Date on Master's Thesis/Doctoral Dissertation

12-2007

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Physiology and Biophysics

Committee Chair

Anderson, Gary

Author's Keywords

Skeletal muscle; Ischemia; Fusogenic lipid vesicles; Tissue preservation; Reperfusion

Subject

Reperfusion injury; Ischemia

Abstract

The purpose of this dissertation was to demonstrate that a new method of direct intracellular energy delivery was effective in maintaining viability of in vitro Human Umbilical Vein Endothelial Cells (HUVECs) when exposed to chemical ischemia for prolonged periods of time and in preventing in vivo ischemia reperfusion injury in a composite tissue transplantation model. Ischemia and reperfusion injury is a well recognized phenomenon that occurs in situations like organ transplantation, shock, cardiac surgery, etc, where tissues are temporarily deprived of nutrients and oxygen for energy production. When exposed to short periods of ischemia, cells utilize their stored energy to survive. If ischemia is extended beyond certain limits, cellular energy stores become depleted leading to metabolic and structural changes. One of the effects of this lack of energy is the malfunctioning of the membrane adenosine triphosphate (ATP)-dependent ionic pumps which are ultimately responsible for maintaining cellular volume, intracellular pH and ionic homeostasis. Thus it is hypotesized that the negative effects of energy depletion on cellular homeostasis could be overcome by delivering energy directly into the cell. This method of direct energy delivery into cells is based on fusogenic lipid vesicles (FLVs) composed of lipids very similar to those that form the cellular membrane. These vesicles are very small and when put in contact with cell membranes, rapidly fuse to the cell membrane delivering their content into the cell interior. Making use of this phenomenon, the vesicles are loaded with ATP magnesium chloride (Mg-ATP) that is delivered directly into the cell, providing it with a readily usable form of energy. We hypothesized that, under chemical hypoxia, endothelial cell viability could be preserved and the Na + K +- ATPase pump activity could be maintained by delivering Mg-ATP directly into the cells. Further, we hypothesized that ischemia reperfusion injury due to tissue transplantation could be reduced by perfusing tissues ex vivo before reimplantation with a solution containing Mg-ATP-loaded FLVs. The first two hypotheses were tested in the following manner. First, we determined the optimal concentration of Mg-ATP that the FLVs needed to be loaded with to maintain the viability of HUVECs exposed to 4 hours of chemical hypoxia. Second, we tested if the Na + K + ATPase pump activity could be maintained by delivering Mg-ATP to HUVECs exposed to chemical hypoxia. The results of these experiments demonstrated that direct delivery of Mg-ATP using FLVs was effective in maintaining cell viability and Na + K + ATPase pump activity in cells exposed to chemical hypoxia. The third hypothesis was tested using a rat hind limb transplantation model. Hind limbs were harvested and perfused ex vivo with a preservation solution containing Mg-ATP-loaded FLVs. After 13 or 21 hours of ischemia limbs, were transplanted to the recipient. Limbs perfused with the Mg-ATP FLV solution had a greater survival rate than limbs perfused with control solutions. The application of this method of direct cellular energy delivery could have great implications in clinical situations where tissues are exposed to long periods of ischemia such as transplants or limb amputations. Further investigation is needed to optimize this preservation solution to allow its use as routine therapy in these situations.

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