ABSTRACT
OBJECTIVE: To observe the effect of wrist-ankle acupuncture (WA) stimulation at "R4"- "R5" - "R6" on the expression of glutamate (Glu) and phosphorylated protein NMDAR1(p-NMDAR1) of the spinal dorsal horn in spared nerve injury (SNI) rats, so as to explore its mechanism underlying improvement of SNI. METHODS: A total of 36 SD rats were randomly divi-ded into sham operation, model and WA groups, with 12 rats in each group. The SNI procedure comprised an axotomy and ligation of the tibial and common peroneal nerves leaving the sural nerve intact. Rats of the WA group were treated by acupuncture at "R4"-"R5"-"R6" points from the 5th day to the 14th day after modeling. The mechanical pain thresholds were measured before and 5, 10 and 14 d after SNI, respectively. The cold allodynia was dectected by Acetone solution dropped onto the lateral plantar surface of the paw. Glu content and p-NMDAR1 expression of spinal dorsal horn were detected by 1H-MRS, ELISA and immunohistochemistry Methods. RESULTS: Compared with the sham operation group, the mechanical pain threshold of the model group was significantly decreased (P<0.01), the duration of cold stimulation foot contraction was increased (P<0.01), and the Glu content and p-NMDAR1 expression in the spinal dorsal horn were significantly increased (P<0.05, P<0.01). After WA intervention, the mechanical pain threshold was significantly increased (P<0.01), the duration of cold stimulation was significantly shortened (P<0.01), and Glu content and p-NMDAR1 protein expression of spinal dorsal horn were decreased significantly (P<0.05, P<0.01) in the WA group compared with the model group. CONCLUSION: WA can reduce pain sensitivity in rats with neuropathic pain, possibly by inhibiting the expression of Glu and p-NMDAR1 in the spinal dorsal horn.
ABSTRACT
Aim To study the effects of salidroside on the caspase-9, GSK-30, NMDAR1, GluR2 in MCAO ratsand to further explore the mechanism of neuroprotection of salidroside. Methods For the first part, 36 healthy male Sprague-Dawley rats were randomly divided into sham operation (Sham) group, model (MCAO) group, and salidroside (MCAO + Sal) group. Rats were administered salidroside, or vehicle, daily for 1 day, after middle cerebral artery occlusion (MCAO) 2 h and reperfusion 1 h. The protein expression of GSK-3β, NMDAR1, GluR2 and caspase-9 was detected by Western blot. For the second part, rats were randomly divided into Sham group, SB216763 + Shamgroup, MCAO group, SB216763 + MCAO group, MCAO + Sal group, and SB216763 + MCAO + Sal group. After 30 minutes of injection of GSK-3βinhibitor SB216763 or artificial cerebrospinal fluid into the lateral ventricle, the other groups were subjected to MCAO modeling except for the sham operation group. After the modeling, the administration group was given salidroside, and the material was taken after 1 day. The protein expression of GSK-3β, NMDAR1, GluR2 andcaspase-9 was detected by Western blot. Results Compared with MCAO group, salidroside could reduce the protein expression of cleaved caspase-9 protein in mitochondria and promote the expression of pGSK-3β, NMDAR1, GluR2 protein after 1 day salidroside treatment. And the treatment of salidroside and GSK-3β inhibitor did not show remarkable additive effects. Conclusions Salidroside has a neuroprotective effect on MCAO rats, mainly by promoting GSK-3β phosphorylation, thereby inhibiting caspase-9 and promoting protein expression of NMDAR1 and GluR2.
ABSTRACT
Aim: To investigate the effects of Pien-Tze-Huang (PZH) on mRNA and protein expression of NMDAR1 and GluR2 in cortex of MCAO rats. Methods: Forty healthy adult male Sprague-Dawley rats were randomly divided into three groups: sham, MCAO, MCAO + PZH groups. The rats were subjected to focal cerebral ischemia/reperfusion with suture-occluded method. Neurological deficit testing was performed with Zea Longa scale. The volume of cerebral infarction was evaluated by TTC staining. The mRNA and protein expression of NMDAR1 and GluR2 in cortex of side cerebral ischemic tissues were determined using qPCR and Western blot analysis. Results: Compared with MCAO group, PZH significantly improved the neurological deficit, decreased the volume of cerebral infarction, and up-regulated the mRNA and protein expression of NMDAR1 and GluR2. Conclusions: PZH attenuates the down-regulation of mRNA and protein expression of NMDAR1 and GluR2 after focal cerebral ischemic injury in rats, which may be associated with the cerebral protective effect of PZH.
ABSTRACT
ABSTRACT This study aimed to investigate the expression and mechanism of N- methyl -D- aspartate receptor 1 (NMDAR1) in the pathogenesis of Alzheimer disease (AD). Eighty adult Wistar rats were randomly divided into 4 groups (n=20 each) to receive an injection of 0, 5, 7 and 10 μl of 1 μg/μl amyloid-β 42 (Aβ1-42) in the hippocampus. Twenty rats in normal control group were injected with equal volume of saline. After 10 days, the hippocampus was isolated from 5 randomly selected rats in each group. The NMDAR1 protein and mRNA expression was determined by immunohistochemical staining and qRT-PCR. The aquaporin-1 (AQP-1) mRNA expression was also measured by qRT-PCR. We found that both NMDAR1 and AQP-1 expression in Aβ1-42 groups was increased in a dose-dependent manner. NMDAR1 and AQP-1 expression in 7 and 10 μl Aβ1-42 groups was significantly higher compared with 0 μl Aβ1-42 group (P <0.01). Further, the 10 μl Aβ1-42 group was randomly divided into 3 subgroups: AD-NMDA, AD-MK-801, and AD-Ctrl subgroup, which was given an intraperitoneal injection of NMDAR agonist NMDA, NMDAR antagonist MK-801 and saline, respectively. The relative APQ-1 expression in each subgroup was determined by qRT-PCR and Western blot analysis after 24 h. The AQP-1 expression was significantly decreased in AD-MK-801 group (P < 0.05), but was markedly increased in AD-NMDA group when compared with AD-Ctrl group (P <0.01). Our study suggested that expression abnormity of NMDAR1 is involved in the pathogenesis of AD. NMDAR1 might regulate the pathogenic process through stimulating the expression of AQP-1.
ABSTRACT
This study aimed to construct prokaryotic recombinant plasmids for expression of the extracellular domains of NMDAR1 protein, purify and characterize the immunoreactivity of the recombinant proteins. Based on the mRNA sequence of human NMDAR1 gene, we predicted the structure of the antigenic domains in the extracellular part of the protein using the "phyre2" software. Primers were designed to amplify the nucleic acid fragments encoding the NMDAR1 extracellular antigenic domains by RT-PCR. The amplified gene fragments were cloned into pCold-SUMO vector to construct the recombinant plasmids which were transformed into Escherichia coli DH5α. The positive colonies harboring the recombinant plasmids were picked and verified by PCR and DNA sequencing. Then, the recombinant plasmids were transformed into E. coli BL21(DE3) strain and induced by IPTG for protein expression. The recombinant proteins were purified by Ni-NTA affinity chromatography. The target proteins were further purified by removing the 6 His-SUMO tag using enzyme excision followed by gel filtration chromatography using AKTA purifier. The purity of the recombinant proteins were evaluated by SDS-PAGE and the immunoreactivity were characterized by Western blotting. Three DNA fragments encoding the extracellular domains of NMDAR1 protein, including NR1-M1 (encoding 19-393 aa), NR1-S1 (encoding 394-544 aa) and NR1-S2 (encoding 663-800 aa), were amplified by RT-PCR. The NR1-S1 and NR1-S2 were linked with G (arginine) and T (threonine) amino acid as a combined fragment. The NR1-M1 and NR1-S1-GT-S2 fragments were cloned into pCold-SUMO vector and two recombinant plasmids, pCold-SUMO-M1 and pCold-SUMO-S1-GT-S2, were generated and expressed in E. coli. SDS-PAGE analysis showed that the recombinant plasmids expressed soluble NR1-M1 and NR1-S1-GT-S2 proteins in bacterial. After affinity chromatography and gel filtration chromatography, we obtained high purity target proteins. Western blotting assay showed that the recombinant proteins NR1-M1 and NR1-S1-GT-S2 can bind specially with their corresponding antibodies, suggesting the recombinant proteins retained antigenic reactivity. We constructed a prokaryotic expression system for expressing the NMDAR1 protein extracellular parts that had immunoreactivity successfully, and the purified proteins can be used for studying NMDAR1 function and testing the autoantibodies.
ABSTRACT
Objective To observe the expression of cAMP -response element binding protein (CREB) and N-methyl-D-aspartic acid receptor (NMDA ) after intracochlear electrical stimulation in the auditory cortex and inferior colliculus in infant rats with auditory deprivation .Methods Sixty six SD infant rats were randomly divided into 6 groups (11 rats each group):4 weeks ,and 6 weeks after injection of ototoxic drug ,the control group ,and 3 weeks and 5 weeks after injection of ototoxic drug with intra -cochlear electrical stimulation for one week .Gentami-cin sulphate (350 mg/kg body weight) and frusemide (200 mg/kg body weight) were injected subcutaneously in the skin folds on the lateral abdominal side and the dorsal neck area ,respectively .The expression of CREB and NMDAR1protein were detected by immunohistological staining .Results The results of immunohisto -chemistry revealed that protein expression of CREB and NMDAR1 in 4 week group of injection increased as compared to the control group ,while decreasing as compared to intracochlear electrical stimulation group ,significantly .However ,protein expression of CREB and NMDAR1 in 6 week group of injection decreased as compared to the control group and in-tracochlear electrical stimulation group ,significantly .Conclusion Auditory deprivation could result in the expres-sion of protein of CREB and NMDAR1 in auditory cortex and inferior colliculus increasing in an early stage and then de-creasing in infant rats .Intracochlear electrical stimulation could result in the expression of proteins of CREB and NMDAR 1 in auditory cortex and inferior colliculus increasing in infant rats .The dynamic variation of CREB and NMDAR1 expression in rat auditory cortex and inferior colliculus reflects synaptic plasticity in neurons of auditory pathway .
ABSTRACT
Objective To investigate the expression of NMDAR1 in the hippocampus and cerebral cortex of old rats after 30-min -inhalation of 2% isoflurane, and to investigate the effects of isoflurane on the learning and memory functions of old rats and the underlying mechanism. Methods The healthy old male Sprague Dawley rats (n = 36) were randomly divided into the control group, the oxygen group, the 2-hour post-recovery group, the 1-day post-recovery group, the 3-day post-recovery group, and the 7-day post-recovery group. The morris water maze was used to detect the ethological effect of 30-min inhalation of isoflurane , and the immunohistochemistry assay was used to detect the expression of NMDAR1 in the hippocampus (CA1, CA3) and the cerebral cortex. Results The 30-min inhalation of 2% isoflurane inhibited the learning and memory abilities of old rats at 2 h post-recovery. On 1 d post-recovery, the inhibition of learning and memory began to reduce, then on 3 d and 7 d post-recovery, the learning and memory abilities continously recovered. The expression of NMDAR1 in the rat hippocampus and cerebral cortex decreased at 2 h post-recovery, and reversed on 1 d post-recovery and reached the normal level on 3 d and 7 d post-recovery. Conclusion 30-min inhalation of 2%isoflurane had an inhibitory effect on the learning and memory abilities of old rats, and the attenuation of NMDAR1 in the hippocampus and cerebral cortex may involve in this process.
ABSTRACT
Objective To investigate the impact of tanshinone Ⅱ A on the level of brain NMDAR1 protein in rats after cardiopulmonary resuscitation and its effects of brain function protection.Methods Seventy-eight SD male rats were randomly (random number) divided into three groups:group A (sham group,n =6),group B (control group,n =36) and group C (Tanshinone Ⅱ A intervention group,n =36).All animals were induced to be models of cardiac arrest by choking.The rats of group C received intravenous injection of Tanshinone Ⅱ A in dose of 15 mg/kg immediately at initiation of resuscitation,while rats of group B were intravenous injected same amount of normal saline instead.Brains tissues of all rats were taken at 1,6,12,24,48 and 72 h after the restoration of spontaneous circulation (ROSC).Immunohistochemical staining method was applied for measuring the levels of brain tissue NMDAR1 and Caspases-3,while water content of the brain was detected by wet and dry weight ratio.The experimentaldata were analyzed by using one-way ANOVA.Results (①)The level of brain NMDAR1 protein in group B increased at 1 h and reached its peak at 6 h after ROSC,then its level gradually declined and dropped below normal at 48 h,72 h,and there were significant difference in variation of NMDAR1 protein levels in comparison with the group A (P ＜ 0.05) ; the NMDAR1 protein levels at 1,6,12 h in group C were significantly lower than those in group B at the same intervals (P ＜ 0.01),but no significant differences were seen at 24,48,72 h (P ＞ 0.05).(②)The level of brain Caspases-3 in group B increased after ROSC,and reached its peak at 48 h after ROSC,then declined and maintained above normal at 72 h,and this variation was significantly different from that of the group A (P ＜ 0.01) ; while the levels of caspase-3 at 1,6,12,24 h in group C were significantly lower than those in group B (P ＜ 0.01),but thses differences at 48,72 h were still significant (P ＜ 0.05).(③)The water content of brain tissue in group B increased at 1 h and reached its peak at 24 h after ROSC,then gradually decreased from 48 h,but maintained above normal at 72 h,and this trend of variation was significantly different from that of group A (P ＜ 0.01 or P ＜ 0.05).Compared with group B,water content of brain tissue in group C decreased more significantly (P ＜ 0.01).Conclusions Tanshinone Ⅱ A down-regulates brain NMDAR1 protein level at early stage in rats as well as significantly inhibits the level of Caspases-3 thereby ameliorating brain edema after cardiopulmonary resuscitation.
ABSTRACT
Objective This study examined the effects of Midazolum-ketamine oral solution (MKOS) on the expression of N-methyl-D-aspartate receptor 1 (NMDAR1) and gamma-aminobutyric acid (GABA)A receptors (GABAAR) mRNA in the cerebral cortex of rat, in order to investigate the sedation mechanism of MKOS. Methods Fifty Sprague-Dawley(SD) rats were divided into ten groups according to the observed time after MKOS administration (0,5,10, 15,30,60,90,120,240 and 360 minutes, n =5 each). The 0 minute group(control group) received 0.9% saline instead. Immunohistochemical staining and in situ hybridization were used to detect the expressions of NMDAR1 and GABAAR mRNA in the cerebral cortex. Results Both GABAAR and NMDAR1 all expressed in the glial cells of cerebral cortex. The expression of NMDAR1 in control group was strong. The expression of NMDAR1 became weaker during 15 to 90 minutes after administration of MKOS (P＜0.05). The expression of GABAAR mRNA in control group was weaker,while became stronger during 30 to 90 minutes after administration of MKOS (P ＜0. 05). Conclusion MKOS may play sedation by strengthening the expression of GABAAR and suppressing the expression of NMDAR1 in the cerebral cortex.
ABSTRACT
Objective: To observe the effect of auricular needling on abilities of learning and memory as well as expression of NMDAR1 in rats with vascular dementia(VD).Methods:VD rat model was established by blocking 4-vessel.The immunohistochemistry method was used to detect the changes of NMDAR1 expression,the Y-type maze test was used to measure the abilities of learning and memory.Results:There were slight expression of NMDAR1cells in the brain tissue of normal group.Compared with the normal group,the expression of NMDAR1 positive cells in model group were significantly enhanced,but the average optical density is obvious reduced(P
ABSTRACT
Objective To study the effect of infrasound on the changes of expression of NMDAR1 in hipp- ocampal cells.Methods Eighty-eight male Sprague-Dawley rats were randomized into eleven groups:control group,90 dB/1 d,7 d,14 d,21 d and 28 d infrasound exposed groups;130 dB/1 d,7 d,14 d,21 d and 28 d infra- sound exposed groups.All the animals in the test groups were put in an infrasound field with 8 Hz,90 dB or 130 dB for 2 hours daily.Immunohistochemistry methods were used to detect the changes of intracellular expression of NMDARI in hippocampal cells.Methods The expression of NMDAR1 in hippocampus after the rats were exposed to infrasound of 8 Hz,90 dB SPL showed a procedure from reducing on the 1st day to rising on the 7th and peaked on the 14th day,then to descending on 21st day and returning to the standard level on the 28th day.Exposure to infra- sound of 8 Hz,130 dB SPL induced opposite effects on the expression of NMDAR1 compared with 90 dB SPL,which showed a process of increasing,descending,reaching to the lowest,then ascending and returning to the normal.The lowest expression of NMDAR1 occurred on the 14th day in every observed hippocampal area.Conclusion 8 Hz, 90 dB/130 dB infrasound induced certain reversible reaction in the expression of NMDAR1 of hippoeampal cells in rats,which may disturb their learning and memory function.
ABSTRACT
OBJECTIVES: ECS could have therapeutic effects on psychiatric illnesses by inducing IEGs, which in turn regulates expression of their target genes. We observed AP-1 binding activity and identified AP-1 binding proteins in NMDAR1, late response gene of IEGs, which considered as the candidate gene for schizophrenia. METHODS: By gel shift assay and supershift assay, we observed binding activities and AP-1 binding proteins in NMDAR1. Because IEGs are induced rapidly but transiently by external stimuli, there is a possibility that the expression of IEGs is negatively feedbacked by their own products via their AP-1 binding sites. For that purpose, we also observed AP-1 binding activity of c-fos and c-jun via gel shift and supershift assay. RESULTS: ECS increased AP-1 binding activities of NMDAR1 gene, contributed by c-Fos and its related proteins. Peak of the increased binding was 60 minutes in both hippocampus and cerebellum. Though expression of c-Fos and c-Jun were increased by ECS, there were no changes in AP-1 binding activities after ECS. AP-1 sites of IEGs were binded by CREB, regardless of ECS. CONCLUSION: There is a possibility that ECS induced IEG expression, and then incresed expression of NMDR1 by binding of expressed IEGs to the AP-1 site of NMDAR1. ECS did not increase AP-1 binding activities of IEGs. This suggests that the regulation of IEGs' expression can not be influenced mainly by AP-1 site.
Subject(s)
Animals , Rats , Binding Sites , Brain , Carrier Proteins , Cerebellum , Electroshock , Hippocampus , Schizophrenia , Transcription Factor AP-1ABSTRACT
Objective To explore the effect of Acorus gramimeus and its major components ?-asarone on N-methyl-D-asperate recepter 1 (NMDAR1) mRNA levels of hippocamp neurons in epileptic animal models induced by pentylenetetrazol (PTZ). Methods PTZ 60 mg/kg was injected to the abdominal cavity of three weeks Wistar rats to prepare epileptic animal models. The animal models were randomly divided into three groups: epilepsy control, A. gramimeus and ?-asarone as well as the normal control. Each group was administered through intraperitoneal injection. Both of two control groups were injected physiologic saline 1.0 mL/d, and the rest groups were injected A. gramimeus 2 350 mg/(kg?d) or ?-asarone 29 mg/(kg?d) for 7 d, respectively. All rats were treated by PTZ 60 mg/kg through intraperitoneal injection next day. After their behaviors were observed for 24 h, animals were sacrificed. The expressions of NMDAR1 mRNA in hippocamp CA1 and CA3 of all rats were detected by in situ hybridization and semiquantitative RT-PCR. Results The results of in situ hybridization showed that positive staining granules were located in the cyptoplasm of hippocamp neurons. The number of positive cells and average absorbance of A. gramimeus group or ?-asarone group was markedly less than that of epilepsy control (P
ABSTRACT
Objective To explore the effect of chronic low lead exposure on molecular pathological mechanisms of learning and memory of rats at the developmental stage Methods The changes of N methyl D aspartate receptor 1 (NMDAR 1) mRNA expression in rat hippocampus were observed by using digoxigenin labled anti sense oligonucleotide probes in situ hybridization technique Results The changes in NMDAR 1 mRNA levels in Pb exposed rat hippocampus were found to be dependent upon the developmental period of exposure and the region of the hippocampus analyzed Hippocampal NMDAR 1 mRNA levels increased significantly in the CA1 (18 75%) and CA3 (13 2%) ( P