HISTOLOGICAL EVALUATION AFTER RADIOFREQUENCY TREATMENT FOR FACE REJUVENATION AND CELLULITIS

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1 HISTOLOGICAL EVALUATION AFTER RADIOFREQUENCY TREATMENT FOR FACE REJUVENATION AND CELLULITIS B. Palmieri 1, V. Rottigni 1 Department of General Surgery and Surgical Specialties, University of Modena and Reggio Emilia Medical School, Surgical Clinic, Modena, Italy Radiofrequency and face rejuvenation Multiple therapies involving ablative and nonablative techniques have been developed for rejuvenation of photodamaged skin. Monopolar radiofrequency (RF) is emerging as a gentler, nonablative skin-tightening device that delivers uniform heat to the dermis at a controlled depth. The treatment effectiveness can be demonstrated by not invasive skin quality methods, but evidence based on hystology gives more reliable documentation not only of the RF treatment outcome but also of the action mechanism. Unfortunately the literature reports before and after RF treatment by skin biopsy examination are quite scanty. In a recently study of 2011 El-Domyati and collegues (1) tested the procedure in 6 individuals with Fitzpatrick skin type III to IV submitted to 6 sessions at 2-week intervals (3 months) for facial rejuvenation. They used a monopolar RF skin-tightening device, consisting of RF generator, computerized automatic resistance test technology, a continuous cooling system, and a 3-cm 2 tip. The RF generator produces a 6-MHz alternating current. Skin biopsy specimens were obtained at baseline, at 3 and 6 months after the start up and quantitative evaluation of total elastin, collagen types I and III, and newly synthesized collagen were performed. All 6 volunteers completed the monopolar RF study, and showed clear clinical improvement of 1

2 skin tightening and rhytides in the periorbital and forehead regions, even if the number of patients involved in the study was small. Figure 1 shows representative photographs of periorbital and forehead areas at baseline, end of treatment, and 3 months posttreatment without any kind of adverse effect (erythema, edema, and hypopigmentation or hyperpigmentation). Only one volunteer developed slight erythema and mild transient hyperpigmentation 2 days after the fourth session, which subsided 5 days later and no scarring was observed at all. Figure 1: representative photographs of periorbital and forehead areas at baseline, end of treatment, and 3 months posttreatment Microscopic examination of hematoxylin-eosine stained sections showed epidermal hyperplasia at the end of treatment, which continued to increase 3 months after treatment; a significant increase in the intermediate skin layers was in fact detected 3 months later. This was associated with overall morphologic and structural modification of the epidermis scaffold with marked undulations of the dermo-epidermal junction. Furthermore a significant increase in granular layer thickness (Figure 2) was observed. 2

3 Figure 2: microscopic examination of hematoxylin-eosine stained sections at baseline, end of treatment and 3 months post treatment RF treatment on total dermal elastin by immunohistochemical staining showed a slight decrease in elastin level which was furtherly reduce three months after the treatment. This elastin content breakdown was associated with translocation of the solar elastotic material far away from the epidermis, balanced by the restoration of normal-appearing elastic fibers within the papillary and upper reticular dermis (Figure 3, 4). Figure 3: immunohistochemical staining for elastin levels at baseline, end of treatment and 3 months post treatment 3

4 Figure 4: immunohistochemical staining for elastin levels at baseline, end of treatment and 3 months post treatment Evaluation of immunohistochemical staining for total collagen revealed a narrow collagen band at the dermo-epidermal junction in volunteers before treatment. This band of collagen increased at the end of treatment and 3 months posttreatment there was a significant increase in the thickness of the collagen band (Figure 5) Quantitative assessment of the percentage of collagen showed significant increase in content of type I and III collagen. 4

5 Figure 5: immunohistochemical staining for total collagen levels at baseline, end of treatment and 3 months post treatment Authors concluded that monopolar RF is an effective and valuable procedure that can be used to tighten and rejuvenate photoaged skin, and contour facial skin laxity. This modality stimulates the repair process, and reverses the clinical and the histopathological signs of aging, with the advantage of relatively risk free procedure and with little downtime. Ahn and collaborators (2) treated with bipolar radiofrequency face and neck lifting in 42 patients with face and neck laxity. 5

6 Treatment area RF Power, W Total amount of RF energy Neck W 6-10 kj Nasolabial folds W 2-3 k J Lower face W 2-3 kj Cheeks W kj Forehead and lower lids 10 W 1-2 kj Table 1: treatment parameters in Ahn study The aesthetic outcome revealed an improvement of the position and shape of the cheek, lower lidcheek junction, jawline and neck. The histological analysis of the treated areas showed selective coagulative necrosis of the subcutaneous fat (lacunas and coagulated membranes in sub-dermal space). The deep reticular dermis in the dermis-fat junction exhibited a thermally induced collagen and fibrous tissue coagulation and a non-coagulative thermal restructuring of the adjacent. It was also evident a coagulative necrosis of small blood vessels (Figure 6, 7). 6

7 Figure 6: controlled and localized coagulative necrosis of the reticular dermal collagen fibers, the sub-dermal adipocytes and fibrous septa 7

8 Figure 7: localized coagulative thermal necrosis of the hypodermal adipose tissue and deep reticular dermis. Biesman (3) evaluated the safety of a novel 0.25-cm 2 shallow treatment tip for noninvasive tightening of eyelid skin in a tripartite study that began with an animal model to evaluate soft tissue effects and temperature change at the ocular surface. Findings were then extrapolated to ex vivo evaluation of human eyelids and ultimately to an in vivo human eyelid safety study. A commercially available RF device (uses a 6-MHz RF generator to capacitively couple energy through a monopolar treatment tip to produce volumetric tissue heating) was used. This heating leads to an immediate conformational change in dermal collagen, tightening of the fibrous septae extending from the dermis to the subcutaneous tissue, and stimulation of dermal neocollagenesis. The animal studies demonstrated that the 0.25-cm 2 treatment tip could be used safely on eyelid but with appropriate ocular protection. Clinical changes were not noted after treatment of the excised human eyelid skin. The epidermis remained intact without whitening or discoloration. Histopathological evaluation of the nine treated lids revealed no change in six of nine lids. Limited regions of one lid treated at 68 J/cm 2 and two lids treated at 76 J/cm 2 showed some mild thermal injury (Figure 8). Thermal damage cannot thus be advocated as the main action mechanism of RF being absent in the majority of the rescued specimens. An artifact of tissue processing or handling could not be ruled out. 8

9 Figure 8: ex vivo human eyelid skin treated at 76 J/cm2. At low power (a) an area of slightly increased eosinophilia is observed although the epidermis remains intact. Examination of the designated area under higher power (b) reveals changes that my be consistent with early coagulative necrosis. The in vivo human studies confirmed that, at the tested settings, the novel treatment tip did not injure the eyelids or eyes concluding that if used properly, the 0.25-cm 2 treatment tip can be safely used on human eyelids. Kulick (4) used the Polaris WR, a device that combines laser energy with radiofrequency (RF) treatment to provide more focused RF energy on the skin to reduce wrinkles and/or tighten skin. Skin biopsy analysis was performed before and 1 and 3 months after the treatment of facial wrinkles. Biopsy specimens in the group of patients that were defined as improved by the device showed a greater dermal thickness and interfibrillar spacing. Radiofrequency and cellulitis Recent studies show that bipolar radiofrequency can produce mild skin tightening of cellulite and that it offers faster treatment reducing tissue trauma and improving safety. Divaris and collegues (5) performed on 53 patients the radiofrequency-assisted liposuction (RFAL), a technique based on bipolar radiofrequency. The mean effective energy delivered to treat each zone (arms, abdomen, hips and flanks) was about 10 kj with a power of W. Skin fragments were recovered for the histological analysis. Skin analysis of fragments after RFAL showed coagulation of blood vessels and fat near the tract of the electrode (Figure 9). 9

10 Figure 9: histological results of untreated skin, and immediately following RFAL treatment, showing coagulated vessels and adipolysis. All patients showed a significant modification of collagen fibers with a decrease of space between collagen fibers in the superficial-mid dermis (Figure 10). Figure 10: histological results of untreated skin and after RFAL. 10

11 After the treatment the destruction of collagen fibers was observed in the deep dermis and in the dermal-hypodermal junction (Figure 11). Figure 11: histological results of untreated skin and immediately after RFAL treatment The histological quantification of dermal elastic and collagen fibers revealed an increase in collagen and elastic fibers levels (6% and 28% respectively) (Figure 12 and 13). 11

12 Figure 12: histological results of untreated skin and of RFAL-treated skin after 10 days in survival culture 12

13 Figure 13: histological results of untreated skin and of RFAL-treated skin after 10 days in survival culture Even the study of Blugerman and collaborators (6), which involved 23 patients in a bipolar radiofrequency treatment (power of the device W, skin temperature of 40 C) demonstrated that RFAL treatment cause the rupture and fragmentation of the fatty tissue and the development of fatty channels. They observed also an immediately coagulation of small blood vessels (Figure 14). 13

14 Figure 14: histological samples showing baseline collagen structure (left) and its changes with radiofrequency-assisted liposuction (right). (Above, right) Note fragmentation of collagen fibers (red arrows) and extravasation of red blood cells (black arrows) after radiofrequency-assisted liposuction. Subdermal blood vessels are surrounded by extracellular matrix at baseline, and detachment from the extracellular matrix is observed after radiofrequency-assisted liposuction (below, right) (black arrows). Paul and Mulholland (7) performed in their study RFAL in 20 patients. The energy introduced into the treated area was 100 J/cm 2. In order to analyze the RFAL treatment effect on fat and skin, biopsies were taken at the end of the treatment. The histological evaluation showed a massive destruction of the adipose tissue after the RFAL treatment if compared with a control biopsy (Figure 15, 16). 14

15 Figure 15: adipose tissue after RFAL treatment Figure 16: adipose tissue of a control biopsy Histological analysis of blood vessels in the RFAL-treated zone showed coagulation of small and medium-sized blood vessels, in according with the previous works, whereas the subdermal plexus vessels were not damaged (Figure 17). 15

16 Figure 17: blood vessel from RFAL-treated subcutaneous layer Histological observation of connective tissue in the treated area showed significant change in its structure, with coagulation of deep, reticular dermal collagen (Figure 18, 19). Figure 18, 19: collagen structure before and after the treatment 16

17 Goldberg (8) enrolled 30 subjects, with Nurnberger-Muller Scale III IV upper thigh cellulite, all treated, every other week, with a unipolar radiofrequency device for a total of six treatments. A total of 150 to 170 W of energy was delivered through a unipolar RF handpiece, with a cooled tip, for 30 seconds duration. Three such passes were undertaken so as to maintain a measured epidermal skin temperature of 40 to 42 C. Twenty-seven subjects showed evidence of clinical improvement. The mean decrease in thigh circumference was 2.45 cm. Although posttreatment erythema was noted in all treated subjects for 30 to 120 minutes, no blistering, scarring, or pigmentary changes were seen. Histological evidence of posttreatment dermal fibrosis, without any gross changes in the subcuticular layer, was noted (Figures 20, 21). Figure 20: pretreatment biopsy 17

18 Figure 21: posttreatment biopsy showing dermal fibrosis In conclusion upper thigh cellulite can be improved even with the use of a new noninvasive skintightening device that utilizes unipolar RF with epidermal contact cooling. Treatment results last at least 6 months. CONCLUSIONS The update literature review on RF cosmetic and clinical outcome at histology gave us some interesting perspective on skin and dermis turnover; after the treatment a moderate increase of the middle epidermal layers size was detected, thus rendering the macroscopic appearance of the skin surface smoother and apparently more tonic and tense. In the short and long (three months) run, the elastic fibers replacement is moderate, but some elastic changes of the stroma are cleared out from the dermis. On the other hand the thermic effect of RF is not responsible of such morphologic changes, rather a rearrangement of collagen and elastic fibers might be due a complex biochemical effect of the high frequency electric current across the skin surface. Also the deep subcutaneous tissues, like in cellulite areas of the thigh there is RF induced modification of the collagen bundles with a more cosmetic rearrangement of the fat lobules and reduction o f inflammatory cells infiltrate. In RF assisted liposuction, the synergy between mechanical fat tissue destruction and electric energy delivered achieves a substantial improvement of the cosmetic results in terms of more homogenous fat tissue remodeling and better skin tightening over the lipoaspirated subcutaneous areas. In the study of El-Domyati (1) and Divaris (5) there has been a marked improvement of elastic and collagenic fibers, after preliminary collagen bundles damage and fragmentation. Quite interesting the microvascular deep damage of RF, with red blood cells release in the interstitial fluid and basal membrane damage of minor deep vessels, leaving intact the dermal circulation. Summarizing, the literature overview confirms that the macroscopic skin surface cosmetic improvement, especially in the face and neck area, involves the microscopic background of the 18

19 mesenchymal support in dermis and subcutis, with qualitative-quantitative changes of collagen and elastic fibers and of the epithelial thickness and microvascular network remodeling. Addressing the RF either mono or bipolar across deep subcutaneous tissues, with major energy administration over 100 J/cm 2, a more dramatic effect is achieved with microcirculation vessels and direct fat cells membrane damage: the energy delivered modulation is thus critical to achieve the expected cosmetic goal at the level required. REFERENCES 1) El-Domyati M, el-ammawi TS, Medhat W, Moawad O, Brennan D, Mahoney MG, Uitto J. Radiofrequency facial rejuvenation: evidence-based effect. J Am Acad Dermatol Mar;64(3): ) Ahn DH, Mulholland RS, Duncan D, Paul M. Non-Excisional Face and Neck Tightening Using a Novel Subdermal Radiofrequency Thermo-Coaugulative Device. Journal of Cosmetics, Dermatological Sciences and Applications, 2011, 1 3) Biesman BS, Pope K. Monopolar radiofrequency treatment of the eyelids: a safety evaluation. Dermatol Surg Jul;33(7): ) Kulick MI, Gajjar NA. Analysis of histologic and clinical changes associated with Polaris WR treatment of facial wrinkles. Aesthet Surg J Jan-Feb;27(1): ) Divaris M, Boisnic S, Branchet MC, Paul M. A Clinical and Histological Study of Radiofrequency-Assisted Liposuction (RFAL) Mediated Skin Tightening and Cellulite Improvement. Journal of Cosmetics, Dermatological Sciences and Applications, 2011, 1, ) Blugerman G, Schavelzon D, Paul D. A Safety and Feasibility Study of a Novel Radiofrequency- Assisted Liposuction Technique. Journal of the American Society of Plastic Surgeons. March Volume Issue 3 - pp

20 7) Paul M, Mulholland RS. A new approach for adipose tissue treatment and body contouring using radiofrequency-assisted liposuction. Aesthetic Plast Surg Sep;33(5): ) Goldberg DJ, Fazeli A, Berlin AL. Clinical, laboratory, and MRI analysis of cellulite treatment with a unipolar radiofrequency device. Dermatol Surg Feb;34(2):204-9; discussion 209. Epub 2007 Dec