The Effect Of PF On ACC Pyramidal Neurons After Acute Hypoxia In Rats

Hypoxic-Ischemic Encephalopathy(HIE) is a common disease in neonate during the perinatal period,which is mainly caused by asphyxia leading to hypoxic-ischemic brain damage.Its main clinical performances are edema,necrosis and other symptoms in brain tissue.The lives of newborns are threated seriously. HIE have a higher morbidity, which lead to motor dysfunction,mental retardation, cerebral palsy and other neurological sequelae.Therefore,the research of preventive measures and protective treatment on HIE has very important social significance and clinical value.Almost all the energy of the brain is provided by aerobic glucose metabolism.The brain tissue contains very little glycogen,so all the glucose and oxygen are supplied by the brain blood circulation.Newborn cerebral metabolism is very strong.Oxygen consumption of the brain is almost half of the body.The normal metabolism of brain is firstly affected hypoxia. Hypoxia is the main pathological basis of HIE,which leads to neuronal necrosis and brain edema.Anterior Cingulate Cortex (ACC) is the part of archicortex and paleocortex of the cerebral cortex,which has an important role in emotional adjustmen and higher cognition.ACC is considered the neural basis of executive function.Hypoxia-ischemia can lead to brain edema and neuronal death with the condensation and tar stainde of ACC pyramidal nucleus.The results are a series of neurological sequelae,such as language barrier and hemiplegic paralysis.Therefore,the experiment select the study of ACC pyramidal neurons.The Chinese peony belongs to Ranunculaceae with character of bitter and acid and property of slightly cold.Traditional Chinese Medicine on the efficacy of Peony has a clear understanding. Paeoniflorin (PF) is the main chemical constituents of Peony.At present,many literatures have reported that PF have the roles of reducing blood viscosity,dilating blood vessels, improving microcirculation,bringing high blood pressure down,relieving spasm and eliminating pain,anticonvulsant, etc. Some animal studies have also proved that PF have the roles of inhibitting intracellular calcium overload,blocking sodium channel,inhibitting apoptosis, anti-excitatory amino acid toxicity, antioxidation,etcIn summary,we speculate that the children of HIE occur a series of neurological sequelaes,particularly cognitive impairment.These diseases are caused by hypoxia which leads to ACC pyramidal neurons damage.Through recovering the normal function of injured ACC pyramidal neurons,PF will be to the effect of neuroprotection.Therefore,the ACC pyramidal neurons of normal young SD mouses are used as research objects in this experiment. The model of acute hypoxia is built to study acute hypoxia on the brain neuronal injury with 95%N2/5%CO2.By the technology of Infrared visual patch clamp whole-cell,we will record the ACC pyramidal neurons miniature excitatory postsynaptic current (mEPSC) in acute hypoxia,and the impact of PF on ACC pyramidal neurons mEPSC after acute hypoxia.This experiment focuses on the impact of PF on ACC pyramidal neurons mEPSC after acute hypoxic brain injured to explore neuroprotective mechanism of PF.1 Main Content1.1 ExperimentⅠ:R ecord the electrophysiological properties of ACC pyramidal neurons with the technology of Infrared visual patch clamp whole-cellBy the technology of Infrared visual patch clamp whole-cell,we observed and analysed the morphology and electrophysiological properties of ACC pyramidal neurons determined the recorded neurons in the normal young SD mouses.1.2 ExperimentⅡ:The affect of acute hypoxia on mEPSC of isolated ACC pyramidal neurons in young ratsACC pyramidal neurons mEPSC is recorded by whole-cell patch clamp technique in the normal and acute hypoxia.The affect of acute hypoxia on the ACC pyramidal neurons are observed.1.3 ExperimentⅢ:The affect of PF on the ACC pyramidal neuron after acute hypoxiaUsing whole-cell patch clamp technique,we record changes of the ACC pyramidal neuron mEPSC by perfusion with PF(300μmol/L)in ACSF after acute hypoxia 5～6min.we observe the affect of PF on the ACC pyramidal neuron mEPSC after acute hypoxia and explore the neuroprotective effect of PF. 2 Main Results2.1 We can clearly observed the morphology of pyramidal neurons with the technology of Infrared visual patch clamp whole-cell.ACC pyramidal neurons line up in order.Cell body is pyramid and triangle.Its boundary is clear.According to the different characteristics of recorded Action potential,ACC pyramidal neurons can be divided into three categories.The first category is RS (regular spiking cell) neurons(54.1%).Its features is a single action potential following a marked afterhyperpolarization. The second category is IB (intrinsic bursting cell) neurons(29.7%).It features is a single action potential following a afterhyperpolarization and a large afterdepolarization,and produce a burst of action potentials.The third category is IM (intermediate cell) neurons(16.2%).Its features is a single action potential following a afterhyperpolarization and a small afterdepolarization(less than 2mV).2.2 The numbers and amplitudes of ACC pyramidal neuron mEPSC are increased after acute hypoxia compared with the normal state.The numbers of mEPSC increase from 33.14±0.46 to 38.52±0.44(n=10,P<0.01).The frequences of mEPSC increase from (3.31±0.06)Hz to (3.85±0.06)Hz (n=10,P<0.01),and show an increasing trend.2.3 PF(300μmol/L) is applied after acute hypoxia 5～6min, The numbers and amplitudes of ACC pyramidal neuron mEPSC are reduced compared with acute hypoxia. The numbers of mEPSC increase from 38.52±0.44 to 30.66±0.49(n=10,P<0.05).The frequences of mEPSC increase from (3.85±0.06)Hz to (3.07±0.06)Hz(n=10,P<0.05),and show an decreasing trend. 3 Main conclusions3.1 According to different firing patterns,ACC pyramidal neurons are divided into three types of neurons.That showed the diversity of the ACC pyramidal neurons.3.2 The frequences of the ACC pyramidal neurons mEPSC are recorded,which showed that acute hypoxia can injury the ACC pyramidal neurons.3.3 In order to neuroprotection,PF may inhibite the frequences of the ACC pyramidal neurons after acute hypoxia,and regulate the plasticity of synaptic activity.