Characterization of the glial scar using Raman spectroscopy, microindentation, and immunohistochemistry
Date of Award
Doctor of Philosophy (PhD)
Biomedical and Chemical Engineering
Julie M. Hasenwinkel
Glial scar, Spinal cord injury, Microindentation
Biomedical Engineering and Bioengineering
At the site of injury in the spinal cord (SC), a glial scar (GS) forms. This scar has been classically hypothesized to be a physical and chemical barrier to nerve regeneration. Current research on spinal cord injury (SCI) focuses on treating the GS as a chemical barrier due to the remodeled extracellular matrix in the GS. The objective of this research was to elucidate structure-property relationships in injured SC. Thus, this study investigated the biochemical and physical properties of the injured SC, in its native physiological state , using two unique approaches: Raman spectroscopy (RS) and microindentation based stress relaxation, for biochemical and viscoelastic characterization, respectively. Lateral hemisection injury and a moderate contusion injury models were characterized using RS. For stress relaxation experiments, SCI was investigated using the lateral hemisection model. Immunohistochemical analysis was performed to corroborate Raman spectra and understand the physical structure of the injured cord. Adult female Sprague-Dawley rats were sacrificed 4 days, 2 weeks, and 8 weeks post-injury (PI) for the hemisected groups and at 2 weeks PI for the contused groups. Animals were perfused with isotonic saline and extracted cords were used without further preparation. Relaxation behavior of SCs was modeled using a unique empirical model based on a spring-dashpot system and a stretched exponential. Results from the RS showed that injured SCs could easily be distinguished from uninjured controls, and the changes were systematic. Specifically, spectral differences were observed in peaks corresponding to the aggregation and hydrolysis of glycosaminoglycans and proteoglycans, and demyelination. Microindentation showed that the stiffness of cords decreased, and that the viscous behavior was more pronounced PI. The injured cords two weeks PI displayed significantly different viscoelastic behavior, by virtue of having the largest time constant and viscosity, as ascertained from the model parameters. These results correlated with peak proteoglycan expression, and complete demyelination at two weeks PI. As a pioneering study, this work lays the foundation for the development of RS as an in vivo probe, in SCI research. In summary, this study demonstrated that RS can be employed to distinguish uninjured SCs from injured SCs, and that the GS is not simply a stiffness barrier but rather, it displays complex viscoelastic behavior likely indicating changes in tissue permeability and diffusivity.
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Saxena, Tarun, "Characterization of the glial scar using Raman spectroscopy, microindentation, and immunohistochemistry" (2010). Biomedical and Chemical Engineering - Dissertations. 59.