The Role Of EphA4-ephrinA3 System In Dylayed Neuronal Death Of Rat Hippocampal CA1 Area Induced By Transient Forebrain Ischemia-reperfusion

Background and purpose:Stroke has become the third largest cause of death and first cause of disability. According to statistics, the incidence of stroke as high as 120/10 million,and disability up to 75%. Among them ischemic stroke (cerebral infarction) in total is 60% to 80%. Transient ischemic attack is cerebral ischemic cause by bloke of one or more blood vessels, which causes focal brain dysfunction, focal neurological signs and symptoms continue from a few minutes to several hours, and may relieve without treatment. But 1/3 of the TIA patients will develop ischemic stroke, while the onset of stroke often relapse. Prolonged cerebral ischemia will lead to broad, non-selective neuronal necrosis. And transient ischemic attacks, although the blood supply has resumed, but in humans and some animal models can lead to selective neuronal death, mainly affects pyramidal neurons in hippocampal CA1 region. This kind of death can occur even 28 days after reperfusion, called delayed neuronal death (Delayed neuronal death, DND). The degree of disability is closely related with the DND. But the occurrence and development of DND is a complex process involving a variety of pathological mechanisms, which mechanisms are not yet fully elucidated, and thus lack of a better therapy strategy.The mechanism of DND has not yet been fully understood, but the study found that apoptosis plays an important role in this processing. Pulsinelli established rat transient forebrain ischemia/reperfusion model, it became a commonly used model to study the post-ischemic brain injury and secondary neuronal death. The vertebral artery were permanently occluded by surgery, the next day carotid artery was closed by clips for a short time (15 minutes) and them released to reperfusion, because the blood supply through the Willis ring is retained, no significant effect on the brain stem. In this model, specific pathological changes occured after reperfusion in hippocampus: delayed neuronal death in CA1 pyramidal cells (ie, only pyramidal cells death in CA1 area can be seen under the light microscope 2 to 3 days after reperfusion), while the pyramidal cells in CA3 region and granule cells in dentate gyrus are almost intact. Many studies have found that in experimental cerebral ischemia, the neuronal apoptosis plays an important role in the hippocampal delayed neuronal death.Eph-ephrin system is a member of receptor tyrosine kinase family. Eph receptors include total of 16 members, divided into two subtribes: EphA and EphB, the corresponding ephrin ligands are divided into ephrinA (A1 ~ A5) and ephrinB (B1 ~ B3). ephrinA has a conserved extracellular binding region ,and anchored to the membrane by glycosyl phosphatidyl inositol (GPI). Eph receptors contain the extracellular ligand binding domain, cysteine-rich region and two fibronectin typeⅢarea, including the intracellular tyrosine kinase domain, SAM, and PDZ binding. EphA receptor priority binding ephrinA, EphB priority with ephrinB, but EphA4 and ephrinB2 may also be combined with the ephrinB1. Eph-ephrin signaling complex depend on oligomerization, free monomer does not have a physiological role. In neural development and synaptic plasticity this system plays an important role. Recent studies have found that they may involved in ischemic injury. Study found that ephrinA3 is the most abundant Eph ligands in adult hippocampus, mainly located in the GFAP-positive astrocytes, EphA4 distributed in the hippocampus, especially in pyramidal neurons of the CA1 region. In pathological conditions, CA1, CA3 area and DG of the pyramidal cell layer showed a strong ischemia-induced expression of ephrinA3. In the neonatal rats, ischemia and hypoxia decreased EphA4, but other study also found that chronic cerebral ischemia induced EphA4 increases, but what is the role of EphA4-ephrinA3 in these processes, whether they involved in neuronal apoptosis and what are its signaling pathways and not clear.Signaling involved in the activation of Eph receptor including MAPK, PI3 kinase and other downstream signaling pathways. Among them, the large number of studies focused on that MAPKs, which including ERK1/2, JNK and p38 MAPK, these molecules are widely involved in the regulation of neuronal survival and apoptosis, including ischemia / reperfusion injury. So if MAPKs involved in the ischemia/reperfusion induced activation of EphA4-ephrinA3 and modulates neuronal apoptosis is also not clear.Variety of factors involved in the regulation of cell proliferation and apoptosis. Cell proliferation, apoptosis, and some basic physiological processes are accompanied by changes in cell volume. Ion channels, such as K + channels and Cl-channel, are involved in volume regulation of cell volume. Recent studies show that the volume regulation of Cl-channels play an important role in cell volume, membrane potential, and other mechanisms involved in the regulation of apoptosis. But the mechanism of regulation of chloride channel is not fully understood, studies suggest protein tyrosine phosphorylation signal involved in the volume regulation of chloride channels, and in the application of chloride channel blockers or tyrosine kinase inhibitors can significantly reduce the transient cerebral ischemia / reperfusion induced apoptosis of hippocampal neurons. EphA4-ephrinA3 belongs to receptor tyrosine kinase family and whether they involved in the regulation of neuronal apoptosis by chloride channels?In summary, by establishment of transient forebrain ischemia / reperfusion model, we performed the following studies: 1. Observe the expression of EphA4-ephrinA3 in ischemia / reperfusion injury, and cell apoptosis related assays were performed simultaneous; 2. The effects of EphA4-ephrinA3 in ischemia/reperfusion induced neuronal apoptosis; 3. whether EphA4-ephrinA3 involved in the ischemia / reperfusion induced apoptosis in hippocampal CA1 neuronal by the regulation of chloride channels. 4. By observing the alterations of MAPKs expression, to investigate if MAPKs involved in EphA4-ephrinA3 signal pathway after reperfusion injury.PartⅠIschemia/reperfusion induced delayed neuronal death and expression of EphA4 and ephrinA3 in rat hippocampusObjectiveTo investigate the role of EphA4 and ephrinA3 in ischemia/reperfusion induced apoptosis of hippocampus CA1 neurons, rat transient cerebral ischemia/reperfusion model was established, intraventricular administration of ephrinA3-Fc, the EphA4 receptor agonist, or EphA4-Fc, the antagonist of EphA4 receptor, was performed respectively. The neuronal damage of hippocampus CA1 area and the change of EphA4-ephrinA3 expression were observed.Methods1. The adult male Wistar rats were divided into the following groups at random:control group (n=6), normal healthy rats;Vertebral artery occlusion group (n=6), only occlusion of vertebral artery was performed; Sham group (at each point, n=6), bilateral vertebral arteries occlusion was done first, the neck incision was opened the next day, but carotid occlusion was not performed;Ischemia / reperfusion group (at each point, n=6), bilateral vertebral arteries occlusion was done first, at the following day after 15 minutes of bilateral carotid artery occlusion by clips, them clips were released to achieve reperfusion, detection were performed 6, 24,48 hours after reperfusion;Vehicle + ischemia / reperfusion group (at each point, n=6), intraventricular injection of sterile PBS 10μl, detection were performed 6, 24, 48 hours after reperfusion;ephrinA3-Fc + ischemia / reperfusion group (at each point, n=6), intraventricular injection of ephrinA3-Fc, detection were performed 6, 24, 48 hours after reperfusion;EphA4-Fc + ischemia / reperfusion group (at each point, n=6), intraventricular injection of e EphA4-Fc, detection were performed 6, 24, 48 hours after reperfusion;2. The transient cerebral ischemic/reperfusion were made by 4-VO model. Soluble fusion protein ephrinA3-Fc was given intraventricular to active EphA4 receptor, or EphA4-Fc was given intraventricular to block EphA4 receptor. Corresponding drug administration was performed before vertebral artery occlusion. The hippocampi were removed. Hippocampus neurons damage was observed by Cresyl violet staining; neuronal apoptosis was determined by in situ TdT mediated dUTP nick end labeling (TUNEL) and measurement of Caspase 3 activity in hippocampus; ephrinA3 EphA4 expression changes were measured by Western blot.Results1. A transient cerebral ischemia / reperfusion can lead to hippocampal area CA1 delayed neuronal death.At different time points after ischemia, progressive loss of hippocampal CA1 neurons was observed by Nissl staining. 7 days after reperfusion, neuron soma in CA1 area almost disappeared. This means most of the ca1 pyramidal cells were dead. Caspase 3 activity increased to peak 6 hours after reperfusionthen decreased, but after 48 hours reperfusion it did not recover to normal levels (OD: 2.3±0.19,2.2±0.28,1.9±0. 15, vs sham: 1.1±0.017, P<0.05). The results showed that transient cerebral ischemia/reperfusion can lead to Caspase 3 activity increased significantly in hippocampal CA1 area; TUNEL detection also indicate progressive neuronal apoptosis.2. Transient cerebral ischemia / reperfusion in rat hippocampus ephrinA3 and EphA4 expression were significantly increasedWestern-blot assay by the hippocampus ephrinA3 and EphA4 expression, the results show that compared with normal control group, 6 hours after reperfusion in hippocampal CA1 area ephrinA3 expression was significantly higher (IOD: 2.05±0.12 vs 0.78±0.02, P<0.05), 6 hours after ischemia the expression a peak, then decreased gradually, Until 48 hours after there is not significant defference compared with controls. EphA4 expression in CA1 area has increased 6 hours after reperfusion (2.21±0.12 vs 0.72±0.03, P<0.05), and then decreased gradually.3. Pretreatment by ephrinA3-Fc before ischemia may induce increasing of Caspase 3 activity and it is significantly higher than ischemia/reperfusion group without ephrinA3-Fc treatment. Neuronal apoptosis have also increased, suggesting the occurrence of apoptosis after reperfusion may be enhanced by activation of EphA4.4. After intraventricular injection of EphA4-Fc, the blocker of EphA4, Caspase 3 activity decreased, and neuronal apoptosis are significantly reduced, suggesting that activation of EphA4 is involved in neuronal apoptosis after reperfusion. Summary1. Transient ischemic / reperfusion lead to delayed neuronal death of hippocampal CA1 neurons. 2. Caspase 3 activity significantly increased 6 hours after reperfusion, then decreased, but not back to normal level until 48h reperfusion which indicate that caspase dependent apoptosis may be an important factor of delayed neuronal deaths.3. Transient cerebral ischemia / reperfusion induced ephrinA3 and EphA4 significant upregulation in hippocampal CA1, the time course consistent with the increasing of Caspase, TUNEL detection also confirmed the results. It suggesting that increased EphA4 and ephrinA3 expression in CA1 neurons may play important role in reperfusion injury.4. EphA4 and ephrinA3 involved in the reperfusion induced neuronal apoptosis, activation of EphA4 can lead to increased neuronal apoptosis, and blocking of EphA4 partially inhibited reperfusion induced neuronal apoptosis.PartⅡThe effect of EphA4-ephrinA3 system to outwardly rectifying chloride channel in hippocampal CA1 neuronsObjectiveStudies showed that in the central nervous system, the chloride channel activity and neuronal damage, apoptosis are closely related. after ischemic injury outwardly rectifying chloride channel (ORCC) of hippocampal CA1 pyramidal neurons enhanced, caused neuronal apoptosis possibly by changes of cell volume. The application of chloride channel blockers can inhibit brain ischemia induced delayed neuronal death. Studies have shown that of ORCC can be activated by tyrosine kinase receptors. EphA4 is one of tyrosine kinase receptor abundant in the hippocampus, Present study performed on hippocampal slices obstained from ischemia / reperfusion model and normal rat. The ORCC of pyramidal neurons was recorded by whole cell patch clamp, treated with EphA4 receptor blocker EphA4-Fc and EphA4 receptor agonist ephrinA3-Fc, we attempted to confirmed the effects of EphA4-ephrinA3 system to the chloride channel in the ischemic / reperfusion injury.Methods25-21 day postnata healthy male Wistar rats were choosed. Weight: 180 ~ 250g. randomly grouped as follows:Pseudo-operation group, n=6;Ischemia / reperfusion 6 hours group, n=6, the recording was performed 6 houes after reperfusion, without other treatment;Ischemia / reperfusion 24 hours group, n=6, the recording was performed 24 houes after reperfusion, without other treatment; EphA4-Fc + sham group6 hours, n=6, the recording was performed 6 hours after operation, the slice was incubate in 10μg/ml EphA4-Fc for 10 minutes;EphA4-Fc group + ischemia / reperfusion 6 hours group, n=6. he recording was performed 6 hours after reperfusion, the slice was incubate in 10μg/ml EphA4-Fc for 10 minutes;ephrinA3-Fc + sham group 6 hours + sham group, n=6, the recording was performed 6 hours after operation, the slice was incubate in 10μg/ml ephrinA3-Fc for 10 minutes;ephrinA3-Fc + ischemia / reperfusion 6 hours group, n=6, the recording was performed 6 hours after reperfusion, the slice was incubate in 10μg/ml ephrinA3-Fc for 10 minutes.Results1. Transient cerebral ischemia may induce enhance of outwardly rectifying chloride channels in rat hippocampal CA1 pyramidal neurons.Transient cerebral ischemia induces outwardly rectifying chloride currents in rat hippocampus CA1 pyramidal neurons increasing. Whole-cell current 6 hours and 24 hours after ischemia/reperfusion were increased from the sham 9.11±1.2pA/pF (-80mV) and 64.2±6.9pA/pF (80mV) to: 15.80±1.70, 17.70±1.93 pA / pF ( -80mV) and 120.16±12.3,129.82±11.830 pA / pF (+80 mV) (n = 15, p <0.05), compared with the control gro the increasing was significantly.2. Effects of ephrinA3-Fc and EphA4-Fc on ORCC of hippocampal CA1 pyramidal neurons10 minutes pre-incubated with ephrinA3-Fc enhance the increased ORCC current induced by ischemic/reperfusion, respectively, 16.980±1.9pA/pF (- 80mV) and 132.35±10.33 (80mV).10 minutes pre-incubated with EphA4--Fc decrease the ORCC enhanced by ischemic/reperfusion 13.30±1.3pA/pF (-80mV) and 113.35±7.33 (80mV), the difference was significant compared with ischemia / reperfusion group.ephrinA3-Fc pre-incubated to brain slice from sham may slightly increase chloride current, but compared with normal controls, the difference was not significant. EphA4-Fc pre-incubated to brain slice from sham were not affect the chloride currentsSummary1. Cerebral ischemia/reperfusion induced outwardly rectifying chloride channel activity enhancement in rat hippocampal CA1 pyramidal neurons.2. Given EphA4 receptor agonists and antagonists can affect the ischemia / reperfusion induced increasing of chloride current, given EphA4-Fc can inhibit the ischemia/reperfusion induced increasing of outwardly rectifying chloride currents, and ephrinA3-Fc can increase the outward rectifying chloride currents, suggesting EphA4-ephrinA3 system may involve ischemia / reperfusion injury through regulation of chloride channel.PartⅢThe downstream signaling pathway of EphA4-ephrinA3 system involved in the ischemia/reperfusion induced neuronal apoptosis–investigate of MAPKs pathwayObjectiveThe signaling way involved in ephrin activate Eph receptor including Rho GTPases, MAPK, PI3 kinase and other downstream signaling pathways. MAPKs, including ERK1 / 2, JNK and p38 MAPK, involved in a variety of pathological conditions and regulate neuronal survival and apoptosis after injur.Therefore, by establishment of transient forebrain ischemia / reperfusion model and administrate of EphA4-Fc or ephrinA3-Fc, we proposed to observed the expression alteration of MAPKs signaling pathways in P38, ERK1 / 2 and JNK, to analysis is MAPKs signal pathway is involved in the signal transduction of EphA4-ephrinA3 system.Methods1.Adult male Wistar rats were selected, grouped seemas partⅡ2.Cerebral transient ischemic/reperfusion model were established, soluble fusion protein ephrinA3-Fc was given intraventricular to active EphA4 receptor, or EphA4-Fc was given intraventricular to block EphA4 receptor. At different time points after reperfusion, the hippocampi were removed. Then P38, ERK1/2 and JNK expressions were determined and analyzed by western blot.Results1 ischemia / reperfusion can increase p-P38 expression in the CA1 area. Treatment with EphrinA3-Fc increase ischemia / reperfusion-induced p-P38 expression, and EphA4-Fc does not affect expression of p-P38 after reperfusion.2 ischemia / reperfusion induced ERK1 / 2 activation in hippocampal CA1 area, but given EphA4-Fc and lateral ephrinA3-Fc did not affect this ischemia / reperfusion induced upregulation..3 ischemia / reperfusion induced p-JNK activation in hippocampal CA1 area. It was significantly increased 6 hours after reperfusion and then decreased gradually, returned to normal levels at 48 hours after reperfusionr, and treatment with EphA4-Fc or ephrinA3-Fc did not affect this ischemia / reperfusion-induced increasing. SummaryEphrinA3-EphA4 system is involved in ischemia / reperfusion injury and associated with MAPKs, especially p-P38 activation, activation of the receptor can be lead to activation of p-P38 was significantly increased, but blcoked EphA4 by EphA4-Fc did not inhibited the activation of p-P38 significantly, it is also need further study.Conclusions:1. Transient ischemic / reperfusion can lead to delayed death of hippocampal CA1 neurons. In this process, ephrinA3 and EphA4 expression in the hippocampal CA1 area was significantly increased, the time course is consistent with the increasing of Caspase and TUNEL, suggesting that ephrinA3 with increased expression of EphA4 in the CA1 area may be play an important role in neuronal ischemia/reperfusion injury.2. EphA4 and ephrinA3 involved in reperfusion induced neuronal apoptosis, activation of EphA4 can lead to increased neuronal apoptosis, and EphA4 blocking may partially inhibited the reperfusion induced neuronal apoptosis.3 Cerebral ischemia / reperfusion in rat hippocampus CA1 pyramidal neurons may lead to enhancement of outwardly rectifying chloride channel activity. Given EphA4 receptor agonists and antagonists can affect the ischemia / reperfusion enhanced chloride channel currents, ephrinA3-Fc activation of EphA4 receptor can increase neurons outward rectifying chloride currents after ischemia and reperfusion. Giving EphA4-Fc block EphA4 receptor can inhibit the ischemia/ reperfusion induced increasing of outwardly rectifying chloride currents, suggesting EphA4-ephrinA3 system may be affecte neuronal apoptosis after ischemia / reperfusion by regulation of chloride channel.4. EphrinA3-EphA4 system may be involved in ischemia / reperfusion in hippocampal CA1 neurons through the P38 pathway, activation of the receptor can lead to significant increase in p-P38, but EphA4-Fc block the receptor did not alter the expression of p-P38, it is needs further study.