Mechanism Of Injury In Brain And Spinal Cord Of Gerbil And Rabbit Infected With HEV

There are more and more reports about neurological disorders such as encephalitis,meningitis,myelitis and Guillian-Barre syndrome caused by HEV infection.In order to uncover the pathogenesis of central nervous system dysfunction induced by HEV infection,gerbils and rabbits experimentally inoculated with genotype four swine HEV were used as animal models.RT-nPCR,real-time qPCR,immunohistochemistry,histopathology and ultrastructural histopathology,Transwell chamber test,western-blot and immunofluorescence staining methods were adopted to investigate the replication and distribution of HEV in central nervous system,and the resultant pathological changes.HEV inoculate human primary microvascular endothelial cells in-vitro test was carried out to determine the approach of HEV used to get through the blood-brain-barrier and reveal the mechanism of BBB structural damage.The results are as follows:1.The positive-and replicative negative-strand of HEV RNA were detected in the central nervous system of gerbils intraperitoneally inoculated with HEV during the acute infection;the viral load in the spinal cord was higher than it in the brain;HEV ORF2 positive signal was observed in the perivascular area,choroid plexus,ependymal cell and some neurons and neurogliocytes;optical microscope observation revealed microglia proliferation,neuron degeneration and necrosis,lymphocyte infiltration in the perivascular region,choroid plexus and below the ependyma;ultrastructural observation showed decreased number of organelles,mitochondrion swelling with less crista,myelin sheath degeneration,blood vessel disorganization with swollen endothelial cells,absence of junction complex,et al.2.Rabbits were successfully infected with genotype four swine HEV via both intraperitoneal injection and filling stomach,but central nervous system HEV positive was only determined in rabbits intraperitoneally inoculated with HEV,which indicating that oral-fecal transmission pathway is less likely to cause HEV infecting brain and spinal cord compared with intraperitoneal injection.Real-time qPCR,immunohistochemistry and histopathological analysis results were consistent with gerbil experiments.3.The significant decrease(P<0.05 or P<0.00)of microvascular endothelial cell tight junction associated proteins expression caused by HEV infection was proved in vivo and in vitro,which led to the increase of BBB permeability.As a result,HEV was capable of geting through the BBB and invading the brain and spinal cord parenchymal tissue.HEV inoculating human primary brain microvascular endothelial cells transwell chamber test showed that the endothelial endocytosis and transcytosis of HEV can be inhibited by the clathrin inhibitors.In addition,western-blot analysis showed that HEV promoted NOX4 expression and inhibited ATP5A1 expression in microvascular endothelial cells.4.The microglia and astrocytes proliferation accompanied with upregulation of substance P,TNF-a,IL-1? and VIP in HEV infected gerbil brain and spinal cord were determined by immunohistochemistry analysis.In addition,the over expression of Bax,Bcl-2,Caspase-9 and Caspase-3 in the brain tissue infected with HEV was proved with western-blot assay.All of the above may participate in the mechanism of brain and spinal cord injury induced by HEV infection.In conclusion,HEV may enter the brain microvascular endothelial cells through the clathrin-mediated endocytosis,affect synthesis of ATP and cause oxidative stress in endothelial cells,which led to BBB damage.Consequently HEV passed the BBB and infected the CNS parenchymal tissue via both transcellular and paracellular appoaches,induced the activation and proliferation of microglia and astrocytes.Proinflammatory factors including TNF-? and IL-1? released by the neurogliocytes may result in brain and spinal cord inflammation damage and activate mitochondrial apoptosis pathway,eventually leading to brain and spinal cord injury.