| Objective(1) To examine the protective effect of exogenous vascular endothelial growth factor (VEGF) on the cognitive impairment induced by chronic brain ischemia and its possible electrophysiological and histological mechanisms.(2) Using in vitro hippocampal brain slice, to highlight the neuroprotective effect of VEGF on oxygen and glucose deprivated hippocampal neurons and its possible cellular and electrophysiological mechanism.Methods(1) In vivo:30male Wistar rats were randomly divided into3groups:Sham operation group (Sham), Ischemic group (Ischemia) and VEGF treated group (Ischemia+VEGF). A global model of cerebral ischemia was established by two vessel occlusion (2-VO), The performances of learning and memory were examined by the Morris water maze (MWM). The morphology of neurons in hippocampus was determined by hematoxylin and eosin (HE) staining, and VEGF content in hippocampus was evaluated by ELISA. The LTP from hippocampus CA3region to CA1region was recorded. Before the LTP induction, the local field potentials (LFPs) were collected.(2) In vitro:An oxygen and glucose deprivated model was established. There were4groups:Control group (Control), Oxygen and glucose deprivated group (OGD), VEGF treated group (OGD+VEGF) and VEGF and receptor antagonist group (OGD+VEGF+SU5416). In the neuropathological and viability study, PI staining was applied to observe neuronal death in hippocampal regions. In the neuro-electrophysiological study, membrane potential, spike frequency and sEPCSs were recorded10min before and after the ODG treatment.Results(1) In the Morris water maze test (MWM) study, it was found that, the escape latencies were prolonged significantly in the ischemia group compared with those in the sham group (Ischemia:13.5±1.9s vs. Sham:8.1±1.2s, P<0.05) from day3. The escape latencies were shortened significantly in the VEGF group compared with those in the ischemia group (Ischemia+VEGF,8.5±1.1s, P<0.05vs. Ischemia). On day6, animals in the ischemia group spent less time in the target quadrant (Ischemia:34.2±1.6%vs. Sham:42.9±1.5%, P<0.05) compared to that in the sham group, while animals in the VEGF group spent nearly the same time in the target quadrant (Ischemia+VEGF:40.1±2.6%) compared to that in the sham group.(2) In LTP recording, the normalized slope of the fEPSP was significantly reduced by chronic ischemia (Ischemia:117.8±6.23%vs. Sham:136.2±5.96%, P<0.01). But in the VEGF treated group the fEPSP slope was significantly reversed to the sham levels (130.6±5.36%vs. Ischemia:117.8±6.23%,P<0.01).(3) In field potential recording we found that cerebral ischemia could induce decreased phase synchronization between hippocampal CA3and CA1area and reduce the strength of CA3theta phase modulating CA1low gamma (LG,30-60Hz) amplitude. While VEGF could recovery the reduced coupling.(4) In HE staining, pyramid neurons in hippocampal CA1region appeared to be normal in the sham group. While neurons in the Ischemia group was arranged loosely, and edema and karyopyknosis were also observed. In the VEGF group, the neurons were recovered to the normal morphology to some extent.(5) In ELISA study, the VEGF content in the ischemia group was significantly lower than that in the sham group (Sham:745.33±42.34pg/g vs. Ischemia:420.09±40.82pg/g, P<0.01), while in the VEGF group it was significantly higher than that of ischemia group, though still lower than that of the sham group (592.83±38.76pg/g vs. Ischemia, P<0.05).(6) In the in vitro study, we found that20minutes after OGD treatment, the neuronal death rate of the hippocampal CA1region was significantly increased (Control vs. OGD, P<0.05), while in VEGF treated group it was significantly decreased to the similar level of the Control group, and VEGFR-2antagonist SU5416could eliminate the effect of VEGF.(7) In our electrophysiological experiment, we found that the time window of the reversibly changed electrophysiological character after OGD treatment in the hippocampal neurons was around10minute, during which time most of the membrane potential of the neurons could return to normal after washout.(8) In the electrophysiological experiment, we found that the membrane potential of the hippocampal neurons was depolarized14.7±1.23mV(n=5) mV after10min OGD treatment, while with VEGF treatment,the membrane potential was depolarized6.1±1.57mV (OGD vs. OGD+VEGF, P<0.01). In OGD+VEGF+SU5416group, the membrane potential was depolarized12.1±1.92mV (n=6). Using the current-clamp, we found that OGD+VEGF treatment increased the spike frequency (510±60%), and the increasement was far less than that of the OGD treatment (920±56%, P<0.01, n=5), In OGD+VEGF+SU5416group, the increasement of freqency was810±47%, which is far larger than that of the VEGF treatment (P<0.01, n=5), but was not significantly different compared to that of the OGD treatment.(9) In our electrophysiological experiment, we found that after OGD treatment, the frequency and amplitude of the sEPSCs were increased in a time-dependent manner. That increasement was attenuated by VEGF treatment and was not effected by VEGF+SU5416, suggesting that VEGF play its role through activating the VEGFR-2receptor.Conclusions(1) Exogenous VEGF could improve impairment of learning and memory in2-VO rats. The underlying mechanism was associated with the improvement of the edema around pyramidal neurons and the nuclear shrink induced by ischemia.(2) Intranasal delivery was proved to be an effective way to administrate exogenous VEGF. It can effectively increase the VEGF content in the hippocampus of ischemic brain of rats.(3) VEGF could improve the decreasement of synaptic plasticity caused by ischemia in the hippocampus. Its mechanism could be related to that VEGF improved the coupling mode of theta and gamma rhythm between CA3and CA1(4) By alleviating the neuronal apoptosis caused by oxygen-glucose deprivation, exogenous VEGF could protect the hippocampal neurons through activating VEGFR-2receptor in the in vitro brain slice ischemic model.(5) VEGF could attenuate the increasement of neuronal excitability caused by ischemia and maintained the normal neuron function, which could be the possible electrophysiological mechanism for the neuroprotective effects of VEGF in the early stage of the ischemia.(6) VEGF could attenuate the abnormal increasement of neuronal synaptic transmission caused by ischemia and maintained the normal neuronal synaptic function, which could be the possible mechanism for the neuroprotective effects of VEGF on the synaptic plasticity. |
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