Bhertha Miyuki Tamura1, Luiz Carlos Cucé1
Keywords: AXILLARY, HYPERHIDROSIS, IMMUNOLOGICAL RESPONSE, BOTULINUM TOXINS, BOTULINUM TOXIN, HIPERHIDROSIS TREATMENT
The most frequent complications with the use of botulinum toxin type A (BoNTA) and its application for cosmetic purposes described in the medical literature include ecchymosis, hematoma, pain, discomfort or a weight sensation in the forehead, acneiform eruptions, headache, paresthesia, eyebrow ptosis, keratoconjunctivitis, blurred vision, photophobia, diplopia, edema, transient erythematous papule, foreign body sensation in the eyes, migraine, strabismus, dysphagia and lacrimation.1,2
Complications that can occur due to the treatment of hyperhidrosis include muscle weakness, paresthesia, compensating hyperhidrosis, ecchymosis, hematoma, pain and complications from anesthetic techniques chosen and used by the physician.3-5
Complications related to other treatments include hypochromia,6 pain, facial palsy, epiphora, diffi culty chewing, ectropion, entropion, keratoconjunctivitis, blurred vision, photophobia, diplopia, conjunctivitis, ecchymosis, hematoma, edema, foreign body sensation in the eyes, diarrhea, migraine, cough, dysphonia, epistaxis, urinary infection, strabismus, lacrimation, muscle weakness (muscle fi brosis and atrophy), muscle changes due to wrong application, metal taste, transient neuronal trauma, hypotonia, cervical pain, respiratory obstruction, xerostomia and bladder dysfunction.7-13
Finally, the systemic symptoms described include general weakness, dry mouth and eyes, urinary retention, cold-like symptoms, botulism syndrome symptoms,14 fatigue, nausea, vomit, skin exanthema, antibody formation, albumin allergy, myasthenic crisis, neuromuscular syndrome,10,12,13,15-18 type I reaction12 and necrotizing fasciitis.19
Some medications have human albumin and the risk of contamination and transmission of viral diseases, including Creutzfeldt-Jakob disease, cannot be ruled out.20 The large-scale use of this medication for the treatment of several dermatological diseases, including the use for improvement of hyperkinetic wrinkle lines, has aroused our curiosity regarding the immunological changes that might occur in the skin.
We decided to study and compare the immunological repercussion of botulinum toxin type A in the skin, before and after its application in the treatment for axillary hyperhidrosis; for this study, we used the immunohistochemical markers anti-CD1a, CD4+, CD8+, CD25, TNF-á, HLA-DR, ICAM-1, IFN-ã and IL-4. The selection of this disease and of the treatment site are due to the simple and fast technique, low morbidity, response from all patients to treatment and the possibility of performing skin biopsy in an unexposed area. Therefore, the patients accepted to participate in this study.
This is a non-randomized, coorte, quasi-experimental study. Fifteen patients with axillary hyperhidrosis were selected for treatment after being informed about therapy, how biopsies would be performed, follow-up visits and signing the informed consent term. There were 14 females and one male, age range of 19-56 years (mean of 33.5 years). Following the diagnosis of hyperhidrosis, we demarcated the compromised axillary area with the Minor test.1
Botulinum toxin was diluted with 4 mL of saline solution immediately before treatment. We injected (using 1-mL syringes and 30 G½ needles) two units per injection site in the intradermal region.1,3,24,26-29 This dilution corresponds to the ratio of two units of medication to each 0.08 mL of solution. The distance maintained between the injection sites was 1.5 cm30-33 and the injection technique used is shown in Figure 1.
In the first stage of the research, we injected botulinum toxin in the left axilla and performed a normal skin biopsy in the axillary fold of the right axilla. Thirty days after the first injection, we treated the right axilla and performed a skin biopsy in the axillary fold of the previously treated left axilla.
Freezing Technique
The biopsy fragment to be sent for immunohistochemical evaluation was placed on a cork support and coated with a cryoprotective solution (Tissue-tek – Milles Laboratories, USA). Next, it was gradually frozen in a bottle containing liquefied isopentane (Reagen) in a liquid nitrogen environment, wrapped in aluminum paper and maintained in liquid nitrogen until processing.
Immunohistochemical Technique
Imunoperoxidase technique on cryopreserved tissue was used and adjusted. It is the current technique applied by the Tropical Dermatology Laboratory of Sao Paulo (HCFMUSP).
The frozen skin fragments underwent cryomicrotomy to obtain 4-micrometer thick fragments, collected with glass slides coated with adhesive solution of 3-aminopropyltrietoxisilane (Sigma Chemical Co., St Louis, MO/EUA, code A3648) followed by fixation with anhydrous acetone, pro-analysis, for 10 minutes at room temperature. After fixation, the specimens were submitted to immunohistochemical reaction with the method of streptavidin-peroxidase complex (modified from HSU, 1981).34 The material was washed with distilled water and submerged in the Tris-HCL (hydroxymethyl aminomethane – chloridric acid) buffer at pH 7.4, for 5 minutes, at room temperature. Subsequently, the endogenous peroxidase was blocked with hydrogen peroxide (3% in Tris-HCl) and the fragment was submitted to three immersions in the Tris-HCl buffer for 5 minutes. The area surrounding the fragment was dried and the primary antibody diluted in bovine serum albumin (BSA) was added by drops. The dilutions of primary antibodies were standardized with the use of skin fragments of psoriasis (for antibodies anti-ICAM-1 and anti-CD1a) and tonsillar extracts, obtained after elective tonsillectomies for the antibodies anti-CD4+, anti-CD8+ and anti-HLA-DR. The material was incubated overnight in a humid chamber and, on the next day, the specimens were submitted to three immersions in Tris-HCL, for five minutes. After drying the area surrounding the fragment, the secondary biotinylated antibody was dripped, that is, the mouse anti-immune globulin G produced in rabbits for the following antibodies: anti-ICAM-1 (DAKO, code M7063, titer 1/100), anti-HLADR (DAKO, code M704, titer 1/150), anti-CD4+ (DAKO, code M0716, titer 1/100), anti-CD8+ (DAKO, code M7103, titer 1/100), anti-CD1a (DAKO, code M721, titer 1/100), anti-CD25 (DAKO, code M0731, titer 1/20), anti-TNF-á (Santa Cruz, code SC-1350, titer 1/50, IFN-ã (R&D, code AF285-NA, titer 1/20) and for the anti-LihL-4 antibody (R&D, code AF204-NA, titer 1/20) (Table 1).
After immersion of the specimens in Tris-HCL (three changes in five minutes), the area surrounding the fragment was dried. The tertiary complex streptavidin-biotin- peroxidase (StrepABC complex/HRP duet, mouse/rabbit, DAKO, code KO492) was placed in a 1:500 dilution, and the specimens were incubated in a humid chamber, for one hour, at 35-37°C. After this stage, they were immersed in Tris-HCL three times within five minutes.
Development of immunoperoxidase reaction was performed with a solution of 0.006% 3,3’ diaminobenzidine hydrochloride (DAB) (Sigma Chemicals, code D-5637/D-8001) in a Tris-HCL buffer, added by 600 ìL of hydrogen peroxide 20 volume (H2O2/20v) for three minutes.
After staining, the specimens were rinsed in distilled water. Counterstaining was performed with Carazzi’s hematoxylin for one minute, and they were rinsed again for 5 minutes.
The material was dehydrated in an ascending chain of ethyl alcohol, diaphanized in xylol, coated with a Permount synthetic resin (Fisher Scientific Fair Lawn, NJ/USA, code SP15-100) and a cover slip.
The positive control of the reactions was obtained by observing the control fragments (psoriatic skin and tonsilla) concomitantly with the material being studied. The negative control of the reactions was indicated by the absence of primary antibodies that were replaced by the Tris-HCl buffer.
Histomorphometric Evaluation of the Specimens
Collected
A total of 32 slides with hematoxylin-eosin staining for control were prepared, and 288 slides were prepared for immunohistochemical reactions. Histomorphometric evaluations were performed using the Kontron Eletronic 300 Image Analysis System. The workstation was composed of a binocular optical microscope (Zeiss), a color video camera (SONY CCD-Iris), image digitalizing plaque, a microcomputer with a 133 MHz Pentium processor, IBMPC compatible, operating in Windows 95 – 32bits.
Images were digitalized with the aid of a specific program for image analysis (Kontron 300), which allowed sharing of data with the text processor (Microsoft Word) and electronic spreadsheet (Microsoft Excel). The use of these programs determined the treatment analysis and interpretation, acquisition of measurement values of the structures with all possible variables and automatic distribution of data generated by the image analysis station for electronic spreadsheets and text processor.
The histological fragments were processed in the image analysis system using 2x and 4x objective lenses, and 10x ocular lenses. This quantification was performed in the smallest spatial unit called pixel. The calibration factor (CF) was automatically calculated in pixels and this factor was used by the program for calculations in corresponding micrometers (ìm) according to calibration.
Skin represented in the histological sections of the specimens was analyzed with random evaluation of the dermis and epidermis. The area fraction was analyzed with a 20x magnification (anti-CD1a, anti-ICAM-1 and anti-TNF-ã). The cell count was performed with counts per field with a 40X magnification and a total of 10 fields per slide were counted (anti-CD25, anti-LihL-4, anti-CD4+, anti-CD8+, anti-IFN-ã and anti-HLA-DR). The mean of these fields was counted for obtaining the final results.
The count of structures that could be well individualized, such as CD4+ and CD8+ lymphocytes in the dermalepidermal junction, Langerhans cells in the epidermal area, dermal ICAM-1 and HLA-DR, was oriented by the cell nuclei. Stained vascular endothelium was disregarded in the count of ICAM-1 and HLA-DR due to the occurrence of this event in normal skin.
The count of individualized structures such as CD-25, anti-LihL-4, anti TNF-á and anti-IFN-ã was oriented by immunohistochemical labeling in cytoplasm. In such cases, the stained nuclei were disregarded. The reading and count were carried out by a skilled and well-trained observer.
Statistical Analysis
The statistical analysis involved the use of a statistical package called SPSS 10.0 for Windows. Descriptive statistical analysis was performed in order to observe the frequency, mean, median, standard deviation and other parameters; that is, the descriptive statistics illustrates more clearly the data distribution and behavior.
For evaluation of the possible immunological changes in the skin according to the immunohistochemical aspect, before and after injection of the BoNTA, the non-parametrical statistical analysis was used by means of the Wilcoxon sign test.35,36
Slides stained with hematoxylin-eosin showed in all cases the presence of normal skin that was compatible with the area studied.
Figures 2 through 8 show the evaluation and number of structures that could be well individualized; the photographs are representations of the labeled specimens indicating the cells or areas that were evaluated by the digitalized photograph system described in the methods section. The structures are exhibited as follows: CD4+ (Figure 2) and CD8+ (Figure 3) lymphocytes in the dermo-epidermal junction; Langerhans cells (Figure 4) in the epidermal region; dermal ICAM-1 (Figure 5) and HLA-DR (Figure 6) oriented by the cell nuclei. CD-25 (Figure 7), anti- LihL-4 (Figure 8), anti TNF-á and anti-IFN-ã, were oriented by the immunohistochemical labeling in the cytoplasm.
Initially, a descriptive statistical analysis was performed for each marker with values that were comparable (Table II). Afterwards, the data were analyzed with the Wilcoxon sign test (Tables III, IV and V). The evaluation of fragments obtained from the patient’s skin, before and after the treatment for axillary hyperhidrosis, regarding the immunohistochemical aspect resulted in the following data:
The anti-CD1a pre-treatment immunohistochemical labeling showed a minimum of 0.19 labeled cells/field and a maximum of 9.3 cells/field (mean of 3.51 cells/field); anti-CD4+ with a minimum of 12 and a maximum of 199 cells/ field (mean of 87.6 cells/field); anti-CD8+ with a minimum of 8 and a maximum of 293 cells/field (mean of 84.1 cells/field).
The anti-CD1a post-treatment immunohistochemical labeling showed a minimum of 0.41 labeled cells and a maximum of 7.5 cells/field (mean of 3.5 cells/field); anti- CD4 with a minimum of 20 and a maximum of 131 cells/ field (mean of 89 cells/field); anti-CD8+ with a minimum of 0 and a maximum of 293 cells/field (mean of 83.6 cells/field).
The immunohistochemical evaluation of the slides using anti-ICAM-1 marker in pre-treatment skin showed a minimum value of 0.045 and a maximum value of 1.9 cells/ field (mean of 0.62 cells/field) and post-treatment values of 0.11 (minimum) and 1.55 cells/field (maximum); mean of 0.7 cells/field. In Table II, the level of significance considered was 0.05 in the Wilcoxon sign tests.
The anti-CD25, anti-TNF-á, anti-HLA-DR, anti-LihL-4 and anti-IFN-ã pre-treatment immunohistochemical labeling showed only one positive case, with a pre-treatment cell count of 30 and a post-treatment count of 28 cells/field for anti- CD25, and the other pre-and post-treatment values were 2.05 and 6.85; 58 and 64; 0 and 85; 22 and 26 cells/field, respectively.
The pre-and post-treatment data analysis obtained with anti-CD4+ and CD8+ markers with Wilcoxon sign tests shows that there was no significant difference between the pre-and post-treatment stages (Table III).
When analyzing the data in detail, we observe a decrease in variability (see standard deviation) for the markers, except for anti-CD1a expressed in the dermis. Another way to observe this result when only the anti-CD1a marker is analyzed is that the minimum and maximum values significantly changed, thus reducing data dispersion. However, if we apply the test to analyze the means, we come to the conclusion that the response for the anti-CD1 immunohistochemical marker in the dermis before and after treatment is the same (Table VI). Applying the Student’s t-test, we also conclude that there is no significant difference between the results obtained.
For anti-TNF-á, anti-CD25, anti-HLA-DR, anti- LihL-4, IFN-ã and anti-ICAM-1 markers in the epidermis, no conclusion was achieved based on the data collected, since the results obtained were negative in almost all slides analyzed.
The analysis of pre- and post treatment data obtained with anti-ICAM-1 marker in the dermis using the Wilcoxon sign tests did not show any significant difference in the both stages (Table 5).
>Immune reactions were approached under different aspects according to the literature review, resulting in an update of current and past research.
Our study aimed to evaluate possible changes in immunity in the skin of patients receiving BoNTA injection. To this date, we have not found in the medical literature any study that addressed this issue regarding the action of this drug in the skin. We found a report of one patient who was treated for facial hyperkinetic wrinkles with BoNTA four times; one week after each injection, including the first one, the patient developed a herpes simplex in the area treated.
Despite its large-scale use for treatment of expression wrinkles and hyperhidrosis, there is no clinical and laboratory observation regarding what really happens in the skin. These facts aroused our interest in evaluating the immune behavior in the skin before and after the use of this medication.
The second biopsy in this study was performed 30 days after injection of the drug, based on studies in the medical literature suggesting that the process of recognition and activation of the immune system takes 20 to 30 days. This time period is respected and the recommendation so far is that reinjection of BT should be avoided before this interval in order to minimize the occurrence of sensitization 16,37.
We demonstrated that the skin does not present any immunological abnormality caused by the injection of the dosages used by most physicians to treat hyperhidrosis, when analyzing anti-CD4+, CD8+, HLA-DR, ICAM-1, TNF-á; CD25; LihL-4 and IFN-ã and anti-CD1a markers in the epidermis and dermis.
Immune markers were chosen with the purpose of defining a profile of cellular immunity and to know if there is any humoral stimulation in this kind of treatment. When analyzed separately, patients 3 and 4 showed increased reactions after treatment and patients 7, 8 and 11 showed decreased reactions (Table 2).
Would there be individual differences in immunological response of the skin after treatment of hyperhidrosis, both in terms of T-cell mediated immune response and humoral response in some individuals? And what would be the behavior of these patients after the second injection of the medication? A more detailed evaluation would be very interesting, applying the protocol used by Larsen et al. (1990)37, Lukas et al. (1996)38 and Sugiura et al. (2003),39 tracking cell migration with biopsies after 24 and 48 hours in addition to evaluation after 30 days. We believe this study should be performed starting from our research to try to determine if there is a local inflammatory response that is dependent or non-dependent on host mediators or the level of involvement of epidermal dendritic cells in intensity of the skin immune response to chemical products according to Lappin et al. (1996).40
Therefore, in our work we conclude that:
There was no change in the skin before or after treatment of hyperhidrosis with botulinum toxin type A when anti- CD4+, anti-CD8+, anti-HLA-DR, anti-ICAM-1, anti- TNF-á, anti-CD25, anti-LihL-4, anti-IFN-ã and anti-CD1a markers in the epidermis and dermis were evaluated according to the techniques proposed in this study.
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