Date of Award

5-2009

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Engineering and Imaging

Research Advisor

M. Waleed Gaber, Ph.D.

Committee

Frank A. DiBianca, Ph.D. Lisa K. Jennings, Ph.D. Thomas E. Merchant, D.O., Ph.D. Christopher M. Waters, Ph.D.

Abstract

Radiation is one of the principal treatments for adults and children with brain tumors, and is one of the oldest established treatments for tumors of all types. Currently, the limiting factor for the use of radiation is the effect on normal tissue adjacent to the tumor. Toxicity, including early and late effects from radiation, limits the dose administered to the tumor and reduces the probability of cure. This work has three aims in its attempt to understand and limit early radiation damage: characterize the role of the inflammatory molecules tumor necrosis factor alpha (TNF) and intracellular adhesion molecule-1 (ICAM-1) in the radiation response; evaluate the role of a novel anti-inflammatory agent as an interventional therapy to limit the radiation-induced inflammatory response; begin to characterize the acute damage following radiation using the shift in the gene expression.

Fluorescence intravital microscopy and a mouse cranial window model were used to quantitatively measure: permeability of the blood-brain barrier, leukocyte adhesion, and changes in vessel diameter following a single dose of 20-Gy localized cranial irradiation. Immunohistochemistry and immunofluorescence staining were used to evaluate the number of activated astrocytes and the protein expression of TNF and ICAM-1. Antibodies to TNF and ICAM-1 were administered to investigate their role in the early radiation response. In addition, a novel anti-inflammatory agent, KZ-41, which is thought to work through NF-κB was evaluated as a non-specific inhibitor to the early radiation response. Microarray analysis was used to characterize the molecular shift that occurs in the brain at 2-hrs after 20-Gy radiation.

Our results show that the inflammatory molecules TNF and ICAM-1 are involved in the early radiation response. Radiation induces an increase in permeability of the BBB and in the number of adhering leukocytes at 24- and 48-hrs post-irradiation. It also causes a decrease in the average diameter of arterioles at 48-hrs post-irradiation. Immunohistochemistry showed a significant increase in the number of activated astrocytes at 24-and 48-hrs post-irradiation, while immunofluorescence verified the expression of TNF and ICAM-1 protein following radiation. When TNF expression was inhibited via specific antibodies, all of the radiation-induced effects were abrogated. When ICAM-1 was inhibited via specific antibodies, most of the radiation-induced effects were abrogated. Treatment with ICAM-1 mAb in conjunction with radiation, completely inhibited the radiation-induced vascular effects (BBB permeability, leukocyte adhesion, and arteriole diameter changes), but did not inhibit activated astrocytes, however it did significantly reduce them compared to radiation alone.

KZ-41 was evaluated as a potential agent for interventional therapy aimed at reducing the radiation-induced inflammatory response. Using our radiation model, KZ-41 was found to protect the vasculature in a manner similar to the specific antibodies to TNF or ICAM-1, but was unable to prevent the radiation-induced astrocytic response. Permeability of the BBB and leukocyte adhesion was inhibited, while there was no observed change in arteriole diameter at any time point. Immunofluorescence showed that treatment with KZ-41 limited the radiation-induced expression of ICAM-1 protein, but did not alter the expression of TNF protein. In addition, the number of activated astrocytes, following treatment with KZ-41, was significantly higher than in radiation only animals.

Microarray analysis was used to investigate the shift in the genetic profile of the brain following radiation. It was seen that, 2-hrs following irradiation, there is: a paracrine response exists that is primarily initiated by chemotactic cytokines through the cytokine-cytokine receptor interaction pathway; vascular andparenchymal damage whose response can be distinguished when gene function is considered; and an alteration in genes known to be related to neurological/neurodegenerative diseases.

Our overall aim is to elucidate the role of inflammation in the acute radiation response and its effects on the microvasculature of the brain. Understanding this role will allow for the development of treatment strategies to limit the acute radiation damage which we believe will lead to a reduction in the long-term side effects of radiation.

DOI

10.21007/etd.cghs.2009.0353

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