The objective of our grant application is to develop an in vitro model of the blood-brain barrier and assays to examine permeability by using isolated brain microvessels. Unlike capillaries in other organs, brain capillaries establish a barrier that is largely impermeable to polar and larger chemicals, which are due to tight junctions, the absence of both fenestrae and pinocytosis. Although, the permeability of most chemicals to cross the blood-brain barrier is directly associated to its'oil/water partition coefficient, many lipophillic chemicals fail to enter the brain because of the expression of the multidrug transporters. In contrast, polar nutrients that would be predicted to be less permeable display higher uptake because of the expression of nutrient transporters. Many chemicals that are potentially toxic to the brain fail to cross the blood-brain barrier. Additionally, many drug candidates for treating mental illnesses and neurological diseases fail because of the blood-brain barrier. Consequently, a model to study the blood-brain barrier is needed for the new paradigm in which testing chemicals for toxicity will be accomplished with in vitro and in silico methods. Additionally, a model is needed to aid chemists in their attempts to design neurotrophic drugs. Current cell culture models fail to display the tightness and the many transporters found in vivo. In contrast, isolated brain microvessels retain all of the transporters and display a tight barrier, and have been used in the past 40 years in research studies on blood-brain barrier permeability. The major obstacles in using isolated brain microvessels have been the laborious procedure for their preparation and their short life span. A commercial source of brain microvessels will overcome these obstacles. The overall objective of our study is to develop cryopreserved BM as a model of the blood-brain barrier and optimize reagents and assays to measure permeability and transport that will either be conducted by our company or sold to others. To accomplish the objective, in Specific Aim 1 conditions will be standardized for preparing, cryopreserving viable bovine brain microvessels and assure consistency in different batches of microvessels sold. The functionality of the brain microvessels will be assessed by conducting assays to measure amino acid and sugar transport, multidrug transporters, and viability. Assays achieving Z-factors closest to 1.0 will indicate the best preparation conditions.
In Specific Aim 2, assays will be developed to examine chemical toxicity of the blood-brain barrier by measuring non specific transport. Sensitivity and specificity will be assessed by computing receiver operating curves. In the phase 2 grant application, assays will be developed to measure multi drug efflux pumps and the luminal and abluminal transporters. By establishing a commercial source of reagents for assessing the blood-brain barrier, pharmaceutical companies and chemical testing laboratories will have a readily available model to screen test chemicals for permeability and toxicity. When considering the importance of the blood-brain barrier in neurological functions, these products will potentially have wide distribution and commercial success.
The lack of an effective model for measuring the effects of drugs and chemicals on the blood-brain barrier (BBB) has been an impediment to evaluating chemicals for toxicity to the brain as well as to the successful development of drugs to treat diseases of the brain18,29. The proposed model of the blood-brain barrier, isolated brain microvessels (BM) from cows, and assay for blood-brain barrier permeability, has the potential for enormous impact in the field of neurotoxicity testing and drug development. The innovation here is in taking a successful research model, freshly isolated BM, and converting it into a cryopreserved, marketable product as well as translating its use into assays that address the previously unmet need for evaluating BBB permeability.
|O'Driscoll, Cliona M; Coulter, Jonathan B; Bressler, Joseph P (2013) Induction of a trophoblast-like phenotype by hydralazine in the p19 embryonic carcinoma cell line. Biochim Biophys Acta 1833:460-7|