This project aims to develop a human cell-culture model of the blood–brain barrier, to replace the use of some animal studies.
Encephalitis (inflammation and swelling of the brain) can be caused by a range of viruses, and the results are usually devastating. The blood‒brain barrier (BBB) is the specialised barrier that prevents viruses, bacteria and toxins from entering the brain, and a number of studies have implicated a disruption of the BBB during encephalitis. However, most studies of the BBB are done in animals or animal cell lines; most commonly rat and mouse models. These animals have symptoms such as leg paralysis, hunched back, and seizures following infection. A model of the BBB using human cells would represent an important scientific advance and replace the use of some animals used in BBB experiments.
Research details and methods
Building on existing 2D cultures, a 3D flow-based model which mimics blood flow in brain capillaries will be developed and characterised. Primary or immortalised human brain endothelial cells will be seeded on the blood side of the model and astrocytes or pericytes on the brain side. Serum from patients will be pumped over the cells and viruses will be introduced to the system to simulate infection and investigate the role of the BBB.
Viral encephalitis is one of the most important types of neurological infection, because of its devastating impact on the quality of life of those affected, and its disproportionately large burden on the healthcare systems and community with large health economic and social costs. Encephalitis such as Japanese encephalitis (JE) is a massive burden on many developing countries. In the UK, encephalitis caused by herpes simplex virus and human immunodeficiency virus is a major problem. The blood-brain barrier (BBB) is the protective physiological barrier that prevents pathogens such as viruses entering the central nervous system (CNS). Studies have shown that BBB damage due to inflammation is a final common pathway in many brain infections including viral encephalitis, allowing pathogens and leukocyte entry into the brain. Currently studies that investigate the immunopathogenesis of viral brain infections are very dependent on animal models because of a lack of good in vitro human models. The most commonly used animal models for encephalitis are mouse and rat models. In mice, signs of infection include leg paralysis, a hunched back, ruffled fur, minimal activity and seizures. A human cell culture model of the BBB can give physiologically relevant data and will lead to a reduction of animals used for this kind of research. During my postdoctoral research over the last two years, I have developed a reliable two-dimensional cell culture model of the human BBB using immortalised human brain endothelial cells (iHBEC) and primary human astrocytes. I am investigating the mechanisms of Japanese encephalitis virus (JEV) entry through the BBB. However, there are certain limitations in this model; achieving a tight monolayer is difficult (compared with animal models) and the in vivo blood flow (important for leukocyte transmigration) cannot be simulated. I would now like to take this work forward with a David Sainsbury Fellowship by developing a multi-culture three-dimensional flow-based human BBB model for studying viral encephalitis in collaboration with researchers from the Cleveland Clinic Lerner Research Institute, USA. The model will mimic the blood flow in the brain capillaries. Primary HBEC or iHBEC will be seeded on the blood side and CNS cells such as astrocytes (main inducers of BBB phenotype on endothelial cells) and microglia (release cytokines/chemokines in response to CNS infection) in the brain side. Cell culture medium or serum from patients will be pumped through the lumen of the model to add shear stress on the endothelial cells. I will have access to JE serum and CSF samples through the Liverpool Group's extensive collaborations across Asia, funded by MRC and the Wellcome Trust and other samples from patients in the NIHR Encephalitis Programme Grant in Liverpool, headed by Professor Tom Solomon. These modifications will allow an even better approximation to the real situation in humans. I will then use the model to understand the immunopathogenesis of JE. In particular, I will study the pathways of JEV-infected leukocyte trafficking through the BBB (not possible in the current model) and the effects of pro-inflammatory cytokines on this model. This model will be useful in the future for studying viral CNS infections including human immunodeficiency virus and herpes simplex virus, and for testing potential therapeutic drug candidates for viral encephalitis. Several UK and international biotech companies have expressed their strong interest on this as the model will be a useful tool for reducing/replacing the number of animals used in the first phase of drug discovery.
Patabendige A et al (2018). Brain microvascular endothelial-astrocyte cell responses following Japanese encephalitis virus infection in an in vitro human blood-brain barrier model. Molecular and Cellular Neuroscience 89: 60-70. doi: 10.1016/j.mcn.2018.04.002.
Ferguson MC, Saul S, Fragkoudis R, Weisheit S, Cox J, Patabendige A, Sherwood K, Watson M, Merits A, Fazakerley JK (2015). Ability of the encephalitic arbovirus semliki forest virus to cross the blood-brain barrier is determined by the charge of the E2 glycoprotein. J Virol 89(15): 7536-49. doi: 10.1128/JVI.03645-14.
Patabendige A, Abbott NJ (2014). Primary porcine brain microvessel endothelial cell isolation and culture. Curr Protoc Neurosci 69: 3.27.1-17. doi: 10.1002/0471142301.ns0327s69.
Patabendige, A. (2012) The value of in vitro blood-brain barrier models and their uses. ATLA 40:335-338.
Patabendige, A. (2012) Toward a humanised alternative to the use of laboratory animals for blood-brain barrier research. ATLA 40, PiLAS 1(1):12-13.