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Excitatory toxicity(ET) is an important factor of neuropathic pain(NPP) induced by central sensitization(CS), and the association of pannexin-1(Panx1)-Src-N-methyl-D-aspartate receptor subunit 2 B(NMDAR-2 B) is an important new pathway for ET to initiate CS. The present study confirmed whether the central analgesic effect of Chuanxiong Rhizoma extract(CRE) was achieved through the synchronous regulation of the brain and spinal pathways of Panx1-Src-NMDAR-2 B. In this study, dynamic and simulta-neo-us microdialysis of the brain and spinal cord in vivo combined with behavioristics, high performance liquid chromatography(HPLC)-fluorescence detection, microdialysis analysis(ISCUS~(flex)), ultrasensitive multifactorial electrochemiluminescence immunoassay, ELISA, and Western blot was employed to investigate the protein expression of NMDAR-2 B, Src, and Panx1, extracellular excitatory amino acids, cytokines, energy metabolites, and substance P in spinal dorsal horn(SDH) and anterior cingulate cortex(ACC) after CRE intervention with the rat model of spared sciatic nerve injury(SNI) as the experimental tool. Compared with the sham group, the SNI group exhibited diminished mechanical withdrawal threshold(MWT)(P<0.01), increased cold spray scores(P<0.01), glutamate(Glu), D-serine(D-Ser), and glycine(Gly) in extracellular fluids of ACC, and Glu, D-Ser, interleukin-1β(IL-1β), and lactic acid(Lac) in extracellular fluids of SDH(P<0.05), dwindled tumor necrosis factor(TNF-α)(P<0.05), and elevated protein levels of NMDAR-2 B, Src, and Panx1 in ACC(P<0.05). Compared with the SNI model rats, high-and medium-dose CRE(CRE-H/M) could potentiate the analgesic activity as revealed by the MWT test(P<0.05) and CRE-M enabled the decrease in cold spray scores(P<0.05). CRE-H/M could inhibit the levels of Glu, D-Ser and Gly in the extracellular fluids of ACC(P<0.05), and the levels of Glu in the extracellular fluids of SDH(P<0.05) in SNI rats. CRE-M significantly increased the levels of glucose(Gluc), Lac, interferon-gamma(IFN-γ), keratinocyte chemoattractant/human growth-regulated oncogenes(KC/GRO), and IL-4 in extracellular fluids of SDH in SNI rats(P<0.05). CRE-H/M/L could also inhibit the levels of NMDAR-2 B, Src and Panx1 in ACC and SDH in SNI rats(P<0.05). The central analgesic effect of CRE is presumedly related to the inhibited release of excitatory amino acid transmitters(Glu, D-Ser and Gly) in ACC and SDH of SNI rats, decreased protein expression of NMDAR-2 B, Src and Panx1 in the two regions, and the regulation of the Panx1-Src-NMDAR-2 B pathway in the spinal cord and brain. The above findings partially clarified the scientific basis of clinical analgesic effect of Chuanxiong Rhizoma.
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Animals , Rats , Central Nervous System Sensitization , Neuralgia/drug therapy , Rats, Sprague-Dawley , Receptors, N-Methyl-D-Aspartate/metabolism , Signal Transduction , Spinal Cord/metabolismABSTRACT
Objective To investigate the expression of excitatory neurotransmitter glutamate (Glu),inhibitory neurotransmitter gamma-aminobutyric acid (GABA),glutamate receptor R1 (GluR1) and gammaaminobutyric acid receptor A (γ-aminobutyric acid A receptor,GABAA) in the brain of rats with intrauterine growth retardation.Methods Thirty-two healthy Wistar female virgin rats and eight healthy Wistar male rats were purchased from the Laboratory Animal Center of China Medical University.The pregnant rats were randomly divided into IUGR group and control group according to the order of conception.The control group was fed with normal diet,while the IUGR group was fed with low-protein diet.The offspring were named IUGR group and control group respectively.The expressions of positive cells of Glu,GluR1,GABA and GABAA in cerebral cortex of normal offspring and IUGR offspring were observed by immunohistochemistry.Results Immunohistochemical staining showed that the glutamate positive field of vision in IUGR group was more than that in control group (x2 =82.69,P ＜ 0.05).The glutamate receptor GluR1 positive field of vision in IUGR group was more than that in control group (x2 =76.91,P ＜0.05),while the gamma-aminobutyric acid and its receptors in IUGR group were lower than those in control group (x2 =91.51,x2 =24.05,respectively).The difference was statistically significant (P ＜ 0.05).Conclusion Intrauterine growth retardation can increase the expression of excitatory amino acids and receptors while decrease the expression of inhibitory amino acids and their receptors.Increased expression of excitatory amino acids and receptors and decreased expression of inhibitory amino acids and their receptors may be one of the mechanisms of epilepsy in patients with intrauterine growth retardation.
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@#Objective To analyze the physiologically active substances, named amino acid neurotransmitters, ATP and Ca2+ in rat spinal synaptosomes. Methods The spinal synaptosome was extracted by discontinuous Percoll density gradient centrifugation from a female Sprague-Dawley rat. The amino acid neurotransmitters were determined with high performance liquid chromatography, ATP content, Na+-K+-ATPase activity, Ca2+-Mg2+-ATPase activity and total ATPase activity with ultraviolet spectrophotometer, and Ca2+ concentration with fluorescence spectrophotometer. Results The amino acid neurotransmitters contained mainly aspartic acid, glycine, glutamic acid and r-aminobutyric acid. The concentration of ATP was about 414.7461 μmol/mg, total ATPase activity > Na+-K+-ATPase activity > Ca2+-Mg2+-ATPasese activity. The concentration of Ca2+ could be calculated from absorbancy directly. Conclusion The rat spinal synaptosome contains a variety of physiologically active substances, which can be accurately analyzed to explore their activities.
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Background: N(2-propylpentanoyl) urea (VPU) is a new valproic acid (VPA) analog with higher anticonvulsant activity than its parent compound in various animal models including seizure acutely induced by pilocarpine. Objective: Investigate its effects on hippocampal amino acid neurotransmitters in spontaneous recurrent seizure (SRS) rats. Methods: Pilocarpine hydrochloride was used to induce status epilepticus (SE). Animals were visually observed for two hours/day for an episode of SRS for six weeks. Microdialysis experiment was performed to detect hippocampal amino acid neurotransmitters on those rats that developed SRS. Results: In comparison to normal rats, hippocampal glutamate, gamma-aminobutyric acid (GABA), and glycine, significantly increased in SRS rats. Occurrence of SRS in the faces of increased level of inhibitory neurotransmitters suggests the key role played by glutamate in the genesis and control of SRS. Based on the observation in pilocarpine-induced SE, the level of glutamate in SRS rats significantly decreased by a clinically effective anticonvulsant, VPA (300 and 600 mg/kg, i.p). Similar profile on hippocampal glutamate was also exhibited by VPU (50 and 100 mg/kg, i.p.). Conclusion: The possible role of VPU in controlling seizure in SRS rats and subsequently human temporal lobe epilepsy as VPA was suggested.
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Se estudiaron los efectos de la exposición prenatal al paraquat (PQ), sobre el desarrollo postnatal de la transmisión sináptica aminoacídica de la corteza cerebral parietal del ratón. Las ratonas NMRI preñadas del grupo experimental recibieron 5 dosis de 10mg/kg de peso corporal de PQ, entre el día de gestación (G)12 y G20, y el grupo control recibió solución salina. Mediante HPLC, se determinaron los niveles de aspartato, glutamato, glicina, GABA y taurina de las crías, entre la edad postnatal (P)1 y P30. Entre P3 y P15, se observó un incremento significativo de los neurotransmisores excitatorios, aspartato y glutamato, en los ratones expuestos a PQ. Con respecto a la neurotransmisión inhibidora, los cambios más importantes se observaron en glicina: sus niveles se mantuvieron significativamente por debajo del grupo control entre P1 y P7, y significativamente por encima en P11 y P15. Para taurina, entre P1 y P7 sus niveles se mantuvieron significativamente altos con respecto al grupo control. En P30, los niveles de todos los neurotransmisores se encontraron significativamente por debajo del grupo control. En conclusión, podríamos decir que la exposición prenatal a PQ tuvo efectos tóxicos que se reflejaron en una alteración de los niveles basales de los neurotransmisores aminoacídicos durante el desarrollo postnatal de la corteza parietal del ratón, predominando la excitación sobre la inhibición durante todo el período estudiado. Estas alteraciones podrían indicar la ocurrencia de importantes daños corticales, tales como la disminución de algunas poblaciones neuronales, la inadecuada formación de los circuitos corticales y alteraciones en el proceso de sinaptogénesis.
The effects of prenatal expossure to paraquat (PQ) were studied on postnatal development of mouse parietal cerebral cortex, in particular, the ontogenesis of amino acid synaptic transmission. Pregnant NMRI mice were separated into two groups: the experimental group received 5 doses of 10mg PQ/kg body weight, between days of gestation (G)12 and G20, whereas the control group received physiological saline solution. Levels of neurotransmitter amino acids: Asp, Glu, Gly, GABA and Tau were determined by HPLC between postnatal (P) days P1 and P30. Between P3 and P15, a significant increment in the levels of excitatory amino acids, Asp and Glu, were observed in mice exposed to PQ, as compared with the control group. With respect to the inhibitory neurotransmitter levels, in the group exposed to PQ, the more important changes were observed in Gly between P1 and P15. In relation to taurine, its levels remained significantly higher between P1 and P7 with respect to the control group. It is important to emphasize that at P30, the levels of all neurotransmitters in the experimental group were significantly lower than those of control. In conclusion, prenatal exposure to PQ caused neurotoxicity in the developing mouse parietal cortex, as shown by the alterations in the basal levels of amino acid neurotransmitters, with the excitatory predominating over inhibitory neurotransmission, throughout the studied developmental period. These alterations could indicate the occurrence of important cortical injuries, such as decrement in some neuronal populations, inadequate formation of intrinsic cortical circuits and alterations in synaptogenic processes.
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Animals , Mice , Amino Acids, Neutral , Cerebral Cortex/growth & development , Pesticides , Paraquat/adverse effects , Animals, LaboratoryABSTRACT
Objective To explore contents of amino acid neurotransmitters and expression of GABAAreceptor subunits'mRNA in subareas of basal ganglia in unilateral rat model of Parkinson's disease (PD).Methods The rat model of PD was established through right unilateral intranigral microinjection of 6-hydroxydopamine(6-OHDA)in this study.Thawed samples were taken form neostriatum(Str).globus pallidus internus(Gpi),globus pallidus externum(Gpe)and subthalamic nucleus(STN).then contents of amino acid neurotransmitters were analyzed by established high performance liquid chromatography (HPLC)-electrochemical detection methods.The subunits α1,α2,β2/3 and γ2 of GABAA receptor in Str,Gpi,Gpe and STN wre examined with Northern Enzyme linked immunosorbent assay(ELISA).Results The content of GABA in Str,Gpi,Gpe and STN of diseased side were significantly increased as compared with undiseasedside.The level of glutamic acid(Glu)in Str,Gpe and STN and contents of aspartic acid (Asp)and glycine(Gly)in STN of the diseased side were significantly increased.In Str.there was a significant decrease of mRNA expression either in the subunit α1(105.3±24.5)or in the subunit β2/3(113.7 ±15.3)of GABAA receptor in the diseased side as compared with the undiseased 8ide(186.7 ±37.2,157.4±32.4,t=5.16,3.45;P<0.01).In Gpi,there was a significant increase of mRNA expression in the subunit α1(P<0.05)and α2,β2/3(P<0.01)of GABAA receptor in lesion side.In Gpe,there was a significant decrease of mRNA expression in the subunit α2(179.1±26.8)andβ2/3(154.7 ±37.8)of GABAA receptor in the diseased side(219.3.±19.7,231.1±55.8,t=3.42,3.21:P<0.01).In STN of right unilateral 6-0HDA lesion rat.there was a significant decrease of mRNA expression both in the subunitα1,α2 and β2/3(P<0.01)of GABAA receptor and in the subunit γ2(P<0.05)of GABAA receptor in the diseased side.Conclusions Changes of amino acid neurotransmitter contents and GABAA receptor subunits'mRNA expressional level in subareas of basal ganglia may be involved in PD.
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Objective To examine the dyllamic changes in excitatory and inhibitory amino acid neurotransmitter release in the spinal cord in a rat model of incisional pain.Methods Twelve healthy adult male SD rats weighing 250-300g were anesthetized with intraperitoneal chloral hydrate 300 mg/kg.A loop microdialysis catheter was implanted into the subarachnoid space via the atlanto-occipital membrane and advanced for 8.5 cm candad until lumbar region.The animals were randomly divided into 2 groups(n=6 each): control group(C) and incisional pain group(I).Incisional pain was produced by the plantar incision in the tight hindpaw under 1.2% isoflurane in group I while group C received only anesthesia with 1.2% isoflurane.The microdialysis samples were collected before incision(To,baseline)at 3 h,1 d,2 d and 3 d after incision(T1-4) for determination of amino acid using HPLC.The pain behavior was assessed and scored (O=no pain,2=severe pain) at the above time paints.Results In group I the aspartate and glutamate concentrations in the microdialysis samples were significantly increased at 3 h after incision(T1) as compared with the baseline value at To and returned to the baseline level at l d(T2);the glyeine and r-amino butyric acid concentrations were signifieantly increased at ld (T2)and returned to the baseline level at 2 d(T3).The cumulated pain scores were significantly increased at 3 h,1 d and 2 d after incision and returned to baseline level at 3 d (T4) in group I.Conclusion The increased release of excitatory amino acid neurotransmitter in the early phase after incision may be involved in hyperalgesia while the increased release of inhibitory amino acid neurotransmitter in the later phase may be involved in the pain relief.
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To investigate whether changes of amino acid neurotransmitter releases in the rostral ventrolateral medulla (rVLM) were related to acupuncture and to the antihypertensive effect of melatonin (Mel) microinjected into the anterior hypothalamic area (AHA) of rats with stress-induced hypertension (SIH), as well as to compare and analyze the relationship between the both antihypertensive mechanisms of acupuncture and of Mel in the AHA. Methods: Animal model of SIHR was made by electric foot shocks combined with noises. Electroacupuncture (EA) was used and Zusanli acupoint of both side was selected. The technique of drug microinjection into the brain was used to observe the change of blood pressure (BP), and synchronously, brain microdialysis was performed for collecting dialysate samples, and then the concentration of amino acid neurotransmitters in the dialysate samples was determined by high performance liquid chromatography combined with fluorescent detection (HPLC-FD). Results: After the animal received stress treatment, the BP elevated, synchronously, the release of glutamate (Glu) in the rVLM increased, and when EA was performed, the elevated BP of the rats with SIH decreased,simultaneously, the release of Glu in the rVLM decreased also. After Mel was microinjected of into the AHA of the SIHR, the elevated BP attenuated, meanwhile, the release of Glu decreased,and those of γ-aminobutyric acid (GABA) and taurine (Tau) increased in the rVLM.Administration ofbicuculline, an antagonist of GABAA receptor, into the rVLM prior to microinjection of Mel into the AHA could partially block the depressor effect of Mel in the AHA. Conclusion: The decrease in the release of Glu in the rVLM contributes to the antihypertensive effect of both acupuncture and Mel in the AHA in the rats with SIH, and the increase in the release of GABA and Tau in the rVLM is also important to Mel in the AHA.
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Objective:To investigate the mechanism of the reversal effect of acupuncture on the fall of blood pressure induced by angiotensin-(1-7)[Ang-(1-7)] in the caudal ventrolateral medulla(CVLM) of rats. Methods: Ang-(1-7) and its selective receptor antagonist (D-Ala7), Ang-(1-7) and Ang-(779) were microinjected into the CVLM respectively to induce the change of blood pressure, during which, the concentrations of amino acid neurotransmitters were detected by means of microdialysis and high performance liquid chromatography(HPLC)combined with fluorescent detector, on the other hand, the effects of electro-acupuncture (EA) on the level of blood pressure and the concentrations of amino acid neurotransmitters were also observed. Results:Unilateral microinjection of Ang-(1-7) into the CVLM could decrease the blood pressure (BP), which was accompanied by an increase in the release of Glu and a decrease in the release of Tau in the same site; Whereas microinjection of Ang-779 into the CVLM could elevate BP, which was accompanied by a decrease in the release of Glu and an increase in the release of Tau; EA at acupoint Zusanli (ST 36) for 20 min could inhibit the fall of blood pressure induced by microinjection of Ang-(1-7) into the CVLM and the elevation of blood pressure induced by microinjection of Ang-779 into the CVLM, and in the meantime, it could inhibit the changes of the release of Glu and Tau induced by microinjection of Ang-(1-7) and Ang-779 into the CVLM. Conclusion: The reversal effect of EA on the fall or elevation of blood pressure induced by microinjection of Ang-(1-7) or Ang-779 into the CVLM might be related to the changes of Glu and Tau release.
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【Objective】To investigate the influence of acupuncture on the power of brain electrical activity mapping(BEAM) and hippocampal amino acid neurotransmitters in acute epilepsy rats.【Methods】Forty SD rats were randomized into groups A(normal),B(model),C(treated with acupuncture) and D(treated with sodium valproate).Group C was pretreated with acupuncture on acupoints of Dazhui and Fenglong and with needling through Ganshu toward Danshu.Group D was pretreated with sodium valproate by gastric gavage.The treatment in groups C and D lasted 3 days.Except group A,the mice in other 3 groups received intraperitoneal injection of sodium penicillin to induce acute epilepsy.Within seventy to eighty-five minutes after modeling,different frequency-band power of BEAM in rat brain middle Pz region was detected,and rats hippocampal contents of excitatory amino acids such as glutamic acid(Glu) and aspartic acid(ASP) as well as inhibitory amino acids such as gamma-aminobutyric acid(GABA),glycine(Gly),alanine(Ala) and taurine(Tau) were examined.【Results】Power of frequency band ?,?,?_1,?_2,?_1 and ?_2 in group B increased(P
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AIM: To study whether the excitatory amino acids (EAAs)-triggered excitotoxicity contribute to the evolution of hyperbilirubinemia-associated brain injury. METHODS: Newborn rabbits with hyperbilirubinemia were decapitated and then, Na~+-K~+-ATPase activities, neurotransmitters and non-neurotransmitters concentration in brains were determined. RESULTS: It was found that the activities of Na~+-K~+-ATPase both in brains and cytomembrane and the amounts of glutamate (P
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Object To study the effect of total alkaloids of Equisetum pratense Ehrh. (TAEP) on the contents of amino acid neurotransmitters and Ach in rat brain to reveal the mechanism of TAEP inhibitory action on the central nervous system (CNS). Methods Contents of amino acid neurotransmitters and Ach in rat brain were determined by double-wavelengh scan and GPI mensuration. Results TAEP could not influence four kinds of content of amino acid neurotransmitter (glutamic acid, glycin, ?-aminobutyric acid, aspartic acid), but TAEP could significantly lower the content of Ach in striatum of rat. Conclusion The inhibition of TAEP to CNS is attained by lowering the content of Ach in striatum to affect DA-2 receptor, and it is irrelevant to the amino acid neurotransmitters (glutamic acid, glycin, ?-aminobutyric acid, aspartic acid).
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Objective To determine the effects of isoflurane on the amino acid neurotransmitter contents in rat cerebral cortex, hippocampus and spinal cord. Methods Sixteen male SD rats weighting 220-280g were randomly divided into two groups: isoflurane group (A) and control group (B). Animals in group A were killed after 30min inhalation of 1.3% isoflurane and cerebral cortex, hippocampus and spinal cord were removed immediately for determination of glutamic acid (Glu), aspartic acid (ASP), glutamine (Gln), GABA and glycine (Gly) levels by high-performance liquid chromatography (HPLC), whereas in group B O2 was inhaled instead of isoflurane. Results As compared with control group, Asp and Glu levels in cerebral cortex and hippocampus decreased markedly while Gly level increased significantly in hippocampus and spinal cord in isoflurane group. Conclusions The inhibition of excitatory amino acid synapse transmission and augmentation of inhibitory amino acid synapse transmission may be involved in the mechanism of isoflurane anesthesia.