Hippocampal µ-opioid receptors on GABAergic neurons mediate stress-induced impairment of memory retrieval.

Stressful life events induce abnormalities in emotional and cognitive behaviour. The endogenous opioid system plays an essential role in stress adaptation and coping strategies. In particular, the µ-opioid receptor (µR), one of the major opioid receptors, strongly influences memory processing in that alterations in µR signalling are associated with various neuropsychiatric disorders. However, it remains unclear whether µR signalling contributes to memory impairments induced by acute stress. Here, we utilized pharmacological methods and cell-type-selective/non-cell-type-selective µR depletion approaches combined with behavioural tests, biochemical analyses, and in vitro electrophysiological recordings to investigate the role of hippocampal µR signalling in memory-retrieval impairment induced by acute elevated platform (EP) stress in mice. Biochemical and molecular analyses revealed that hippocampal µRs were significantly activated during acute stress. Blockage of hippocampal µRs, non-selective deletion of µRs or selective deletion of µRs on GABAergic neurons (µRGABA) reversed EP-stress-induced impairment of memory retrieval, with no effect on the elevation of serum corticosterone after stress. Electrophysiological results demonstrated that stress depressed hippocampal GABAergic synaptic transmission to CA1 pyramidal neurons, thereby leading to excitation/inhibition (E/I) imbalance in a µRGABA-dependent manner. Pharmaceutically enhancing hippocampal GABAA receptor-mediated inhibitory currents in stressed mice restored their memory retrieval, whereas inhibiting those currents in the unstressed mice mimicked the stress-induced impairment of memory retrieval. Our findings reveal a novel pathway in which endogenous opioids recruited by acute stress predominantly activate µRGABA to depress GABAergic inhibitory effects on CA1 pyramidal neurons, which subsequently alters the E/I balance in the hippocampus and results in impairment of memory retrieval.

Parthenolide attenuates cerebral ischemia/reperfusion injury via Akt/GSK-3ß pathway in PC12 cells.

Parthenolide (PN), a sesquiterpene lactone isolated from the herbal medicine feverfew (Tanacetum parthenium), was reported to possess neuroprotective activity. However, the neuroprotective effect of PN against cerebral ischemia/reperfusion (I/R) injury remains unclear. Therefore, the aim of the present study was to explore the neuroprotective effects of PN against oxygen-glucose deprivation (OGD)-induced apoptosis in PC12 cells and the underlying mechanisms. Our results demonstrated that PN ameliorated OGD/R-evoked neuronal injury and oxidative stress in PC12 cells. In addition, PN notably decreased HIF-1α expression, as well as inhibited apoptosis in PC12 cells after OGD/R. Furthermore, PN pretreatment significantly enhanced the phosphorylation of Akt and GSK-3ß in PC12 cells exposed to OGD/R. In conclusion, the present study demonstrated that PN exhibits a neuroprotective effect against OGD/R through activation of the Akt/GSK-3ß signaling pathway. Our findings suggest that PN has the potential to serve as a novel therapeutic agent for cerebral I/R injury.

MicroRNA-25 Negatively Regulates Cerebral Ischemia/Reperfusion Injury-Induced Cell Apoptosis Through Fas/FasL Pathway.

MicroRNA-25 (miR-25) has been reported to be a major miRNA marker in neural cells and is strongly expressed in ischemic brain tissues. However, the precise mechanism and effect of miR-25 in cerebral ischemia/reperfusion (I/R) injury needs further investigations. In the present study, the oxygen-glucose deprivation (OGD) model was constructed in human SH-SY5Y and IMR-32 cells to mimic I/R injury and to evaluate the role of miR-25 in regulating OGD/reperfusion (OGDR)-induced cell apoptosis. We found that miR-25 was downregulated in the OGDR model. Overexpression of miR-25 via miRNA-mimics transfection remarkably inhibited OGDR-induced cell apoptosis. Moreover, Fas was predicted as a target gene of miR-25 through bioinformatic analysis. The interaction between miR-25 and 3'-untranslated region (UTR) of Fas mRNA was confirmed by dual-luciferase reporter assay. Fas protein expression was downregulated by miR-25 overexpression in OGDR model. Subsequently, the small interfering RNA (siRNA)-mediated knockdown of Fas expression also inhibited cell apoptosis induced by OGDR model; in contrast, Fas overexpression abrogated the protective effects of miR-25 on OGDR-induced cells. Taken together, our results indicate that the upregulation of miR-25 inhibits cerebral I/R injury-induced apoptosis through downregulating Fas/FasL, which will provide a promising therapeutic target.

[A simple method for assessment of RNA integrity in laser capture microdissection samples].

OBJECTIVE: To develop a simple method for assessment of RNA integrity in laser capture microdissection (LCM) samples. METHODS: The total RNA were isolated from the LCM samples and the sections before and after microdissection and examined by agarose gel electrophoresis. Real-time PCR was employed to assess the RNA from LCM samples, and the quantity of RNA was theoretically estimated according to the average total RNA product in mammalian cells (10 ng/1000 cells). RESULTS: When the total RNA from the sections before and after microdissection was intact, the RNA from LCM samples also had good quality, and the 28S and 18S rRNAs were visualized by ethidium bromide staining. Real-time PCR also showed good RNA quality in the LCM samples. CONCLUSION: A simple method for quantitative and qualitative assessment of the RNA from LCM samples is established, which can also be applied to assessment of DNA or proteins in LCM samples.

[Research on the clinical features and effective factors of 249 diabetic patients with deep foot infection].

OBJECTIVE: 249 diabetic patients with deep foot infection were retrospectively studied. Their clinical features and effective factors related to wound healing were analyzed. METHODS: 249 patients team (physician, surgeon and diabetes-specific nurse) were divided into 3 groups. Group A: patients healed without amputation (n = 107), group B: patients healed after amputation (n = 114), and group C: patients didn't heal after amputation. All patients' clinical features, lab examinations and foot wound features were compared. RESULTS: The group A patient were (59 +/- 12) years old, significantly younger than the group B (67 +/- 11, P < 0.01). Hypersensitive C reactive protein (hs-CRP, 18 +/- 5 mg/L) and plasma albumin (32 +/- 7 g/L) of group A were significantly higher than those of group B (13 +/- 5 mg/L and 29 +/- 5 g/L, respectively, P < 0.01). The duration of diabetes mellitus of group B (17 +/- 11) year was significantly longer than that of group A (10 +/- 6 year, P < 0.05). The possibility of probing bone, purulent secretion, necrosis, bone exposure, cacosmia, edema and critical limb ischemia of group B were more frequent (P < 0.01 or P < 0.05) compared to group A. The age of group B (67 +/- 11) are younger than that of group C (72 +/- 9, P < 0.05). In group B, temperature (38.1 +/- 1.1) degrees C, white blood cell (WBC) count (10 +/- 3) x 10(9)/L and hs-CRP (13 +/- 5) mg/L were higher than those of group C (37.4 +/- 0.8 degrees C, 8 +/- 2 x 10(9)/L and 7 +/- 6 mg/L, respectively, all P < 0.05). Critical limb ischemia of group B (37%) was more frequent than that of group A (7%, P < 0.05), but less frequent than that of group C (77%, P < 0.01). hs-CRP and plasma albumin were protective factors for wound healing. Age, the possibility of probing bone, purulent secretion, necrosis, bone exposure, cacosmia, edema and critical limb ischemia were risk factors for wound healing. CONCLUSION: diabetic patients with deep foot infection are difficult to be diagnosed in early stage since they often have no significant clinical syndrome such as fever, redness, swelling and pain, and their WBC count does not increase. Though multidisciplinary team manages these patients, more than half of them need amputation. Wound healing in the patients is related to multiple factors, including age, duration of diabetes mellitus, hs-CRP, plasma albumin, WBC count, level of limb ischemia and wound features.

[In vivo labeling of cells in the ependyma/subventricular zone of adult rats].

OBJECTIVE: To identify the labeled cells in the ependyma/subventricular zone (SVZ) of normal adult rats by DiI injected into the lateral ventricle. METHODS: Fifty male Sprague-Dawley rats were divided randomly into five groups (10 per group). All of the rats were injected with 10 microL of 2 g/L fluorescence dye DiI into the right lateral ventricle. The five groups of rats were sacrificed at 6 h, 12 h, 24 h, 36 h or 48 h after injection respectively. Hoechst 33258 staining was used to identify nuclei and laser confocal microscopy was used to detect the DiI-labeled cells and to measure the thickness of the tissue with DiI fluorescence in the wall of the left lateral ventricle. RESULTS: After injection of the DiI into the right lateral ventricle, DiI- Hoechst 33258 double positive cells were found in the ependymal layer of the left lateral ventricular wall at 24 h and in the SVZ at 48 h as well. The thickness of the tissue with DiI fluorescence in the left ependyma/septal subventricular zone (SVZspt) and ependyma/postnatal equivalent of the ganglionic eminences (SVZge) remained unchanged at 12 h and 24 h after DiI injection. The thickness of the tissue with DiI fluorescence in the left ependyma/SVZge was significantly greater than that in the ependyma/SVZspt at all of the time points (P<0.05). CONCLUSION: The ependyma/SVZ cells can be labeled by Dil 24-48 h after injection (10 microL of 2 g/L) into the lateral ventricle.

[Effects of ligustrazine on hippocampal dentate gyrus cell proliferation after focal cerebral ischemia in adult rats].

OBJECTIVE: To explore the effects of ligustrazine on cell proliferation in hippocampal dentate gyrus subgranular zone (SGZ) after focal cerebral ischemia in adult rats. METHODS: Middle cerebral artery occlusion (MCAO) model was established in adult rats by placement of an intraluminal filament at the origin of the MCA. Ligustrazine was administered intraperitoneally at a daily dose of 80 mg/kg starting at 2 h after MCAO, and BrdU (50 mg/kg daily) was also injected intraperitoneally starting at 4 h after MCAO. BrdU-positive cells in the SGZ were counted 7, 14 and 24 days after MCAO, respectively. RESULTS: Compared with sham operation group, ischemic ipsilateral BrdU-positive cells in the ischemic model group increased 7 days after MCAO, reaching the peak on day 14, and decreased on day 21 (P<0.01). The number of ischemic ipsilateral BrdU-positive cells in ligustrazine group was significantly greater than that in the ischemic model group on days 7, 14 and 21 (P<0.01), and maintained the high level on day 21. CONCLUSION: Ligustrazine possesses long lasting effect of promoting cell proliferation in the SGZ after focal cerebral ischemia in adult rats.

[Effect of ligustrazine on cell proliferation in subventricular zone of lateral cerebral ventricle after adult rat suffering from focal cerebral ischemia].

OBJECTIVE: To explore the effect of ligustrazine on cell proliferation in subventricular zone of lateral cerebral ventricle after middle cerebral artery occluded (MACO) in adult rat. METHODS: SD male rats were randomly divided into three group: sham operation group, ischemic model group and Ligustrazine group. The model of the middle cerebral artery occlusion was established by placement of an intraluminal filament at the origin of left MCA. Ligustrazine was administered intraperitoneally with a dose of 80 mg/kg daily starting at 2 hours after MCAO. BrdU (50 mg/kg) was injected once a day intraperitoneally starting at 4 hours after operation. Number of BrdU-positive cells and expression of doublecortin (DCX) in subventricular zone (SVZ) were measured by immunohistochemistry on day 7, 14, 24 after operation. RESULTS: Compared with sham operation group, BrdU-positive cells in ischemic model group increased on day 7, reached the peak on day 14, then decreased on day 21 after operation. On day 7 and 14, the numbers of BrdU-positive cells in Ligustrazine group were markedly augmented and significantly more than those in ischemic model group (P < 0.01), but decreased on day 21. The expressions of DCX in SVZ in ischemic model group were enhanced on day 7 with lasting into day 14, and reduced on day 21, but still higher than those in sham operation group on day 7, 14, and 21, respectively. The expressions of DCX in SVZ in Ligustrazine group increased gradually along with prolong of ischemia and kept the high level up to day 21 after operation, and were higher than those in ischemic model group on day 14 and 21. CONCLUSION: Our results suggest that Ligustrazine may promote the cells of SVZ, which the adult rats suffer from the focal cerebral ischemia, to go into cell proliferation.

Effects of ketamine-midazolam anesthesia on the expression of NMDA and AMPA receptor subunit in the peri-infarction of rat brain.

BACKGROUND: Activation of N-methyl-D-aspartate (NMDA) receptors and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors play an important role in the neurons death induced by ischemia. The mitigating effect of intravenous anesthetics on ischemic neuron injury is related to their influence on NMDA receptors. This study was performed to investigate the effect of ketamine-midazolam anesthesia on the NMDA and AMPA receptor subunits expression in the peri-infarction of ischemic rat brain and explore its potential mechanism of neuroprotection. METHODS: Thirty Sprague Dawley (SD) rats were subjected to permanent middle cerebral artery occlusion under ketamine/atropine (100/0.05 mg/kg) or ketamine-midazolam/atropine (60/50/0.05 mg/kg) intraperitoneal anesthesia (n=15 each). Twenty-four hours after ischemia, five rats in each group were killed by injecting the above dosage of ketamine or ketamine-midazolam intraperitoneally and infarct size was measured. Twenty-four and 72 hours after ischemia, four rats in each group were killed by injecting the above dosage of ketamine or ketamine-midazolam intraperitoneally. After staining the brain tissue slices with toluidine blue, the survived neurons in the peri-infarction were observed. Also, the expression level of NMDA receptors 1 (NR1), NMDA receptors 2A (NR2A), NMDA receptors 2B (NR2B) and AMPA (GluR1 subunit) were determined by grayscale analysis in immunohistochemical stained slices. RESULTS: Compared with ketamine anesthesia, ketamine-midazolam anesthesia produced not only smaller infarct size [(24.1+/-4.6)% vs (38.4+/-4.2)%, P<0.05], but also higher neuron density (24 hours: 846+/-16 vs 756+/-24, P<0.05; 72 hours: 882+/-22 vs 785+/-18, P<0.05) and lower NR2A (24 hours: 123.0+/-4.9 vs 95.0+/-2.5, P<0.05; 72 hours: 77.8+/-4.1 vs 54.2+/-3.9, P<0.05) and NR2B (24 hours: 98.5+/-2.7 vs 76.3+/-2.4, P<0.05; 72 hours: 67.2 +/-7.5 vs 22.2+/-2.6, P<0.05) expression level in the peri-infarction following ischemia. CONCLUSION: The protective effects of ketamine-midazolam anesthesia on ischemic brain injury may related to decreasing NR2A and NR2B expression.

[Effect of histamine on intracortical blood vessels of rats].

OBJECTIVE: To investigate histamine-induced changes of the intracortical vessels in the cortical slice of rat brain. METHODS: Immunohistochemistry was employed to detect the expression of H1 and H2 receptors in the intracortical blood vessels of rats. Histamine-induced constriction of the intracortical blood vessels of the brain slices was observed with differential interference contrast microscope. Measurements of the luminal diameter were made on-line during the course of the experiment and confirmed off-line from the stored images. In order to observe whether histamine H1 and H2 receptors affected histamine-induced constriction, the intracortical blood vessels in the brain slices were pre-treated with H1 receptor antagonist diphenhydramine and H2 receptor antagonist cimetidine. RESULTS: Expression of H1 and H2 receptors was detected in the intracortical blood vessels of the rat brain. Histamine (1-100 micromol/L) induced a concentration-dependent constriction from (1.48-/+0.67)% to (32.91-/+7.91)%. The reactions to each histamine concentration were significantly (P<0.01) different from each other, with the exception of the highest histamine concentrations (30 and 100 micromol/L) when maximal constriction due to histamine were observed (P>0.05). With pre-treatment of the slice with 10 micromol/L diphenhydramine, application of histamine did not elicit constriction. Pre-treatment of the slice with 10 micromol/L cimetidine did not completely inhibit but somehow significantly weakened vascular constriction in response to histamine treatment at 10 and 30 micromol/L (P<0.05). CONCLUSION: Histamine can induce constriction of the intracortical blood vessels, which is mediated by H1 receptor.

[Effect of citicoline on spatial learning and memory of rats after focal cerebral ischemia].

OBJECTIVE: To investigate the effects of citicoline on spatial learning and memory of rats after focal cerebral ischemia. METHODS: The rats were randomly divided into sham-operation group, ischemia control group and citicoline group. In the later two groups, focal cerebral ischemia model was established by introducing an intraluminal filament into the left middle cerebral artery, and citicoline (500 mg/kg) or 0.9% NaCl was administered intraperitoneally once a day for 2 weeks after the operation. The rats in the sham-operation group were not subjected to middle cerebral artery occlusion (MCAO) with intraluminal filament. The spatial learning and memory functions of the rats were evaluated by Morris water maze test 15 days after MCAO for 5 days. RESULTS: The rats in ischemia control group exhibited serious spatial learning and memory deficits in both place navigation test and spatial probe test. In the former test, the mean escape latency of citicoline-treated rats were significantly shorter than that of ischemia control rats (P<0.01), and in the latter test significant diffidence was noted between citicoline and ischemia control groups in the percentage time spent in the former platform quadrant and frequency of crossing the former platform (P<0.05). CONCLUSION: Citicoline can improve the spatial learning and memory function of rats after focal cerebral ischemia.

[The effect of ligustrazine on cells proliferation in cortex and striatum after focal cerebral ischemia in adult rats].

OBJECTIVE: To explore the effect of Ligustrazine on cells proliferation in cortex and striatum after focal cerebral ischemia in adult rats. METHODS: Focal cerebral ischemia was induced by left middle cerebral artery occlusion (MCAO) with suture method. Two hours later, injection of Ligustrazine (80 mg/kg, 1 time/d) was performed peritoneally. Four hours after the ischemia, 5-bromodeoxyuridine (BrdU) (50 mg/kg, 1 time/d) was injected peritoneally. At 7d, 14d and 21d after ischemia, BrdU-Labeled cells in the cortex and striatum were observed by immunohistochemical staining. RESULTS: In ischemia model group, at 7 day, BrdU-labeled cells were observed in the ipsilateral cortex and striatum. With the prolongation of ischemia, the number of BrdU-labeled cells increased, reached the peak at 21d. In Ligustrazine group, BrdU-labeded cells were observed with an intense distribution in ischemic penumbra of the cortex and striatum. With the prolongation of ischemia, the number of BrdU-labeled cells increased significantly at 14d, and reached the peak at 21d. The numbers of BrdU-labeled cells at 7d, 14d and 21d were more than those in ischemia model group respectively. CONCLUSION: Ligustrazine increase the proliferated cells in the cortex and striatum after focal cerebral ischemia in adult rats. The results suggest that it may be useful for promoting self-repair after ischemia.

Ependymal/subventricular zone cells migrate to the peri-infarct region and differentiate into neurons and astrocytes after focal cerebral ischemia in adult rats.

OBJECTIVE: To investigate the migration and differentiation of ependymal/subventricular zone cells after focal cerebral ischemia in rats, and reveal the origin of the newly generated neural cells in the peri-infarct region. METHODS: Normal adult male Sprague Dawley rats weighing 250-350 g were used in this study. Before middle cerebral artery occlusion (MCAO), 10 microl of 0.2% DiI was injected into the lateral ventricle for prelabeling the ependymal/subventricular zone cells. After ischemia, cumulative BrdU labeling was employed to detect the newly generated cells and double immunofluorescent staining to identify cell differentiation. The labeled cells were observed with laser confocal microscopy. RESULTS: In the non-ischemic control rats, DiI-labeled cells resided in the ependyma/subventricular zone. After focal cerebral ischemia, DiI-labeled cells were found in the corpus callosum, adjacent striatum and cortex, and some DiI/BrdU/glial fibrillary acidic protein (GFAP)-positive cells or DiI/BrdU/ neuronal nuclear antigen (NeuN)-positive cells were observed in the peri-infarct region in the striatum or cortex since day 14 after MCAO. CONCLUSION: After focal cerebral ischemia, ependymal/subventricular zone cells migrate into the peri-infarct region where they differentiate into neurons and astrocytes. This finding may be important for understanding the source of adult neural stem cells and for developing new therapeutic intervention strategy through enhancing endogenous neurogenesis after brain injury.

[Effects of ketamine and midazolam combination anesthesia on focal cerebral ischemia injury in rats].

OBJECTIVE: We investigated the effects of anesthesia with ketamine and midazolam on focal cerebral ischemia injury, and evaluated the neuroprotective properties of this combination during surgical procedures. METHODS: Thirty male Sprague-Dawley rats were subjected to permanent middle cerebral artery occlusion (MCAO) under ketamine or ketamine-midazolam intraperitoneal anesthesia (n = 15 each). The neurological scoring was performed to determine functional impairment of the brain at 4 h after MCAO. TTC staining of brain slices was used to measure infarct size of the brain at 24 h after MCAO. At day 3 after MCAO, TUNEL staining was employed to detect apoptotic cells in the penumbra. RESULTS: Although neurological scoring (1.57 +/- 0.65 vs 1.74 +/- 0.52) was not significantly different between ketamine and ketamine-midazolam groups (P > 0.05), infarct size and apoptotic cell density in the penumbra were significantly lower in the ketamine-midazolaml group than those in the ketaminel group [infarct size: (24.1 +/- 4.63)% vs (38.48 +/- 4.18)%; apoptotic cells density: (178 +/- 23) cells/mm2 vs (258 +/- 15) cells/mm2, P < 0.05], respectively. CONCLUSION: The above findings suggested that ketamine and midazolam combination might provide neuroprotection during surgical procedures that pose risk for ischemic cerebral injury.

[Effects of ketamine-midazolam anesthesia on focal cerebral ischemic injury in rats].

OBJECTIVE: To compare the effects of anesthesia with ketamine and ketamine-midazolam on focal cerebral ischemic injury in rats and evaluate the neuroprotective effect of ketamine-midazolam anesthesia during surgical procedures. METHODS: Thirty male Sprague-Dawley rats were subjected to permanent middle cerebral artery occlusion (MCAO) under anesthesia with intraperitoneal administration of ketamine alone or in combination with midazolam (n=15). Neurological scoring was conducted the infarct size measured at 4 h and 24 h after the surgery. Histological examination was performed using TUNEL staining 72 h after MCAO. RESULTS: After MCAO, the neurological scores showed no significant difference between ketamine and ketamine-midazolam groups (1.57+/-0.65 vs 1.74+/-0.52, P>0.05), but the rats in the latter group had significantly smaller infarct size (38.48/+/-4.18/ vs 24.1/+/-4.63/, P<0.05) and lower apoptotic cell density (258+/-15 cells/mm2 vs 178+/-23 cells/mm2, P<0.05) in the penumbra. CONCLUSION: Compared with ketamine used alone, anesthesia with the combination of ketamine and midazolam may provide neuroprotection from ischemic cerebral injury by reducing the infarct size and lowering the cell apoptosis rate following MCAO in rats.

[Genetic polymorphism of 9 STR loci in Han nation population in Shandong Province].

OBJECTIVE: To study genetic polymorphism of 9 STR loci in Han nation population in Shangdong Province. METHODS: We investigated 100 unrelated individuals of Han nation population in Shandong Province and studied their genetic distribution of 9 STR loci and Amelogenin locus. Primers of 10 loci were labeled with the NHS-ester fluorescent dye 5-FAM (blue), Joe (green) or NED (yellow). The data of Han nation population were generated by multiple amplification and subjected to GeneScan, genotype and genetic distribution analysis. RESULTS: 83 alleles and 220 genotypes were observed, with the corresponding frequency of 0.0050-0.4050 and 0.0100-0.2100. The average of heterozygosity was 0.7778, the accumulated discrimination power was 0.9999. The accumulated probability of exclusion paternity was 0.9999. The polymorphism information content was 0.5823-0.8396. CONCLUSIONS: Chi-Square test indicated that the distribution of genotypes agreed with Hardy-Weinberg equilibrium.