Assessment of equivalence of adipose tissue treatment with a noncontact field RF system delivering 200 W for 30 min and 300 W for 20 min: An in vivo porcine study

Abstract

Background and aims

Abdominal circumferential reduction with noncontact high frequency apoptosis-inducing field RF (AiRF) is becoming very popular. The present study compared the treatment results from two different sets of parameters giving the same dose from the same system in an in vivo porcine model.

Materials and methods

Two 10 cm × 10 cm areas were symmetrically marked on both sides of the midline (total of 4 areas) over the rectus abdominis muscle of two anesthetized female micropigs. In Animal A (G1), 27.12 MHz AiRF treatment was given at 200 W for 30 min, and 300 W for 20 min in Animal B (G2). Four sessions were performed at weekly intervals. Gross observation by a veterinary specialist was performed on a daily basis. Temperature measurements (fat and skin), clinical photography and ultrasound imaging were carried out at each session. In addition, blood chemistry was performed before each session to check lipid levels, any adverse changes in markers for liver damage in addition to an enzyme-linked immunosorbent assay (ELISA) for raised levels of TNF-α and IL-1β. Biopsies were taken and routinely processed for hematoxylin and eosin, Toluidine blue and oil red O stains to examine for tissue damage at baseline and after each treatment. TUNEL assays were performed to check of apoptotic-related DNA damage. Follow-up assessments included photography, ultrasound, ELISA tests and biopsies which were taken regularly up to 90 days after the final treatment.

Results

The maximum adipose tissue temperatures at and over the apoptotic threshold of 43°C were reached and maintained in both G1 and G2. The skin surface temperature was slightly higher in G2 after 20 min than in G1 after 30 min, but was still below 43°C. Gross and magnified observation revealed no appreciable differences or thermally-mediated damage between the skin of either of the two groups after the treatments or during the 90-day follow-up period. No lasting erythema or any other adverse event was seen in either group. The liver enzyme markers showed very similar patterns over the 4 weeks of treatment compared with baseline with no levels outside of the normal range. Triglycerides were all within normal rage with no significant differences between the groups. Remarkably similar patterns were noted for the ELISAs in both groups performed over the 4 weeks of treatment and at periods during the 90-day follow-up with no notable abnormal changes in levels. Staining patterns for both G1 and G2 specimens were similar for all stain types during treatment and the 90-day follow-up, showing decreased numbers of adipocytes by the 90-day point. The ultrasound findings revealed a 44.8% and 55.6% decrease in the adipose layer for G1 and G2, respectively, at the 90-day assessment.

Conclusions

The 200 W AiRF treatment for 30 min (G1) and the 300 W AiRF treatment for 20 min (G2) produced very similar results in the porcine model for all assessments and at all assessment points during and up to 90 days after treatment, with slightly better findings suggested for G2. Based on the above findings, the two different settings, delivering the same dose, produced good results with no skin damage and no adverse events. This has implications in busy clinics for AiRF treatment, where the shorter treatment time could represent time saving for the clinic and the patient without compromising safety and giving equal if not better efficacy.

Introduction

Body sculpting is becoming a patient-driven procedure, but the conventional methods based on mechanical liposuction are associated with unwanted side effects or in extreme cases, death. ¹⁾ Other methods have been explored for less invasive removal of fat from the abdominal area including interstitial laser-assisted lipolysis (LAL), ²⁾ cryolipolysis, ³⁾ contact-type radiofrequency (RF) ⁴⁾ and various iterations of ultrasound. ⁵⁾ LAL has proved effective, but is very technique- and clinician-dependent. Cryolipolysis delivers good but variable effects, with the potential, however, of tissue damage through frostbite. Contact-type RF cannot deliver enough heat deep into the adipose tissue. Ultrasound, such as focused ultrasound, can also achieve good results, but is clinician-intensive to apply and can leave an uneven skin surface with indurations. More recently a different type of RF has been developed to deliver a rapidly oscillating and powerful electromagnetic field in a noncontact mode, known as field RF, ⁶⁾ at a much higher frequency compared with conventional contact RF, namely 27.12 MHz compared with 1–7 MHz.

Conventional contact-type RF depends on a current being passed through tissue from one or more delivery electrodes to one or more return electrodes. As the RF current passes through tissue, it generates an electrothermal reaction depending on the resistivity of the target tissue (so-called Joule heating): the higher the resistivity or impedance, the higher the temperatures generated in the target tissue which for conventional RF indications is normally connective tissue. On the other hand, adipose tissue as a dielectric substance has a very low conductivity, more or less in fact an insulator, therefore conventional RF has very little effect on heating up adipose tissue due to its lack of conductivity. ⁷⁾

At 27.12 MHz, on the other hand, delivered via an oscillating electromagnetic RF field generated via bipolar electrodes in an applicator which is not in contact with tissue, resistivity no longer plays a role and instead of Joule heating as with conventional RF, a form of heating known as dielectric or dipole heating occurs. Adipose tissue comprises many units of oppositely-charged atoms called dipoles, which are randomly arranged in the tissue thus making it an insulator, a dielectric. This confers on adipose tissue the property of poor permittivity. ⁸⁾ Connective tissue, blood and muscle tissue on the other hand have varying degrees of higher permittivity. In the case of a tissue with poor permittivity such as adipose tissue, the dipoles are forced to align themselves in rapidly changing directions which are opposite to the polarity of the rapidly oscillating RF field. This generates a great deal of rapid movement during which the dipoles interfere with each other, colliding with and cannoning off each other. This generates rapidly increasing kinetic energy which in turn generates heat, but selectively in those tissue with poor permittivity, i.e., adipose tissue, whereas skin, muscles and vasculature are not directly heated. In a recent study on human subjects in vivo treated with noncontact apoptosis-inducing radio frequency (AiRF), the recorded skin temperature was measured in real time with a thermocamera at a maximum average of 42.3°C after 30 min at 200 W, whereas subcutaneous adipose tissue measured with an implanted fiberoptic thermocouple reached temperatures of 45°C and over. ⁸⁾

Many cell types, including adipocytes, have a threshold temperature above which the cells are forced to enter apoptosis, programmed cell death, which occurs over time: this apoptotic threshold is 43°C, which needs to be maintained for several minutes. ⁹⁾ This deliberate creation of heat in tissues and cells is known as hyperthermia. It is known that hyperthermia significantly affects proteins with a knock-on effect throughout the cell, but with the nucleus as a specific target resulting in DNA damage. In addition, a calcium spike in the cytosol results from alteration of the integrity and permeability of cellular membranes, resulting in detrimental changes to the cell redox status through shifts in the membrane potential of the mitochondria. ¹⁰⁾ These alterations combine to induce apoptosis in affected cells. Apoptosis results in a gradual deterioration and final disintegration of the cellular membranes, allowing the contents of the cytoplasm, lipid droplets in the case of adipocytes, to escape into the extracellular tissues. The higher the temperature generated in the target cells above 43°C up to a certain limit, usually agreed to be around 47°C to 48°C, the more rapidly the cells reach the final stage of apoptosis. Above 48°C the cells enter increasingly necrotic rather than apoptotic stages with much more rapid destruction than is the case with apoptosis. ^{10, 11)} The appearance of cellular debris and freed lipid in the adipose tissue matrix triggers a foreign body reaction, and macrophages are recruited into the area to phagocytose the debris and lipid, move them to the lymphatic system, thence to the blood, and to final excretion from the body mostly via the kidneys.

The number of adipocytes in human body white adipose tissue (WAT) remains fairly constant up to a certain stage. ¹²⁾ As energy is diverted to fat storage rather than being expended, adipose tissue first expands through enlargement of adipocytes through the incorporation of larger and larger amounts of lipid in each adipocyte until they are about 4-fold their original size (hypertrophy), whereupon they divide and the number of adipocytes also increases (hyperplasia). ¹³⁾ The removal of adipocytes is therefore a fairly final process, and results in the removal of fat and a subsequent decrease in the volume of the adipose tissue which can be seen as weight loss and/or circumferential reduction of the waist measurement.

More so than weight loss per se, reasonable abdominal circumferential reduction of from 3–5 cm has been well-associated with prophylaxis against developing the metabolic syndrome (METs), and to helping reduce the severity of components of METs in patients already having METs. ^14,15) The goal of circumferential reduction could therefore offer a solid health benefit, in addition to its aesthetic component. A recent paper by Kim showed circumferential reduction of some centimeters in all 27 patients participating in a study on the efficacy of AiRF. ¹⁶⁾

Taking the results of that study and the one by Goo and Kim referenced above in ref ⁸⁾, the authors noted that the treatment parameters for the noncontact AiRF system used in these studies involved an output power of 200 W and a treatment time of 20–30 min with an ideal apoptosis-inducing temperature reached in the adipose tissue without raising the skin temperature above the apoptosis threshold of 43°C. The same system is, however, capable of delivering 300 W. The authors wanted to examine the possibility of delivering AiRF at 300 W over 20 min, compared with 200 W over 30 min (same energy delivered of 360,000 J), which would enable shorter treatment times as a convenience to both patients and clinics. The authors therefore devised the current animal study using a porcine model, recognized as having more similar characteristics to human skin compared with loose-skinned animal models. The equivalence of the two sets of AiRF parameters in both safety and efficacy was examined with a variety of assessments.

Materials and methods

Animal model

Because of their size and manageability, two female micropigs were used (Medi Kinetics Co., Ltd, Seoul, South Korea), 68 and 77 months old, weighing approximately 100 g each, with an adipose tissue thickness of from 2∼2.5 cm. Following anaesthesia (induction with Zoletil 50 [VIRBAC, France, 5 mg/kg] and xylazine [Rompun®, Bayer AG, Germany, 2.5 mg/kg], both IV) the animals were restrained in the supine position, and two sets of 10 cm × 10 cm squares were marked on either side of the midline over the rectus abdominis muscle area, designated A, B, C and D. A rectangle was marked over the midline, denoting the area for temperature measurement (Figure 1). All of the squares A–D would serve for photographic and other imaging; ultrasound imaging would be captured over squares A and C; and biopsies would be taken from squares B and C in both animals. One animal, designated G1, would undergo 4 weekly sessions of AiRF at 200 W for 30 min, and G2 would undergo 4 weekly AiRF sessions at 300 W for 20 min. Animals were handled and cared for by professional veterinary experts and given food and water ad libitum, throughout the treatment and follow-up periods. At the end of the study, the animals were humanely killed by exsanguination under anesthesia. The pigs were handled in a dedicated experimental animal center in accordance with the US National Institute of Health (NIH) Guidelines for the Care and Use of Laboratory Animals.

Fig. 1:

Diagram showing the area to be treated on the ventral surface of the minipigs for G1 and G2. Also shown are the 4 marked areas A – D and the measurements/imaging to be taken from them, as well as the zone for temperature measurement.

AiRF System

The apoptosis-inducing radio frequency system was the enCurve from Lutronic Corporation, Goyang, South Korea. The system comprises a caster-mounted control console, connected by an articulated arm and umbilical control cables to the applicator. The applicator has a slightly curved large central fixed panel with two hinged adjustable “wings” at either end, enabling treatment of the abdomen and flanks in one session with the ability to adjust the contour to best match the contour of the tissue being treated. Figure 2 shows the applicator in place over the rectus abdominis area of one of the animals. The system is controlled and time and power parameters are set from a touchscreen panel on the control console.

Fig. 2:

The apoptosis-inducing RF system applicator set up over the treatment area on the minipig belly area, being careful to avoid contact between skin and applicator. Note the adjustment of the lateral ‘wings' on the applicator to enable treatment of both the central abdomen and flanks with even distribution of the RF field.

Treatment protocol

All treatment sessions were performed under anesthesia. Following induction (see above) and intubation, anesthesia was maintained with 1–2% isoflurane/O₂ mixture via a ventilator.

Animal G1 received 4 AiRF sessions at an output power of 200 W for 30 min, with one week between sessions. Four weekly sessions at 300 W for 20 min were delivered to animal G2. In both animals the applicator was set up as in Figure 2 approximately 1 cm from the target area, ensuring that no part of the target tissue was in actual contact with the applicator. Once the parameters were set, the system was activated. Nothing else was required from the operator until the system automatically shut off at the end of the preset exposure time.

Assessments

A variety of assessments were performed during the 4-week treatment period at baseline, and pre- and post-treatment (while the animals were under general anesthesia), and the subsequent 90-day follow-up period at days 1, 7, 15, 30, 60 and 90 after the final treatment session, with the animals under IV anesthesia only. Animals were examined visually on a daily basis by a veterinarian to check for any damage and general skin condition from baseline and throughout the treatment and follow-up periods. Tables 1 and 2 provide the schedules for assessments during the 4-week treatment periods and the 90-day follow-up periods, respectively.

Table 1: Assessment schedule during the 4-week treatment period.

Item	Baseline	Tx 1	Tx 2	Tx 3	Tx 4
Treatment	No	Yes	Yes	Yes	Yes
Visual examination	Gross visual inspection was performed daily throughout the treatment period
Blood chemistry	Yes	--	PreTx	PreTx	PreTx
Biopsy	Yes (+TUNEL)	PostTx	postTx	postTx	postTx
Photography (Incl. dermoscopy and folliscopy)	Yes	Yes	Yes	Yes	Yes
Temperature (Skin & fat)	No	PreTx∼ PreTx∼	PostTx PostTx	PreTx∼ PreTx∼	PostTx PostTx
Ultrasound	No	PreTX	PreTX	PreTX	PreTX

TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling assay

Table 2: Assessment schedule during the 90-day follow-up period.

Item	Day 1	Day 7	Day 15	Day 30	Day 60	Day 90
Visual examination	Animals were inspected visually on a daily basis throughout the follow-up period
Blood chemistry	Yes	Yes	Yes	Yes	Yes	Yes
Biopsy	Yes	Yes	Yes (+TUNEL)	Yes	Yes	Yes (+ TUNEL)
Photography (Incl. dermoscopy and folliscopy)	Yes	Yes	Yes	Yes	Yes	Yes
Ultrasound	Yes	Yes	Yes	Yes	Yes	Yes

TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling assay

Photography

Digital macrophotography of the marked areas A–D was captured using a SLR digital camera, a dermoscope and folliscope at baseline and after each session to check for any adverse changes in skin color and condition. The dermoscope and folliscope were also used during the follow-up assessment days.

Temperature assessment

During each treatment, skin temperature was constantly monitored and data captured in real time with an extremely sensitive video thermocamera (FLIR A3255, FLIR Inc, OR, USA). A fiberoptic-based thermocouple was implanted in the subcutaneous layer to capture temperature in the adipose layer (FOB100 Series, OMEGA, Manchester, UK), and connected to a laptop computer to record the data in real time.

Blood chemistry

Blood chemistry was performed with samples taken from multiple points at baseline and before treatments 2, 3 and 4. Assays were performed on serum triglyceride levels, total cholesterol, high density lipoprotein and low density lipoprotein to check for any abnormal rise in free lipids. The serum levels of liver enzymes (alkaline phosphatase [ALP], alanine transaminase [ALT] and glutamic-oxaloacetic transaminase [GOT]) were assayed to ascertain if any liver damage was caused by having to deal with the free lipids in the bloodstream from the lymphatics. An enzyme-linked immunosorbent assay (ELISA) was performed at baseline and before treatments 2,3 and 4 to check for raised levels of proinflammatory cytokines, namely tumor necrosis factor alpha (TNF-α) and interleukin 1-beta (IL-1β) suggestive of any liver damage caused by excessive serum levels of free lipids. The ELISA was also performed on the follow-up assessment days.

Histology

Biopsies were taken at baseline, before and after treatment sessions 2–4, and at post-treatment follow-up days 1, 7, 15, 30, 60 and 90, and routinely processed for staining with hematoxylin and eosin, Toluidine blue and oil red O, the first two to identify any damage or other changes in the connective and adipose tissues, and the third to check for the release of lipids from the adipocyte cytoplasm. In addition, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were performed at baseline, and at the 30-day and 90-day followup points to check for the degree of apoptotic cell death through detection of DNA fragmentation.

Ultrasound

Ultrasound scans were taken to visualize morphological changes in the adipose layer and overlying skin, and to check for any adverse changes in architecture associated with excessive heat. At baseline a SIEMENS Acuson P300. Munich, Germany was used.: A Bionet Co., Ltd, (Seoul, Korea) MU1V veterinary model was used before and after each treatment session and on the 90-day follow-up assessment days, and at the 90^th post-treatment day the Acuson system was used again.

Results

All tests and photographic records were successfully completed on both the G1 and G2 animals during the treatment and the 90-day follow up periods.

Photographic assessment

Gross photography at baseline, after treatment and during the 90-day assessment showed no remarkable difference between any of the post-baseline images and the baseline photos for either G1 or G2, which echoed the gross visual inspection (Figure 3a–h). This was also true for the dermoscopy images (Figure 3j–s) which were used to examine for any adverse changes to the skin and perifollicular areas at a magnification ×12 under a white light source. No adverse changes were seen to the skin during the 4 treatments and in the 90-day follow-up period. A similar damage-free pattern during the treatment and follow-up assessments was observed in the folliscopy images (data not shown).

Fig. 3:

Photographic images taken during treatment period and the 90-day follow-up period. (a–d): gross SLR photography of a G1 marked area at baseline (BL), immediately after the first treatment session (Post), and the 15th and 90th day after the 4th and final treatment session. The same timings for G2 are seen in e–h. (j–n): Dermoscopy images (x12) taken of G1 treated skin under a white light source at baseline and immediately after the first treatment, and at the 7th, 60th and 90th day in the follow-up period. The same timings are shown for G2 skin in o–s.

Temperature assessment

Table 3 and Figure 4 show the skin and adipose tissue temperatures for the G1 (200 W for 30 min) and G2 (300 W for 20 min) animals averaged over the 4 treatment sessions. The apoptotic threshold temperature of 43°C is indicated on the graph. As can be seen, in both G1 and G2 the temperature in the adipose tissue exceeded the apoptotic threshold for both G1 and G2, reaching a maximum of 45.7°C and 44.9°C, respectively at 20 min for both, then dropping slightly in G1 to end at 43.6°C. The averaged maximum temperatures at the skin surface were 41.8°C and 42.1°C for G1 and G2, respectively, both being below the apoptotic threshold. It should be noted that the average baseline temperatures in G2 were approximately 1.5°C lower than in G1.

Table 3: Temperature assessments averaged over 4 treatment sessions for G1 (200 W for 30 min) and G2 (300 W for 20 min) measured at the skin surface with a thermocamera and in the adipose tissue with a fiberoptic probe. The standard error of means (SEM) is shown in the brackets.

Animal	Time (min)
	BL	5	10	15	20	25	30
Thermocamera: Skin
G1 °C	33.1	35.9	37.9	41.1	41.3	41.8	41.2
(SEM)	(0.8)	(1.7)	(2.1)	(1.2)	(1.9)	(1.6)	(1.2)
G2 °C	31.7	34.8	37.2	40.9	42.1
(SEM)	(0.4)	(1.7)	(1.7)	(1.4)	(1.3)
Fiberoptic: Adipose tissue
G1 °C	35.7	39.8	43.0	44.4	45.7	44.4	43.6
(SEM)	(0.6)	(0.3)	(0.2)	(0.3)	(0.2)	(0.2)	(0.4)
G2 °C	34.2	37.4	40.1	42.6	44.9
(SEM)	(0.3)	(0.2)	(0.4)	(0.7)	(0.7)

Fig. 4:

Real time temperatures measurements in °C taken of abdominal fat and the skin surface for G1 and G2 measured over the 4 treatment sessions (see also Table 3).

Blood Chemistry

Figure 5 shows the serum levels of the enzyme markers of liver damage, ALP, ALT and GOT, at baseline (before the 1^st treatment) and then before treatments 2, 3 and 4 for G1 and G2. Blood was drawn from a number of sites and the results averaged as there was only 1 animal each for G1 and G2. The representative normal maximum levels/ranges for human subjects are indicated on the figure. There were nonsignificant fluctuations in the levels of each of the enzymes over the 4 sessions, but all readings were within normal levels, and differences between G1 and G2 were simply owing to the individual characteristics of each animal. In other words, G2 with 300 W for 20 min did not display significantly higher reading than for G1 (200 W for 30 min).

Fig. 5:

Peripheral blood chemistry assays for the liver enzymes ALP, ALT and GOT for each of the 4 treatment sessions comparing G1 and G2. Normal maximum values and ranges for humans shown where indicated for reference.

The serum lipid levels are compared in Figure 6 between G1 and G2 for the 4 treatment sessions, namely triglycerides (Tg), total cholesterol (Chol), high-density lipoprotein (HDL) and low-density lipoprotein (LDL). As with the liver enzyme levels, no item reached over the maximum level recommended for human subjects. In the case of the Tg readings, an unexplained spike was noted before the first treatment in G1, and in both G1 and G2, the levels before the 4^th treatment showed a noticeable rise. Nothing notable was seen between G1 and G2 for Chol, HDL and LDL levels.

Fig. 6:

Lipid levels from peripheral blood for triglycerides (Tg), total cholesterol (Chol), high-density lipoprotein (HDL) and low-density lipoprotein (LDL) before each of the 4 treatment sessions, compared for G1 and G2.

The results of the ELISA are seen in Figure 7, with the levels of TNF-α and IL-1β as markers for any potential liver damage-related inflammatory response caused by the stress of dealing with excessive levels of freed lipids in the bloodstream. In both G1 and G2, and in the levels of TNF-α and IL-1β, a small peak in levels was noted on day 7 in the follow-up period, but it was not significant: otherwise the levels more or less echoed each other both within the group and the enzyme being assayed with no significant difference between the animal treated at 200 W for 30 min or at 300 W for 20 min.

Fig. 7:

ELISA results compared for G1 and G2 assaying the inflammatory enzymes tumor necrosis factor-alpha (TNF-α) (left panel) and interleukin-1 beta (LI-1β) (right panel) before each treatment session and on follow-up days 1, 7, 15, 30, 60 and 90.

Histology

Figure 8 illustrates the histological findings for G1 (upper panel) and G2 (lower panel) specimens. The two stains used for morphological assessment were hematoxylin and eosin (H&E) and Toluidine blue, while oil red-O was used to stain for triglycerides and free lipid. The G1 and G2 H&E stains showed a similar progression. At baseline, tightly ordered lipocytes were seen through positive staining of the membranes. As treatment progressed, small areas of necrosis could be seen between some cells, and by the 4^th treatment session, some denaturation of the membranes could also be seen. By the 15^th day after the final session, some strands of fibrotic tissue were interspersed among the fibrocytes which had decreased somewhat in number compared to the baseline findings. At the 90^th post-treatment day, fibrous strands were more evident and the arrangement of the lipocytes was much less tight than in the baseline images, suggesting loss of lipocytes with some shrinkage of the structure. Similar findings were seen for the G2 specimens at the same stages with no notable differences attributable to the higher power in G2. The Toluidine blue results echoed findings demonstrated in the H&E stains, with no notable differences between the G1 and G2 specimens (data not shown).

Fig. 8:

Histological specimens (all at original magnification ×100) from G1 (upper panel) and G2 (lower panel) showing H&E staining (upper and lower panels, first row) and oil red O staining (upper and lower panels, 2nd row) at baseline (Pre), immediately after the 4th treatment session (4Tx) the 15th and 90the days in the follow-up period.

The oil red O-stained specimens at baseline showed tight concentrations of lipid droplets enveloped in the lipocyte membranes. By the 4^th treatment session. The borders between lipocytes and lipid were much less clear, suggesting the extracellulation of the lipid from the cells entering apoptosis. This progressed further in the post-treatment followup period, and by the 90^th day the staining indicated lower oil densities, with fee oil indicating apoptosis was till occurring although less than in the 15-day specimen. Once again there were no clear differences between the G1 and G2 specimens.

Some specimens were subjected to a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay to identify apoptotic cells, and the extent of apoptosis. The results are seen in Figure 9. With the baseline percentage of apoptotic cells in the G1 and G1 normalized to zero, the apoptotic activities for G1 and G2 TUNEL specimens peaked at 40 ± 4.1% and 42.4 ± 9.2%, respectively, at 15 days after the final treatment, dropping back to 16 ± 4.4% and 18.1 ± 10.4%, respectively, at 90 days. In other words, at 90 days after the final treatment session, adipocytes were still in various stages of apoptotic cell death so in theory at least, circumferential reduction could still be occurring.

Fig. 9:

(Upper panel): Images of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays for G1 (a–c) and G2 (d–f) at baseline (a,d), 15th follow-up day (b,e) and 90th follow-up day (c,f). (Lower panel): Graphical comparison of adipocyte apoptotic activity at baseline, 30 and 90 days after the final treatment session for G1 and G2, expressed as increased percentages of baseline apoptotic activity normalised to 0%.

Ultrasound imaging

Two different ultrasound systems were used: a high-frequency top-end system at baseline and at the 90-day assessment for a more accurate assessment of measurement of reduction in the thickness of the adipose layer, and a portable veterinary US system at all treatment sessions and during the follow-up period to give a general idea of changes in the tissue layers, and to give early warning of any adverse changes in tissue morphology associated with tissue damage. At 90 days after the final treatment session, repeated images from G1 showed a 44.8% decrease compared with a 55.6% decrease in G2. (Figure 10). The images captured during the treatment and follow-up periods showed a steady reduction in the adipose layer in both G1 and G2, without any signs of treatment-related damage such as vacuole or cyst formation associated with overheating of the connective or adipose tissue (Figure 11).

Fig. 10:

(Upper panel): High-frequency high resolution ultrasound imaging findings of treated skin and adipose tissues at baseline (a,c) and at the 90th follow-up day (b,d) for G1 (a,b) and G2 (c,d). (Lower panel): Decreased thickness of the adipose tissue in G1 and G2 expressed as a percentage and shown graphically. Results are averaged values of multiple readings.

Fig. 11:

Lower resolution US images than in Fig 10 above to assess general changes in tissue layer thicknesses and identify any thermal damage-related tissue artefacts taken after the 1st and 4th treatments, and at 15, 30 and 60 days after the 4th treatment for G1 (upper images) and G2 (lower images).

Discussion

The present study was designed around a porcine model to demonstrate the safety and efficacy of two sets of noncontact field RF parameters delivered by a hands-free applicator giving the same total dose, namely 360,000 J delivered by 200 W for a 30 min exposure and 300 W for a 20 min exposure. The minipig as an ideal model for human subjects to test the safety and efficacy of apoptosis-inducing radiofrequency (AiRF) has been well reported, with the 2013 study by Weiss and colleagues as a good example: ¹⁷⁾ we therefore modelled our present study on that one, including the same set of assessments. Pig skin is much more similar to human skin than the loose-skinned models such as rats and dogs, and the structure is larger to allow more accurate observation and measurement following intervention.

The minipig is much smaller than its larger species farmyard cousins, making it easier to handle in the laboratory setting, and an experimental-specific species is bred for laboratory use. However it is still a large animal, the specimens used in the present study weighing in at 100 kg, and with fatty tissue with thicknesses of around 2–2.5 cm allowing noninvasive visualisation with modalities such as ultrasound. The size and husbandry-related expense were factors which limited the number of the study animals to two, and thereby lies a limitation to the study since the two sets of parameters were applied to only one animal each. To attempt to improve the range of the results, 4 target areas were therefore marked on the belly of each animal (Figure 1) to attempt to allow duplication of various assessments. It is still the case, however, that there were only 2 animal subjects, each with its own different metabolic rates, so it is a possible limitation.

AiRF, in order to be effective for circumferential reduction, needs to induce a temperature rise in the target adipocytes which is high enough to lead to apoptotic cell death. The accepted apoptotic threshold is 43°C, ⁹⁾ and that temperature was reached and exceeded by both sets of parameters in the present study. The major concern is that the adjacent tissues will also be heated up beyond the apoptotic threshold by conducted heat, since such tissue as overlying skin, underlying muscle and associated vasculature have high permittivity and will therefore not be directly and efficiently affected by a high frequency oscillating RF field, such as 27.12 MHz as used in the system in the present study, and in the study by Weiss and colleagues mentioned above. At this frequency, it is tissues with poor permittivity, such as adipose tissue, which are selectively heated through dipole action associated with the rapidly oscillating field. ⁸⁾ Thus two sensitive real-time temperature measurement systems were employed: a fiberoptic-based thermocouple implanted in the adipose tissue, and a fine-plate thermographic video camera to measure the temperature rise at the surface of the skin. As illustrated in Figure 4, the skin temperature for G1 and G2 remained below the 43°C mark at 30 and 20 min, respectively, whereas the adipocytes were heated to over 43°C. The skin showed no ill effects at all for either set of parameters in both gross and macroscopic assessment (Figure 3), whereas adipocyte apoptosis was clearly demonstrated through TUNEL staining up to the 90 day assessment (Figure 9), and with H&E and oil red O stains (Figure 8). This was matched by reduction in the thickness of the subcutaneous fatty layer as assessed with ultrasound (Figure 10) and no adverse changes in the tissue layers associated with possible overheating (Figure 11). The TUNEL assay in the study therefore demonstrated that the apoptotic reaction was not limited to the treatment period of 4 weeks, but continued out beyond that into the 90-day followup. In fact a peak of over 40% greater activity compared to baseline in both G1 and G2 was seen at the 30-day point compared to the activity at 90 days, however at over 15% in both G1 and G2 that was still higher than at baseline, so lipid was still being released from dying adipocytes even at 90 days after the final treatment session.

Of some concern is what happens to the lipid freed up as the adipocytes die and release their encapsulated fat into the extracellular matrix, to be phagocytosed by macrophage activity, delivered to the lymphatic system, thence to the blood and excreted via the liver and kidneys. The worry is that liver function could be compromised by having to deal with the excessive load of free lipids caused by release of lipid from the adipocytes. Firstly, monitoring of the level of lipids in the peripheral blood (triglycerides, total cholesterol, high-density lipoprotein and low-density lipoprotein) during the treatment period did not reveal dangerously high increases, although once again there were fluctuations as seen in Figure 6. It would be possible that the higher power delivered over the shorter exposure time in G2 might have shown higher levels than in G1, but this was not the case. Secondly, none of the liver enzymes used as markers for liver damage (ALP, ALT and GOT) showed remarkably increased serum levels, although there were some fluctuations during the 4 treatment sessions (Figure 5), but of no significance to liver function impairment or damage. Once again, the higher treatment power associated with G2 did not elicit higher levels than G1, in fact for ALP the G1 levels were higher than for G2 but still below the maximum level for human adults. Over the treatment period, and the longer term of the 90-day follow-up, potential liver damage was again monitored through the level of activity of the inflammatory enzymes TNF-α and Il-1β. Both groups showed similar small fluctuations with no remarkably dangerous rise in levels, although a peak was seen in both G1 and G2 at post-treatment day 7 for TNF-α and Il-1β.

Table 4: Results of the enzyme-linked immunosorbent assay (ELISA) for levels of inflammatory markers tumor necrosis factor-alpha (TNF-α) and IL-1β. during the treatment period (before the first treatment, and before treatments 2 – 4), and on selected days during the 90-day follow-up period.

Group	Treatments				Post-Tx follow up in days
	1	2	3	4	1	7	15	30	60	90
TNF-α
G - Avg	98.664	95.416	99.747	136.637	159.017	187.460	216.554	174.610	208.901	155.118
G1 - SD	1.327	23.482	1.021	1.123	4.390	0.715	10.005	6.432	1.634	1.940
G2 - Avg	177.209	171.578	148.549	117.867	128.552	283.908	135.843	147.033	186.305	174.899
G2 - SD	24.809	3.573	11.639	2.552	16.233	14.804	0.204	8.882	15.212	0.715
IL-1β
G1 - Avg	300.7	320.8	334.6	277.3	323.9	546.5	340.8	377.4	406.6	358.7
G1 - SD	2.2	29.0	5.4	10.4	4.6	2.1	19.1	4.1	13.9	19.7
G2 - Avg	348.4	351.9	374.1	285.5	303.6	470.7	350.7	305.5	328.1	267.1
G2 - SD	4.1	4.1	14.5	8.7	16.5	4.8	11.0	3.1	6.5	4.9

Looking beyond the porcine model in the present study, of interest to those patients looking for circumferential reduction, and to a lesser extent, weight loss, it can be suggested that the loss of adipocytes seen in the histological stainings in the present study is beneficial to that aim, without any concern regarding tissue damage or impairment of liver or kidney function associated with AiRF treatment. Once a reduction in adipocytes and their encapsulated lipid occurs through apoptotic cell death, it will take some time for adipocytes to increase again in number. Combining AiRF for abdominal circumferential loss with a mild exercise program and some form of dietary restriction could only be beneficial to such patients, to maintain or even improve the results of field RF treatment. Furthermore, the results of the present study would appear to suggest that a higher treatment power delivered over a shorter exposure time had equal if not better results than the same dose delivered over a longer exposure with lower power, with no compromise to patient safety from the aspect of tissue damage, potential induction of hyperlipidemia or liver function impairment through having to scrub higher levels of lipid from circulating blood. This has implications for the busy clinic wishing to fit in more patients, or for the busy patient for whom treatment time is money.

There are other options for abdominal fat removal, conventional liposuction being one, but this has been associated with medium to severe side effects, including death. ¹⁸⁾ A more elegant and less traumatic approach for both the patient and the surgeon has been the evolution of laser-assisted lipolysis (LAL), ¹⁹⁾ which has involved interstitial application of laser energy through a canula-held optical fiber at a number of wavelengths, namely 910 nm diode laser, 1064 nm, 1320 nm and 1440 nm, all based on the Nd:YAG laser.^{20, 21)} In theory, the micropulsed laser beam destroyed the lipocytes through both thermal and photoacoustic effects in a much gentler way that the mechanical effect of the conventional liposuction cannula. ²⁰⁾ Although the laser-assisted approach was much less invasive compared to mechanical liposuction, it was still associated with specific hands-on surgical skills, and still required both the invasive insertion of the canula and fiber into the fat, and some effort on the part of the surgeon. ²¹⁾ LAL was also associated with specific side effects including over- and under fat reduction, tissue and fat necrosis, pain and induration post surgery. ²²⁾ The hands-free aspect of the field RF system and the total noninvasive nature of the RF energy are much more surgeon- and patient-friendly. Indeed field RF could be applied by a trained therapist under the clinician's guidance. In its favor, LAL required usually a single session, whereas multiple sessions should required with apoptosis-inducing field RF to obtain the desired result. From the patient's viewpoint, the comfortable treatment procedure and total noninvasive aspect of field RF will probably be more appealing than the invasive nature of an LAL procedure, and the associated follow-up required post-surgery compared with no downtime following an apoptosis-inducing field RF session.

An alternative to LAL has been the recent reports on non-contact laser and light-emitting diode systems for fat reduction. ^{23, 24)} However, it has been suggested that much more is required by way of rigourous scientific evidence before either of these LLLT modalities can be recommended as stand-alone approaches for circumferential reduction, without some form of co-therapy, and both approaches require several treatments.

Conclusions

In the present study based on a porcine model for adipocyte reduction using noncontact high frequency field RF at 27.12 MHz, the same dose delivered by 200 W for 30 min was compared with 300 W for 20 min. Using a number of tissue, cellular and metabolic assessments showed that apoptosis-inducing RF treatment with a shorter time and higher power was as effective as the longer time and lower power treatment, or even slightly more effective, but with equal safety. The optimistic results of the present porcine model study merit larger controlled and more detailed studies in human subjects.

Conflict of Interest

“The authors hereby declare no conflict of interest exists regarding the RF system used in the present study.”