The Effects Of Sevoflurane On The Blood-brain Barrier And The Role Of Wnt/β-catenin-Annexin A1 Pathway In This Process

Objective: We investigate the effects of sevoflurane on the blood-brain barrier and the role of Wnt/β-catenin-Annexin A1 pathway in this process.Contents: We engage our research in the sevoflurane inhalation in vivo model and the rat brain microvessle endothelial culturing in vitro model.Methods: methods were divided into 3 parts. In part 1, 18~20 month male Wistar rats,weighing 500~600g, were randomly divided into control group(C) which received continuous inhalation of 30% O2 and sevoflurane group(S) which received sevoflurane of different concentrations(2.4%, 3.1%, 3.6%) and durations(2h, 4h, 6h).Evan’s blue staining and fibrinogen immunofluorescence staining in hippocampus were given at 24 h after inhalation. In part 2, Immunofluorescence staining was taken to make double staining of Annexin A1 with Neu N(biomarker of neuron), Iba-1(biomarker of microglia) and CD31(biomarker of endothelial). Rats were given 3.6%sevoflurane inhalation for 6h. After inhalation, western blot was engaged to test the expression of tight junction protein occludin, claudin-5, claudin-3 and ZO-1, and the expression of adhesion junction VE-cadherin and cytoskeleton protein F-actin at 6h,12 h and 24 h. In addition, western blot and immunofluorescence were executed to investigate the alteration of Annexin A1. Then animals were randomly devided into 3groups,(1) control group(Group C): continuous inhalation of air containing 30% O2(2) sevoflurane group(Group S): continuous inhalation of 3.6% sevoflurane+30% O2,6h and(3) human recombinant Annexin A1+sevoflurane group(Group AS):continuous inhalation of 3.6% sevoflurane+30% O2 for 6h, pre-treated with Annexin A1 through one-time injections(0.67μg/kg) via tail veins at 1h before sevoflurane inhalation. Western blot was taken to detect the expression of tight junction proteins occludin, claudin-3, claudin-5 and ZO-1, as well as adhesion junction protein VE-cadherin and cytoskeleton protein F-actin in hippocampi at 24 h after anesthesia.Immunofluorescence staining was adopted to detect the deposition of fibrinogen in hippocampi area at 24 h after anesthesia. Y maze test and fear conditioning test were engaged to detect the cognitive function at 1d and 3d after sevoflurane. In part 3, 7d male Wistar rats were adopted. The brain microendothelial cells were extracted and then cultured in vitro to mimic the BBB model. The total and nucleus β-catenin protein and total GSK-3β were measured at 6h, 12 h and 24 h after 3.6% sevoflurane treating for 6h. Then we took use of Wnt-3a and DKK1 as agonist/inhibitor of the Wnt/β-catenin signalling pathway under the circumstances of with/without sevoflurane and discuss the alteration of Annexin A1 via western blot and immunofluorescence staining.Results : Compared with group C, rats administered by 3.6% sevoflurane for 6h showed increased Evans blue staining and increased fibrinogen deposition area in hippocampus at 24 h after inhalation. The Annexin A1 expressed in hippocampus of the aged rats was highly colocalized with CD31, rather than with Neu N and Iba-1.Compared with those before anesthesia, it showed that the expression of occludin,claudin-3 and F-actin in group S presented significant decrease at 24 h after sevoflurane. Compared with those before anesthesia, the expression of Annexin A1 in hippocampal of the aged rats decreased at 24 h after sevoflurane inhalation. Compared with group S, rats in AS group showed increased expression of occludin,claudin-3,VE-cadherin and F-actin at 24 h after sevoflurane inhalation. The fibrinogen deposition area decreased compared with animals from group AS. Compared with those from group C, animals in group S showed decreased arm visits, novel arm visiting durations and decreased contextual freezing percentage as well as decreased cued freezing percentage. And compared with those of group S, animals in group AS showed increased arm visits, novel arm visiting durations and increased contextual freezing percentage as well as increased cued freezing percentage. Compared with the untreated endothelial cells, there was no change at 6h,and 12 h after sevoflurane treatment; and at 24 h after sevoflurane treatment the protein level of total and nucleusβ-catenin decreased and the total level of GSK-3β increased. Compared with the control group, Wnt-3a/DKK1 significantly increased/decreased the expression of Annexin A1 in brain microendothelial cels of rats.Conclusion : Our findings indicate that in endothelial cells, treatment with 3.6%sevoflurane for 6 h inhibits the Wnt/β-catenin signalling pathway, thereby increasing GSK-3β and decreasing β-catenin. By inhibiting this pathway, the gas anaesthetic sevoflurane down-regulated Annexin A1, which consequently breached the BBB and induced POCD.