The Molecular Selectivity of Non-Thermal Irreversible Electroporation and Tissue Regeneration In Vivo

Non-thermal irreversible electroporation (NTIRE) is new minimally-invasive surgical technique for tissue ablation that utilizes molecular selectivity to ablate tissue tumors. Short, microsecond electrical pulses are applied to the tissue, selectively targeting the cell membrane, causing pores to form within the membrane and leading to cell death. This tissue ablation technique has potential for a variety of medical applications, and has shown great promise as a method for treating cancer tumors. NTIRE has many promising attributes as a treatment modality, such as the preservation of tissue scaffolding and the blood vessels. Very little work, however, has been done in examining how the molecular selectivity of NTIRE affects tissue regeneration./nThis work examines how tissues regenerate and recover after NTIRE, with a focus on those critical tissues that are particularly susceptible to collateral damage from treating an adjacent tumor. Two important tissues are examined: the artery and the small intestine. The artery may be embedded within a tumor. Although complete tumor ablation is desired, it is important that the artery can recover quickly in order to aid in overall tissue regeneration at the treated site. It is also important to understand how the molecular selectivity of NTIRE affects the regeneration of the small intestine, especially for the application of abdominal cancer treatment. Damage to the small intestine is often the limiting factor in other types of cancer treatments such as localized radiation therapy, causing pain and discomfort and even resulting in stopping the treatment early. Understanding how the small intestine recovers after NTIRE is essential in developing this technology for treating abdominal cancers such as pancreatic cancer./nFinite element models were utilized to design electrical parameters for both the artery and the small intestine that would cause irreversible electroporation to occur within the tissue while avoiding thermal damage due to Joule heating effects. These electrical parameters were then applied in vivo. Electrical parameters chosen to apply to the artery were an electric field of 1750 V/cm, 90 pulses of a pulse length of 100 μs, and a frequency of either 1 or 4 Hz. The chosen small intestine electroporation protocol consisted of 2000 V/cm, 50 pulses of 70 μs each, and a frequency of 4 Hz. Additional finite element analysis was used to examine the effect of the heterogeneity of tissues such as the small intestine, indicating that changes in electrical conductivity from layer to layer is an important factor that should be accounted for in clinical treatment planning, and future work should include quantifying these electrical conductivity values./nBy applying NTIRE to the rat carotid artery, the recovery of the artery over the week following treatment was observed. It was demonstrated that the electroporation protocol preserved the native tissue extracellular matrix. Three days after NTIRE treatment, the ablated cells had been naturally removed from the tissue, leaving a decellularized construct. By one week after electroporation, new endothelial cells were seen lining the artery lumen. This endothelial layer indicates that normal recellularization is taking place and that the artery is beginning to recover within 7 days of treatment./n/nIn a similar fashion, NTIRE was applied to the rat small intestine in vivo, and the recovery of the small intestine was observed during one week post-treatment. The electrical parameters used were shown to be strong enough to initially cause complete cellular destruction. The extracellular matrix, however, appeared undamaged, and the structure of the small intestine remained intact. The intestine showed signs of recovery, developing an epithelial layer at 3 days post-treatment and regenerating mucosa, submucosa, and muscular layers within a week. These results suggest that the small intestine is only temporarily affected by NTIRE, indicating that this procedure can be utilized for abdominal cancer treatment while minimizing collateral damage to adjacent tissues./nIn addition to examining the recovery of the artery for cancer treatment applications, the potential use of NTIRE to develop a decellularized arterial scaffold was also investigated. The tissue scaffold is a key component for tissue engineering, and the extracellular matrix is nature's ideal scaffold material. Two different methods for applying NTIRE to the artery were compared; the results obtained when plate electrodes were applied across the rat carotid artery were compared to the case when endovascular electrodes were applied to the rabbit iliac artery in a minimally invasive fashion. Both methods were shown to preserve the native extracellular matrix and produce a scaffold that is functional and facilitates recellularization. At 3 days post NTIRE, the immune system had decellularized the electroporated tissue, leaving behind a functional scaffold. The endothelial regrowth at 7 days after treatment indicates that the extracellular matrix still maintained its important components to support cell growth. In addition, this endothelial layer shows promise for the tissue scaffold, helping it to avoid issues such as thrombogenicity that many small diameter scaffolds face.

Advisor: Rubinsky, Boris