Protective effect of ethyl pyruvate on brain tissues in neonatal rats with hypoxic-ischemic brain damage

AIM：To study the effects of ethyl pyruvate (EP) on brain tissues in neonatal rats with hypoxic-ischemic brain damage (HIBD) and its underlying mechanisms. METHODS：A total of 165 seven-day-old Sprague-Dawley (SD) rats were randomly divided into 3 groups：sham operation group (n=43), HIBD group (n=61) and HIBD+EP group (n=61). The rats in HIBD+EP group were intraperitoneally injected with EP (50 mg/kg) 30 min before operation, and once a day after surgery. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in brain homogenate, water content of brain and apoptotic cells in cortex were detected 3 days later. Ischemic and non-ischemic brain tissues were weighed to assess the extent of brain atrophy 14 days later. RESULTS：Higher level of SOD ［(125.78±18.35)×103 U/(g protein)］ and lower level of MDA ［(4.42±1.04) μmol/(g protein)］ in HIBD+EP group than that in HIBD group ［(97.84±15.50)×103 U/(g protein) and (6.02±0.89) μmol/(g protein), respectively］ was observed (P<0.05)．In addition, the water content of ischemic hemisphere was significantly higher than that of non-ischemic one in HIBD group (P<0.05), and was indistinguishable from that of non-ischemic one after EP treatment (P>0.05), indicating the protective effect of EP against brain edema. The apoptotic cells in cortex and hippocampus in HIBD+EP group ［(96.63±10.08)/field and (41.91±9.96)/field, respectively］ were obviously decreased compared with HIBD group ［(111.54±1.64)/field and (51.73±1.77)/field, respectively］, but still higher than those in sham operation group (P<0.05). The atrophy ratio of ischemic hemisphere in HIBD+EP group was (13.25±5.19)%, significantly lower than that in HIBD group ［(20.32±5.10)%, P<0.05］. CONCLUSION：Ethyl pyruvate is neuroprotective against HIBD in neonatal rats via increasing SOD level, decreasing MDA level, attenuating brain edema, decreasing apoptotic cells in cortex and alleviating atrophy of hypoxic-ischemic hemisphere.     

Establishment of intrauterine hypoxic-ischemic brain damage model in near term fetal rabbits

AIM: To establish intrauterine hypoxic-ischemic brain damage (HIBD) model in near term fetal rabbits at 29 d gestation age for the investigation of the pathogenesis and treatment of newborn HIBD. METHODS: Twenty-four pregnant New Zealand white rabbits at 29th gestational day were chosen for this project. Under combined general anesthesia and spinal anesthesia, a 4F Fogarty arterial embolectomy catheter was introduced into the left femoral artery. The blood supply of uterus in experiment group was blocked by inflating the catheter balloon with 0.3 mL saline for 20 min, 25 min, 28 min, 30 min and 40 min (n=4 for each experimental time group). The catheter balloon was not inflated in control group (n=4). All pregnant rabbits were subject to cesarean section 24 h after the experimental procedure to induce hypoxia-ischemia to the fetus. The general conditions of the newborn rabbits were recorded, and the neurobehavioral damage and histology of the brain tissue were assessed. RESULTS: During the entire procedure, the pregnant rabbits had stable vital signs, no hypoxia happened,and had a good tolerance to the anesthesia program. When the balloon was inflated, the pulses of right femoral artery disappeared and the right leg blood pressure became non-detectable in experimental groups. In contrast, no fluctuation of the right leg blood pressure in control group (P>0.05) was observed. Intrauterine hypoxia-ischemia caused neonatal and fetal rabbit death, neurobehavioral damage and brain cell death. When the balloon was inflated for 20 min, all fetal rabbits were alive and had no obvious neurologic damage. For 25~28 min, the stillbirth rates were 12.9% and 40.6%, respectively, while the live neonatal rabbits manifested neurobehavioral damage, edema neural cells, activated microglia cells and apoptotic brain cells. When blocking time beyond 30 min, above 80% fetal rabbits died. CONCLUSION: Continuous blockage of uterine blood supply in pregnant rabbits causes neonatal rabbit death, neurobehavioral damage and brain cell death. Different blocking time arouses different levels of brain damage. Continuous blockage of uterine blood supply for 25-28 min can establish fetal generalize hypoxic-ischemic brain damage rabbit model, which is a good animal model for the investigation of newborn HIBD.     

Effects of A2a adenosine receptor on hypoxic pulmonary hypertension in rats treated with salidroside

AIM: To study the protective effect of A_2a adenosine receptor (A_2aAR) on hypoxic pulmonary hypertension in the rats treated with salidroside. METHODS: Sprague-Dawley rats were randomly divided into 6 groups: normal control group, hypoxia group, hypoxia+salidroside (low dose) group, hypoxia+salidroside (median dose) group, hypoxia+salidroside (high dose) group, and hypoxia+CGS-21680 (a selective agonist of A_2aAR) group. Pulmonary hypertension in the rats was produced for 4 weeks. Mean pulmonary artery pressure (mPAP), mean carotid arterial pressure (mCAP) and the weight ratio of right ventricle/(left ventricle+septum)［RV/(LV+S)］ were measured. The expression of A_2aAR in the pulmonary arterioles was determined by immunohistochemistry and in situ hybridization. The mRNA expression of A2aAR in the lung tissues was detected by real-time RT-PCR. The protein level of A_2aAR in the lung tissues was analyzed by Western blotting. RESULTS: The mPAP in hypoxia group was significantly higher than that in normal control group. The mPAP in hypoxia+salidroside (high dose) group and CGS-21680 group was significantly lower than that in hypoxia group. RV/(LV+S) in hypoxia group were significantly higher than that in normal control group. RV/(LV+S) in hypoxia+salidroside (median dose) group, hypoxia+salidroside (high dose) group and CGS-21680 group were lower than that in hypoxia group. The ratio of vessel wall area/vessel total area (WA/TA) in hypoxia group was significantly higher than that in normal control group. WA/TA in hypoxia+salidroside (low dose) group, hypoxia+salidroside (median dose) group, hypoxia+salidroside (high dose) group and CGS21680 group were obviously lower than that in hypoxia group. The expression of A_2aAR was significantly higher in hypoxia group than that in normal control group. The expression of A_2aAR in hypoxia+salidroside (high dose) group and CGS-21680 group was obviously higher than that in hypoxia group. CONCLUSION: The A_2aAR attenuates pulmonary vessel remodeling and pulmonary hypertension induced by hypoxia. Salidroside protects the pulmonary vessel from remodeling and inhibits the development of hypoxia-induced pulmonary hypertension by up-regulation of A_2aAR expression.     

Expression of p-p38 MAPK in partial cerebral tissues after hypoxic-ischemic brain damage in neonatal A2AR knockout mice

AIM： To study the expression of p-p38 MAPK in partial cerebral tissues after hypoxic-ischemic brain damage (HIBD) in the neonatal adenosine A2A receptor knockout (A2AR-/-) mice. METHODS：Base on the modified Rice method, the model of HIBD was established. The total 64 C57/BL6 neonatal mice (7 days old) of A2AR-/-(KO) and corresponding wild type (A2AR+/+, WT) were randomized into sham-operated group and model group. The mice in model group were divided into 3 subgroups： 1 d after HIBD, 3 d after HIBD and 7 d after HIBD (n=8 for each group). The cortex and hippocampal CA1 region were used as the study areas. The neuronal apoptosis was detected using TUNEL assay combined with Nissl staining. The expression of p-p38 MAPK and activated caspase-3 was determined by the method of immunohistochemistry. The KO mice and WT mice were also taken from sham-operated group (SKO and SWT, n=10) and model group (MKO and MWT, n=30) 1 d after HIBD to assess the early neurological behavior. RESULTS：The apoptotic neurons, activated caspase-3 and p-p38 MAPK increased after HIBD and peaked at 1 d after HIBD in the cortex and the hippocampal CA1 region. The apoptotic neurons and the expression of activated caspase-3 in KO mice were significantly higher than those in WT mice at the same time point after HIBD. The expression level of p-p38 MAPK in KO mice were significantly higher than that in WT mice at 1 d and 3 d after HIBD. The expression of activated caspase-3 was positively correlated with the expression of p-p38 MAPK in neonatal mice after HIBD (in the cortex：r=0.957, P<0.01; in the hippocampal CA1 region： r=0.939, P<0.01). CONCLUSION：p-p38 MAPK might be involved in the aggravated neuron apoptosis and brain damage induced by A2AR knockout after neonatal HIBO.     

Lipoxin A4 reduces lipopolysaccharide-induced expression of cyclooxygenase 2 and prostaglandin E2 in human bronchial epithelial cells

AIM: To explore the effects of lipoxin A₄ on the expression of cyclooxygenase 2 (COX-2) in human bronchial epithelial cells (HBECs). METHODS: HBECs were incubated with various concentrations (0.1, 1 and 10 mg/L) of lipopolysaccharide(LPS) for 9 h, or 1 mg/L LPS for different time (3 h, 6 h and 9 h). The levels of COX-2 mRNA in HBECs and prostaglandin E₂ (PGE₂) in the culture supernatant were measured. In addition, the HBECs were exposed to lipoxin A₄ at concentration of 0, 100 and 400 μmol/L after stimulated with LPS at concentration of 1 mg/L for 9 h, and the supernatant of the culture cells was collected for determining the content of PGE₂ by ELISA. The cells were also harvested, and the mRNA and protein levels of COX-2 were analyzed by RT-PCR and Western blotting, respectively. RESULTS: LPS increased the mRNA expression of COX-2 and production of PGE₂ in a dose and time dependent manners in HBECs. Induction of COX-2 mRNA and protein by LPS were inhibited by lipoxin A₄ in a dose-dependent manner. Lipoxin A₄ also significantly decreased LPS-induced production of PGE₂. CONCLUSION: Lipoxin A₄ down-regulates LPS-induced expression of COX-2 and consequently inhibits the production of PGE₂ in HBECs.     

Effect of 5-HT1A receptor in hippocampal CA1 region on spatial memory of PTSD rats

AIM: To investigate the change of long-term potentiation (LTP), and the expression of 5-hydroxytryptamine 1A receptor (5-HT_1A receptor) and postsynaptic density protein 95 (PSD-95) in the hippocampus of the rats with posttraumatic stress disorder (PTSD), and to explore the mechanism of 5-HT_1A receptor in the regulation of spatial memory in the PTSD rats.METHODS: Healthy adult SD rats (n=36) were randomly divided into control group and model group, with 18 rats in each group. The rats in model group were treated with single prolonged stress to construct the model of PTSD. Morris water maze (MWM) was used to test the learning and memory ability. The LTP induced by high-frequency stimulation (HFS) was detected by electrophysiological method. The protein expression of 5-HT_1A receptor and PSD-95 in the hippocampus was determined by Western blot and immunofluorescence. RESULTS: The MWM analysis showed that the latency of the rats searching for the underwater platform in model group was significantly longer than that in control group (P<0.01). The results of electrophysiological analysis showed that the amplitude of the evoked potential in both groups were significantly increased after HFS in the hippocampus, but that in model group was significantly lower than that in control group (P<0.01). The results of Western blot and immunofluorescence analysis showed that compared with control group, the protein expression of 5-HT_1A receptor was obviously increased (P<0.05), while the expression of PSD-95 was obviously decreased in model group (P<0.05).CONCLUSION: The spatial memory impairment in the PTSD rats may be associated with the increase in the expression of 5-HT_1A receptor and the decrease in the expression of PSD-95 in the CA1 region of hippocampus.     

Microinjection of AT1 and AT2 receptor antagonists into PVN affects renal sympathetic nerve activity in chronic heart failure rats

AIM: To examine the renal sympathoexcitation affected by microinjection of angiotensin Ⅱ type 1 (AT₁) receptor antagonist L-158809 and angiotensin Ⅱ type 2 (AT₂) receptor antagonist PD123319 into paraventricular nucleus (PVN) in heart failure rats.METHODS: Left anterior descending coronary artery ligation was used to induce rat heart failure (HF) . Four weeks after operation, the left ventricular end-diastolic pressure (LVEDP), the ratios of heart weight/body weight and lung weight/body weight, and the ratio of infarct area of the left ventricle were observed. Under anesthesia, SD rats were fixed into the brain stereo controller to locate PVN for microinjection and the artificial cerebrospinal fluid (ACSF) was used for control. The left kidney was exposed by retroperitoneal approach and the renal sympathetic nerve was separated under surgical microscope. The heart rate, blood pressure and the activity of renal sympathetic nerve discharge (RSNA) were recorded by POWERLAB 8/30 system. RESULTS: Microinjection of AT₁ receptor antagonist into PVN induced a decrease in RSNA in both HF rats and sham rats. The RSNA responses to L-158809 in the HF rats were significantly greater (P<0.05) than those in the sham rats. However, microinjection of AT₂ receptor antagonist and ACSF into PVN induced no change of RSNA in both HF and sham rats. CONCLUSION: There are some differences of sympathetic nerve outputs between using AT₁ receptor antagonist and AT₂ receptor antagonist on PVN, indicating the up-regulation of AT₁ receptors in PVN during HF. The central renin-angiotensin-aldosterone system(RAAS) may be affected by AT₁ receptor, not by AT₂ receptor.     

Sphingosine-1-phosphate receptor 2 attenuates influenza A virus-induced viral pneumonia through PI3K/AKT/eNOS pathway

AIM: To investigate the effects of sphingosine-1-phosphate receptor 2 (S1PR2) on influenza A virus-induced viral pneumonia.METHODS: The animal model of influenza A virus pneumonia was established by infecting wild-type C57BL/6 mice and S1pr2^-/- mice with influenza virus subtype FM1 mouse lung adaptable strain through nose drops. The pathological changes of the lung tissues of wild-type mice (model group), JTE-013 (S1PR2 effective antagonist)-challenged mice and S1pr2^-/- mice were observed, and the protein concentration, total cell number, and interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α) levels were determined in the bronchoalveolar lavage fluid (BALF) at 4 d and 6 d after virus infection. The phosphorylation levels of AKT and eNOS in the lung tissues were determined by Western blot. RESULTS: Compared with the wild-type mice of control group, the influenza A virus pneumonia in JTE treatment group and S1pr2^-/- mice were more serious, and the protein concentration, total cell number and inflammatory cytokines in the BALF were remarkably increased. Moreover, the phosphorylation levels of AKT and eNOS, the downstream targets of PI3K, were significantly increased (P<0.01). CONCLUSION: S1PR2 mediates PI3K/AKT/eNOS signaling transduction pathway to regulate NO generation, and inhibit vascular permeability and inflammatory cytokine release, thus attenuating the viral pneumonia induced by influenza A virus.     

Effects of P2X4 receptor in spinal microglia on rrTNF-induced pathological pain

AIM: To investigate the effects of P2X₄ receptor on peri-sciatic administration of recombinant rat TNF-α (rrTNF)-induced mechanical allodynia. METHODS: Male Sprague-Dawley rats (180~200 g) were used in the experiments. The levels of P2X₄ receptor on day 3, day 7 and day 14 after peri-sciatic administration of rrTNF were examined by Western blot, and the location of P2X₄ receptor in the spinal dorsal horn was observed by double immunofluorescence staining. The changes of 50% paw-withdrawal thresholds of the rat were detected by behavioral test, and the level of TNF-α in the spinal dorsal horn was also examined by Western blot when TNP-ATP was intrathecally injected before the administration of rrTNF. RESULTS: Compared with control group, the expression of P2X₄ receptor in the spinal dorsal horn on the ipsilateral side significantly increased on day 3, day 7 and day 14 (P<0.01) after rrTNF (100 ng/L) administration. P2X₄ receptor was co-localized only with microglia, but not with neurons or astrocytes. Intrathecal injection of TNP-ATP before rrTNF administration prevented mechanical allodynia induced by rrTNF and inhibited the upregulation of TNF-α in the spinal dorsal horn. CONCLUSION: P2X₄ receptors in microglia may be involved in rrTNF-induced mechanical allodynia by the upregulation of TNF-α in the spinal dorsal horn.     

Effect of Zhengtian pills on P2X3 receptor expression in trigeminal gan-glion of migraine rats

AIM: To explore the effect of Zhengtian pills on P2X₃ receptor expression in trigeminal ganglion (TG) of migraine rat. METHODS: Sprague-Dawley (SD) rats were randomly divided into control group, migraine group, Zhengtian pills (ZTP) group and A-317491 group. After given corresponding drugs for 7 d, migraine rat model was established by subcutaneously injection of nitroglycerin (10 mg/kg), while the control rats were injected with saline. The beha-vioral manifestations of the rats were observed. The expression of P2X₃ receptor in rat TG was detected by the methods of immunofluorescence, Western blot and rea-time PCR. RESULTS: About 5 min after subcutaneousl injection, the behavioral manifestations such as scratching head and climbing cage were observed. The behavioral manifestations were observed within the first 30 min in control group, but the erythroid ears did not appear. After 2 h of molding, the behavioral manifestations disappeared in ZTP group and A-317491 group, while those in migraine group lasted for 3 h. Immunofluorescence results showed that the expression of P2X₃ receptor in TG was positive in each group. The expression level in migraine group was significantly higher than that in other groups. The P2X₃ receptor protein and mRNA levels in the TG of migraine group were higher than those in control group (P<0.01), while those in ZTP group were lower than those in migraine group (P<0.01). No difference of the P2X₃ receptor expression between ZTP group and A-317491 group was observed. CONCLUSION: Zhengtian pills may effectively alleviate migraine by inhibiting the expression of P2X₃ receptor in TG.