| BackgroundThe average annual incidence of cerebrovascular disease in China is about19.3-77.9per million, and the mortality differs from8.1to44.1per million. The cerebrovascular disease is the first cause of death and disability in China. Ischemia stroke is the most common one of cerebrovascular diseases。 The mortality of massive cerebral infarction, especially caused by the obstruction of middle cerebral artery, is up to80%. The incidence and mortality are getting worse, which will lay heavy burden on social and families.How to reduce the morbidity and mortality of massive cerebral infarction is a hot topic and still a difficult task in the field. Cerebral infarction is caused by interruption of blood flow or oxygen and sugar supply to the brain, which led to brain cells death, such as neuron cells, vascular endothelial cells and glial cells. Damage of cells after cerebral ischemia is the consequence of a series of ischemic cascade events, including mitochondrial dysfunction, overloaded intracellular calcium, sodium, and decreased K+, increased production of ROS and NO, excessive release of excitatory amino acid, glutamate receptor activation, released inflammatory cytokines and adhesion molecules, microvascular obstruction, blood-brain barrier damage; the induction of activation of protein caspase family. In a few minutes to a few days after infarction, different pathological processes play roles and overlap each other. According to the degree of ischemia, cerebral lesions can be divided into ischemic core area and ischemic penumbra area. Cells of core area die due to entirely ischemia, excessive activation of excitotoxicity and other reasons within minutes. Cells of ischemic penumbra area survive due to partly ischemia and reperfusion surrounding collateral circulation. Free radicals generated by reperfusion damage cells, led to most of cells death mainly via apoptosis pathway. In the reperfusion time window, the best treatment is thrombolytic therapy. Since reperfusion time window is only within4.5h, many patients visiting the hospital could not receive thrombolytic therapy. Therefore, protecting the targeted cells of ischemic penumbra area is the main goal of the current experimental research and clinical treatments. It is important to reduce morbidity and mortality of cerebrovascular disease and improve the quality of life of patients to early protect ischemic penumbra cells, to promot the recovery of cells function.At present, therapeutic hypothermia is a clinically proven protective measure for brain damage. Hypothermia refers to the temperature range from32to35℃, and widely used in clinical is at33-34℃. Hypothermia has achieved remarkable results in cardiopulmonary resuscitation, neonatal hypoxic-ischemic encephalopathy apnea and treatment of traumatic brain injury. However, low-temperature applied in the cerebral infarction in recent years has just started. The evidences from laboratory and clinic studies are insufficient. Our previous studies have shown that hypothermia reduced infarct size in MCAO rats, but did not improve neurological function. Therefore it is need to resort to another treatment to further improve the effects of hypothermia. And hypothermia also has many side effects, for example:shiver, restraining of respiratory function, and prolonged hypothermia increasing complications of lung infection and deep vein thrombosis. Therefore, combination of drugs and hypothermia may be an alternative treatment for ischemic stroke. We intend to find the drugs that may further improve the effect of hypothermia on cerebral ischemia, finally improve the outcome of ischemic stroke.A lot of reports have shown the protective effects of neuroprotective agents in vitro or in vivo experiments. These drugs can be divided into following kinds according to mechanism, including glutamate receptor blockers, γ-aminobutyric acid receptor agonists, calcium antagonists, free radical scavengers, antibiotics and immunosuppressive agents, growth factors,5-serotonin receptor agonists, membrane component and stabilizers, cerebral metabolic agents, neuropeptides, anti-diabetic drugs, hormones, tradition medicines and so on. But they encountered obstacles when were transformed to clinic. Up to now, none of these neuroprotective agents showed to be effective in phase III clinical trials. The reasons may include design flaws of the animal experimental, racial differences, different timing of drug used in the clinical and in the experiments and so on. But one of the most reasons may come from the single target and the single act of a drug in the ischemic cascades. Based on the complexity and diversity of the pathological mechanisms of cerebral infarction, combinations with a variety of drugs or treatments (especially non-pharmacological neuroprotective treatment) are likely to be a synergistic effect.Recently, the combination of mild hypothermia and neuroprotective agents were covered. Our previous and other studies all demonstrated that hypothermia combined with drugs, such as brain-derived neurotrophic factor or magnesium sulfate, had a synergistic effect in the treatment of cerebral artery infarction. However, systematically and extensively study for combination of hypothermia and neuroprotective agents has not appear. We selected26kinds of neuroprotective agents based on the working mechanism, availability, toxicity, usage, and so on. Compared with the animal models and human studies, oxygen-glucose deprivation/reoxygenation (OGD/R) model can simulate different levels of ischemia/reperfusion status in vitro, get more consistent experimental conditions, and perform high-throughput drug screening, consuming less cost, with incomparable superiorities. Neuron is a decisive brain cell which determines the overall prognosis of brain tissue, so that we firstly set up the model of severe OGD/R primary cortical neuron, and then use this model screening26kinds of candidate medicines with hypothermia with the cell viability and apoptosis, the former reflected the number of living cells, the latter reflected the extent of the damaged. In addition, ischemic cascade processes after infarct relating to neurons include:intracellular calcium overload, decreased mitochondrial membrane potential, ROS generation, NO production, activation of Caspase-3. Therefore, we finally further validated the synergism of the screened drugs with hypothermia on these sites.Objects(1) Screening the appropriate concentrations of neuroprotective agents and synergistic neuroprotective agents with hypothermia in OGD/R primary cultured cortical neurons model.(2) Verifying the synergism of neuroprotective agents and hypothermia.Methods(1) Culturing primary cortical neurons, and estimating the purity of neurons by neuron-specific anti-β-Tubulin Ⅲ staining.(2) Establishing the hypoxia-reperfusion cells model of primary cortical neurons suffered OGD/R in vitro.Cultured primary cortical neurons were randomly divided into five groups:OGD1.5h, OGD3h, OGD4.5h, OGD6h and normal control groups, each group with four bottles of cells. OGD groups were given OGD1.5h,OGD3h, OGD4.5h and OGD6h, respectively, then reoxygenation, no dealing for the normal control group. The apoptosis rates were measured using the TUNEL Assay after48hours. The duration of oxygen deprivation led to40-60%apoptosis rate was chosen for severe OGD/R model.(3) Establishing the duration of hypothermia treatment for primary cortical neurons suffered OGD/R in vitro. Cultured primary cortical neurons were randomly divided into10groups:HT33℃1.5h, HT33℃3h, HT33℃4.5h, HT33℃6h, HT34℃1.5h, HT34℃3h, HT34℃4.5h, HT34℃6h, OGD/R control group and normal control, each group with4bottles of cells. Besides the normal group, other groups were OGD for4.5h and reoxygenated, then treated with hypothermia at33℃for1.5h,3h,4.5h,6h, or at34℃for1.5h,3h,4.5h,6h, OGD/R control group no treatment. The apoptosis rates were determined by TUNEL assay. The duration and temperature leading to the lowest apoptosis percentage was chosen as the best one of hypothermia treatment.(4) Screening the optimal working concentration of neuroprotective agents:the primary cortical neurons were randomly divided into3-5groups, each group six complex wells. Each group was treated by different doses of neuroprotective agent after OGD/R. The protective effects on neurons of neuroprotective agents were measured by rate of cell viability change (ACV%) with CCK-8kit. We selected the dose led to lowestΔCV%as the best dose for the next experiments.(5) Screening the neuroprotective agents with synergistic effects on OGD/R primary neurons applied with hypothermia. The primary cortical neurons were randomly divided into five groups:neuroprotective agents group, hypothermia(HT) group, neuroprotective agents+HT group, OGD/R control group and normal control groups, each group six wells (for cell viability) or four bottles (for apoptosis of cells). In addition to the normal group, each group was given oxygen-glucose deprivation/reoxygenation for4.5h, and then was given a neuroprotective agent, hypothermia, or a combination of a neuroprotective agent and hypothermia, OGD/R control group no treatment, the normal control group cultured in normal condition. We detected ΔCV%with CCK-8kit, and total apoptosis (early plus late apoptotic rate) of cells with Annexin V/PI double staining flow cytometry detecting.(6) Verifying the joint protective effects of combination of a neuroprotective and hypothermia on major aspects of the ischemic cascade. The primary cortical neurons were randomly divided into five groups:a neuroprotective agent group, hypothermia group, a combination of neuroprotective agent and hypothermia group, OGD/R control group and normal control group, each group4bottles of cells. In addition to the normal group, each group was given OGD/R, and then was given a neuroprotective agent or hypothermia or neuroprotective agents plus hypothermia treatment. OGD/R control group did not receive any treatment, and the normal control group cultured in normal condition. Mitochondrial membrane potential(MMP) was measured by JC-1staining with flow cytometry, ROS generation by DCF-DA staining with multifunctional microplate reader, intracellular calcium ion concentration by FLUO-3staining with flow cytometry, NO levels by DAF-FM DA staining with flow cytometry, and Caspase3expression determined by Western Blotting.Statistical AnalysisNeurons were divided into several groups using random numbers. Statistics values were described as mean±standard deviation (x±S). Multiple comparisons were conducted by One-Way ANOVA LSD method if homogeneity of variance, otherwise by One-Way ANOVA Games-Howell method. Factor analysis was used for factorial design to analyze the interaction between the two factors, and the interaction map was drawn. The individual effect of each factor was analysed by independent samples t test. P<0.05was considered statistically significant.Results(1) The body and synapses of primary cortical neurons were stained by anti-β-Tubulin III into red fluorescence, and the nuclei were stained by DAPI into blue fluorescence. The purity of cultured primary cortical neurons was about97.312±0.831%(2) With the extension of the time oxygen-glucose deprivation, the rate of TUNEL-positive primary neurons increased gradually. The percentage of TUNEL-positive primary neurons after OGD for1.5h、3h、4.5h、6h are9.048±3.128%,25.383±5.492%±45.217±20.778%and74.524±11.199%, respectively. The apoptosis rate of OGD1.5h neurons did not differ from normal controls (5.117±4.157), but not among the other groups(F=79.986, P=0.000). Despite the high apoptosis percentage of neurons after OGD6h (74.524±11.199), the total number of cells was significantly reduced as well. Consequently we chose OGD4.5h as the most appropriate duration for primary cortical neurons to imitate the severe ischemic/reperfusion situation in vitro.(3) The interaction between level and duration of temperatiure exerted effect on the apoptosis rate (F=4.108, P=0.009). The apoptosis percentage of neurons after OGD/R was significantly higher than the control group, increasing from2.659±1.613%to58.227±9.773%(t=19.433, P=0.000). The apoptosis percentages of neurons treated by34℃hypothermia for4.5h were lowest (15.390±3.343%), compared with the other groups (34℃1.5h:38.886±9.982%,3h:25.253±5.769%,6h:42.938±9.266%;33℃1.5h:40.771±11.723%、3h: 35.895±7.122%、4.5h:27.752±7.654%、6h:39.825±12.228%)(F=22.374, P=0.000)(Games-Howell). The optimal level of hypothermia was at34℃for OGD/R primary neuron, the best time of hypothermia was4.5h.(4) The best working concentrations of26neuroprotective agents are as follows: albumin:5%(F=28.805, P=0.000); atorvastatin:0.1μM (F=9.996, P=0.002); baclofen:10μM (F=128.964, P=0.000); BDNF:25ng/ml (F=13.812, P=0.000); bumetanide:50μM (F=7.220, P=0.006); CDPC:100mM (F=108.716, P=0.000); CsA:0.01μM(F=4.383,P=0.032);8-OH-DPAT:100μM (F=96.434, P=0.000); DFO:100μM (F=17.072, P=0.000); edavarone:100μM (F=85.527, P=0.000); GBC:μM (F=34.426, P=0.000); glicalzide:10μM (F=113.337, P=0.000); GM1:1μM(F=10.804,P=0.001); HUK:0.0015PNA/ml (F=20.374, P=0.000); MgSO4:3mM (F=66.204, P=0.000); minocycline:0.1μM (F=16.638, P=0.000); MK-801:1OμM (F=8.469, P=0.003); MP:10μM (F=11.968, P=0.001); Ngb:50nM (F=16.194, P=0.000); nimodipine:10μM (F=3.281, P=0.066); NXY-059:250mM (F=5.821, P=0.013); pegesterone:O.1μM (F=0.565, P=0.580); liluzole:100mM (F=8.506, P=0.003); pyruvate:10μM (F=36.500, P=0.000); a-tocopherol:1μM (F=79.606, P=0.000); VAS2870:2μM (F=19.489,P=0.000)。(5) Δ CV%in primary neurons after OGD/R increased to some degree (t=-64.754～-16.003, P=0.000). Hypothermia associated with baclofen (F=8.685, P=0.008)、BUM (F=37.521, P=0.000)、CDPC (F=14.148, P=0.001)、 DPAT (F=5.576, P=0.028)、edavarone (F=17.712, P=0.000)、gliclazide (F=27.411, P=0.000)、GM1(F=51.121, P=0.000)、MP(F=26.277, P=0.000)、 nimodipine (F=6.404, P=0.020)、 pyruvate (F=10.279, P=0.004) exerted efffects on Δ CV%. Separate effects analysis:five drugs associated with hypothermia reduced Δ CV%:HUK (9.028±6.527), Ngb (16.768±5.249), MK-801 (16.965±5.085), GBC (24.784±13.203), BDNF (9.028±6.527), better than treated with single-drug (HUK:34.994±5.021, Ngb:45.101±7.956, MK-801:44.227±5.570, GBC:39.176±.044, BDNF:32.628±8.221)(BDNF:t=5.507, P=0.000; GBC:t=2.553, P=0.044; HUK:t=3.563, P=0.006; MK-801: t=8.853, P=0.000; Ngb:t=7.282, P=0.000)or hypothermia(35.792±.633or45.283±13.591)(BDNF:t=7.043, P=0.000; GBC:t=2.650, P=0.024; HUK:t=3.600, P=0.009; MK-801:t=5.518, P=0.000; Ngb:t=5.509, P=0.000).(6) OGD/R increased the total apoptosis of neurons from12.138±0.658%to26.430±1.706(t=-15.633, P=0.000). Interactions between HUK、NGB、GBC and hypothermia exerted on total apoptosis rate (F=47.458,15.572,9.504; P=0.000,0.002,0.002). Among five potential drugs, a combination of human urine kallikrein (HUK) or MK-801or neuroglobulin (Ngb) and hypothermia (HT) lowered the total apoptosis rate in primary cortical neurons after OGD/R (from26.430±1.706%to13.108±1.006%,15.508±0.938%and11.138±0.893%), better than hypothermia alone (24.933±1.822%)(HUK:F=11.360, P=0.000; MK-801:F=9.196, P=0.000; Ngb:F=13.595, P=0.000) or single drugs (HUK26.270±2.055%, MK-801:18.725±1.292%, Ngb18.175±1.181%)(HUK:F=11.504, P=0.000; MK801:F=4.031, P=0.008;Ngb:F=9.504, P=0.000).(7) The proportion of cells with decreased mitochondrial membrane potential(MMP) in primary cortical neurons after OGD/R increased from16.703±0.546%to31.880±1.877%(t=15.531, P=0.000).There were interactions between MK-801or Ngb and hypothermia (F=42.474,8.188; P=0.000,0.014). Combination of MK-801or Ngb and hypothermia exerted effect on MMP (F=42.474,8.188; P=0.000,0.014). Separate effects analysis:A combination of either the three drugs and hypothermia reduced the proportion of cells with decreased MMP (to12.848±0.448%,19.443±0.706%and13.183±0.613%), better than single drugs (HUK:18.130±0.717%, MK-801:20.973±0.423%, Ngb:32.198±1.235%)(t=12.495,20.911,17.933; p=0.000) or hypothermia alone (27.165±0.737%)(F=33.213,29.176,15.138, P=0.000).(8) ROS-relative ODs values increased in primary cortical neurons after OGD/R from normal19.639±2.688up to201.596±3.939(t=-93.452, P=0.000). There were interaction between MK-801or Ngb and hypothermia on ROS-relative Geo Mean(F=858.166,285.321,973.659; P=0.000). Separate effects analysis: HUK, MK-801and Ngb when used in combination with hypothermia reduced ROS-relative ODs value to29.542±2.604,31.712±3.328and33.270±4.458, lower than a single drug (56.630±7.815,89.363±11.731,69.022±4.667)(F=8.055,11.581,13.569; P=0.000) or hypothermia (48.377±5.279).(F=7.839,6.542,5.356; p=0.000)(9)[Ca2+] i-dependent Fluo-3/AM mean fluorescence intensity in normal primary cortical neurons was about1693.000±23.678, and increased up to3104.250±90.112after OGD/R (t=-30.294, P=0.000). There were interaction between HUK or Ngb and hypothermia on [Ca2+] i-dependent Fluo-3/AM mean fluorescence intensity.A combination of HUK or Ngb and hypothermia exerted effects on [Ca2+] i-dependent mean fluorescence intensity (F=18.743,45.911; P=0.001,0.000). Separate effects analysis:every drug combined with hypothermia decreased mean fluorescence intensity to1817.500±51.157(HUK),1794.250±26.837(MK-801), and2213.750±23.908(Ngb), better than single-drug (HUK:2339.250±47.822, MK-801:2561.000±34.186and Ngb:2576.250±73.781)(F=14.901,35.284,9.374; P=0.000) or hypothermia (2330.500±22.986)(F=18.294,30.352,7.101; p=0.000). (10) NO-relative fluorescence intensity in normal primary cortical neurons was about1146.500±73.831, after OGD/R significantly increased to1374.250±110.328(t=-3.431,P=0.017). There were interactions between one of the three drugs and hypothermia. Combinating one of the three drugs and hypothermia exerted no effects on NO-relative mean fluorescence intensity (HUK:F=0.310, P=0.588; MK-801:F=1.555, P=0.593; Ngb:F=0.302, P=0.236). Separate effects analysis:Ngb (1253.250±100.590) or a combination of Ngb and hypothermia (1180.500±76.116) reduced NO production, and the combination is better than hypothermia alone (1352.750±82.140)(t=3.076, P=0.022), but not Ngb alone (t=1.153, P=0.296). Other treatments exert no effects on NO-relative fluorescence intensity.(11) Activation of caspase3increases in primary cortical neurons after OGD/R. Combination each of the three drugs and hypothermia decreased the activation of caspase3, prior to the agents alone or hypothermia alone.ConclusionA combination of human urinary kallikrein, neuroglobulin or MK-801and hypothermia improved cell viability and decreased total apoptosis percentage in primary cortical neurons after OGD/R. We verified the synergism of the combinations in the mitochondrial pathway apoptosis including decreased intracellular calcium overload, ROS generation, and improved caspase-3expression and mitochondria membrane potential. |
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