School: Martha is a graduate student in the Master of Science in Biology for Teachers program at the University of Washington

Principle Investigator: Nephi Stella, Ph.D.
Department: Pharmacology, Psychiatry and Behavioral Sciences
Institution: University of Washington

Project Description
The current focus in Dr. Nephi Stella’s lab is the study of neuroinflammatory responses and their associated neuropathologies, such as, Alzheimer’s, multiple sclerosis. AIDS dementia and acute brain injuries. The molecular basis of these neuroinflammatory mechanisms are unclear. The Stella lab is involved in unraveling the complexity of these interactions between cells of the CNS (neurons, microglia and astrocytes) and their roles in disease. The Stella lab is a pharmacology research laboratory and has expertise in studying drugs and their involvement in inflammatory responses of the CNS.  Specifically, the research group is interested in the effects and interactions of cannabinoid compounds and the cell receptors they agonize and antagonize. Neurons, microglia and astrocytes express cannabinoid receptors, and it is accepted that cannabinoids, such as THC (delta⁹ tetrahydocannabinol), a bioreactive component of marijuana induce effects that are meditated by a G-protein coupled receptor system.  It is also known that THC produces an adverse psychoactive effect mediated by the G- coupled receptor CB1. The presence of endogenous ligands (endocannabinoids) in humans and the effects of exogenous cannabinoids (natural and synthetic) indicate a likely avenue for unique therapeutic approaches to health related diseases of the CNS. Dr. Stella’s lab is in pursuit of clarifying these cannabinoid mechanisms that modulate inflammatory responses while circumventing the known adverse psychoactive effects of drug therapy.

This summer, I participated in the characterization of an in vitro model of an astrocyte cell culture system. A key to understanding the biology of any system is in the development of scientific models. These models should produce results that are reliable, valid, testable and reproducible in any laboratory.... Ideally, the in vitro model should mimic the in vivo metabolic processes. Current research reveals that astrocyes are involved in several varied and fascinating functions in the brain. My research involved growing astrocytes in a variety of media to characterize their growth and differentiation using current cell staining procedures and known markers of astrocyte differentiation. I also assessed interactions of cannabinoids with these primary cell lines in a cytotoxicity assay.

Growing the primary cell cultures on coverslips allowed for subsequent staining of the nuclei with DAPI (4’6-Diamidine-2-phenylindole dimethylsulfoxide) and characterization of apoptotic cells with TUNEL (Terminal deoxynucleotide transferase mediated dUTP nick end labeling).  Cells dying in an apoptotic pathway differ from cells dying in a necrotic manner. Apoptosis is important in the study of diseases including neuroinflammation and has been shown to be down-regulated in astrocytomas and conversely, up-regulated by cannabinoids. I found no significant differences in cell growth or number of apoptotic cells among the different media within an eight day period.  However, Western blot analyses of the cultures revealed two noteworthy results.  First, cells grown in a cyclic AMP analogue (DBcAMP, N6-2’ dibuteryladenosine-3’5’ monophosphate) showed increasing levels of the protein glutamine synthetase (GluS) over the eight days.  Second, cells grown in a minimal essential media (MEM) expressed steady levels of glial fibrillary associated protein (GFAP).  I then completed a cell cytotoxicity assay (WST-1) by adding various cannabinoids to these primary astrocyte cell cultures and measuring the relative levels of respiration and metabolic activity.  The dye (WST-1) is cleaved by ATP in the mitochondria of live cells to produce a measurable metabolic or respriratory spectrophotometric response.  Some interesting preliminary results show that cells grown in all media receive a boost in their metabolic rates when given THC.  Finally, two synthetic cannabinoid compounds showed toxic effects on cells grown in MEM (presumably the least differentiated cells of any media).  Some interesting future questions might be; “do cannabinoids prevent or induce in vitro astrocytes to differentiate?”, “do GFAP or GluS levels change with cannabinoid treatment?” and “do cannabinoids induce or reduce apoptosis in this in vitro cell culture system?”.

It has been my good fortune to work in the Stella lab this summer as part of the StarNet Research Program for Teachers.  This experience has aided me immeasurably in my efforts to complete my Master’s of Science in Biology for Teacher’s degree. As a result, I hope to join the teaching profession this fall. Working in the Stella lab gave me the opportunity to conduct hands-on research and helped me validate my skills as a technician.  In addition, the realization that the results of the in vitro model raised provoking questions is exciting to me because I know the value of encouraging my future students to look beyond expected results and into the world of limitless discovery.

I would like to express my gratitude to Dr. Stella, Allyn Franklin, the entire Stella Lab, and everyone in the StarNet program for mentoring and encouraging me through such a great research experience.