The Neuroprotective Mechanisms Of The Intermittent Hypothermia On Oxygen-glucose Deprivated Neurons

The discrepancy in results for neuroprotective agents in animal experiments compared to clinical trials is a major problem. While many neuroprotective agents have been proven effective in a variety of animal ischemic stroke models, none of them have been shown to work in phase III clinical trials. We retrospectively summarizes the neuroprotectants selected for human randomized controlled trials (RCT) and explores the reasons behind the clinical translational failure of these agents. Here, we suggest that there are many factors (model selection, anesthetic choice, physiological monitoring, model success criteria, embolus property, reperfusion damage, infarction area, therapeutic time window, drug penetration, blood concentration, gender difference, outcome evaluation) responsible for this phenomenon. Ultra-early treatment using a "home run" drug and multi-target therapy may be the most promising for future consideration.Hypothermia is the most effective way of neuroprotection through inhibiting multi-target mechanism of ischemic injury. Intracarotid cold saline infusion (ICSI) is the fastest hypothermia induction. Therapeutic time window of hypothermia is broader than cerebral artery flushing in carotid saline infusion after transient focal ischemic stroke in rats. Continuous ICSI to maintain local brain hypothermia is unrealistic in clinic for massive infusion fluid volume. Intermittent ICSI can greatly reduce the difficulties in liquid capacity management and brain temperature fluctuation. Compared with traditional ICSI, the intermittent method has a longer duration of hypothermia and less effect on hematocrit and offers more potentially improved neuroprotection.Although primary studies showed that intermittent hypothermia did work on MCAO models in rats. However, the neuroprotective mechanisms of the intermittent multiple hypothermia following ischemic stroke was still unknown. The fetal rat cortical neurons after an oxygen glucose deprivation (OGD) in vitro was established to mimic ischemic stroke cellular model. The high throughput of cell model is used to investigate the the possible neuroprotective mechanisms of intermittent hypothermia for screening optimal intermittent hypothermia pattern, as well as a theoretical basis for future clinical applications.Chapter one. Primary culture of cortical neuronsThe primary culture of fetal rat cortical neurons is widely used in cell models of many neurological disorders. However, owing to the complicated procedures involved in dissection and culture, a universally accepted protocol for their derivation has not yet been determined. Diverse techniques make it difficult to readily compare results obtained from different cell models and to repeat experiments in other laboratories. Therefore, it is essential to develop a simple and reproducible protocol for the study cell models. SPF-class E18Sprague Dawley pregnant rats were sacrificed by cervical dislocation. The uterus was removed by rotating clockwise from the left lower quadrant. The placenta was removed, and the color, fetal movements and number of stillbirths were recorded. In order to reduce the metabolism of neurons and guarantee its vitality, the following anatomical process was carried out on crushed ice. We improved traditional microscopic anatomy path to bilateral symmetry separation via nasal approach on E18SD fetal rats. The next was to dissect the skull and dura mater. Take care when removing the pia mater and blood vessels to reduce the interference of the meningeal and vascular cells. Owing to the long process, microscopic anatomy was in a petri dish with cold HG-DMEM containing10%fetal bovine serum (FBS) and glutamine to ensure the energy metabolism. Clean-stripped fetal rat cortex was put into a3.5cm petri dish containing cold FBS-free HG-DMEM and then cut into1mm with sterilized scissors. We have modified the procedure to a sequential digestion of papain and DNase I for taking into account the fast traditional trypsin digestion. Cell suspension was harvested by pipetting and cell sieve, and stained by trypan blue to ensure the dead was less than1%and the clum was less than10%. The neurons were cultured on0.1mg/ml LPP-precoated vessels with the density of50000per cm2. The most neurons were adhered after4hours when the culture medium was replaced by neurobasal with B27and glutaminate. Nearly all the cells were DAPI-and β-tubulin Ⅲ-positive. The determination of neuronal purity using dark field imaging suggests that the percentage of β-tubulin Ⅲ-immunostained neurons was over95%; accordingly96.8%were identified as neurons when assessed by flow cytometry.Chapter two. OGD model and hypothermia interventionThe decrease in blood supply caused OGD in focal zone after an ischemic stroke. It is the important reason to form "core" infarction and ischemic penumbra. And this process can be simulated in vitro via cell model. The dead neurons in core infarction can not be saved, while the ones in ischemic penumbra still have chance. We focus the latter one. The closer to the core of the infarct, the heavier oxygen-glucose deprivation, but relatively mild in ischemic penumbra zone. So we can precisely control the sugar and other nutrients in culture medium to simulate glucose deprivation. Anaerobic incubator was used to control the oxygen concentration to mimic oxygen deprivation. when responsible vascular recanalization or collateral circulation restores the blood supply, it will face ischemia-reperfusion injury. We can restore neuronal culture medium and oxygen supply to study this important pathophysiological process. Therefore, intermittent hypothermia was administrated on neuronal OGD model. Specific programs are as follows:Glucose deprivation in the culture solution was replaced with phosphate buffered saline (PBS). Oxygen deprivation was applied by an anaerobic culture glove box, oxygen-glucose deprivation time was90minutes. There are7groups in the experiment:normal group, OGD group, continuous hypothermia1group (CHI), continuous hypothermia2group (CH2), intermittent hypothermia1group (IH1), intermittent hypothermia2group (IH2) and intermittent hypothermia3group (IH3). Hypothermia intervention contained continuous hypothermia (CH) and intermittent hypothermia (IH). Hypothermia was provided by33℃cellular incubator (Thermo, USA) while the normothermia by37℃(Both cellular incubators were supplied with5%CO2). The hypothermia runtime of CHI group was6hours which were consistent with the sum of hypothermia runtime in other IH groups. The hypothermia runtime of CH2group was12hours which were consistent with the total time in other IH groups. IH1group ran1-hour hypothermia and1-hour normothermia alternately. The intermittent cycle of IH2group was1.5hour, and IH3group was2hours. The endpoint was48hours later after hypothermia intervention, while the normal and OGD groups were served as controls. The effects of hypothermia were assessed from different angles afterwards. Chapter three. The neuroprotective mechanisms of the intermittent hypothermia on OGD neuronsThis chapter aims to explore the potential targets of intermittent multiple hypothermia and compare the differences between CH and IH. Fetal rat cortical neurons were cultured as chapter one, OGD and hypothermia phase as chapter two. The indicators were observed at the endpoint. Cell morphology between each groups were observed under a inverted microscope (Olympus, Japan) with bright field. By the neuronal microenvironmental metabolism angle, we compared the cell viability change, enzyme-labeled substance of cell injury, and excitatory amino acids released into the supernatant in each group. Intracellular acidosis, calcium overload, oxidative damage, mitochondrial depolarization, and apoptosis were detected then.First, we compared the morphology of neurons in each group. Normal neurons refracted three-dimensional shape, cell body, axon growth was strong, and with the surrounding interconnected network shape. In OGD groups, neuronal density decreased significantly for dead floating and appeared axon disintegration or Waller degeneration. The neurons recovered well in hypothermia groups, only a small number of axons fractured. Compared with the normal control group, the morphological changes were not obvious. The microstructure of some seemingly "normal" neurons has changed. Next we will seek for the potential evidence of intermittent hypothermia.Judging from the point of view of the neuronal vitality detected by CCK-8kit, the data was as follows:normal group (0.2984±0.0017), OGD group (0.2205±0.0215), CH1group (0.2617±0.0015), CH2group (0.2535±0.0052), IH1group (0.2329±0.0026), IH2group (0.2724±0.0033), IH3group (0.2814±0.0025). Neuronal vitality drops after OGD. In continuous hypothermia, both6-hours and12-hours continuous hypothermia were helpful to restore the vitality of neurons after OGD.6hours pattern was better than12hours. In intermittent hypothermia,1-hour intermittent pattern did not work.1.5-hour and2-hours intermittent hypothermia were helpful to restore the vitality of neurons after OGD.2-hours intermittent hypothermia was better than1.5-hour intermittent hypothermia. Comparison of intermittent and continuous hypothermia:except for1-hour intermittent hypothermia, the1.5-hour and2-hours intermittent hypothermia were more effective than continuous hypothermia,2-hours intermittent hypothermia was the best.From the point of view of neuronal enzyme injury markers, we used a rat neurons microtubule associated protein-2(MAP-2) enzyme-linked immunosorbent assay (ELISA) kits to detect the supernatant MAP-2. The data was as follows:normal group(1.0780±0.1366), OGD group (1.3461±0.0966), CHI group (1.1858±0.0881), CH2group (1.2893±0.0747), IH1group (1.3251±0.0616), IH2group (1.3325±0.1618), IH3group (1.1808±0.1593). Statistical analysis indicated that neuronal microenvironmental MAP-2level increased after OGD. In continuous hypothermia,6-hours continuous hypothermia could inhibit MAP-2release after OGD,12-hours continuous hypothermia had no such effect. In intermittent hypothermia,2-hours intermittent hypothermia could inhibit MAP-2release after OGD,1-hour and1.5-hour intermittent hypothermia had no such effect. Comparison of intermittent and continuous hypothermia, the neuroprotection of2-hours intermittent hypothermia and6-hours continuous hypothermia was same.From the point of view of the excitatory amino acid glutamate and its oxidase, we detected the glutamic acid and glutamate oxidase activity in the supernatants with the Amplex(?) Red kit. The results showed glutamic acid in normal group (2698.91±206.74), OGD group (2719.35±195.53), CHI group (2763.77±227.90), CH2group(2703.73±278.95),IH1group(2697.83±203.14), IH2group(2714.32±195.63), IH3group (2653.49±230.16). Glutamate oxidase in normal group (478.62±52.08), OGD group (490.14±70.19), CH1group (494.33±92.01), CH2group (450.42±54.26), IH1group (462.52±95.59), IH2group (468.53±33.50),IH3group (455.95±46.10). There was no difference between groups. By analyzing the reason, we found that it due to the settings on the endpoint which missed detection time window. The early dynamic monitoring of glutamate release with microdialysis may be more reasonable. Thus, the microenvironmental metabolism changes may not be sensitive to response inner neuronal changes. Next, we will use the technology of fluorescent probes to explore the interior of the cell targets after the intermittent hypothermia.Prior to this, we must first clear whether intermittent hypothermia was protective on oxygen-glucose deprivated neurons. Qualitative stained with Hoechst33342after OGD neurons found that normal nucleus was calamine blue but the apoptosis showed small high-lighted blue. Quantitative detection of each group was finished by TUNEL kit, the data was as follows:normal group (6676.2±91.84), OGD group (8327.6±177.19), CHI group (7524.8±310.71), CH2group (6092.4±85.09), IH1group (4121±124.83), IH2group (7570±118.53), IH3group (5628.20±699.82). Compared with the control group, neuronal apoptosis increased significantly after48hours of90minutes oxygen-glucose deprivation. In continuous hypothermia,6-hours continuous hypothermia could reduce neuronal apoptosis after OGD but the effect was weaker than12-hours. In intermittent hypothermia, all the intermittent hypothermia reduced neuronal apoptosis after OGD.1-hour and2-hour intermittent hypothermia were better than1.5-hour pattern. Comparison of intermittent and continuous hypothermia,1-hour intermittent pattern was more effective than6-hour and12-hour continuous hypothermia. The neuroprotection of1.5-hour intermittent pattern was same as6hour, but weaker than12hour.2-hour intermittent hypothermia was better than6hour, but same as12hour. Based on the above,1-hour intermittent hypothermia was the most effective way to reduce apoptosis after OGD. Our retrospective analysis found that, oxidative damage, acidosis, calcium overload, mitochondrial failure were the main pathways of the damage to the neurons. Next, we will test the corresponding intracellular targets.The generation of reactive oxygen species is the main reason of oxidative damage. DCFH-DA fluorescent probe measurement of intracellular reactive oxygen species (ROS) found normal group (397.67±49.34), OGD group (1954±69.94), CHI group(424.67±21.36),CH2group(395.33±33.47),IH1group(562.67±92.79), IH2group(331±26.06), IH3group(8098.33±1033.02). Statistical analysis indicated that, neuronal reactive oxygen species (ROS) had a significant increase at48hours after OGD. In continuous hypothermia, both6-hours and12-hours continuous hypothermia could inhibit ROS after OGD, and the effectiveness was same. In intermittent hypothermia, both1-hour and1.5-hour intermittent hypothermia could inhibit ROS after OGD, The effectiveness of former two were same. The data of2-hours intermittent hypothermia varied greatly, it was not included in analysis. Based on the above, the effectiveness of intermittent hypothermia and continuous pattern were same.The generation of superoxide anion radical oxidative damage was also an important factor, we used dihydroethidium (DHE) fluorescent probe to detect superoxide anion radical. The data was as foloows:normal group (69.4±3.36), OGD group (167.75±15.59), CHI group (64.2±2.28), CH2group (114.4±7.54), IH1group (43.6±2.30), IH2group (52.8±1.79), IH3group (52.6±1.14) Intraneuronal superoxide anion radical increased after OGD. In continuous hypothermia, both6-hours and12-hours continuous hypothermia did work. In intermittent hypothermia, all the intermittent hypothermia could inhibit superoxide anion radical after OGD,1-hour pattern was best, and the rest1.5-hour or2-hours pattern were same on effectiveness. Comparison of intermittent and continuous hypothermia, all the intermittent hypothermia groups were better than continuous hypothermia,1-hour intermittent hypothermia performed best.We used BCECF AM fluorescent probe to measure intracellular pH changes. The data was as follows:normal group (994.2±58.87), OGD group (67.8±24.45), CHI group (92.2±10.26), CH2group (104.2±8.14), IH1group (70.8±43.3), IH2group (90.0±51.87), IH3group (84.4±12.86). Intra-celluar pH level decreased significantly. The pH level appeared recovery trend after both continuous and intermittent hypothermia, but there was no statistically significant improvement.FLUO-3AM fluorescent probe was applied to measure intracellular free calcium. Due to the limited amount of the same batch of fetal rat cortical neurons, we firstly measured the mean fluorescence intensity of the oxygen-glucose deprivated group and the hypothermia groups to ensure the sample homogenicity. The neuronal fluorescence of different batches varied largely, the results were not comparable. So another batch of neuronal supplemental measure was applied to test normal group and OGD group. The data of first batch were as follows:OGD group (1706.8±40.3), CHI group (2586.8±97.91), CH2group (741.4±13.46), IH1group (797.4±12.82), IH2group (403.8±15.61), IH3group (688.2±13.22). The supplemental-measure results were as follows:normal group (58±1.58), OGD group (84.2±1.64). Intraneuronal free calcium level increased after OGD. In continuous hypothermia,6-hours continuous hypothermia had no effect on decreasing cellular free calcium after OGD, while12-hours pattern worked. In intermittent hypothermia, all the intermittent hypothermia could decrease cellular free calcium after OGD. The1.5-hour intermittent hypothermia is the best. Comparison of intermittent and continuous hypothermia,1-hour intermittent hypothermia was less effective than12-hours continuous hypothermia.1.5and 2-hours pattern was prior to12-hours continuous hypothermia.1.5-hour intermittent pattern was the most effective on inhibiting calcium overload after OGD.JC-1fluorescent probe was applied to measure mitochondrial membrane potential changes. The data was as follows:normal group(0.21±0.03), OGD group (1.85±0.16), CHI group (2.23±0.23), CH2group (0.98±0.05), IH1group (1.15±0.14), IH2group (0.61±0.14), IH3group (0.93±0.05). Oxygen-glucose deprivation can lead to the mitochondrial membrane potential depolarization. In continuous hypothermia,6-hours continuous hypothermia was not enough to inhibit mitochondrial membrane potential depolarization, but12-hours pattern worked. In intermittent hypothermia, all the intermittent hypothermia could be helpful, and the effectiveness was same. The intermittent hypothermia was as effective as12-hours continuous pattern.In conclusion, the study found that intermittent hypothermia could inhibit apoptosis of fetal rat cortical neurons after OGD. The neuroprotective mechanisms were as follows:inhibiting ROS and superoxide anion radical generation, alleviating intracellular calcium overload, protecting mitochondrial membrane potential damage. Both continuous hypothermia and intermittent hypothermia had a neuroprotective effect, but intermittent hypothermia was a potentially clinical strategy.