L] compared with control group [(6.27 +/- 1.27) nmol/L] (P < 0.01). And it was increased in immature rats exposed to CZP [(8.15 +/- 1.67) nmol/L] or PB [(8.07 +/- 1.27) nmol/L] for long term compared with controls [(6.02 +/- 1.20) nmol/L] (P < 0.01). But there were no significant differences between AEDs-treated adult rats and control rats. (4) The expression of Bcl-2 and Bax protein in mature brain did not change at therapeutic level. In contrast, expression of Bax protein in the frontal lobe was increased significantly in immature rats receiving CZP and PB for long period compared with control. (5) The number of TUNEL positive cells in immature rats exposed to CZP or PB for long term was obviously increased.

CONCLUSIONSPB and CZP may result in remarkable histological abnormalities, neuronal apoptosis and necrosis in immature brain. The brain damage induced by PB was more serious and persistent than that induced by CZP.

Age Factors ; Animals ; Anticonvulsants ; adverse effects ; Apoptosis ; Brain ; pathology ; ultrastructure ; Brain Diseases ; chemically induced ; pathology ; Clonazepam ; adverse effects ; Microscopy, Electron, Transmission ; Neurons ; pathology ; Phenobarbital ; adverse effects ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Rats, Sprague-Dawley ; Valproic Acid ; adverse effects ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo explore the role of glial cell metabolism in the generation and regulation of central respiratory rhythm.

METHODSThe medulla oblongata slices (600-700 microm) containing the medial region of the nucleus retrofacialis (mNRF) with the hypoglossal nerve rootlets retained from 12 neonatal (0-3 days) Sprague-Dawley rats were prepared and perfused with modified Kreb's solution (MKS). Upon recording of respiratory rhythmical discharge activity (RRDA) of the rootlets of the hypoglossal nerve, the brain slices were treated with glial cell metabolism antagonist L-methionine sulfoximine (L-MSO, 50 micromol/L) for 20 min followed by application of glial cell metabolism agonist L-glutamine (L-GLN, 30 micromol/L) for 20 min, or with L-MSO for 20 min with additional L-GLN for 20 min. The changes in the RRDA of the rootlets of the hypoglossal nerve in response to the treatments were recorded.

RESULTSL-MSO prolonged the respiratory cycle (RC) and expiratory time (TE), and reduced the integral amplitude (IA) and the inspiratory time (TI) in the brain slices. L-GLN induced a significant decrease in RC and TE, but IA and TI showed no obvious variations. The effect of L-MSO on the respiratory rhythm was reversed by the application of L-GLN.

CONCLUSIONGlial cell metabolism may play an important role in the modulation of RRDA in neonatal rat brainstem.

Animals ; Animals, Newborn ; Glutamine ; pharmacology ; In Vitro Techniques ; Medulla Oblongata ; metabolism ; physiology ; Methionine Sulfoximine ; pharmacology ; Neuroglia ; metabolism ; Periodicity ; Rats ; Rats, Sprague-Dawley ; Respiration

The high expression of tissue factor (TF) is related to the coagulation disorder in acute leukemia. TF in blood circulation is mainly expressed in cells, microparticles (MP) and alternatively spliced human tissue factor (asHTF). To elucidate the role of TF in the coagulation disorder of acute myeloid leukemia (AML), RT-PCR was performed on 6 common AML cell lines NB4, HL-60, Kasumi-1, U937, K562 and THP-1. The results showed that only NB4 and U937 cells expressed baseline full-length TF and asHTF which were proved by sequencing. The flow cytometric detection, TF activity and TF antigen tests in NB4 and U937 cells revealed that the asHTF was expressed in trace amount and almost had no activity, while the TF antigen and activity in microparticles were significantly higher than that in asHTF. It is concluded that asHTF may play an unimportant role in the coagulation disorder of AML. Microparticle associated tissue factor (MP-TF) is the predominant source of TF activity released from AML cells.
Alternative Splicing ; HL-60 Cells ; Humans ; Leukemia, Promyelocytic, Acute ; genetics ; metabolism ; Thromboplastin ; genetics ; metabolism ; Tumor Cells, Cultured ; U937 Cells

OBJECTIVETo explore the role of group II metabotropic glutamate receptors in the modulation of basic respiratory rhythm.

METHODSNeonatal (0-3 days) SD rats of either sex were used. The medulla oblongata brain slice containing the medial region of the nucleus retrofacialis (mNRF) and the hypoglossal nerve rootlets was prepared, and the surgical procedure was performed in the modified Kreb's solution (MKS) with continuous carbogen (95% O2 and 5% CO2) within 3 min. The brain slices were quickly transferred to a recording chamber and continuously perfused with oxygen-saturated MKS at a rate of 4-6 ml/min at 27-29 degrees celsius. Eighteen medulla oblongata slices were divided into 3 groups and treated for 10 min with group II metabotropic glutamate receptor-specific agonist 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate (APDC) (at concentrations of 10, 20, 50 micromol/L), group II metabotropic glutamate receptor antagonist (2S)-alpha-ethylglutamic acid (EGLU) (300 micromol/L), or APDC (50 micromol/L)+EGLU (300 micromol/L) after a 10 min APDC (50 micromol/L) application. Respiratory rhythmical discharge activity (RRDA) of the rootlets of the hypoglossal nerve was recorded by suction electrodes.

RESULTSAPDC produced a dose-dependent inhibitory effect on the RRDA, prolonging the respiratory cycle and expiratory time and decreasing the integral amplitude and inspiratory time. EGLU induced a significant decrease in the respiratory cycle and expiratory time. The effect of APDC on the respiratory rhythm was partially reversed by the application of APDC+EGLU.

CONCLUSIONGroup II metabotropic glutamate receptors are probably involved in the modulation of the RRDA in isolated neonatal rat brainstem slice.

Animals ; Animals, Newborn ; In Vitro Techniques ; Medulla Oblongata ; physiology ; Rats ; Rats, Sprague-Dawley ; Receptors, Metabotropic Glutamate ; physiology ; Respiratory Center ; physiology

OBJECTIVETo investigate the effects of doxapram on the respiratory rhythmical discharge activity (RRDA) in the brainstem slices of neonatal rats.

METHODSThirty neonatal SD rats (of either sex, 0-3 days old) were randomly divided into 6 equal groups (groups I-VI), and the brainstem slices which contained the medial region of the nucleus retrofacialis (mNRF) were prepared. All the slices were perfused with modified Kreb's solution (MKS), and in group I (control group), the slices were perfused with MKS only; in groups II to IV, the slices were perfused with doxapram in MKS continuously at the concentrations of 2, 5, and 10 micromol/L, respectively; in groups V and VI, the slices were perfused with 20 micromol/L propofol and 20 micromol/L propofol plus 5 micromol/L doxapram, respectively. The RRDA in the hypoglossal nerve was recorded by suction electrode. The discharge time course of the inspiratory (TI), expiratory (TE), respiratory cycle (RC) and integral amplitude of the inspiratory discharge (IA) were recorded at 1, 3, 5, 10, 15, and 30 min after the application of the drugs.

RESULTSThe hypoglossal nerve in groups I, II and VI showed no significant changes of RRDA in the entire course of the experiment (P>0.05). In groups III and IV, the TI, IA increased and TE decreased significantly 5 min after doxapram application (P<0.05), and the RC was shortened only at 10 min. In group V, the TI and IA decreased and the RC and TE increased significantly after the drug application (P<0.05).

CONCLUSIONDoxapram (>5 micromol/L ) can directly stimulate the RRDA and prevent propofol-induced inhibitory effects in the brainstem slice of neonatal rats, and the effects are mediated by its actions upon the inspiratory neurons in the mNRF.

Animals ; Animals, Newborn ; Doxapram ; pharmacology ; Electrophysiological Phenomena ; drug effects ; Female ; In Vitro Techniques ; Male ; Medulla Oblongata ; physiology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Respiration ; drug effects ; Respiratory System Agents ; pharmacology