Does microneedle fractional radiofrequency system inactivate botulinum toxin type A?

The combination of botulinum toxin type A (BoNT/A) and energy equipment have been widely used in the clinic.


| INTRODUC TI ON
Botulinum toxin type A (BoNT/A) is one of the most widely used biological toxin in clinical practice. It weakens muscle contraction by inhibiting the release of acetylcholine from the neuromuscular junction (NMJ), resulting in local weakness and paralysis. 1,2 At present, people have a higher demand for self-image, and BoNT/A also shows an increasingly prominent role in aesthetic medicine. BoNT/A injection is currently the most effective nonsurgical treatment to improve facial dynamic wrinkles, which significantly improves people's appearance, social psychology, self-confidence, and reduces anxiety. 3,4 Escalating demand for cosmetic treatment to reduce facial wrinkles and laxity has stimulated the search for more effective nonsurgical techniques. A combined treatment between BoNT/A and various energy devices benefits many patients because these treatments target various regions of the face and supplement each other, demonstrating superior cosmetic effects and higher patient satisfaction.
Currently, all clinicians agree with the idea that associating BoNT/A and energy equipment presents better efficacy. But the optimal strategy for combining these two treatments is elusive, and whether the thermal effects of energy devices affect the activity of botulinum toxin is still a problem. Considering safety and recovery of patients, clinicians generally recommend that the time of BoNT/A injection should be 1 week after energy device therapy.
Microneedle fractional radiofrequency (MFR), a minimally invasive treatment, has been rapidly developed in recent years. This treatment induces the production of new collagens via creating perforations in the epidermis and delivering radiofrequency-generated thermal energy into the underlying dermis. At the same time, the mechanical effects of microneedles on the skin also improve transcutaneous absorption of topical products, and promote secretion of growth factors to stimulate migration and proliferation of adjacent keratinocytes and fibroblast for skin remodeling. 5,6 At present, MFR has also presented good efficacy in the treatment of skin rejuvenation, acne vulgaris, acne scars, surgical scars, axillary hyperhidrosis, striae, and more. 5 In this study, we aim to determine whether the energy of MFR affects the efficacy of BoNT/A, and to provide an optimal strategy for energy device combined with BoNT/A therapy in the clinic. Xinruan Information Technology, China) conducted running training for 1 week, and the terminal event was that the mice ran the whole course stably at the preset maximum speed. One day in advance, the hairs of the double legs were removed from mice using an electric shaver and depilatory protocol.

| Subjects and Treatment
After hair removal, BALB/c mice were randomly divided into eight groups (n = 6/group), including the control group, the MFR group (MFR treatment alone), the BoNT/A group (BoNT/A injection alone), the MFR + BoNT/A (immediately) group (BoNT/A was injected immediately after MFR treatment), the MFR + BoNT/A (interval of 24-h) group (BoNT/A was injected 24 h apart after MFR treatment), the MFR + BoNT/A (interval of 2-day) group (BoNT/A was injected 2 days apart after MFR treatment), the MFR + BoNT/A (interval of 3-day) group (BoNT/A was injected 3 days apart after MFR treatment) and the MFR + BoNT/A (interval of 7-day) group (BoNT/A was injected 7 days apart after MFR treatment).

| Intervention
After full exposure of the quadriceps femoris muscle, we used a

| Patient effectiveness and satisfaction
Therapeutic reaction was measured using the Crow's Feet Grading Scale (CFGS). 7 the CFGS is a 5-point photonumeric rating scale designed to objectively quantify the severity of crow's feet. This scale was applied to 2 separate evaluations of crow's feet: at rest (static) and with expression (dynamic). The scale ratings are 0 for no wrinkles, 1 for very fine wrinkles, 2 for fine wrinkles, 3 for moderate wrinkles, and 4 for severe wrinkles. The main purpose of this study is to observe the improvement effect of dynamic wrinkles in each treatment group. each subject completed a self-assessment questionnaire and rated their condition with a score from 0 (aggravated) to 4 (much improved).

| Muscle strength determination
To quantify BoNT/A-induced focal muscle paralysis, a survey system composed of a horizontal rod, a drive motor and pulley system and a trip switch below each segment for measuring the latency to fall from the beam was used. 8 We set two rotational speeds (15 rpm and 20 rpm), and placed the mice on the rotating beam at a slow speed setting (5 rpm) to continuously increase the speed to train the balance and running ability until they reach stable performance levels at 15 rpm and 20 rpm and leave mice in place for a further 3 min.
After this phase was completed, the mice were given a 15 min rest period before continuing the next training session, which was repeated three times. Running tests were performed at 1,3,7,14,21, and 28 days after BoNT/A injection, and the system will automatically record the time and distance of the mouse fall.

| Muscle mass determination
After administration of BoNT/A or MFR for 28 days, body weight (BW) of each mouse was measured before sacrificed. Then, the injected quadriceps femoris muscle was dissected and weighed. The degree of injected muscle atrophy was expressed by the ratio of the right quadriceps femoris muscle weight to BW. Day-28 was chosen as the final time point because previous studies confirmed that this was the point of maximal BoNT/A-induced muscle atrophy.

| Quantitative real-time polymerase chain reaction (qRT-PCR)
Mouse muscle samples were lysed in Trizol reagent (Invitrogen.) according to the manufacturer's instructions. the CDNAs were synthesized using a PrimeScript® RT reagent Kit (TaKaRa, Shiga, Japan) in a volume of 10 μL, and products were detected using a SYBR® Premix Ex Tap™ Kit (TaKaRa). Data were collected and quantitatively analyzed on an M × 3000 P Real-Time PCR System (Stratagene). The main primer sequences of related genes are listed in Table 1.

| Hematoxylin-eosin (HE) staining
After BoNT/A injection or MFR treatment for the indicated time, mice were sacrificed by cervical dislocation to harvest the quadriceps femoris muscle for histological examination. It was fixed in 10% neutral formalin and embedded in white paraffin. Serial paraffin sections of 4μm thickness were prepared and stained with hematoxylin-eosin (H&E). The tissue slides were converted into digital images and analyzed through iViewer image analysis software (UNIC Technologies).

| Statistical analysis
The results are expressed as mean ± standard deviation (SD). All statistical analyses were performed using SPSS version 23.0 (IBM).
All data that followed a normal distribution were tested with a two tailed Student t-test or one-way ANOVA. Statistical significance was defined as p < 0.05.

TA B L E 1
The primer sequence of related genes involved in the current study.

| Patient satisfaction and efficacy of different treatments
The severity of periorbital crow's feet was evaluated by CFGS before and after treatment. As shown in Table 2 Table 3. As shown in Figure 1, the efficacy of the BoNT/A group and the MFR + BoNT/A (interval of 7-day) group was consistent, whereas the MFR + BoNT/A (immediately) was less effective.

| Effect of BoNT/A on inducing muscle paresis
The latencies period (Time that the mouse remains on the bean) was

| Induction of muscle atrophy after BoNT/A injection
In addition to BoNT/A's effect on muscle strength, we further explored its role on muscle morphology. As shown in Figure 3A, we found that, on the 28th days after BoNT/A injection, the ratios of quadriceps femoris mass to BW in each BoNT/A injection groups were significantly reduced (p < 0.05) compared with the control group. And there was no significant difference between the MFR group and the control group. It is worth noting that the ratio of the

| AchRα , MuSK, NCAM, and MRF4 gene expression by real time qPCR
AchRα, MuSK, NCAM and MRF4 followed the same pattern, with an increase in all denervated groups compared with the control group ( Figure 5). The subtle reduction of AchRα mRNA was observed among

| DISCUSS ION
A combined treatment between BoNT/A and various energy devices brings more benefits for many patients because they target various regions of the face and supplement each other. This view is accepted by all clinicians and is widely used in aesthetic medicine. Our study firstly compared the clinical efficacy in improving periorbital dynamic wrinkles among BoNT/A injection alone, BoNT/A injection followed by MFR treatment immediately, and BoNT/A injection followed by MFR treatment at an interval of 7 days. We found that all patients in each group had high satisfaction (p > 0.05). Surprisingly, clinicians scoring periorbital crow's feet with CFGS showed that immediate BoNT/A injection followed by MFR treatment also improved dynamic wrinkles, but less effective than the other two treatment modalities (p < 0.05). Therefore, we speculated that injection of were similar with the BoNT/A group. These evidences indicate that MFR therapy has some antagonistic effects on the toxic effects of BoNT/A, and this effect will disappear after 3 days.
Botulinum neurotoxin is a 150-kDa polypeptides, in which a 100-kDa heavy chain links with a 50-kDa light chain via heat-sensitive disulfide bonds and noncovalent forces. [16][17][18] The pure BoNT/A is a white crystal powder and soluble in water. But it has poor stability, easily degraded by heat, mechanical force and oxygen. Although botulinum toxin is known to have optimal biological activity at around 37°C and be inactivated at around 85°C for 5 min, the effect of heat on the toxin in tissue is unclear. 19,20 The energy of MFR can selectively targeted on the dermis. In order to achieve the optimal denaturation and regeneration of dermal collagen, the tip's temperature of the MFR is usually set at we will explore related mechanism in the future, so as to achieve the optimal efficacy in clinical medicine.

| CON CLUS ION
In summary, the combination of energy devices and BoNT/A has been widely used and recommended in aesthetic medicine. In this study, MFR combined with BoNT/A was applied to treat periorbital wrinkles at different intervals that benefited clinical practice to some extent. The results showed that MFR combined with botulinum toxin in a short time would have a certain reduction effect on the activity of BoNT/A, and this reduction effect would last for 3 days. Therefore, we recommend that energy devices therapy and BoNT/A injection should be performed at least 3 days apart in clinical practice, so as to ensure the optimal efficacy of BoNT/A.

ACK N OWLED G M ENTS
We want to thank all participants in this study for their enthusiastic cooperation. We also thank the patients in this manuscript who have given written informed consent to the publication of their case details.

FU N D I N G I N FO R M ATI O N
This study was supported by the National Natural Science Foundation of China (No.82073442).

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors report no other conflicts of interest in this work.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

I R B A PPROVA L S TATUS
Reviewed and approved by Southwest Hospital of Army Medical University IRB; approval #KY2022181.

PATI E NT CO N S E NT O N FI LE
Consent for the publication of recognizable patient photographs or other identifiable material was obtained by the authors and included at the time of article submission to the journal stating that all patients gave consent with the understanding that this information may be publicly available.