Document Type
Dissertation
Date of Award
1-31-2014
Degree Name
Doctor of Philosophy in Biomedical Engineering - (Ph.D.)
Department
Biomedical Engineering
First Advisor
Bryan J. Pfister
Second Advisor
Patricia Soteropoulos
Third Advisor
Cheul H. Cho
Fourth Advisor
Treena Livingston Arinzeh
Fifth Advisor
Steven W. Levison
Abstract
The integration of long nerve fibers throughout the body is distinctive among somatic tissues. Single, individual motor neurons grow to a meter in length or more as they maintain connectivity with sacral areas of the spinal cord from the brain. Similarly, homologous growth also occurs via spinal ganglion neurons within the peripheral nervous system. The most widely studied aspect of nerve development has been the migration and extension of growth cones towards synaptic targets during early development. However, following growth cone extension, axonal fibers continue to grow in synchrony with the expansion of limbs and mit otic tissues throughout childhood and adolescence. The preeminent regulatory mechanism for such symbiotic interaction is through the biomechanical stretching of axons, a known stimulus of neuronal growth. While technical barriers have limited study of axon stretch growth (ASG), the in vitromethodology is refined here for investigation of the underlying mechanisms.
Utilizing custom designed live imaging bioreactors, ASG of rat dorsal root ganglion (DRG) neurons is investigated using long duration time-lapse recording. Unidirectional ASG of dissociated cultures is optimized for both embryonic and adult neurons at rates of 3 and 2 mm/d, respectively, which could be sustained without disconnection. Further, whole embryonic explants are capable of the highest sustained growth, which reach a continuous 4 mm/d. Reversible seeding techniques are established to enable imaging and manipulation of soma or growth cones during ASG. At the maximum established rates, ASG results in normal somatic morphology, whereas a 50% enlargement of the cytoplasmic cross-sectional area is found in stretch-axotomized neurons. Stretch grown neurons also maintain normal levels of spontaneous electrophysiological activity during stretch as measured by whole-cell patch clamp recording. These results support that stretch grown neurons maintain normal physiology, and do not resemble the phenotype associated with neuronal injury.
Following establishment of optimal ASG methods, genome-wide microarray analysis is performed on embryonic DRGs undergoing persistent week-long ASG, which reach 1.7 cm in length. Bioinformatic analysis using Ingenuity® pathway analysis (IPA) software reveals significant molecular and cellular changes in lipid metabolism, molecular transport, and small molecule biochemistry. In addition to the expression of developmental genes, specific stress-response and regenerative associated genes such as SPRR1a and ATF3 are found, which suggests an alternate role to their transient expression following injury. Relationships between significant genes are identified using IPA to build a network model of ~50 highly putative findings. Results suggest that ASG is a process whereby neuronal expansion is regulated by developmental stress, which leads to specific and non-injurious levels of gene transcription that promote axon growth.
Recommended Citation
Loverde, Joseph Rocco, "In vitro modeling and analysis of axon stretch growth for the sustained growth of long nerve fibers" (2014). Dissertations. 1862.
https://digitalcommons.njit.edu/dissertations/1862
