TitleDevelopment of an organotypic model for characterizing axonal responses to In vitro strain injuries
NameDollé, Jean-Pierre (author), Yarmush, Martin l (chair), Schloss, Rene (internal member), Shreiber, David (internal member), Firestein, Bonnie (internal member), Morrison, Barclay (outside member), Rutgers University, Graduate School - New Brunswick,
Brain--Wounds and injuries--United States,
DescriptionTraumatic brain injuries are the leading cause of disability each year in the US. The most common and devastating consequence is the stretching of axons caused by shear deformation that occurs during rotational acceleration of the brain during injury. These injuries often lead to unconsciousness and long-term impairment and unfortunately the effects on axonal molecular and functional events are not fully characterized. We have developed a strain injury model that maintains the three dimensional cell architecture and neuronal networks found in vivo with the ability to visualize individual axons and their response to a mechanical injury. The advantage of this model is that it can apply uniaxial strain injuries to axons that make functional connections between two organotypic slices and injury responses can be observed in real-time and over long term. This is accomplished using microfabrication techniques to produce micron-sized channels that direct axon growth originating from the periphery of organotypic hippocampal slices. This guidance of axonal growth allows for two organotypic slices to connect to each other. The dimension of these channels can be manipulated to control the number of entering axons allowing for observation of injury effects on both individual axons and axon bundles in real-time and long term following injury. These injury effects are assessed through the use of morphology, molecular, biochemical, and cellular techniques. This uniaxial strain injury model was designed to be capable of applying an array of mechanical strains at various rates of strain, thus replicating a range of modes of axonal injury. These applied uniaxial strains are reproducible and verified through finite element analysis. Long term culture, preservation of slice and cell orientation, and slice-slice connection on the device was demonstrated. The fidelity of the model was verified by observing characteristic responses to various strain injuries which included axonal beading, delayed elastic effects, microtubule degeneration, axonal transport, axonal degeneration and mitochondrial membrane potential. The axonal beading and delayed elastic effect responses to a strain injury are dependent on both the applied strain and the bundle diameter. As the diameter increases the number of beads that form decreases and the delayed elastic effect increases. Axonal bundle unraveling and primary axotomy at lower applied strains than previously reported are observed. The induced strain injury leads to a breakdown in axonal cytoskeleton resulting in a failure in axonal transport of essential proteins as seen by accumulations of amyloid precursor protein along the length of the axon. Both of these responses result in axonal degradation and are proportional to the degree of applied strain and time following injury. We observe an applied strain injury threshold with respect to mitochondrial membrane potential response, below which there is a delayed hyperpolarization and above which immediate depolarization. Using EIPA, a sodium / hydrogen exchanger inhibitor, we were able to attenuate both mitochondrial membrane hyperpolarization and depolarization, resulting in a decrease in axonal degeneration following injury. This model could prove to be a powerful tool in assessing strain injury effects on functional axon-axon connections by further characterizing axonal responses to controlled uniaxial strain injuries and the testing of additional potential traumatic brain injury therapeutics.
NoteIncludes bibliographical references
Noteby Jean-Pierre Dollé
CollectionGraduate School - New Brunswick Electronic Theses and Dissertations
Organization NameRutgers, The State University of New Jersey
RightsThe author owns the copyright to this work.