54 EQUIPMENT I body language Pyroptosis of fat DR DIANE DUNCAN discusses the BodyFX treatment and how cell mechanisms affect clinical results

hen we talk about have programmed we ways to destroy fat, would be massive creatures. we loosely use the By definition there’s no inflam- Wterms and mation at all when an apoptotic because these are the process occurs. mechanisms that are most com- Necrosis, on the other hand, monly known. Here I’d like to is severely inflammatory, but it is highlight the importance of bear- not the ideal mechanism for fat ing in mind the particular way in reduction either. Necrosis causes which a cell dies. Consideration an instant demise of the affected of how the cell dies and how this cell. The cell membrane ruptures, mechanism of death will affect the causing the release of lysozymes clinical outcomes is an important into the surrounding tissue and the and somewhat overlooked premise. involved cells undergo significant swelling. Another word for the pro- Ways to kill fat cess is “oncosis”, as necrosis is more Until recently we’ve only thought correctly used as identifying the of the two polar opposites of ways cell when it is dead. to kill fat. Apoptosis is really silent cell death and this is what happens Necrosis and apoptosis when our cells die in order keep Why is neither necrosis nor ap- the total population of cells stable. optosis an ideal mechanism when About 100,000 cells per second contouring the face, neck, or body? undergo apoptosis in a human be- In apoptosis there is no inflam- cause our cells also divide and un- mation, and in necrosis, the swell- has a thick, sturdy fibrovascular dergo mitosis. A balance must be ing and bruising cause significant network that holds the fatty layer kept. As you know embryos tend to down time. Controlled inflamma- together and binds it to the under- have a tail, and it’s apoptosis that tion is desirable, as with time, the lying fascia and overlying skin. A makes the cells of that tail disap- body loses its support system for 44 year old has lost about 50% of pear without a scar. If we didn’t soft tissue (figure 1). A 23 year old this support network, allowing re- gions of fat to become pendulous and quite saggy. By age 60, about CELL DEATH 85% of the fibrous tissue binding Apoptosis is a normal, always ongoing process in humans that programs fat cells together has eroded away. cells to die in order to offset the number of new cells created by mitosis. When we use the term “skin lax- Necrosis is immediate, sudden, and very inflammatory, so patients treated ity”, we are actually talking about with a necrosis inducing regimen—such as injection lipolysis—will see a lot a combination of the skin and adi- of swelling and bruising. Both necrosis and pyroptotic-like mechanisms are pose layer. The loss of attachment pro-inflammatory. One has poration and one does not. One has cell shrink- to the underlying fascia, plus the age and one is characterized by rupture. loss of a scaffold that is knitting Apoptosis and necrosis are not the only mechanisms of cell death; others the fatty layer together, cause the include , pyroptosis, independent apoptosis, paraptosis, flabby character of ageing skin Wallerian degeneration, cornification and anoikis. and soft tissue. In many cases, the nature of the soft tissue is actu-

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scar tissue formation. A desired outcome would be restoration of the youthful fibro- vascular support system to the adipose layer. This would require a fractional and somewhat uni- form response—layers of tendrils of collagen that are interspersed 23 years old within the fatty layer. A multilevel response would be needed, and the response would need to be able to be controlled—more fibrous sup- port in some areas, like the jowl or lower abdomen and a bit less in areas that do not tend to be pendu- lous, like the cheek, or outer thigh. The fibrous support response alone 44 years old can be generated with a moving external radiofrequency device like Forma or Plus.

Results and temperature I did a study in which I looked at serial scanning electron microsco- py sections of fat, treated over time 60 years old with Forma at different maximum Figure 1: Young adipose tissue has a strong fi- temperatures: 40, 41, 42, and 43 brovascular support framework. Middle aged degrees Celsius. We looked at adi- tissue has lost part of the binding and struc- tural tissue. Older tissue has retained very little pose cells immediately following remaining connective tissue, allowing the soft the eighth weekly treatment, at one tissue to appear lax and pendulous month following treatment cessa- tion, and again at three months fol- ered, and no fat death was noted lowing treatment cessation. We had with Forma treatments. two questions: one, could moving Contrary to the belief that bulk RF alone kill fat? And two, does a heating, especially radio frequency hotter temperature which may not bulk heating, doesn’t work, I have be well tolerated by the patient cre- proof that it does. However, the ate a better tissue response? Figure level of fibrous ingrowth into the 2 shows a matrix of SEMs showing adipose layer did not vary as sig- tissue response over time at differ- nificantly with temperature level ent temperatures. Temporary cell as originally thought. We’ve been deformation increases with higher instructed to get the tissue as hot ally more important than that of temperatures. If you read the ther- as possible, however even at 40 de- the skin. mal literature, fat must get to about grees, there was a significant tissue 55 degrees Celsius in order to cause response in treated areas. Patients Soft Tissue Contouring necrosis. It is extremely difficult to tend not to come back to repeat The ideal mechanism for soft tissue create that level of heat transcuta- painful experiences. Clinical re- contouring would be somewhere neously with external RF alone. In- sults are just as good with lower between apoptosis—with no in- terestingly, at one month and three temperatures and longer treatment flammation, and necrosis’ rampant months, all adipocytes had recov- times.

40 41 42 43

Figure 2: Effect of temperature difference on fibrous tissue response at three months post treatment with moving external RF only. All tissue has fibrous and vascular in- growth. More cell deformation is present at 43 degrees. If tissue support and molding is the goal, keeping the temperature at 42 degrees or below may be best, as a small amount of fat may die at higher temperatures.

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Repeated Measures Before treatment Mean/ SD After 1 month Mean/ SD After 3 months Mean/ SD ANCOVA US @ 0 degrees 2.63+/- .283 1.74 +/- .202 1.68 +/- .218

US @ 90 degrees 2.83 +/-.160 1.57 +/- .164 1.55 +/- .188 Figure 3: High reso- lution ultrasound US @ 180 degrees 2.60 +/- .249 1.71 +/- .184 1.76 +/- .204 fat thickness meas- US @270 degrees 2.82 +/- .237 1.49 +/- .150 1.57 +/- .179 urements Multi- variate ANCOVA : * p=<.05 The standard for statistical significance 53.25% fat thick- **p=<.001 Stronger statistical significance ness reduction at three months post The repeated measures analysis (ANCOVA) has been adjusted for age and height last treatment

Tissue tightening and fat The importance of death lowers the poration threshold. It reduction over time doesn’t really cause induction of fat A device like BodyFX also has Most people understand that the death—we treat anywhere between moving external radiofrequency. inflammatory process in humans 40°C and 43°C—but it does cause The handpiece is suction-cou- takes time. There is about a six- the induction of fibrous response. pled, which means that the target week healing process after surgery Different devices that claim fat loss tissue is firmly held so that heat and the strength of the repair of a with only moving RF are not very can better get to it. The depth of surgical incision gains about 10% likely to work, given this research. energy penetration is also con- per month. This is a process that I’ve done a lot of scanning of what trolled this way. However, the cannot be hurried. If adipose cells happens when tissues are heated secondary electrical impulse is die over time, there is no discom- using scanning electron micros- the secret ingredient that makes fort, no swelling, no bruising, and copy, SEM, which is much easier the fat die. no down time. With simple exter- for people to understand than his- One of the recent advances in nal RF heat, the pattern of fibrous tology. Adipocytes are notoriously medicine is reversible electropo- infiltration has barely begun at difficult to fix and cut in histologic ration, which is used to introduce the end of the eighth treatment, sections as the cells can easily frac- small molecules into a cell, cause is stronger one month following ture with processing. The SEM cell fusion, or enable genetic al- treatment cessation, and has be- doesn’t have those artefacts. What teration. This causes temporary come quite visible at three months. you see with electron microscopy relaxation of the pores in the A similar process is seen with the cannot be manipulated. cell membrane of target cells, so Body FX; fat takes at least three Using an SEM I have studied that medicine or other “genetic months to optimally remodel. pieces of tissue as a control (figure directions” can get through. A Fractional cell death is seen; that 4) that have been treated with heat newer and rapidly growing field, means not all of the fat cells in a and vacuum alone without the high especially in the arena of given region die, but a significant voltage pulses. These high voltage treatment, is irreversible elec- number do. Fibrous support is re- pulses are the a third step and the troporation. With reversible elec- stored, and fat layer thickness is patient will feel this as a thump, troporation, cells can be altered, reduced by an average of 40% as since a feeling of an electric shock but none die. With irreversible measured by high-resolution ultra- would really be an adverse stimu- electroporation, the holes in the sound (figure 3). lus. Mechanically the large size of pores cannot be reversed, and the With the BodyFX, the external the fat cells is what makes them cells are programmed to die over RF heat is only the introduction— more susceptible to injury. Their time. it sensitises the adipocyte and blood supply is poor, so they are

Figure 4: Tissue treated with external RF plus vacuum but no high voltage Figure 5: Adipocytes treated with Body FX including high voltage pulses, effect pulses shows no adipocytolysis at three months. Cells appear similar to tissue at three months. Note lipid droplet egress continues. Critical volume loss has treated with external RF alone. occurred

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How do apoptosis, necrosis and pyroptosis differ? In looking at the apoptotic process, you can see some cellular budding and eventual separation of these buds (apoptotic bodies). Macrophages will clean up cellular debris. By definition, there is no inflammation. The mechanism is caspase mediated. With necrosis, immediate swelling and bursting of cells causes significant inflammation in the affected area. It is not caspase mediated. Pyroptosis is caspase mediated, which explains why early researchers thought the Body FX mechanism was apoptotic. Also, apoptosis occur over time, so the slow response with no down time was assumed to be apoptotic. With pyroptosis, poration causes the cell to become “leaky”. Cell biology literature notes that when a cell cannot repair the damage to its membrane, it will go on to die.

more easily damaged than other while looking at people who had you can see the fibrocyte trying to smaller cells. infection with salmonella and shi- mend that crack so the cell won’t SEM images (figure 5) show gella and observing that the cell die. In breaking down the mechan- how tissue looks over time after creates little pores in the cell mem- ics of cell death we know that if the treatment with the BodyFX device brane. Some of the cytosol then fibrocyte can mend that tear then with the high voltage pulses. The leaks out, cells shrink and then the cell won’t die—but that it can’t lipid droplets leak out, or leave the cytokines signal the cell to die. Py- survive with many tears and a lot a cell through these membrane de- roptosis is the current best model to lipid droplets that egress, or signifi- fects. In looking at thousands of help explain what is going on in the cant volume loss. treated cells, we note very few have Body FX process, but the process is The current gold standard in actual membrane rupture. The not identical. fat reduction is liposuction. Li- membrane has many micro-inju- In order to see the whole picture posuction removes some, but not ries—and there must be enough, of I took some control samples of fat all of the fat, so that the tissue a degree that cannot be repaired by tissue injected with saline, but no that remains behind still acts like the cell, in order to cause perma- mechanical or electrical impulses, “soft” tissue. After liposuction, the nent cell death. to illustrate what a normal area of remaining tissue can glide over fat cells look like for comparison. underlying structures, the surface Pyroptosis The early effect of the BodyFX is ideally remains smooth, and the Originally discovered by Brad totally different than anything fatty layer is reduced. With Body Cookson in 2001 pyroptosis is a that we’ve seen before. You can see FX, a similar response is seen; fat pro-inflammatory mechanism. some early cracks in the cell mem- reduction occurs, but it may not Cookson discovered pyroptosis brane, which is interesting because be as significant as with liposuc-

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really interesting and we’re barely ideal, as some, but not all of the fat on the verge of understanding is affected. There is some inflam- them. The radio frequency-induced mation, and I believe this is a good mechanism is pyroptosis-like. Cur- way of inducing fibrous scaffold rently we call it “poroptosis” be- restoration. However, cryolipolysis cause the mechanism appears to is also very interesting because this be irreversible poration that doesn’t is also pro-inflammatory. We’re

have any real relationship with the just beginning to study both of Top: 52 year old Figure 6: Adipocyte size reduction 6 weeks infectious process of Cookson’s. these mechanisms and clearly more before and three post cryolipolysis. Average adipocyte size The cryolipolysis mechanism of work needs to be done. months following varies from 50- 200 microns, with a mean action is clearly not inflammation- eight Body FX treat- size of 100 microns. Most adipocytes in this ments. Weight gain field of treated tissue measure less than free; it’s not apoptosis, but it’s not Diane Duncan is a Board Certi- was one pound. 100 microns; all appear viable. necrosis either. We didn’t see a sin- fied Plastic Surgeon and has been in Bottom: 63 year old before and three gle ruptured cell in our SEMs. Cry- practice in Colorado for over 26 years. months following optosis has a very different mecha- She specialises in facial enhancement, eight weekly treat- nism of action than anything we’ve breast surgery, and body contouring ments. Fat reduc- tion and soft tissue seen before. and teaches new surgical and non- laxity correction has surgical techniques around the world. occured. Age differences There are age-related differences as well as ethnic variations in tis- sue type. You can see at age 23 you Figure 7: Four months following cryolipoly- sis, viable fat cells show persistent size re- have a lot more inherent fibrosis or duction in the treatment region, although it fibre support of the fat tissue, at age is less uniform. 100 microns is the average 44 there are patches where the sup- adipocyte diameter. Note extensive fibrosis in this patient, control untreated tissue ap- port tissue is gone. By age 60 the pears on the left. fat cells are barely held together with little threads of fibrotic tis- tion. However, there is not a lot of sue. Darker skin type patients tend residual scar tissue, and tissue lift to have more fibrotic soft tissue, can clearly be seen. which explains the lesser effect of transcutaneous treatments such as Cryolipolysis cryolipolysis and radiofrequency at As another control study, I looked lower settings; the adipose tissue is the mechanism of action of cry- insulated from both heat and cold olipolysis because I wanted to com- by the extra amount of fibrous tis- pare it to that of RF. The officially sue. By knowing these things, we agreed mechanism of cryolipolysis can optimise the clinical outcome. is apoptosis. However, by compar- Fat reduction plus tissue lift, I ing the tissues under the SEM it think, gives the best outcome in becomes apparent that his cannot most patients. be true. The mechanism appears to In looking at before and af- be mechanical in the early stages. ter images from the BodyFX, you In early SEMs you can see little can see some definite tissue lift as crimps of folds in the cell wall, well as fat reduction. This is great most likely due to a combination for older patients who don’t have of the vacuum and the freezing a lot of fibrotic tissue remaining; that creates a crystalline structure they get top results because there within the cell. You get some mem- is a new collagen scaffold that’s brane peeling, probably due to the built up. Paradoxically, with this massage performed immediately treatment, middle aged and older following treatment, but there’s patients may see more results than no poration. The lipid droplets youthful patients with firm tissues. don’t really come out of pores in cryolipolysis. At four months post- Conclusion treatment with cryolipolysis, no The ideal mechanism inducing cell death was seen in these SEM adipose cell death is not necrosis, specimens. A uniform reduction because there’s too much down of adipocyte size is clear (figure 6). time and possible subsequent scar- Fibrosis is also evident, which can ring. Nor is it apoptosis, because if clinically translate as improvement you don’t get any inflammation at of tissue pendulosity and a firmer all you won’t get any correction of character (figure 7). soft tissue and overlying skin lax- These two new mechanisms are ity. Poroptosis is fractional which is

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