The Ability Of Isoflurane-induced Burst Suppression To Detect Local Cortical Hyperexcitability Induced By Bicuculline Methiodide

Objective:Epilepsy is one of the most common brain diseases.Secondary epilepsy occupies a large proportion of adult epilepsy.Previous animal studies have recommended some candidate electrophysiological biomarkers to predict the occurrence of epileptic seizures,but they have not been effectively translated into clinical practice.Epilepsy is generally considered as a disease of excitatory-inhibitory imbalance in the neuronal network,which leads to hyperexcitation.In the early stage of epilepsy,the minor change in excitatory-inhibitory balance is usually concealed by the ongoing neural activity and is difficult to detect.Therefore,it is crucial to find a biomarker that is not affected by the background "noise" and can detect excitatory-inhibitory imbalances in the early stage of epileptogenesis.Calcium signals can reflect the cortical neural electrical activity.Compared with local field potential(LFP)recording of neural electrical activity,wide-field calcium imaging has a higher spatial resolution and spatial coverage.In this study,we use widefield calcium imaging to record the spatiotemporal dynamic of burst suppression(BS).The initiation sites,waveform,and frequency changes were analyzed to evaluate if isoflurane-induced BS can be used to detect the local cortical overexcitation in the brain.Methods:The healthy male SD rats weighing 250?350 g were divided into 4 groups,and a convection-enhanced delivery technique(CED)was used to inject the calcium dye Oregon Green 488 BAPTA-1 AM(OGB-1)into the cerebral cortex to achieve continuous staining in a wide range of the cortex.Then,two 5×8mm2 cranial windows were created over both hemispheres between the bregma and lambda,leaving the dura matter interacted.BS was induced with 1.2~2.0% of isoflurane.calcium signal and intrinsic optical signal were recorded with a charged coupled detector(CCD)camera,and LFP was recorded by an electrophysiological acquisition system.Then,0.5?L Bicuculline methiodide(BMI)in 4 different concentrations(1.0mM,0.5mM,0.25 mM,and 0.125 mM)were injected into the cortex of rats in 4 groups,respectively,to create a cortical local hyperexcitation model.LFP,calcium signal and intrinsic optical signal were collected again.Custom-made Matlab programs were employed for data analysis.The seeded correlation coefficient map was employed to evaluate the spatial synchronicity.The calcium trace at the BMI injection site was chosen as the seed trace(ie the reference point),and the correlation coefficient(0-1)between the seed trace and the calcium signal from the other pixel was calculated.The higher the correlation coefficient,the more synchronized the calcium signal at other points is to the seed trace.The region with a correlation coefficient higher than 0.9 was defined as the synchronization region.The distribution of origin points,waveform characteristics,and occurrence frequency of BS outbreaks in each group before and after BMI injection were compared to evaluate the ability of BS in the detection of focal hyperexcitation.Results:1.The initiation sites of isoflurane-induced BS bursts were mainly randomly distributed around the peripheral visual field.Such random distribution was modified by local injection of BMI into the cortex.The distance between the initiation sites and the BMI injection site was significantly shorter than before the injection.Our result indicates that the initiation cites tended to be distributed close to the BMI injection site.With the decrease of BMI concentration,the initiation sites became less clustered to the BMI injection site.At the concentration of 0.125 mM,the spatial distribution of the initiation sites was no longer affected by BMI application.2.Before BMI injection,the calcium waveforms were similar across the entire field of view.With BMI injection,The waveforms of the burst surrounding the injection site were obviously changed with BMI injection and became significantly different than the distant locations.Analyzing the correlation coefficient maps,BMI injection significantly reduced the correlation coefficient area.The area increases with the decrease of BMI concertation.3.The interval between bursts was also affected by BMI injection.The interval became significantly shorter with BMI injection in the 1.0mM BMI and 0.5mM BMI groups,that is,the frequency of bursts increases after BMI injection in these two groups.In the 0.25 mM BMI group,there was no significant difference in the interval after BMI injection.The interval became slightly longer with BMI injection in the 0.125 mM BMI.Conclusions:1.In isoflurane-induced BS,the initiation site was distributed close to the BMI injection site.2.The calcium signal waveform erupting in the local over-excited region is not synchronized with other positions.3.With the decrease of BMI concentration,the hyperexcitability effect on the cortical area was weakened,and the above changes were less obvious.4.Isoflurane-induced BS can be used as a method to detect cortical hyperexcitability.