The Effect Of Hypoxia On Blood-Brain Barrier And Its Molecular Mechanisms

Hypoxia is believed to play an important role in the development of many diseases in central nervous system (CNS), such as inflammation, tumor, vasculopathy and trauma. Looking into the anatomical and physiological change after hypoxia in CNS will obviously help us to understand these diseases. Many researchers have paid attention to neurons in CNS only. In fact, the blood-brain barrier (BBB) serves as a critical organ in the maintenance of CNS homeostasis and is disrupted in a number of neurological disorders, including hypoxia stress. Before neuron damage BBB has been destroyed with a largely increased permeability. However, it is unclear of how hypoxia affecting BBB as well as the underlying molecular mechanisms. In our study, we used a well-established in vitro BBB model to investigate the impacts of hypoxia of microvascular endothelial cells (BMEC) and astrocytes on the BBB and their possible mechanisms.Methods: 1) A BBB model was established by co-culture of BMEC and astrocytes in vitro. 2) BMECs were observed by HE staining. This model was further examined on its biological functions as following. Enzyme activity ofγ-glutamyl transpeptidase (γ-GT) was measured with a diagnostic assay kit (as U/mg of protein). Restriction of paracellular transport was determined by measurement of the apparent permeability co-efficient (Papp) for sodium fluorescein (FLU MW: 376KDa); 125I-BSA (MW: 66kDa) that passed the polyester-multipore-membrane was then counted in scintillation counter at various time points. 3) To simulate anoxic condition in vitro and monitor time dependent changes of PaO2, PaCO2, PH, osmotic pressure, Glucose and LDH concentration in the medium of hypoxic BMEC cultures, detect cell volume changes, cytoplasmic free Ca2+ concentration in various time points after hypoxia. 4) To evaluation the impacts of hypoxia of blood-brain barrier function by examination concentration of FLU passed the polyester-multipore-membrane after hypoxia. 5) The expression levels of Zonula-1(ZO-1), Occludin in the BMEC were analyzed by semiquatitative RT-PCR and Western-...