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Temperature Measurements for Laser-assisted Cryosurgery in Ex Vivo Mice Hepatic Tissue |
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8/2007-present |
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DESCRIPTION |
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Cryosurgery was first used to treat prostate cancer in the early 1970s but it was not until 1993, when the first published results of percutaneous ultrasound-guided cryosurgery demonstrated the potential advantages of this treatment. Developments in imaging and cryoprobe equipment have improved the surgical outcome of cryosurgery. Nevertheless, prostate cryosurgery is still a surgically challenging procedure because of the strict control required to freeze exclusively the cancerous prostate tissue, while avoiding unintended freezing of the rectal mucosa and urethra. To achieve this goal, the freezing process of current cryosurgical procedures is prematurely stopped resulting in a high incidence of tumor recurrence. Improvements to time-controlled tissue freezing are the urethral warmers and cryoheaters, which intend to protect the urethral and rectal mucosa. Unfortunately, these devices only provide much localized surface heating and, thus, are inefficient in confining the freezing front propagation over a large tissue volume. They also have restrictions in terms of the sites where they can be placed and require a very accurate auxiliary imaging procedure to monitor the growth of the frozen tissue volume or “iceball”. The objective of these studies is to show that regulated laser irradiation may be used effectively to provide volumetric tissue heating with a deeper protection depth compared to that provided by cryoheaters or other superficial heating elements and thus avoid unintended freezing of healthy tissue. Laser-assisted cryosurgery (LAC) better confines the frozen tissue to predetermined, boundaries by establishing a quasi-steady state frozen region that minimizes the demand on accurate imaging. All this while ensuring complete destruction of cancerous tissue and avoiding tumor recurrence. For this purpose, the proposed research method intends to use green fluorescence protein (GFP) for the first time as a viability maker in cryosurgery. By confining with precision the freezing front due to enhanced volumetric laser heating, prostate cancer eradicated through LAC will be possible without the well-known post-surgical complications and malignance tumor recurrence. LAC is performed ex vivo on fresh mice liver samples. The tissue sample is placed in a Plexiglas chamber, and a liquid nitrogen cryoprobe is used to lesion a portion of the liver. A laser optical fiber is used to deliver heat to adjacent tissue, with the goal of limiting the area of damage. The cryoprobe tip and the laser beam are placed at opposite ends of the tissue sample to freeze one portion, while another is protected from cryothermia through the laser heating. Thermocouples are placed between the cryoprobe and laser sight to monitor the gradient of temperature in between. The cryoprobe and the thermocouples are placed with a special stand at even and known distances. Freezing starts as the cryoprobe is turned on, and the temperature histories are recorded during the freezing process while laser heating protects and confines the lesion to a desired location. As a consequence, the freezing front stalls at some distance from the cryoprobe and quasi-steady state is achieved and held. Afterwards, the cryoprobe is turned off, while regulated laser heating prevents the freezing front from further advancing towards the unfrozen side of the sample. After complete thaw of the cryolesion, the cryoprobe and the thermocouples are removed. Figure 1 shows the experimental setup used for the LAC procedures.
Figure 1. Experimental Setup Post-thaw viabilities encompassing the total length of the tissue sample are performed by green fluorescence protein (GFP) expression. GFP is a protein originally isolated from the Aequorea Victoria jellyfish that fluoresces green when exposed to blue light. Since GFP functions as a viability marker in living cells, post-thaw viability assays of the whole organ and slices encompassing the total length of the tissue sample are performed. An advantage of using GFP transgenic mice is that the tissue is assayed without altering the system in any way, other than illuminating with light of the proper excitation wavelength. Macroscopic evaluation of cell viability following a LAC procedure is assessed by detecting fluorescence under UV illumination by collecting images with a CCD camera. The recorded images are then analyzed to determine with precision the extent of dead tissue and to obtain the critical temperatures below which tissue was cryoablated. For cancerous tissue and tissues where necrosis must be produced by freezing, reaching a temperature of -50 0C is the appropriate goal. Therefore, by correlating the limits of cell viability with the recorded temperature histories the value of the lethal temperature is obtained. In order to achieve this, detailed parametric studies are performed to methodically delineate the temporal and physical factors required to consistently produce predictable areas of cell death (see Figures 2-5).
Figure 2. Temperature histories in a LAC procedure.
Figure 3. Quasi-steady state achieved during LAC.
Figure 4. Advancement of the freezing front and laser irradiation for tissue protection during a LAC procedure.
Figure 5. Viability assays using GFP. In Vivo Temperature Measurements Once a basic understanding of the temperature response and interface confinement is achieved, further studies must be performed in vivo. Evidence in previous studies suggests that there is a marked difference between survival of cells treated ex vivo vs. in vivo, and in experiments with implanted tumors, death of all cells in the frozen volume was not immediate. After studying the influence of the freezing and heating parameters in LAC on the ex vivo tissue temperature and viability, in vivo studies will be performed to quantify the influence of blood perfusion in regions where hepatic cryolesions are produced. The importance of the vascular effect will be assessed with experiments in vivo to separate the direct cell injury from that caused by vascular stasis.
In Vivo Temperature Measurements in Human Prostate Cancer Cells in a Xenograft Model Once the ex vivo and in vivo differences have been established using hepatic tissue, the next step in this research project is to perform in vivo experiments with human prostate cancer tissue, and this will be done using a xenograft mice model. The aim is to define the in vivo sensitivities of human prostate cancer cells to LAC and to define the minimum temperature required to cause adequate cryoablation and prevent tumor recurrence.
3-D Experimental Measurements In the fourth part of this study, LAC will be performed to simulate a clinical prostate cryosurgical procedure in a tissue-equivalent prostate phantom. Again, temperature will be monitored with thermocouples to determine the end temperature of the cryosurgical procedure and freezing will be done with liquid nitrogen needle cryoprobes. Two endoscopic optical fibers coupled to the same laser will be introduced via the model’s urethral and rectal canals, to provide the laser heating necessary to confine the growth of the frozen tissue. Experiments will be conducted to compare the temperature variations and thickness of protected tissue. Based on the experimental results, a parametric assessment of the penetration depth and accuracy of the peripheral confinement will be determined. The target lethal temperature in all thermocouple positions will be set based on the viability assays performed in the ex vivo and in vivo studies. Future work in this project requires pre-clinical trials with experimental apparatus that account for realistic clinical applications such as complex prostate geometries, asymmetrical urethra dimensions, and uneven cryoprobe placement. This work must be performed before this procedure can transition from the experimental stage proposed to clinical trials, but this stage is beyond the scope of this work. |
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