Date of Award
12-2017
Document Type
Thesis
Degree Name
Master of Science (MS)
Department
Bioengineering
Committee Member
Dr. Jiro Nagatomi, Committee Chair
Committee Member
Dr. Dan Simionescu
Committee Member
Dr. Delphine Dean
Abstract
Traumatic brain injury (TBI) and subsequent rises in intracranial pressure (ICP) are associated with high mortality and has a number of physical and behavorial consequences to which the specific causes are unknown. We hypothesized that exposure of neuronal cells to elevated pressure can cause neurite retraction and / or apoptosis. Loss of communication between neurons due to these cellular-level events may play a part in complications seen in TBI patients, such as neurodegenerative diseases, increased anxiety, fear, depression, cognitive problems, as well as motor and visual deficits. In the present study, the effect of elevated hydrostatic pressure was evaluated at the cellular level in vitro by examining cell morphology, particularly neurite retraction and extension, as well as apoptosis. Neuro-2A cells (a mouse neuroblastoma cell line) were plated in cell culture dishes, as well as on soft polyacrylamide gel + type I collagen substrates. The cells were imaged using phase contrast microscopy (Nikon) before and after exposure to 25 or 35 mmHg of hydrostatic pressure in a custom-made pressure chamber for 15, 30, 60 min, as well as 4 and 6 hours. Pressure values were chosen to model elevated ICP correlated with moderate to severe TBI. Using the Fiji software, the post-pressure images were compared with the pre-pressure images of the same cells and the data were reported as normalized change in neurite length per cell. Additionally, TUNEL assay was performed using a commercially available kit (TiterTACSTM, Trevigen) to quantify apoptotic cells after exposure of Neuro-2A cells to 35 mmHg pressure. Results indicate that neurite length decreased significantly (p<0.05) when Neuro-2A cells were exposed to a pressure of 35 mmHg. When results are compared from 15, 30, 60 minutes and 4 hours of pressure exposure, it appears that neurite retraction is correlated with exposure time. To further understand these morphological changes, the Rho/ROCK pathway was examined as a potential pathway involved in the mechanotransduction of hydrostatic pressure by neuronal cells. By using Y-27632 as a p160ROCK inhibitor, it was demonstrated that the pressure-induced neurite retraction response was blocked, therefore indicating the pathway's involvement. The results of TUNEL assay indicated that the number of apoptotic cells were similar between the no pressure control and the cells exposed to 35 mmHg of pressure for 24 hours. The results of the present study provide evidence that elevated hydrostatic pressure causes Neuro-2A cell neurite retraction in a time-dependent manner in vitro. The model highlights the importance of urgent ICP clinical management, as elevated ICP caused by TBI may have a negative impact on neuronal tissue and contribute to further consequences.
Recommended Citation
Etten, Kallie, "The Effect of Hydrostatic Pressure on Neuronal Cell Morphology In Vitro" (2017). All Theses. 2798.
https://tigerprints.clemson.edu/all_theses/2798