The following studies were performed at different time points with distinct emphasis. Therefore different cooling devices were used. For the initial exploratory study a simple cold applicator was employed, the dosimetry study used an electronically regulated thermoelectric chiller in order to maintain a well-controlled, preset surface temperature, and the lipid level study was performed with an applicator that had an increased size to facilitate the cold exposure of relative large areas.
Exploratory Cold Exposures (N ¼ 1)
A black Yucatan pig (female, 6 months of age, approximately 25 kg) was used to explore the feasibility of removing subcutaneous fatty tissue by local, non-invasive cold exposure.
This pig was exposed to a laboratory cooling prototype device that had as the cold applicator a slightly convex, circular copper plate (curvature radius approximately 10 cm, and diameter 5.5 cm), designed to be pressed firmly against the skin surface. The copper plate was cooled with a laboratory chiller (Neslab RTE 100, Thermo Electron Corp. Newington, NH) by circulating a cold antifreeze solution at 78C through a heat exchanger chamber attached to the copper plate. The chiller was operating in constant temperature mode.
The cold applicator was mounted to a springloaded handpiece that required a pressure of approximately 150 N to fully compress the spring, providing a pressure of approximately 6 N/cm2 (450 mmHg) upon the skin. Cold exposures were performed under general anesthesia using Telazol/Xylazine 4.4 mg/kg i.m.þ2.2 mg/kg i.m. and inhalant anesthetics (Isoflurane (1.5–3.0%) with oxygen (3.0 L/min)) delivered by mask.
Several test sites were mapped with microtattoos on the flank, buttock and abdomen. All test sites were shaved before exposure. A glycerol/water lotion was applied between the skin and the cooling device to increase thermal contact. The device was applied with such force during the exposure that the spring was fully compressed in an attempt to inhibit blood flow within the test sites.
In 3 out of 11 test sites (see Table 1) the test sites were located within the flank area. At these sites the application pressure was reduced (spring only partially compressed) in order to avoid suppression of the respiration by prolonged mechanical compression of the rib cage. Exposure time varied arbitrarily between 5 and 21 minutes. This pig was observed for 3.5 months for the appearance and persistence of local fat loss, and then euthanized to obtain gross and histological sections stained with hematoxylin and eosin. The amount of fat loss in each test site was estimated relative to adjacent unexposed fat layer thickness at the margins of each cold exposure site.
Dosimetry Study (N ¼ 4)
The study was performed on four female black Yucatan pigs (retired breeders) of 2–3 years of age, body weight approximately ca. 100 kg.
Cryolysis Device for Fatty Tissue
The Zeltiq prototype device (Zeltiq, Pleasanton, CA) contained a thermoelectric TE cooling element assembled with an active surface cooling area of 34 cm2 , and a maximum heat flux of approximately 10 W/cm2 at dT ¼ 08C and 6 W/cm2 at dT ¼ 308C. The excess heat from the TE element was removed by a PolyScience 5306 (PolyScience, Niles, IL) liquid chiller operating as a close loop system.
A variable, preset plate temperature was maintained constant during each cold exposure, by electronic regulation according to temperature sensors embedded within the cooling plate. Most test sites were performed with a single flat cold applicator simply held against the skin surface (Fig. 1a). In this configuration, subcutaneous fat is cooled through the overlying dermis while its deep fascia margin is still in contact with underlying muscle. The deep fascia between fat and muscle is a sliding layer (allowing muscular motion); due to this mobility, a ‘‘fold’’ of skin and subcutaneous fat can be easily created by pulling. Test sites were also exposed in ‘‘fold configuration’’ (Fig. 1b), using two cooling plates of the same size applied on the sides of the skin fold with pressure.
All procedures were performed under general anesthesia as described above. All test sites were shaved before exposure. Approximately 60 cold exposure sites and 10 control sites were mapped and individually marked at the corners with India Ink microtattoos at the fist visit within the flanks, abdomen and buttocks. Each Yucatan pig was exposed within mapped test sites at selected time points (28 days, 14, 7, 2, 1 and immediately 1 hour) before euthanasia with preset plate temperature settings (20, 1, 3, 5, 78C) for 10 minutes. Each exposure temperature was performed in duplicate at each time point. At each procedure day the assignment of the test sites to the different exposure temperature and follow up intervals was randomly assigned to avoid potential anatomic or other systematic bias. To ensure good thermal contact from skin to applicator, an ultrasound gel (Ultrasound Gel, Medline, Mundelein, IL) was applied to the skin before each cold exposure. Flat exposures were performed holding the device with a contact pressure of approximately 1 N/cm2 (75 mmHg) to the skin. Because the ‘‘fold’’ application required stretching the skin into a fold between two cooling plates, these test sites were limited to the abdomen which has sufficient skin laxity in the pig. Substantial pressure was applied to the skin on each side of the ‘‘fold,’’ sufficient to reduce or eliminate blood flow within the skin fold.
Test sites and surrounding areas were clinically assessed and standardized photography of individual test sites was performed. A diagnostic ultrasound imager (SonoSite 180 with a 7.5 MHz linear transducer, SonoSite, Inc, Bothell, WA) was also used for some test sites. After euthanasia, tissue was processed for gross pathologic and histologic evaluation. Deep (skin, fat, underlying muscle) tissue vertical sections were stained with hematoxylin and eosin and evaluated microscopically to assess the level of fat damage as well as potential damage to the dermis or epidermis. In addition to blinded qualitative assessment, the extent of inflammation within the adipose tissue close to the dermal–fat junction was graded by a blinded investigator on a scale from 0 to 3.
Slides exhibited in Figure 5 were used as reference for the grading. Intermediate half grades were allowed in case the clear assignment to a specific level was ambiguous. Besides these test sites, some additional test sites were used in order to measure the tissue temperatures in vivo during cold exposure. Thin thermocouples designed for tissue insertion (Hyp-1 type, 30 gauge, Omega, Stamford, CT) were put at the surface of the skin and inserted laterally just below the dermal–fat junction. Depth and proper location of the tip of the inserted couple was confirmed by the ultrasound device. Superficial and deep thermocouples were sutured into place to minimize displacement and movement artifacts during cold exposure. These test sites were not used for histological evaluation as the inserted thermocouples could have induced tissue alterations.
Repeated-measures mixed model analysis of variance (ANOVA) was used to evaluate differences in fat damage (0–3 ordinal scale) according temperature and time with damage examined at multiple test sites from the same animal. F-tests were used to determine effects of temperature and time on fat damage and Akaike’s information criterion (AIC) was used to assess goodness-of-fit of the statistical model.
Lipid Level Study (N ¼ 6)
Six adult female black Yucatan pigs (retired breeders) 2–3 years old, weighing approximately 100 kg were used. The animals were fasted for 12 hours prior to cold exposure under general anesthesia to minimize the risk of aspiration, and were otherwise maintained on a standard porcine diet with feedings ad lib. Test sites were exposed to a largearea laboratory prototype cooling device consisting of a flat copper plate of 2.5 cm thickness and 11.511.5 cm2 area, mounted to a thermoelectric cooling element. The thermoelectric TE cooler was a LHP-800CP (TECA Corp., Chicago, IL) with a specified maximum heat flux of approximately 1.5 W/cm2 at dT ¼ 08C and 0.5 W/cm2 at dT ¼ 308C. The entire cooling element had a weight of 6 kg. An on-off thermostatic temperature controller (TC3300, TECA Corp.) was used in this system. Embedded within the surface of the copper plate was a temperature sensor connected to a control unit that triggered a relay to provide power to the thermo electric plate, when the sensor temperature was above a preset temperature. Similar to a common house thermostat with on-off rather than proportional power control, there was fluctuation of the copper plate temperature by approximately 18C around the set point temperature. The copper plate was pre-cooled to the set temperature before it was applied to the test sites, therefore causing an enhanced heat flux during the initial phase of the exposure. The excess heat from the TE cooler was removed by a MLA 270 DS-115 Melcor (Melcor Corp., Trenton, NJ) liquid chiller operating as a close loop system. Each Yucatan pig was exposed in approximately 15 skin areas of 11.511.5 cm2 for 10 minutes, equivalent to about 15% body surface area, with the cold plate set at 58C to 88C. The weight of the applicator resulted in a contact pressure of approximately 0.5 N/cm2 or 38 mmHg. No additional manual force was applied to the device. To ensure good thermal contact from skin to applicator, an ultrasound gel (Ultrasound Gel, Medline) was used and the hairs of the pigs were shaved prior to each exposure. Immediately after each of these exposures, a 1 minute massage in the test sites was performed using a commercially available vibrating massager (Brookstone, Inc., Merrimack, NH).
For each animal, all cold exposure procedures were performed on a single day under general anesthesia as described above. The subsequent blood draws were performed after i.m. sedation on animals fasted for 12 hours. Pigs were observed clinically and blood samples were obtained after a 12-hour fast prior to treatment, within 1 hour and 1 day, 1 week, and 1, 2, and 3 months post-treatment. In addition, lipids were evaluated throughout the time course (pre-exposure, post-exposure, 1, 3, 14 days, 1, 2, and 3 months) using repeated-measures ANOVA. Two-tailed values of P<0.05 were considered statistically significant using the post hoc Fisher least significant difference procedure. Data are presented in terms of the mean and standard error of the mean (SEM). Statistical analysis was performed using the SPSS software package (version 16, SPSS, Inc., Chicago, IL).