Low-temperature Plasma Attenuates MCAO-induced Cerebral Ischemic Stroke

Objective:Low temperature plasma plays a crucial role in antioxidant stress and inflammation.Ischemic stroke is a common cerebrovascular disease in clinical practice.The aim of this study was to explore the molecular mechanism of low temperature plasma reducing the stroke injury in MCAO model rats.Methods:1.MCAO model: SD rats were anesthetized 220-230 g with a minor animal anesthesia machine and fixed in supine position.The muscles and fascia was separated along the inner margin of sternocleidomastoid muscle,and the left common carotid artery(CCA),external carotid artery(ECA)and internal carotid artery(ICA)were separated.Wires were hung at the distal end of CCA and ECA for use.The CCA and ECA were ligation proximal to the heart,then a small incision was made 4 mm from the bifurcation of the CCA,and a wire plug was inserted into the ICA to mechanically block the blood supply from the source of the middle cerebral artery(MCA).A wound suture was performed,embolizing for 2.5 h,and the cord was pulled out for reperfusion.The animals were kept in a single cage after operation,and the animals in the sham operation group only simulated the operation without blocking MCA.2.Identification criteria of MCAO model: Behavioral scores were performed on the awake rats after MCAO operation,and the inclusion criteria are set as the neurological injury function ≥3.3.Behavioral and survival detection: MCAO model rats(neurological injury function score ≥3)were randomly divided into untreated group and plasma treatment group,sham operation group as control.The untreated group was provided for normally without any treatment.In the plasma treatment group,the head,abdomen,back,right forelimb and right hind limb were treated with plasma irradiation every day,3 min for each part;twice a day,the 8H interval.Behavioral scores were performed at 8 o ’clock every day and survival rate were observed for 14 days.4.TTC staining: MCAO model rats(neurological injury function score ≥3)were randomly divided into untreated group and plasma treatment group,sham operation group as control.The rats were sacrificed on the third day after operation,and the brain was taken from the decapitated head.The brain tissues were taken general photographs,TTC staining was carried out,and the infarct volume was calculated to grasp the influence of low temperature plasma on the stroke injury in MCAO rats.5.Pathological and molecular biological analysis: MCAO model rats(neurological injury function score ≥ 3)were randomly divided into untreated group and plasma treatment group,sham operation group as control.The patients were sacrificed on the3 rd,7th and 14 th day after operation,and the brains were taken by decapitation for pathological,Immunohistochemy and molecular biological detection.6.The effect of low temperature plasma on the stroke injury in MCAO rats was analyzed from microscopic perspective.HE staining: Brain tissue injury was detected from four aspects: necrosis,inflammatory cell infiltration,hyperplasia and bleeding.TUNEL staining : The apoptosis of brain cells was detected.Immunohistochemy analysis: The expressions of Ki-67 protein(Ki-67),neuron-specific protein(Neun),IBA-1(IBA-1)and myeloperoxidase(MPO)were detected to evaluate the recovery of neurons and the degree of neuroinflammation after MCAO.m RNA level detection: The apoplectic side of the brain tissue was extracted on day 3,day 7 and day 14 after surgery.The total RNA of the samples was reverse-transcribed into c DNA,and the m RNA expression levels of IL-6,IL-1β and TNF-α were detected by real-time quantitative PCR.Results:1.Survival rate analysis showed that plasma treatment group significantly improved the survival rate of MCAO rats compared with untreated group.2.The results of behavioral evaluation showed that plasma treatment group significantly improved the behavioral symptoms of MCAO rats compared with untreated group.3.The results of brain gross anatomy and TTC staining showed that the infarct size in the low-temperature plasma treatment group was significantly decreased compared with the untreated group(P≤0.01).4.HE staining results showed that compared with the untreated group,the cells in the plasma treated group had decreased necrosis,decreased bleeding,increased proliferation,and significantly decreased the trend of inflammatory cell infiltration.5.The immunohistochemical results showed that compared with the untreated group,the TUNEL positive cell rate in the brain tissue of the plasma treated group was significantly decreased,and the Ki-67 and Neun positive cell rate was significantly increased,indicating that cell apoptosis was inhibited and cell proliferation and neuron repair were promoted.6.Immunohistochemical results showed that compared with untreated group,plasma treated group significantly inhibited the activation of microglia and neutrophils,and IL-6(P ≤ 0.05),TNF-α(P ≤ 0.05),IL-1B m RNA levels were significantly decreased,indicating that plasma significantly inhibited the occurrence and development of neuroinflammation in MCAO brain.Conclusion:Low temperature plasma treatment can significantly improve the survival rate and behavioral symptoms of MCAO rats.Pathological and molecular biological analysis show that plasma can reduce the injury of ischemic stroke in rats by inhibiting intracerebral cell necrosis,promoting cell proliferation and neuronal regeneration,and inhibiting intracerebral inflammation.