OBJECTIVETo investigate and compare the effects of Compound Chaihu Shugan Decoction (CHSGD, "treatment from Gan") and Dingxian Pill (DXP, "treatment from the sputum") on the metabolism path of glutamate in the pentylenetetrazol-kindled seizure rats' hippocampus, thus exploring the molecular mechanism of "heterotherapy for homopathy".

METHODSA chronic kindling seizures rat model was established by intraperitoneal injecting pentylenetetrazol (PTZ). Totally 24 fully kindled seizure rats were randomized into four groups, i.e., the model control group, the Sodium Valproate (VPA) group, the DXP group, and the CHSGD group. They were respectively treated with normal saline, VPA, CHSGD, and DXP, respectively. Rats in the control group were treated with normal saline by peritoneal injection and by gastrogavage. After intragastric administration for 4 successive weeks, the glutamate (Glu) levels in the hippocampus were detected by high performance liquid chromatography (HPLC). The expressions of glutamate transporter-1 (GLT-1) proteins were detected by Western blot. The activity of glutamine synthetase (GS) was detected by using GS detection kit.

RESULTSCompared with the control group, the content of Glu in the model group significantly increased, and the expression of GLT-1 and the activity of GS significantly decreased (P < 0.01). Compared with the model group, the content of Glu in each medication group significantly decreased, and the protein expression of GLT-1 as well as the activity of GS significantly increased (P < 0.01). But when compared between the CHSGD group and the DXP group, the content of Glu was lower and the activity of GS was higher in the CHSGD group than in the DXP group (P < 0.01), while there was no statistical difference in the expression of GLT-1 between the two groups (P > 0.05).

CONCLUSIONSCHSGD ("treatment from Gan") and DXP ("treatment from the sputum") could both decrease the level of Glu and raise the expression of GLT-1 and the activity of GS, indicating that CHSGD and DXP both could regulate the metabolism path of Glu to affect the level of the Glu in the brain. But the effects of CHSGD were superior to those of DXP in decreasing the content of Glu and up-regulating the activity of GS, suggesting that there were some different effects targets between the two compounds on the metabolism path of Glu, which may be one of possible molecular mechanisms for treating epilepsy by heterotherapy for homopathy.

Animals ; Excitatory Amino Acid Transporter 2 ; metabolism ; Glutamic Acid ; metabolism ; Hippocampus ; metabolism ; Kindling, Neurologic ; Male ; Medicine, Chinese Traditional ; methods ; Pentylenetetrazole ; adverse effects ; Rats ; Rats, Wistar ; Seizures ; metabolism ; therapy

OBJECTIVEThe present study was undertaken to design antisense oligodeoxynucleotides (AS-ODNs) of glial glutamate transporter-la (GLT-1a) and to evaluate the effectiveness of the designed AS-ODNs on the expression of GLT-1a.

METHODSFive sequences of GLT-1a AS-ODNs were designed according to the C terminus specific sequences of GLT-1a mRNA using antisense design software of IDT Com- pany. Western blot analysis was used to evaluate the inhibition effects of the five GLT-1a AS-ODNs on the expression of GLT-la.

RESULTSThe sequence of GLT-1a AS-ODNs with sequence of 5'-GGTTCTTCCTCAACACTGCA-3' could specifically inhibit the expression of GLT-1a in the hippocampal CA1 subfield of rats, while it had no effect on the expression of GLT-1b. This sequence showed similar inhibition on the expression of GLT-la in sham and ceftriaxone (Cef)-treated rats. It could also significantly inhibit the cerebral ischemic preconditioning (CIP)-induced up-regulation in the expression of GLT-1a. The magnitude of the inhibition in sham, Cef- or CIP-treated rats was similar by more than 60%.

CONCLUSIONFrom the designed five sequences of GLT-1a AS-ODNs, we obtained an effective sequence which can specifically inhibit the expression of GLT-1a.

Animals ; CA1 Region, Hippocampal ; metabolism ; Excitatory Amino Acid Transporter 2 ; antagonists & inhibitors ; metabolism ; Ischemic Preconditioning ; Oligonucleotides, Antisense ; genetics ; RNA, Messenger ; Rats ; Up-Regulation

BACKGROUND AND PURPOSE: The progression of migraine into chronic daily headache involves multiple risk factors, but the main contributor is not known. Glutamate is the major excitatory neurotransmitter in central sensitization, which is an important process in the pathogenesis of migraine transformation. The glutamate transporter protein excitatory amino acid transporter 2 (EAAT2) is the primary modulator of glutamatergic neurotransmission, and genetic polymorphisms of its gene, EEAT2, have been identified. The aim of this study was to determine the effect of EAAT2 polymorphisms on migraine transformation into chronic daily headache. METHODS: We included 74 migraine patients with episodic attack (M-E) and 59 migraine patients with chronic daily headache (M-CDH). After amplifying EAAT2 by polymerase chain reaction, we assessed its genotype frequencies based on restriction fragment length polymorphisms. We reclassified all migraine patients into two groups according to their EAAT2 genotype, either with the A allele (n=62) or without it (n=71), and compared the clinical variables between the two groups. RESULTS: The genotype frequencies of EAAT2 polymorphisms did not differ between the M-E and M-CDH groups. Comparison between EEAT2 genotypes revealed that the frequency of analgesic usage was significantly higher among migraine patients with the A allele (12.9+/-1.6 days/month) than in those without the A allele (8.1+/-1.2 days/month; p=0.019). The other clinical variables of migraine did not differ between the two groups. CONCLUSIONS: The results suggest that EEAT2 polymorphism contributes to the tendency toward frequent analgesic usage in migraine patients. This implies a potential genetic influence on the progression of migraine into chronic daily headache through the development of medication-overuse headache.
Alleles ; Amino Acid Transport System X-AG ; Central Nervous System Sensitization ; Excitatory Amino Acid Transporter 2 ; Genotype ; Glutamic Acid ; Headache ; Headache Disorders ; Humans ; Migraine Disorders ; Neurotransmitter Agents ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Polymorphism, Restriction Fragment Length ; Risk Factors ; Synaptic Transmission

PURPOSE: The aim of this study was to investigate the differences of expression in glycolysis-related proteins such as Glut-1, carbonic anhydrase (CA) IX, and monocarboxylate transporter (MCT) 4 according to the myoepithelial cell (MEC) and basement membrane (BM) status in solid papillary carcinoma (SPC) of the breast. MATERIALS AND METHODS: Immunohistochemical evaluation of Glut-1, CAIX, and MCT4, as well as p63 and type IV collagen, were performed on 23 SPC cases. RESULTS: Six and nine cases of SPC showed the presence and absence of myoepithelial cells, respectively, and eight cases belonged to the borderline status (p63-positive MEC on some areas of the outer tumor surface but not in others). BM was partially or completely absent in 14 cases and present in nine cases. SPC lacking BM more frequently showed high expression of CAIX than SPC with BM (p=0.037). CONCLUSION: In SPC of the breast, a strong expression of CAIX seems to be associated with an increasing degree of loss of BM, which can be interpreted as BM degradation due to the induction of extracellular acidity with increasing expression of CAIX.
Adult ; Aged ; Basement Membrane/*metabolism ; Breast Neoplasms/*metabolism ; Carcinoma, Papillary/*metabolism ; Excitatory Amino Acid Transporter 2/metabolism ; Female ; Glycolysis ; Humans ; Immunohistochemistry ; Middle Aged ; Monocarboxylic Acid Transporters/metabolism ; Muscle Proteins/metabolism ; Tumor Markers, Biological/*metabolism

OBJECTIVETo observe the effects of electroacupuncture (EA) on the structure parameters of synapse and reactive changes of astrocyte in the marginal zone of focal cerebral ischemia in rats at different time zones so as to further explore its underlying mechanisms in the treatment of cerebral ischemia.

METHODSNinety male Wistar rats were randomly assigned to sham-operation, model, and EA groups, with 30 animals in each group. Each group was subdivided into 1 h, as well as 1, 3, 7, and 21 days post-operation groups, with 6 animals assigned to each time point subgroup. Heat coagulation-induced occlusion of the middle cerebral artery was performed to establish a model of focal cerebral ischemia. EA was applied immediately following surgery to the EA group [4/20 Hz, 2.0-3.0 V, 1-3 mA, to Baihui (GV20) and Dazhui (GV14)] for 30 min. Treatment was performed once a day, and experimental animals were sacrificed at 1 h, as well as 1, 3, 7 and 21 days postoperation. The ultrastructure changes in synapse and astrocytes were observed by using transmission electron microscopy. Glial fibrillary acidic protein (GFAP) expression and Ca(2+) of astrocytes were measured by using laser confocal scanning microscope. Excitatory amino acid transporters-2 (EAAT2) and connexin 43 (CX43) expressions were assayed with immunohistochemical method. Canonical correlation analysis was conducted between structure parameters of synapse and parameters of astrocyte in the same time and group.

RESULTSBroken synapses were observed following cerebral ischemia, and the numbers of synapses were significantly decreased. Compared with the model group, synaptic ultrastructure was significantly improved in the EA group. Compared with the sham-operation group, synaptic number density was significantly decreased, as were postsynaptic density thickness, synaptic cleft width and synaptic interface curvature in the EA and model groups. However, compared with the model group, postsynaptic density thickness was significantly increased in the EA group at the same time points post-operation (P <0.05, P<0.01). In addition, synaptic cleft width, synaptic number density and synaptic interface curvature were significantly increased with the passage of time (P <0.05, P<0.01). The expression of GFAP in the EA group were significantly lower than those in the model group at all the time points (P <0.05, P<0.01). OD values of EAAT2 in the EA group were significantly higher than those in the model group at the same time (P <0.05, P<0.01). Compared with that in the model group, the expressions of CX43 in the EA group increased significantly at 3 days and 7 days (P <0.05, P<0.01). Ca(2+) average fluorescence intensity of astrocytes in the EA group was significantly lower than those in the model group at 1 h, 1 day, 3 days and 7 days (P <0.05, P<0.01). The changes in structure parameters of synapse were closely related to the changes of CX43, EAAT2, GFAP, Ca(2+) of astrocytes by EA treatment at all the time points.

CONCLUSIONSEA is helpful for synaptic reorganization, which may be related to its effect on intervening the activation state of astrocytes and promoting the beneficial interaction between astrocytes and synapses. Acupuncture could start the adjustment of neuron-glial network so as to promote the synaptic reorganization, which may be the key mechanism of treating cerebral ischemia.

Animals ; Astrocytes ; metabolism ; pathology ; Brain Ischemia ; pathology ; therapy ; Calcium ; metabolism ; Connexin 43 ; metabolism ; Electroacupuncture ; methods ; Excitatory Amino Acid Transporter 2 ; metabolism ; Fluorescence ; Glial Fibrillary Acidic Protein ; metabolism ; Male ; Post-Synaptic Density ; metabolism ; Rats ; Rats, Wistar ; Statistics as Topic ; Synapses ; metabolism ; pathology

The present study was undertaken to investigate the role of glial glutamate transporter-1 (GLT-1) in the brain ischemic tolerance induced by cerebral ischemic preconditioning (CIP) by observing the effect of GLT-1 antisense oligodeoxynucleotides (AS-ODNs) on the neuro-protection of CIP against brain ischemic insult in rats. Wistar rats with permanently occluded bilateral vertebral arteries were randomly assigned to 7 groups: (1) Sham group: the bilateral common carotid arteries (BCCA) were separated, but without occluding the blood flow; (2) CIP group: the BCCA were clamped for 3 min; (3) Brain ischemic insult group: the BCCA were clamped for 8 min; (4) CIP+brain ischemic insult group: 3 min CIP was preformed 2 d prior to 8 min ischemic insult; (5) Double distilled water group: 5 muL double distilled water was injected into the right lateral cerebral ventricle 12 h before, 12 h and 36 h after the BCCA was separated (but without occluding the blood flow), respectively; (6) AS-ODNs group: 5 microL AS-ODNs solution was injected into the right lateral cerebral ventricle 12 h before, 12 h and 36 h after the BCCA was separated (but without occluding the blood flow), respectively. This group was further divided into 9 nmol and 18 nmol subgroups according to the doses of AS-ODNs; (7) AS-ODNs+CIP+brain ischemic insult group: 5 microL AS-ODNs solution was injected into the right lateral cerebral ventricle 12 h before, 12 h and 36 h after CIP, respectively. This group was also further divided into 9 nmol and 18 nmol subgroups according to the doses of AS-ODNs. The other treatments were the same as those in CIP+brain ischemic insult group. The effect of the AS-ODNs on the expression of GLT-1 was assayed by using Western blot analysis. The profile of delayed neuronal death (DND) of pyramidal neurons in the CA1 hippocampus was evaluated by using thionin staining under light microscope by determining the neuronal density (ND) and histological grade (HG). Western blot analysis showed that AS-ODNs injected into the lateral cerebroventricle inhibited the expression of GLT-1 in the CA1 hippocampus in a dose-dependent manner. Neuropathological evaluation showed that there was no apparent DND in sham and CIP groups. Obvious DND of pyramidal neurons was found in brain ischemic insult group, which was represented by an increase in HG and a decrease in ND. CIP effectively protected the pyramidal neurons in the CA1 hippocampus against DND normally induced by ischemic insult, which indicating that CIP induced ischemic tolerance on the pyramidal neurons in the CA1 hippocampus. However, the injection of AS-ODNs into the lateral cerebroventricle blocked the neuro-protection of CIP against DND induced by brain ischemic insult. These results further proved the role of GLT-1 in the brain ischemic tolerance induced by CIP in rats.
Animals ; Brain ; pathology ; Brain Ischemia ; drug therapy ; CA1 Region, Hippocampal ; pathology ; Excitatory Amino Acid Transporter 2 ; metabolism ; Ischemic Preconditioning ; Oligodeoxyribonucleotides ; pharmacology ; Oligonucleotides, Antisense ; pharmacology ; Pyramidal Cells ; metabolism ; Rats ; Rats, Wistar

OBJECTIVETo investigate the influence of najanalgesin on glutamate-glial transporter 1(GLT-1) in spinal cord of rats after L5 spinal nerve ligation and transection (SNL), and explore the spinal analgesic mechanism of najanalgesin.

METHODOne hundred male SD rats were randomly divided into 6 groups: sham(A), SNL(B), SNL + najanalgesin(C), SNL + saline (D), SNL + najanalgesin + liposome (E), SNL + najanalgesin + liposome + GLT-1 As-ODNs(F) and treated with intrathecal injections of 10 p.L saline (A and D), 40 ng X kg(-1) najanalgesin (C, E and F), qd, respectively. Besides intrathecal administration of najanalgesin the rats were intrathecally injected with 10 microL of GLT-1 antisense oligodeoxynucleotides (As-ODNs) (F) and 10 micdroL of liposome(E) once daily on day 3. The L4-L6 segments of the spinal cord were isolated in 1, 4 and 7 d(A,B,C and D), 7 d(E and F) after surgery. The mRNA and protein of GLT-1 were determined.

RESULTThe SNL model has successfully been set up. Compared to sham group, the expression of GLT-1 mRNA and protein level in group B and D both increased firstly and decreased later, the expression of GLT-1 in group C was significantly increased and kept stable, which were also higher when compared to group D in day 7th. Compared to SNL + najanalgesin group, after intrathecal injection of GLT-1 As-ODNs the GLT-1, expression of GLT-1 in F group significantly decreased. While intrathecal administration of liposome had no significant effect on the spinal GLT-1 expression.

CONCLUSIONNajanalgesin could increase the mRNA and protein expression of GLT-1 in spinal cord, which may be one of its spinal mechanisms of analgesia.

Animals ; Elapid Venoms ; pharmacology ; Elapidae ; Excitatory Amino Acid Transporter 2 ; genetics ; metabolism ; Gene Expression Regulation ; drug effects ; Male ; Neuralgia ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Spinal Cord ; drug effects ; metabolism

In order to investigate the role of spinal glutamate transporter 1 (GLT-1) in the neuropathic pain and morphine tolerance, rat chronic constriction injury (CCI) of sciatic nerve was performed, and the mechanical allodynia was evaluated by mechanical withdrawal threshold (MWT), the expression of GLT-1 was measured by real-time PCR and Western blotting analysis. The results showed that compared to sham group, the MWT of CCI group had decreased approximately 80%. Administration of morphine alone could develop tolerance rapidly in initial two days, and then had no significant difference with CCI group, the expression of GLT-1 was down-regulated. Ceftriaxone sodium alone could improve mechanical allodynia. Co-administration of ceftriaxone sodium with morphine attenuated morphine tolerance and up-regulated GLT-1 expression, and the MWT remained at high level after 6 days. In conclusion, change of spinal GLT-1 expression and function has close correlation with the development of neuropathic pain and morphine tolerance.
Animals ; Drug Tolerance ; Excitatory Amino Acid Transporter 2 ; metabolism ; pharmacology ; Female ; Male ; Morphine ; pharmacology ; Radiculopathy ; metabolism ; pathology ; Rats ; Rats, Wistar ; Sciatic Nerve ; pathology ; Sciatic Neuropathy ; metabolism ; pathology ; Spinal Cord ; drug effects ; metabolism

Neural progenitor cells (NPs) have shown several promising benefits for the treatment of neurological disorders. To evaluate the therapeutic potential of human neural progenitor cells (hNPs) in amyotrophic lateral sclerosis (ALS), we transplanted hNPs or growth factor (GF)-expressing hNPs into the central nervous system (CNS) of mutant Cu/Zn superoxide dismutase (SOD(1G93A)) transgenic mice. The hNPs were engineered to express brain-derived neurotrophic factor (BDNF), insulin-like growth factor-1 (IGF-1), VEGF, neurotrophin-3 (NT-3), or glial cell-derived neurotrophic factor (GDNF), respectively, by adenoviral vector and GDNF by lentiviral vector before transplantation. Donor-derived cells engrafted and migrated into the spinal cord or brain of ALS mice and differentiated into neurons, oligodendrocytes, or glutamate transporter-1 (GLT1)-expressing astrocytes while some cells retained immature markers. Transplantation of GDNF- or IGF-1-expressing hNPs attenuated the loss of motor neurons and induced trophic changes in motor neurons of the spinal cord. However, improvement in motor performance and extension of lifespan were not observed in all hNP transplantation groups compared to vehicle-injected controls. Moreover, the lifespan of GDNF-expressing hNP recipient mice by lentiviral vector was shortened compared to controls, which was largely due to the decreased survival times of female animals. These results imply that although implanted hNPs differentiate into GLT1-expressing astrocytes and secrete GFs, which maintain dying motor neurons, inadequate trophic support could be harmful and there is sexual dimorphism in response to GDNF delivery in ALS mice. Therefore, additional therapeutic approaches may be required for full functional recovery.
Adenoviridae/genetics ; Amyotrophic Lateral Sclerosis/metabolism/mortality/*therapy ; Animals ; Astrocytes/metabolism ; Brain/*embryology ; Cell Differentiation ; Disease Models, Animal ; Excitatory Amino Acid Transporter 2/metabolism ; Female ; Fetal Stem Cells/*metabolism ; Genetic Vectors ; Humans ; Immunoenzyme Techniques ; Male ; Mice ; Mice, Transgenic ; Motor Neurons/*physiology ; Nerve Growth Factors/*metabolism ; *Stem Cell Transplantation ; Superoxide Dismutase/genetics ; Transfection ; Vascular Endothelial Growth Factor A/genetics/metabolism

In recent years, more attention has been paid to the role of the glutamate transporter 1 (GLT-1, EAAT2) in major depressive disorder (MDD). However, experimental data on brain GLT-1 levels are, to some extent, inconsistent in human postmortem and animal studies. These discrepancies imply that the role of GLT-1 in the pathophysiology of MDD and the action of antidepressants remain obscure. This work was designed to study the impact of chronic unpredictable stress (CUS) for 2 sessions per day for 35 days and four weeks of fluoxetine (FLX) on depressive-like behaviors in rats, as well as the concomitant expression of the GLT-1 protein in the hippocampus. Behavioral changes were assessed by the sucrose preference and open field tests. GLT-1 levels were detected by immunohistchemistry and Western blot analysis. Our study demonstrated that the animals exposed to CUS showed depressive-like behaviors and exhibited a significant decrease in GLT-1 expression in the hippocampus. Chronic FLX treatment reversed the behavioral deficits and the CUS-induced decrease in GLT-1 levels. Taken together, our results support the reduction of GLT-1 in human postmortem studies in MDD and suggest that GLT-1 may be involved in the antidepressant activity of FLX. Our studies further support the notion that GLT-1 is an attractive candidate molecule associated with the fundamental processes of MDD and may be a potential, and novel pharmacological target for the treatment of MDD.
Animals ; Antidepressive Agents, Second-Generation ; pharmacology ; Behavior, Animal ; drug effects ; Brain ; metabolism ; pathology ; Chronic Disease ; Depressive Disorder, Major ; drug therapy ; metabolism ; pathology ; Excitatory Amino Acid Transporter 2 ; metabolism ; Fluoxetine ; pharmacology ; Humans ; Male ; Rats ; Rats, Sprague-Dawley ; Stress, Psychological ; drug therapy ; metabolism ; pathology