Establishment Of Cerebral Ischemia Reperfusion Model And The Neuroprotective Effect Of Protocatechuic Acid

Cerebral ischemia-reperfusion injury（CIRI）refers to the phenomenon that the blood supply is restored after a period of cerebral ischemia,and the brain suffers more serious damage.CIRI can seriously affect the level of human life and health,mainly manifested as brain structural damage,metabolic disorders,and dysfunctions.At present,in vitro cell culture models and animal models,which are important new drug development tools,have a long culture cycle and high cost.It requires repeated screening to enter the clinical stage and enter the market finally.This makes new drug development efficiency very low and greatly increases the risk of failure.As an emerging technology,microfluidic technology has the advantages of being able to control the speed and direction of liquid flow,consume less reagent samples,and greatly improve the efficiency of analysis.Therefore,it is expected to become an important experimental platform for new drug development and drug evaluation.This paper designed and processed a CIRI chip based on microfluidic technology to restore the blood-brain barrier（BBB）and the frontal temporal lobe area that was severely damaged by CIRI firstly.Both two layers of chip are made of Polydimethylsiloxane（PDMS）,and the top and bottom layers are made of polymethylmethacrylate（PMMA）splints to fix and support the chip.The two layers of chips are designed with culture holes with a radius of 8mm respectively.The interaction between the culture holes and the polytetrafluoroethylene（PTFE）membrane sandwiched between the chips serves as the space environment for cell growth and development.The chip is also designed inflow and outflow for cell inoculation and the culture medium.Perform fluid dynamics simulation on the designed and processed chip:when the flow rate of the culture fluid is in the interval of 0.956-6.22m/min,the shear force of the culture fluid reaching the nerve cells meets the real shear force of 0.3-2KPa in the human brain;The diffusion of carbon dioxide in the chip is simulated:when the chip is placed in the environment for 500 seconds,the gas environment in the chip is consistent with the external environment,which provides a theoretical basis for subsequent cell culture in the chip.Then this thesis completed the cultivation and selection of nerve cells（NCs）:the NCs of24h newborn rats were extracted and then primary cultured and purified,and the NCs purified for 3 days were subjected to immunofluorescence identification to express their specific markers（β-TubulinⅢ）;subculture PC12 cells,measure the cell growth curve,and select a cell density of 5×10⁴ Cells/m L for inoculation.After comparison,PC12 cells were selected as NCs to be inoculated into the microfluidic chip,low-glycemic culture medium was passed into the hypoxic box to complete the simulation of oxygen and glucose deprivation of nerve cells to simulate the ischemic process,and then the inoculated chip was transferred from the hypoxic box To the CO₂ incubator,change to high-sugar medium to complete the simulation of complex sugar and reoxygenation and then realize the simulation of CIRI.By adding different concentrations of protocatechuic acid（PCA）to investigate PCA（0,1μmol/L,10μmol/L,100μmol/L）neuroprotective effect on CIRI in the chip system.The experimental results show that the designed and processed CIRI microfluidic chip can maintain the growth state of NCs within 24 hours.CIRI will cause the proliferation of NCs in the microfluidic chip to slow down:the number of dead cells increased,and the number of live cells decreased,Lactate dehydrogenase（LDH）leakage increased,TNF-α,Notch1 protein expression increased,and Bcl-2 protein expression decreased;The neuroprotective effect of PCA on NCs is concentration-dependent:with the increase of PCA concentration,the influence of CIRI on NCs gradually decreased,Compared with the CIRI group,the proliferation of nerve cells is increased to a certain extent,the number of dead cells decreased,the number of live cells increased,the amount of LDH leakage decreased,the expression of TNF-αand Notch1 protein decreased,and the expression of Bcl-2 protein increased.In summary,the CIRI microfluidic chip was designed and processed in this paper,and the selected NCs were inoculated into the CIRI microfluidic chip.Five control groups were set up:Control group,CIRI group,PCA1 group,PCA10 group,and PCA100 group.PCA play a neuroprotective effect on CIRI-treated NCs in the microfluidic chip and show a concentration-dependent effect,which can promote cell proliferation,reduce the number of dead cells,increase the number of live cells,reduce lactate dehydrogenase leakage,reduce TNF-α,Notch1 protein expression content,and increase Bcl-2 protein expression content.