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Sham; ### 0.001, # 0.05, vs. (Tyr1472) and up-regulated phosphorylated NR2A (Tyr1325). Furthermore, GLYX-13 treatment along with NR2B specific antagonist (Ro256981) failed Menaquinone-4 to exhibit any additional neuro-protective effects, whereas the application of NR2A antagonist (NVP-AAM007) abolished the neuroprotective effects of GLYX-13, which suggested that the protective action of GLYX-13 should be by its regulation of NMDAR subunit components. Our study provides important insights around the potential protective mechanism of GLYX-13 in ischemia and proposes the glycine site of NMDARs as a novel target for developing therapeutic strategies to store synaptic function in stroke. and respectively, and we explore the possible neuroprotective mechanism of GLYX-13 on ischemic injury, as a glycine site partial agonist of NMDARs through the modulation of NMDARs subunit components. Materials and Methods Animals and Housing Two months aged male C57BL/6 mice (20C25 g) were used in the study. Animals were purchased from the institute of zoology, Chinese academy of sciences. Animals were housed in cages with solid wood shaving bed linens and were maintained on a 12:12 light:dark cycle in the Tianjin Medical university of China-approved animal facility. Animals were given enough food and water throughout the studies unless otherwise noted. All experiments were approved by the Animal Care and Use Committee of Tianjin medical university, in compliance with National Institutes of Health guidelines. Middle Cerebral Artery Occlusion Ischemia Model Transient MCAO was adopted in our study, mainly according to the method of Rousselet et al. (2012). Briefly, 20C25 g male mice were anesthetized with isoflurane. During the whole surgery body temperature of the mice is usually maintained constant by a heating pad (Rousselet et al., 2012). A monofilament suture (about 9C10 mm is usually coated silicon, Doccol Corporation) was used to occlude the right MCA. Heart rate, rectal heat, and blood gases were monitored during the MCAO surgery. All the mice were subjected a 1 h MCAO. For sham group, all procedures of operation were same except that this MCAO suture was not inserted into MCA. For drug treatment experiments, drugs were then performed via intraperitoneal injection 2 h after the onset of MCAO (GLYX-13, Tocris, 10 mg/kg, D-serine, Sigma, 50 mg/kg, Ro256981 Tocris 5 mg/kg, NVP-AAM077 Tocris 2.4 mg/kg). Measurement of Menaquinone-4 Cerebral Blood Flow Cerebral blood flow was measured by Laser Doppler Flowmetry (Perimed, PeriFlux5010, Sweden) with a straight laser Doppler probe (Perimed, Probe 418, Sweden) as previously described (Iwai et al., 2004). The probe was placed in the territory supplied by Menaquinone-4 the MCA (AP-1.0 mm, L5.0 mm from bregma). Relative cerebral blood flow was measured before MCAO (baseline) and during occlusion. Changes of cerebral blood flow were expressed as percentage of the baseline value. Only those mice which ipsilateral regional cerebral blood flow 20% of baseline were used Menaquinone-4 for further study. Neurological Assessment Twenty-four hours Menaquinone-4 after reperfusion, neurological score was assessed. Neurological score contributed to evaluate the success of transient MCAO and to estimate the degree of neurological deficits and injury severity. Neurological assessments were scored as previously described (Jiang et al., 2005). 0: Normal. 1: Mild circling behavior, left forepaw cannot be fully extended. 2: Mild consistent circling, and walking to the paralyzed side. 3: Strong circling and holds a rotation position more than 2 s with its nose almost reaching its tail. 4: Strong circling progresses into severe rotation, can not walk spontaneously and miss the contralateral reflex activity. 5: Coma or dying. Calculation of Infarct Volumes Twenty-four hours after reperfusion, the animals were Rabbit polyclonal to ATF1.ATF-1 a transcription factor that is a member of the leucine zipper family.Forms a homodimer or heterodimer with c-Jun and stimulates CRE-dependent transcription. decapitated and the brains were removed into a mouse brain matrix for 1 mm section. Then sections were immersed in 1% triphenyltetrazolium chloride (TTC, Sigma) in PBS and incubated about 15 min at 37C. The area of unstained brain was identified as the.