Response Characteristics Of PC12 Cell Cluster And Rat Hippocampal Slice To Electro-Stimulate Under The Effect Of Lidocaine Hydrochloride

It is one of the most difficult challenges to reveal the mechanisms of brain. Neuron is a basic unit of brain, between which the complex and orderly neuronal networks were formed through synapses. The information perception, and coding and transmission mechanism of neuronal networks are the foundation of the higher level brain activities. Therefore, the study of neural signaling pathways and intercellular communication via synaptic transmissions is an effective way to understand the mechanisms of brain functions. Previously, our group proposed a voltage threshold measurement method (VTMM) to quickly and quantitatively evaluate the electrical excitability of neuronal networks influenced by external factors such as temperature, ethanol, and acetylcholine, which have indirect impact on neuronal networks. In this thesis, MEA technology was used to study the response characteristics of quasi-neuron network (PC 12 cell network, PCN) and neural tissue (rat acute hippocampal slice, RAHS) to electrical stimulation while under the influence of lidocaine hydrochloride (LH). The effect of LH concentration on electrical excitability of PCN and RAHS was analyzed via threshold-concentration (VTh-CLH) curve. In addition, high-content cellomics experiments were performed on PCN in solutions containing various concentrations of LH, which further validated the generality of VTMM's application.PC 12 cells were cultured and quasi-neuron network were developed on MEA. Then electrical stimulation and response detection experiments were performed acquire voltage thresholds under various concentrations of LH ranging from 0 to 5?g/mL. We analyzed the electrical excitability of PCN affected by different concentrations of LH quantitatively according to the VTh-CLH curve. The result revealed a concentration dependence of the electrical excitability of PCN. LH inhibited the electrical excitability of PCN in the range from 0.1 to 1.2?g/mL, indicating an anesthetizing effect. The anesthetizing effect first exesited positive correlation with the concentration of LH, and reaching a maximum impact at 0.5?g/mL; then the anesthetizing effect exesited negative correlation with increasing concentration of LH until 1.2?g/mL. But beyond 1.2?g/mL, LH enhanced the electrical excitability of PCN, demonstrating a facilitative effect. The facilitation showed positive correlation with the concentration of LH in the range from 1.2 to 1.5?g/mL. Above 1.5?g/mL, the electrical excitability of PCN plateaued at the most excited state, responding to even the lowest stimulation amplitude by the system (1mV). The results agrees with the past literature.Hippocampal slices of 12-14 days SD rats were prepared, and voltage thresholds were acquired in the LH solution with concentrations ranging from 0 to 5?g/mL. The electrical excitability of RAHS was quantified against the LH concentration with the VTh-CLH curve. The electrical excitability of RAHS showed concentration dependence which was similar to those of PCN and the literature. LH inhibited the electrical excitability of RAHS in range from 0.1 to 1.2?g/mL, indicating an anesthetizing effect. The anesthetizing effect on RAHS exesited positive correlation with the concentration of LH, and reached a maximum at 0.6?g/mL; then the anesthetizing effect exesited negative correlation for increasing concentration of LH. Beyond 1.3?g/mL, LH enhanced the electrical excitability of RAHS, and the facilitation showed positive correlation with the concentration of LH between 1.3 to 1.5?g/mL. Above 1.5?g/mL, the electrical excitability of RAHS plateaued at the most excited state.Next, high-content cellomics analysis system was used to study the acute effect in LH on PCN. We used propidium iodide (PI) dye to mark the dead cells after a 15-miniute acute application of 0?1.7?g/mL LH. The death rate of PC12 cells was found to be near zero, indicating that PC 12 cells do not necrotize after acute exposure to LH. Then, TRITIC-tagged phalloidine was used for PC 12 cells cytoskeleton staining to analyze the length and area of neurite. The result showed a distinct difference between the experimental groups and the control group. The neurite length and area of PC12 cells presented LH-concentration dependence, similar to the relationship between voltage threshold and LH concentration. Furthermore, we found that the change in excitability of PC12 cells might be related to the changes of neurite length, which affect the distribution of sodium channel regulating neuronal excitability. Therefore, changes in the sodium channel zone could be the primary cause of the acute effects of LH on the excitability of PC12 cells.