CRYOLIPOLYSIS FOR SUBCUTANEOUS FAT LAYER REDUCTION | Manstein

Our results indicate that non-invasive cooling at the skin surface can cause selective damage and loss of a substantial volume of subcutaneous fat in a pig model. We name this process ‘‘selective cryolysis’’ of fatty tissue. Many important details about selective cryolysis remain to be studied. It is clear that a delayed, cold-induced lobular panniculitis is involved, presumably in response to direct coldinduced injury of adipocytes. In this study, inflammation and adipose tissue loss were well correlated. Both proceeded for many weeks following a single, local exposure to cold, reaching an apparent maximum around 4 weeks after cold exposure and having resolved about 3 months after cold exposure. In its early inflammatory phase, panniculitis may further damage adipocytes. In its later phase, we observed that phagocytosis appears to account for removal of adipocytes and loss of fat tissue.

Temperature and time of application are both important to induce selective cryolysis of fatty tissue. In this study, we used skin surface temperatures near the freezing point of tissue water, applied for 10 minutes to allow time for sufficient transfer of heat from subcutaneous fat though the overlying dermis. A skin surface temperature as high as
18C induced within the various tested anatomical locations in average a mild superficial panniculitis, and the anatomic depth of panniculitis and of fat loss was increased when lower temperatures were applied.

The extent of inflammation was typically greatest near the fat–dermis interface. For the flat application test sites, this band-like distribution of panniculitis extended only about 2–4 mm into the fatty tissue. This probably explains why, in spite of relatively marked inflammation within the superficial fatty tissue, there was no noticeable indentation due to fat loss for the test sites of the flat application. Samples which exhibited much deeper inflammation, also exhibited marked indentations as shown in Figure 8. This test site involved the ‘‘fold’’ exposure geometry, which we tested as a means of optimising the amount and depth of fat cooling. Such test sites exhibited inflammation down to the muscle layer, and the extent of skin indentation in these sites matched nicely the observed loss of fat layer thickness. Taken together, these findings show that the amount of fat loss is related to both severity and thickness of cold-induced panniculitis.

Fat cooling takes time, as shown in Figure 7 during a 78C flat application. More than 3 minutes were needed for the superficial fat to reach 108C at the dermal–fat interface. There are many reasons for the slow cooling of fat. Heat must be extracted from adipose tissue through the overlying dermis, which is several millimeter thick. Dermal blood flow can effectively limit fat cooling by convectional heating of the affected volume. Cold-induced vasoconstriction may reduce dermal blood flow. Applied pressure analogous to a blood pressure cuff can stop local blood flow entirely, eliminating convective heat exchange. For the flat applicators in our dosimetry study, these pressures were not reached. In contrast, the ‘‘fold’’ applicator and the exploratory convex applicator used for the sample shown in Figure 2, were each applied with sufficient pressure to stop or nearly eliminate any cutaneous blood flow. With these high-pressure applicators deep panniculitis and a large volume of fat loss occurred, when compared with the flat applicator used at the same surface temperature. This may also explain the absence of marked indentations for the lipid level study. The pressure used for this applicator was likely not sufficient to suppress blood flow effectively. The exact roles of blood flow in selective cryolysis remain to be determined. Potentially, reperfusion injury may also play a role. Dermal and adipose tissue blood flow were not measured in this study.

Skin and fat can be formed into a fold over most of the body surface because the deep fascia is mobile. Cooling a ‘‘fold’’ of dermis and subcutaneous fat clamped between two cooling elements, further optimizes fat cooling via changing geometry. For flat applicators, there is always a temperature gradient with depth as illustrated in Figure 7. In contrast, when heat is extracted from both sides of a fold in the absence of blood flow, temperature of tissue inside the fold between the cold applicators can become nearly as cold as the skin surface device. We found that selective cryolysis is remarkably selective at both the gross and microscopic levels to the subcutaneous fatty tissue—neither epidermis, dermis nor underlying muscle tissue were affected in any of the test sites. For example, Figure 8 shows adipose tissue inflammation down to the deep muscle layer, with sharply demarcated sparing of the muscle itself. In every test site, skin was colder than the underlying fat.

Therefore, some mechanism exists by which adipose tissue is intrinsically more sensitive to cold, at temperatures that are easily survived by skin. The most likely mechanism, hypothesized when ‘‘popsicle panniculitis’’ was first described, is that crystallization of the cytoplasmic lipids in adipocytes occurs at temperatures well above the freezing point of tissue water. In essence, ‘‘lipid ice’’ forms at much higher temperatures than water ice. Indeed, we have observed at temperatures around 108C that pig fat solidifies, and when observed under cross polarized light, needlelike crystals can be seen within the tissue (Fig. 9). Triglycerides can crystallize around 108C depending on chain length, cooling rate and degree of saturation [19]. Crystallization requires energy, expressed as the latent heat of freezing. It can be seen in Figure 7 that the rate of temperature decrease in superficial fat during treatment with a flat applicator, changes at around 108C. This deflection is consistent with a latent heat exchange due to crystallization of adipocyte lipids. This mechanism is also consistent with the observed need in this study for the time of cold application to be apparently longer than simply related to heat transfer and heat capacity of the tissues involved. It appears that heat must be extracted from the fat, not only to cool it, but to crystallize it.

Taken together, these observations suggest but do not prove that lipid crystallization is responsible for selective injury to adipose tissue. This mechanism may pose challenges for developing selective cryolysis of fatty tissue clinically, as it is known that pigs have a higher percentage of saturated fatty acids compared to humans [20]. Efficient cooling under optimized conditions may be required. On the other hand, the clinical entity of ‘‘equestrian panniculitis’’ [17] suggests that this is possible in at least some adults, and it also indicates that the concurrent application of cold with motion (e.g., massage) may have some synergistic effects. However, the results of the ‘‘lipid level study’’ have shown that cold exposure followed by massage did not result in significant fat loss (for the conditions used). As also a different cooling applicator was used, further studies are necessary to determine the effects of massage or motion during and after cold exposure.

We would like to comment on the observation that the dosimetry study and the exploratory study did not result in any epidermal damage, while the lipid level study resulted in occasional epidermal damage. The reason for this is not known at this point. It can be speculated that the increased thickness of the copper plate may resulted in an initially enhanced heat flux causing a rapid temperature drop and surface freezing at some test sites. This would suggest that future studies should also carefully monitor the heat flux during the procedure. Also the postexposure massage may have contributed to some epidermal trauma.

This study has other implications. For example, the practice of freezing fat grafts at
208C for extended periods prior to reimplantation, is likely to result in graft failure over several months. On the other hand, stem cells and preadipocytes may not be as susceptible to freeze injury. ‘‘Popsicle panniculitis’’ has not been reported to result in noticeable, permanent facial fat loss. However, the number of infants who experienced this rare entity and were specifically examined later in life, may be zero. If cytoplasmic lipid crystallization is the dominant cellular damage mechanism, one would expect stem and preadipocyte cells to survive, because they contain much less lipid than mature adipocytes. On the other hand, these cells may succumb during the subsequent panniculitis, and/or fail to differentiate due to changes in the adipose tissue microenvironment. In our study, there was no evidence that the fat lost after cold exposure could regenerate, although the longest duration of our observations was 3.5 months. At this point, we know little to nothing about the longevity of fat loss induced by selective cryolysis. Alternative mechanisms may explain or potentiate selective cryolysis. These include death of adipocytes mediated by oxidative stress, either as a result of hypothermia as suggested by Rauen and de Groot [21] or as part of reperfusion injury [22]. Nishikawa et al. [23] demonstrated that at normothermic conditions, the fatty tissue is particularly susceptible to reperfusion injury. Localized vasoconstriction which can be triggered by cold exposure may potentially enhance such reperfusion injury. Such mechanism would also be consistent with our observation that firm pressure during application cold exposure appears to enhance the efficacy and depth of fat loss. In adipose tissue, it remains unclear whether the inflammation causes death of adipocytes, or vice versa, whether death of adipocytes causes inflammation, or both. The time course of inflammation which is delayed for a few days, then seen initially as a neutrophilic infiltrate around individual clusters of adipocytes, suggests that piecemeal adipocyte necrosis may initiate the process.

Cryolysis of fatty tissue is possible due to biological selectivity. Hereby we refer to biological selectivity as a specific response (e.g., inflammation) that is confined to a certain tissue (e.g., fat), although other tissues were exposed to a similar or more intense physical stimulus (e.g., colder temperature of the epidermis and dermis as compared to the fat).

Finally, there is the question of where does the fat go, which is lost gradually over weeks to months after a single exposure to cold? In this study, there was no significant rise in serum lipids at any time point after treating 15% of the animals’ body surface. Instead, a significant transient decrease in serum triglycerides was observed, attributable to fasting prior to and during general anesthesia on the day of cold treatment. Possibly, fat loss was so gradual that an increase in circulating lipid levels was not measurable. More likely, the appearance of numerous lipid-laden mononuclear cells at 2 weeks and beyond strongly suggests that the adipocytes are undergoing apoptosis and being removed by phagocytosis, presumably via activated macrophages. This cellular transport process would not be expected to cause any increase in serum lipids. This process is likely to follow usual pathways for adipose tissue turnover. Each year, about 10% of body fat is recycled through adipocyte apoptosis [24].