Assessment of efficacy of different botulinum toxin A concentrations in the treatment of androgenetic alopecia assessed by dermoscopy

Androgenetic alopecia (AGA) is the most common type of progressive hair loss in men and women. AGA is characterized by the miniaturization of the hair follicle, leading to the transformation of terminal hair to vellus hair.


| INTRODUC TI ON
Androgenetic alopecia (AGA) is a common form of hair loss in men and women.AGA is characterized by the miniaturization of the hair follicle, leading to the transformation of terminal hair to vellus hair. 1 This condition affects 80% of men and 40% of women before the age of 70. 2 It is reported that in some affected people, AGA may impact the quality of life and self-esteem of patients. 3r the development of AGA, it is reported that genetic predisposition is the crucial trigger. 4While changes in androgen sensitivity have a critical role in the progression of the disease.Perifollicular micro-inflammation and oxidative stress have also been recognized as significant factors.Since morphology, proliferation, and death of cells in the dermal papilla can be caused by oxidative stress. 5Additionally, bald areas in AGA have some microvascular insufficiency and lower oxygen (O 2 ) levels than non-bald areas, and its stated that testosterone is most effectively converted to dihydrotestosterone (DHT) in low-oxygen environments. 6DHT stimulates the production of transforming growth factor-beta 1 (TGF-ß1) in dermal papilla cells (DPCs), which is crucial for preventing the formation of follicular epithelial cells.TGF-ß1, which promotes apoptosis, causes fibrosis and stiffness in the scalp. 7In addition, hair diversity, yellow spots, the peripilar sign, an increased percentage of vellus hairs, and a large number of follicular units with only one emerging hair shaft are the principal dermoscopic findings of AGA. 8 Botulinum toxin A (BTA) is a neurotoxin derived from the Clostridium botulinum bacterium that inhibits muscle contraction by blocking the release of acetylcholine at the neuromuscular junction.
It has been used to treat different muscular and neuromuscular conditions and for aesthetic purposes. 3In AGA, BTA injections promote relaxation of the scalp muscles, vasodilatation of the blood vessels, and potentially increase oxygen (O 2 ) flow to bald areas.This would lead to a decrease in tissue DHT, resulting in less follicular miniaturization and less TGF-β1 production and secretions. 7e current work aimed to evaluate the effectiveness and safety of injecting two different concentrations of BTA for treating AGA patients who visit the Memorial Souad Kafafi University Hospital (SKUH), Egypt, from June 2022 to November 2022.The treatment effect was assessed clinically and by dermoscopy, and results were reported in mean ± stander deviation at a significance of <0.005.

| Study location
After ethical committee approval and informed consent from the patients, this prospective, cross-sectional study was performed on a total of 32 patients with androgenetic alopecia (AGA) who were diagnosed clinically and by dermoscopy at the outpatient dermatology clinic of the Memorial Souad Kafafi University Hospital (SKUH) starting from June 2022 to November 2022.

| Sample size
The sample size calculation was based on the mean hair count among cases with AGA treated with BTA injections before and after, as described in a previous study. 9Using G*power version 3.0.10 to calculate sample size based on t test = 2.04 to compare between two means, 2-tailed, α error = 0.05, and power = 80.0% with effect size = 0.511, the total calculated sample size was 32 cases.

| Inclusion criteria
All AGA patients (diagnosed through clinical examination and dermoscopy) belonging to Norwood-Hamilton classifications I-VII for males and Ludwig classifications I-III for females aged between 18 and 50 years were included.

| Exclusion criteria
Patient undergoing treatment with topical or systemic treatment for hair fall; severe systemic diseases or hormonal disturbances; neuromuscular diseases; the presence of inflammation or infection on the skin around the site of injections; systemic treatment with corticosteroids, immunosuppressants, antimalarials, and NSAIDs in the past 3 months; phobia from injections, pregnancy and lactation; previous use of any facial Botox injections in the last 3 months were excluded from the study.

| Study procedure
All patients underwent the following procedures.
1. Informed consent: Consent was obtained from all the participants before enrolling in this study and after an explanation of the nature and purpose of the research.
2. Full history: Personal history (name, age, sex, and phone number), present history of AGA (regarding onset, course, and duration), medical history (e.g., PCO-thyroid gland dysfunction) and drug history were obtained before treatment.Family history of similar conditions was also considered prior to the treatment.

Assessment of quality of life (Qol):
The quality-of-life assessment was evaluated using the Hair-Specific Skindex-29 scale.There are three different types of scales in the questionnaire: a function scale (12 items), a symptom scale (7 items), and an emotion scale (10 items). 10 Clinical examination: Before treatment, AGA patients were examined on two levels as follow: • General examination: Full body examination, the patient's general condition (e.g., chronic fatigue may be present in anemic patients), the presence of dyspnea (as a symptom of anemia), vital data: measuring peripheral pulse and arterial blood pressure for thyroid disturbance.
• Local examination: Examination of the hair and scalp and evaluation of hair quantity, quality (e.g., length, density, color, and texture), and any skin conditions affecting the scalp.

| Botulinum treatment
Botulinum toxin injections (100 U) were obtained from Nabota, South Korea, for AGA treatment.Before treatment, the patient's vertex was sterilized with 70% ethyl alcohol and divided into two equal halves from the midline.In the right half of the patient's vertex, the dilution was 100 U/3 mL with 0.9% normal saline; thus, each 1 mL contains 33.3 U of botox.A 100 U insulin syringe was used, and each large mark was 0.1 mL, containing 3.3 U of botox.While in the left half, the dilution was 100 U/4 mL with 0.9% normal saline; thus, each 1 mL contains 25 U of botox.Each large mark of the 100 U insulin syringe was 0.1 mL, containing 2.5 U of botox.
In each half, 15 injection target sites (2-3 cm apart) were marked.A 3.3 U for each target site was injected intradermally in the right half, and 2.5 U for each target site in the left half.One centimeter was spared from the midline on each side to avoid diffusion between both sides (Figure 1).Moreover, patients were instructed to do the following: no pressure or massage on the treated area for 6 h; no lying down for 6 h after treatment; avoid exercise and extensive sun or heat exposure; contact the doctor if there is edema, erythema, inflammation, or blisters; and not to use NSAIDs for headache, which is common after injections.

| Assessment of the efficacy of treatment and follow-up
The assessment was obtained at the four levels as given below:

| The socio-demographic characteristics of the studied cases
The mean age was 29.25 ± 4.94 years.Positive family history of AGA was detected in 21 (65.6%)patients.This study was done on 27 (84.4%)females and five (15.6%) males (Table 1).  2 demonstrates a statistically significant improvement of Ludwig Scale after treatment (p = 0.001); Grade 1 changed from 14.8% (before treatment) to 70.4% (after 6 months of treatment).Norwood-Hamilton scale showed a statistically significant change (p < 0.001) in which grade 2 changed from 0% (before treatment) to 60% (after treatment).3.

| Evaluation of adverse effects
Irritation was more prevalent in four (12.5%) patients on the right side of the vertex.Headache was reported in 10 (31.1%) patients.One (3.1%) reported pain at the site of injections, and one (3.1%)patient-reported nausea, Table 4.

| Patient satisfaction evaluation
Table 5 illustrates that 18 (56.2%) of the studied cases are satisfied with the appearance of their hair overall, and eight (25%) of them found that the right side of the vertex appears better.

| DISCUSS ION
The genetically determined and androgen-dependent condition known as AGA is characterized by a progressive miniaturization of hair follicles.Systemic medications like finasteride and spironolactone, topical therapies like minoxidil, and other methods, including PRP and hair transplants, are used to treat AGA. 9 Botulinum toxin may ameliorate AGA, but the exact mechanisms are still unknown.Theoretical explanations, however, point to at least two possible outcomes: (a) reduced TGF-ß1 activity in DPCs (through intradermal injections) and (b) relaxation of the muscles around the scalp (through intramuscular injections). 11e progression of AGA is thought to be aided by the release of androgen-induced TGF-ß1, especially to DPC-related hair growth suppression, anagen phase shortening, and hair follicle miniaturization. 1 Intradermal injections of botulinum toxin type A lower TGF-ß1 expression in cultured human DPCs, suggesting that decreased TGF-B1 activity may help to explain the rise in hair counts following these injections. 7 is also known that DHT stimulates DPCs production of TGF-ß1, which is crucial for preventing the development of follicular epithelial cells.A proapoptotic molecule with a significant function in the AGA is TGF-ß1.Inhibiting TGF-ß1 production from hair follicles would therefore be another way through which BTA would exert its anti-fibrotic action. 12T-induced paracrine mediator synthesis, including the production of Dkk-1, interleukin-6, and TGF-1 in balding DPCs, may contribute to AGA and offer an alternative therapeutic target.However, there have not been any documented clinical trials focusing on these paracrine mediators. 13tulinum toxin intramuscular injections have shown increase in hair counts, however, there is no mechanistic research to support Moreover, by easing stress across the galea aponeurotica, intramuscular injections may enhance AGA.Anatomically, the galea aponeurotica, which extends across the scalp and rests beneath areas that are vulnerable to AGA, serves as the anchor for the muscles that form the scalp's border. 11creased DHT and TGF-ß1 activity, as well as dose-dependent intracellular oxidation, are all brought on by tissues under pressure and/ or strain.Hic-5/ARA55, an androgen receptor co-activator, may be involved in both the activation of TGF-ß1 and the sensitivity of DPC to androgens, according to in vitro studies.Hic-5/ARA55 builds up in the nucleus, where it can facilitate cell adhesion kinase beta activation, which is triggered by mechanical stretch in vascular smooth muscle cells, as well as the transactivation of androgen-specific genes. 9Emotionally, 24 patients experienced embarrassment and humiliation, which was in agreement with Gupta et al., 15 who also reported how AGA negatively impacts the patient's social life, mental health, and quality of life.Hence, at the end of treatment, patients' satisfaction levels were also assessed.The obtained assessment implied that 18 of our studied cases were satisfied with their hair's overall appearance and improvement, and eight studied cases found that the right side of the scalp appeared better.The patient also reported a boost in their confidence after the treatment.These findings are supported by Zhou et al. 9 study, where patients with BTA injections were satisfied and experienced moderate to a marked reduction in hair loss compared to before treatment.
Currently, studies reported an AGA treatment strategy involving the use of BTA and found a couple of factors (including overdose) that might make this treatment a double-edged sword. 3In the present study, considering differences in races and safety problems, we used a low dose of Botox (33.3 U/ML versus 25 U/UL) diluted with 0.9% normal saline to treat AGA in Egyptian patients.According to Freund and Schwartz, 16 people with AGA have tight scalp muscles that reduce blood flow to the distal extremities of the veins, particularly the vertex and front peaks.The perforating vasculature is less stressed thanks to Botox, which improves blood flow and oxygenation.Freund and Schwartz 16 found that a high oxygen concentration causes more testosterone to be converted into estradiol, which reduces grease secretion and, as a result, causes hair loss.Thus, in our investigation, a lower dose of Botox (compared to doses available in literatures for treatment) was found to be beneficial and safe for Egypt patients.Moreover, a similar result was also reported for Chinese patients when only 50 U of Botox was used for the treatment. 17 The left side 0 0.0

1 .
Clinical evaluation or Clinical staging: Patients were assessed depending on the stages of the Ludwig scale for females and the Norwood-Hamilton scales for males before and after treatment at 0 (baseline), 3, and 6 months.

2 .
Dermoscopic evaluation: Scalp and hair examination for AGA and hair loss regions using the dermoscopy (DermLite DL4): 2 points were set each in the midline: the first point at the frontal region, 18 cm from the tip of the nose, and the other point at the vertex region, 24 cm from the tip of the nose (Figure2).The dermoscopic examination was done for the right half (5 cm from the midline) and the left half (5 cm from the midline).Dermoscopy results included yellow dots, vellus hair, hair diversity, and perifollicular sign at 0 (baseline), 3, and 6 months.

3 .
Patients' satisfaction evaluation: Patients were asked to answer these questions: 1.Compared to the start of the study, which statement best describes your satisfaction with the improvement of your hair condition (Satisfied-Neutral-Dissatisfied).2. Compared to the start of the study, the appearance of which side of the head is better (No difference-Right side-Left side).

4 .
Evaluation of Adverse Effects: Data on systemic adverse effects such as fever, headache, tightness in the chest, and nausea, as well as localized adverse effects such as erythema and irritability, were collected.
Data analysis was performed by SPSS software, version 25 (SPSS Inc., PASW statistics for windows version 25: SPSS Inc.).Qualitative data were described using numbers and percentages.Quantitative data were described using median (minimum and maximum or interquartile range) for non-normally distributed data and mean ± standard deviation (SD) for normally distributed data after testing normality using Kolmogorov-Smirnov test.The significance of the obtained results was judged at the 0.05 level.Kruskal-Wallis and Mann-Whitney U tests were used to compare between two studied groups and more than two studied groups, respectively, for non-normally distributed data.Wilcoxon signed Rank test and Friedman test were used to compare between two studied periods and more than two studied periods.While Spearman's rank-order correlation was used to determine the strength and direction of a linear relationship between two non-normally distributed continuous variables and/or ordinal variables.F I G U R E 1 Sites for injections.

Figure 3
Figure 3 illustrates the change in AGA patient condition after being treated with BTA injections over 6 months.Table2 demonstrates

Figures 4 and 5
Figures 4 and 5 demonstrates the dermoscopic findings of patients treated with two different concentrations of BTA (33.3 U/mL or

F I G U R E 2
Dermoscopic evaluation.TA B L E 1 Socio-demographic characteristics of the studied cases.
these gains.Therefore, two theories have been put up by researchers: (a) the loosening of artery branches that support balding areas indirectly and (b) the lowering of tension across the galea aponeurotica-a dense, fibrous, fascia-like membranes that stretch across the scalp. 11According to study about the scalp anatomy, transcutaneous oxygen levels in frontal balding scalps were 54% lower than those of controls.Particularly, the frontalis, occipitalis, and temporalis muscles might constrict the circulatory networks originating from the supraorbital, supratrochlear, and carotid artery branches, creating a hypoxic environment that favors DHT conversion over estradiol in F I G U R E 3 Clinical findings of patient treated with botox injections (33.3 U/mL at the right side and 25 U/mL at the left side).(A) 0 baseline.(B) 3 months after treatment.(C) 6 months after treatment.The clinical image in (A) indicates diffuse hair thinning in the frontal and middle areas of the scalp.After treatment and follow-up at 3 and 6 months (B, C), clinical photographs showed significant improvement.TA B L E 2 Clinical assessment of studied cases using Ludwig scale and Norwood-Hamilton scale changes before and after 3 and 6 months of treatment.Note: p1: significance between baseline and after 3 months, p2: significance between baseline and after 6 months, p3: significance between 3 and after 6 months.*Statistically significant.balding-prone areas.Local ischemia decreases hair shaft diameter, postpones hair shaft elongation, diminishes hair density, and stops anagen.11

F I G U R E 4 F I G U R E 5
Dermoscopic findings of patient treated with botox injections at the right side (33.3 U/mL) of the scalp.(A) 0 baseline.(B) 3 months after treatment.(C) 6 months after treatment.First visit dermoscopy (A) reveals hair shaft diversity (blue arrows) and vellus hair (red arrows) and yellow dot (black dots), which is suggestive of AGA.Dermoscopy (B, C) showed decrease in the hair shaft diversity, vellus hair and yellow dots.Dermoscopic findings of patient treated with botox injections at the left side (25 U/mL) of the scalp.(A) 0 baseline.(B) 3 months after treatment.(C) 6 months after treatment.First visit dermoscopy (A) reveals hair shaft diversity (blue arrows) and vellus hair (red arrows) and yellow dot (black dots), which is suggestive of AGA.Dermoscopy (B, C) showed improvement but less than the right side.As a result, tension-mediated stretching across the galea aponeurotica may cause inflammation that transactivates to the upper layers of scalp tissue, potentiating increased DHT and TGF-ß1 activity in DPCs as well as the quickening of AGA progression.14Therefore, present study aimed to evaluate the clinical and dermoscopic responses of intradermal injections of two different concentrations of BTA in the treatment of AGA.Before the study, a family history of hair loss was noted in 21 patients, along with their social and emotional satisfaction levels.During the clinical examination, 26 patients reported symptoms such as scalp irritation.In contrast, 17 patients shared how they lost the desire to meet people.

Moreover, improvements inTA B L E 4 1 TA B L E 5
the clinical grades in females and males were also noticed over time after BTA treatment.Regarding Ludwig grading scale, 17 patients were in Grade III, six in Grade II, and four in Grade I.After 6 months, 19 patients' raised to Grade III, seven to Grade II, and one to Grade I. So, the majority of cases were in Grade III on the Ludwig scale for females before treatment, and after treatment, most cases improved by two grades to Gade I.With the exception of two patients, no difference in the results was found between the follow-up visits at three and 6 months.Regarding the Norwood-Hamilton grading scale, three patients were in Grade IV and two in Grade III.After 6 months, Used test: Friedman test.TA B L E 3 Dermoscopic findings change between 0 (baseline), 3 and 6 months' follow-up.Evaluation of Adverse Effects.Patient Satisfaction Evaluation.
Compared to the start of the study, which statement best describes your satisfaction with the improvement of your hair condition