Imaging cryosurgery with EIT: tracking the ice front and post-thaw tissue viability.

Cryosurgery employs freezing for targeted destruction of undesirable tissues such as cancer. Ice front imaging has made controlled treatment of deep body tumors possible. One promising method, recently explored for this task, is EIT, which recovers images of electrical impedance from measurements made at boundary electrodes. However, since frozen tissue near the ice front survives, ice front imaging is insufficient. Monitoring treatment effect would enable iterative cryosurgery, where extents of ablation and need for further treatment are assessed upon thawing. Since lipid bilayers are strong barriers to low frequency electrical current and cell destruction implies impaired membranes, EIT should be able to detect the desired effect of cryosurgery: cell death. Previous work has tested EIT for ice front imaging with tank studies while others have simulated EIT in detecting cryoablation, but in vivo tests have not been reported in either case. To address this, we report 3D images of differential conductivity throughout the freeze-thaw cycle in a rat liver model in vivo with histological validation, first testing our system for ice front imaging in a gel and for viability imaging post-thaw in a raw potato slice.

In vivo inductive phase shift measurements to detect intraperitoneal fluid.

Four different volumes of physiological saline were infused into the abdominal cavity of rats and the resulting inductive phase shift in the bulk of the abdomen was measured with a noncontact electrical induction system, built to measure phase shift in the bulk of the body in the frequency range from 1 MHz to 8.5 MHz. This experimental study shows that inductive bulk measurements of phase shift have the potential to detect changes in intraperitoneal fluid in vivo with measurements made at frequencies higher than approximately 1 MHz. The experiments also show that the bulk phase shift increases as a function of frequency and fluid volume in a way that is qualitatively consistent with earlier theoretical predictions.

In vivo results of a new focal tissue ablation technique: irreversible electroporation.

This paper reports results of in vivo experiments that confirm the feasibility of a new minimally invasive method for tissue ablation, irreversible electroporation (IRE). Electroporation is the generation of a destabilizing electric potential across biological membranes that causes the formation of nanoscale defects in the lipid bilayer. In IRE, these defects are permanent and lead to cell death. This paper builds on our earlier theoretical work and demonstrates that IRE can become an effective method for nonthermal tissue ablation requiring no drugs. To test the capability of IRE pulses to ablate tissue in a controlled fashion, we subjected the livers of male Sprague-Dawley rats to a single 20-ms-long square pulse of 1000 V/cm, which calculations had predicted would cause nonthermal IRE. Three hours after the pulse, treated areas in perfusion-fixed livers exhibited microvascular occlusion, endothelial cell necrosis, and diapedeses, resulting in ischemic damage to parenchyma and massive pooling of erythrocytes in sinusoids. However, large blood vessel architecture was preserved. Hepatocytes displayed blurred cell borders, pale eosinophilic cytoplasm, variable pyknosis and vacuolar degeneration. Mathematical analysis indicates that this damage was primarily nonthermal in nature and that sharp borders between affected and unaffected regions corresponded to electric fields of 300-500 V/cm.

Improved viability and reduced apoptosis in sub-zero 21-hour preservation of transplanted rat hearts using anti-freeze proteins.

BACKGROUND: Freeze-tolerant fish survive sub-zero temperatures by non-colligatively lowering the freezing temperature of their body fluids using anti-freeze proteins (AFPs). We sought to evaluate and compare the effects of prolonged sub-zero cryopreservation of transplanted rat hearts using AFP I or AFP III. METHODS: Two heterotopic rat heart transplantation protocols were used. In Protocol 1 (n = 104), hearts (n = 8/group) were preserved for 12, 18 and 24 hours in University of Wisconsin solution (UW) at 4 degrees C, UW at -1.3 degrees C, UW/AFP I at -1.3 degrees C and UW/AFP III at -1.3 degrees C, with and without nucleation. Post-operative evaluation consisted of visual viability scoring of the hearts after 60 minutes. Protocol 2 (n = 58) involved evaluation of 24-hour post-transplant viability, echocardiography (fractional shortening [FS], left ventricular end-systolic and -diastolic diameter [ESD, EDD] and anterior and posterior wall systolic and diastolic thickness [AWT-S, AWT-D, PWT-S, PWT-D]), TUNEL staining and electron microscopy (EM) findings for hearts preserved for 18, 21 and 24 hours in UW at 4 degrees C or UW/AFP III at -1.3 degrees C. RESULTS: Hearts preserved in UW at -1.3 degrees C with nucleation froze and died. Three of 8 hearts preserved in UW at 4 degrees C for 24 hours died, whereas all hearts preserved at -1.3 degrees C survived. Hearts preserved in UW/AFP for 18 and 24 hours at -1.3 degrees C had superior viability scores compared with those in UW at 4 degrees C. Hearts in AFP III at -1.3 degrees C displayed greater AWT-S and AWT-D (3.5 +/- 0.2 vs 2.4 +/- 0.2, p < 0.05, and 3.5 +/- 0.2 vs 2.2 +/- 0.2, p < 0.05, respectively) after 18-hour preservation. In the 21-hour preservation group, AFP-treated hearts displayed improved echocardiographic systolic contraction indices, including: improved FS (27 +/- 3.7 vs 15 +/- 4, p = 0.04); diminished ESD (0.28 +/- 0.57 vs 0.47 +/- 0.6, p < 0.05); greater AWT-S (3.4 +/- 0.18 vs 2.8 +/- 0.2, p < 0.05); and fewer positively TUNEL-stained nuclei per specimen (35 +/- 14 vs 5.3 +/- 2.7, p = 0.04). Also, improved EM scores were noted compared with UW at 4 degrees C. CONCLUSIONS: In prolonged sub-zero cryopreservation, AFPs protect the heart from freezing, improve survival and hemodynamics, and reduce apoptotic cell death.

Temperature dependence of tissue impedivity in electrical impedance tomography of cryosurgery.

The temperature-dependent impedivity of rat liver, transverse abdominal muscle and full skin was determined in vitro as a function of frequency across the temperature range 5 degrees C to 37 degrees C and from 100 Hz to 10 kHz. This study was motivated by an increasing interest in using electrical impedance tomography (EIT) for imaging of cryosurgery and a lack of applicable data in the hypothermic range. Using a controlled-temperature impedance analyzer, it was found that as the temperature is reduced the resulting increase in tissue impedivity is more pronounced at low frequencies and that the beta dispersion, resulting from cell membrane polarization, shifts to lower frequencies. With these new data a simple case study of EIT of liver cryosurgery was examined, using a finite-element model incorporating the Pennes bio-heat equation, to determine the impact of this behavior on imaging accuracy. Overestimation of the ice-front position was found to occur if the EIT system ignored the effects of the low-temperature zone surrounding the frozen tissue. This error decreases with increasing blood perfusion and with higher measurement frequencies.

Subzero nonfreezing cryopresevation of rat hearts using antifreeze protein I and antifreeze protein III.

The purpose of the present study was to evaluate whether AFPs protect the heart from freezing and improve survival and viability in subzero cryopreservation. Hearts were subject to 5 preservation protocols; University of Wisconsin solution (UW) at 4 degrees C, UW at -1.3 degrees C without nucleation, UW at -1.3 degrees C with nucleation, UW AFP I (15 mg/cm(3)) at -1.3 degrees C with nucleation, and in UW AFP III (15 mg/cm(3)) at -1.3 degrees C with nucleation. Hearts were preserved for 24, 28, and 32 h, rewarmed and connected to the working isolated perfusion system. Data [heart rate (HR), coronary flow (CF), and developed pressure (dP)] was collected 30 and 60 min after reperfusion. Hearts preserved at -1.3 degrees C without AFPs froze, while hearts preserved with AFP did not freeze when nucleation was initiated and survived. Survival and dP of hearts preserved for 24h at -1.3 degrees C using AFP III was better than those preserved at 4 degrees C, (dP; 1.4 vs. 0.8, p<0.05). Four of six hearts and six of six hearts died when preserved at 4 degrees C for 28 and 32 h, respectively, all of the hearts that were preserved at -1.3 degrees C with or without AFPs survived after 28 h (n=18) and 32 h (n=18). CF was higher in UW -1.3 degrees C group without attempted nucleation than in AFP I and AFP III groups after 28 and 32 h (3.4 vs. 1.7, p<0.05, and 3.4 vs. 1.7, p<0.05, respectively). In conclusion, AFPs were found to protect the heart from freezing and improve survival and dP (AFP III) in prolonged subzero preservation.

Prolonged 24-hour subzero preservation of heterotopically transplanted rat hearts using antifreeze proteins derived from arctic fish.

BACKGROUND: Arctic fish survive subzero temperatures by producing a family of antifreeze proteins (AFPs) that noncolligatively lower the freezing temperature of their body fluids. We report 24-hour storage of mammalian hearts for transplantation at subzero temperatures using AFPs derived from arctic fish. METHODS: Forty-two heterotopic transplantations were performed in isoimmune Sprague-Dawley rats. Harvested hearts were retrogradely infused with cold 4 degrees C University of Wisconsin (UW) solution and were preserved in a specialized cooling bath at two target temperatures, 4 degrees C and -1.3 degrees C for 12,18, and 24 hours (6 experiments/group). Preservation solutions were UW alone for the 4 degrees C group, and UW with 15 mg/mL AFP III for the -1.3 degrees C group. After hypothermic storage the hearts were heterotopically transplanted into isoimmune rats. Viability was assessed and graded on a scale of 0 to 6 (0 = no contractions to 6 = excellent contractions). Transplanted hearts were then fixed in vivo and were subject to electron microscopy and histopathologic examination. RESULTS: None of the hearts preserved at -1.3 degrees C in UW/AFP III solution froze. All control hearts preserved at -1.3 degrees C without AFP protection froze and died at reperfusion. Viability of hearts preserved at -1.3 degrees C in UW/AFP III solution was significantly better after 18 hours of preservation, 30 and 60 minutes after reperfusion (median, 5 versus 3 and 6 versus 3, respectively; p < 0.05) and after 24 hours of preservation 30 and 60 minutes after reperfusion (median, 4.5 versus 1.5 and 5 versus 2, respectively; p < 0.05). Histologic and electron microscopy studies demonstrated better myocyte structure and mitochondrial integrity preservation with UW/AFP III solution. CONCLUSIONS: Antifreeze proteins prevent freezing in subzero cryopreservation of mammalian hearts for transplantation. Subzero preservation prolongs ischemic times and improves posttransplant viability.

Preservation of myocyte structure and mitochondrial integrity in subzero cryopreservation of mammalian hearts for transplantation using antifreeze proteins--an electron microscopy study.

OBJECTIVE: Freeze tolerant fish and insects in nature are able to survive subzero temperatures by noncolligatively lowering the freezing temperature of their body fluids using a family of thermal hysteresis proteins (antifreeze proteins, AFPs) specific for each species. Past efforts to cryopreserve mammalian hearts using these proteins were unsuccessful. We report the first successful subzero cryopreservation of rat hearts using fish derived antifreeze proteins with preservation of myocyte structure. METHODS: Heterotopic heart transplantations were performed in isoimmunic Sprague Dawley rats. Donors' hearts were arrested using University of Wisconsin (UW) solution and preserved in UW solution containing AFP I (six experiments) or AFP III (six experiments) at concentrations of 15-20 mg/cc for 2-6 h at subzero temperatures ranging from -1.1 to -1.3 degrees C. Four control experiments were performed by preserving harvested hearts in UW solution alone at -1.3 degrees C for 6 h. In all experiments ice was added in the solution for crystallization. Heterotopic transplantations were performed in the abdomen of the recipient rats. Viability was visually assessed and graded on a scale of 1 (poor contraction) to 6 (excellent contraction). The hearts were then fixed in vivo and processed for electron microscopy study. RESULTS: All hearts preserved at subzero temperatures using AFP I or AFP III survived displaying viability scores of 4-6 1 h after transplantation. Three of the four control hearts that were preserved at -1.3 degrees C without the protective effect of AFP froze and died upon reperfusion. Electron microscopy study of hearts preserved with AFP demonstrated preservation of myocyte structure and mitochondrial integrity. CONCLUSION: Subzero cryopreservation of mammalian hearts for transplantation using AFP I or AFP III is feasible with preservation of myocyte structure and mitochondrial integrity.