Experimental Study Of PTD-BDNF Effect On Electric Activity Of Hippocampal Neurons And Therapy For Cerebral Ischemia Injury In Rats

Part 1 Effects of PTD-BDNF on Excitatory Synaptic Activity ofHippocampal NeuronsObjective: To study the effects of the brain-blood barrier permeable PTD-BDNF on the spontaneous neural discharge of the neurons in rat hippocampus.Method: Hippocampal neurons obtained sterility from the new-born Wistar rats were transplanted at a density of 5～7×10⁴·ml^-1 for primary cultured . 8～9 days late, the cells were treated by different drags. The cells were grouped into the control group that there was no drug in the culture medium, the PTD-BDNF group and the BDNF group with the ultima concentration 1nmol·L^-1 PTD-BDNF and BDNF respectively. 24 hours after the cells treated by drugs, the whole cell patch clamp recording technique was used to observe the impact of PTD-BDNF and BDNF on the spontaneous synaptic activity of the cultured rat hippocampal neurons. Holding potential was -70mV, the spontaneous excitatory postsynaptic current was recorded. After adding 1μmol·L^-1 TTX, holding potential was -70mV, the miniature excitatory postsynaptic current was recorded.Results: Compared with the control group, after treatment of hippocampal neurons with PTD-BDNF or BDNF, the frequency of the spontaneous excitatory postsynaptic current (sEPSC) and the miniature excitatory postsynaptic current (mEPSC) were significantly increased [ the frequency of sEPSC: (1.4±0.5) vs (4.0±0.6), (3.9±0.5) Hz, P<0.01; the frequency of mEPSC: (4.2±1.0) vs (7.9±1.9),(7.6±1.8) Hz, P<0.01]. However, in spite of increment of the amplitude of sEPSC, the amplitude of mEPSC remained unchanged [ the amplitude of sEPSC: (415.7±48.0) vs (743.7±95.7), (693.7±99.9) pA, P<0.01; the amplitude of mEPSC: (34.7±4.7) vs (36.3±6.2), (36.2±4.4) Hz, P>0.05]. The effect of PTD-BDNF was not significantly different from BDNF.Conclusion: PTD-BDNF can enhance the excitatory spontaneous synaptic activity of the rat hippocampal neurons. It implies that PTD-BDNF and BDNF shares the similar electrophysiological effects.Part 2 Effects of PTD-BDNF on Na⁺, Ca²⁺, K⁺ channel incultured rat Hippocampal NeuronsObjective: To study the effects of PTD-BDNF fusion protein on Na⁺, Ca²⁺, K⁺ channels in cultured primary rat hippocampus neurons.Method: The method of cells culture was the same as described before. 8～9 days late, the cells were grouped into the control group that there was no drug in the culture medium, the PTD-BDNF group with the ultima concentration 1nmol·ml^-1 PTD-BDNF. 2～3 days after the cells treated by drugs, using whole-cell patch-clamp techniques in cultured hippocampal neurons record the voltage dependent ion channels currents of sodium, potassium and calcium. Holding potential was -100mV, and sodium currents were elicited by voltage-step at 10mV increment from -60mV to +10mV, and the duration was 400ms. Holding potential was -80mV, and calcium currents were elicited by voltage-step at 10mV increment from -50mV to +20mV, and the duration was 150ms. Holding potential was -100mV, and potassium currents were elicited by voltage-step at 10mV increment from -50mV to +20mV, and the duration was 400ms. Observe the impact of PTD-BDNF fusion protein on the ion channels currents and explore the changes of characteristics of the ion channels.Results: Compared with the control group, PTD-BDNF and BDNF increased the peak amplitude of Na⁺current 39.35% and 41.99% respectively (n=4, P<0.01). Compared with the control group, PTD-BDNF and BDNF increased the peak amplitude of Ca²⁺current 39.20% and 38.72% respectively (n=4, P<0.01). It implies that PTD-BDNF fusion protein significantly increased the Na⁺ inward Na⁺, Ca²⁺ currents. But compared with the control group, at the stimulant voltage of 30mV, PTD-BDNF and BDNF decreased the amplitude of I_A,I_K 29.30%,28.06% and 29.76%,33.38% respectively (n=4, P<0.01). It implies that PTD-BDNF fusion protein significantly decreased the outward K⁺ currents.Conclusion: PTD-BDNF fusion protein and BDNF shares the similar modulation effects on the voltage dependent ion channels. It implies that PTD-BDNF and BDNF shares the similar molecular basis on the resting membrane potential of neurons, nervous excitation.Part 3 Effects of PTD-BDNF on Cerebral Ischemia Injury inRatsObjective: To evaluate the beneficial effects of PTD-BDNF fusion protein in vitro for ischemia reperfusion injury in rats.Method: Twenty 200～300g SD rats were grouped into the PTD-BDNF groups and the normal saline control groups. A model of middle cerebral artery occlusion (MCAO) in rats was established with intraluminal filament occlusion. The filament 0.26mm in diameter was inserted 18mm into arteria carotis interna. PTD-BDNF groups were intravenously injected PTD-BDNF fusion protein 0.5μmol·kg^-1 immediately after operation (inject once every day after this) and the control groups were injected same volume of normal saline at the same time. 48h late, executing four rats every group, brains were taken to TTC stain. 7d late, executing remnant rats, brains were taken to HE stain and TUNEL stain. To analyze neuroprotective effect of PTD-BDNF fusion protein.Results: Compared with the control group, PTD-BDNF decreased the percentage of infarct volume from 18.20% to 10.12% (n=4,P<0.01), and decreased the TUNEL positive rate from 51.24% to 23.54% (n=6,P<0.01). It implies that PTD- BDNF fusion protein significantly decreased the infarction volume, decreased the apoptosis ratio and enhanced neurons survival in focal cerebral injury rats induced by ischemia-reperfusion.Conclusion: Intravenous PTD-BDNF fusion protein can protect neurons from injury across blood brain barrier, reduce ischemical reperfusion injury and improve function of cranial nerve.