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Aug 2020
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Objective Quantitation of Focal Sweating Areas Using a Mouse Sweat-assay Model
小鼠汗液分析模型对局部出汗区域的客观定量   

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Abstract

In vivo sweat quantitation assays are required for the development of drugs for the management of focal hyperhidrosis before clinical trials; however, in vivo assays, particularly mouse models, are rare. Even in sweat assays using mice, sweating is quantitated by manually counting the number of sweating spots, which can contribute to various errors owing to arbitrary judgment. In this study, we developed a mouse sweat-assay model and a method for quantitating the amount of sweating to remove possible errors. The use of the iodine–starch test in the castor oil-covered hind footpad skin of anesthetized mice resulted in the sweating area being stained blue-black. After the anesthesia and treatment with drugs (pilocarpine, glycopyrrolate, botulinum neurotoxin, myricetin, and myricetin-loaded lipid nanoparticles), the remaining area of the footpad skin was eliminated from the acquired footpad images using ImageJ. Blue pixels extracted from the footpad image are automatically adjusted using the Phansalkar method, where the percentage of the blue area was determined based on the whole hind footpad skin area, finally indicating the percentage of the sweating area. Using this mouse model and analysis for sweat assays, a clear difference between the control group and antiperspirant-administered group was observed with respect to the sweating area % with no error. In conclusion, this assay can be used as a preclinical tool to screen potential antiperspirant drugs.


Graphic abstract:



Overview of the mouse-model sweat assay and objective quantitation of the focal sweating area


Keywords: Sweat assay (汗液分析), Mouse model (小鼠模型), Sweat quantitation (汗液定量), Mouse hind footpad perspiration (鼠后足底汗), Non-arbitrarily judged quantitation (非任意判断的定量)

Background

Numerous populations – nearly 3% of people aged 25-64 years – experience focal hyperhidrosis, an idiopathic abnormality characterized by hyper-sweating concentrated in certain regions of the body, e.g., palms, soles, face, and armpits (Haider et al., 2005). It interferes with daily activities; therefore, this condition poses substantial emotional, psychological, social, and professional burden (Strutton et al., 2004) and even leads to social anxiety disorders (Solish et al., 2007). During the development of drugs or medications for the treatment of focal hyperhidrosis, rational assays for evaluating sweating are necessary to evaluate excessive perspiration symptoms. Several tests used in clinical practice and research studies are not sensitive enough to detect subtle perspiration changes resulting from medications (Provitera et al., 2010). The thermoregulatory sweat test stimulates entire body sweating, including central and peripheral components, but does not localize the sweating lesion site (Fealey et al., 1989). Simple colorimetric procedures, such as the Neuropad test, evaluate sweating abnormalities but do not provide detailed analysis (Quattrini et al., 2008). In addition, abundant in vivo preclinical data are necessary for the verification of anti-perspiration efficacy before clinical trials; however, studies, particularly using mouse models, are rare.


The iodine-starch test is a well-known chemical reaction between starch and iodine, which instantly produces an intense blue-black color. The test was first described in 1814 (Colin et al., 1814) and first applied in 1928 as a medical test for visualizing sudomotor function (perspiration or sweating) (Minor, 1928). Polyiodides complexed with the amylose helix in starch form infinite polyiodide homopolymers and produce the blue-black color, whereas non-ionized iodine dissolved in nonpolar solvents and individual iodide (I) do not react with starch (Madhu et al., 2016). The triiodide (I3) ion is the simplest polyiodide, which is yellow-to-brown in aqueous conditions with no starch. In addition, the blue-black color of the triiodide-starch complex is so deep, which can also be detected visually when the concentration of iodine is as low as 2 × 10−5 M at 20°C. Consequently, the iodine-starch test has been used as a well-established diagnostic tool to evaluate underactive (hypohidrosis) and overactive (hyperhidrosis) sweating (Chia et al., 2013).


Even in the mouse sweat assays, various errors may occur owing to arbitrary judgment because the number of sweating spots, attributed to the iodine-starch reaction, is manually counted to quantitate the sweating score (Nejsum et al., 2002; Liu et al., 2017). For the data analysis, image-processing using ImageJ, a free software provided by the National Institutes of Health (NIH), can be utilized to eliminate possible arbitrary judgment and errors. In this protocol, we generated a mouse sweat-assay model and used ImageJ for image analysis (Ban et al., 2020). The sweating area on the skin of hind footpads of anesthetized mice was stained blue-black using an iodine-starch test. The blue-black pixels were extracted from the footpad images obtained, adjusted using the Auto Local Threshold function in ImageJ, and measured to the area fraction (sweating area %). Using the present protocol, a clear difference between the control and the antiperspirant-administered mice was observed with respect to the sweating area % with no error. Moreover, whether expected antiperspirants exploit either circulatory-systematic or focal anti-perspiration efficacy can also be assessed using this protocol.

Materials and Reagents

  1. Pipet tips, 20 µl, 200 µl, and 1,000 µl (Gilson, catalog numbers: F161671, F1739311, and F161451, respectively)

  2. 0.2 μm syringe filter

  3. Conical tube with lid, 50 ml (SPL Life Sciences, catalog number: 50050)

  4. Microtubes, 200 µl and microtube 1.5 ml (Sigma-Aldrich, catalog number: Z374873; Eppendorf, catalog number: EP0030125150)

  5. Sterilized needles for syringe, 17 gauge (Korea vaccine)

  6. Sterilized syringe filter (Sartorius, catalog number: 17764-ACK)

  7. Sterilized syringes for general use, 1 and 50 ml (Korea vaccine)

  8. Mice (6-week-old Institute of Cancer Research, weighing 25-35 g; Koatec Co., Pyeongtaek, Korea)

  9. Myricetin (Sigma-Aldrich, catalog number: M6760)

  10. Glyceryl tristearate (Sigma-Aldrich, catalog number: T5016)

  11. Glyceryl trioctanaote (Sigma-Aldrich, catalog number: T9126)

  12. Brij S100 (Sigma-Aldrich, catalog number: 466387)

  13. Pluronic P-123 (Sigma-Aldrich, catalog number: 435465)

  14. Pilocarpine hydrochloride (pilocarpine) (Tokyo Chemical Industry, catalog number: P0434)

  15. Glycopyrrolate bromide (glycopyrrolate) (Tokyo Chemical Industry, catalog number: G0392)

  16. Neuronox 100U (botulinum neurotoxin type A, BoNT/A) (Medytox)

  17. Alfaxalone solution, 10 mg/ml (Alfaxan) (Alfaxan Multidose, Jurox Animal Health)

  18. Xylazine hydrochloride solution, 23.32 mg/ml (Rumpun, Bayer)

  19. Starch (Millipore, catalog number: 101252)

  20. Castor oil (Sigma-Aldrich, catalog number: 259853)

  21. Iodine (Sigma-Aldrich, catalog number: 207772)

  22. Ethanol (Sigma-Aldrich, catalog number: 02891)

  23. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: 276855)

  24. HyClone phosphate-buffered saline, pH 7.4 (PBS) (GE Healthcare, catalog number: SH30256.01)

  25. Rumpun solution (see Recipes)

  26. Anesthesia solution (see Recipes)

  27. Pilocarpine solution (see Recipes)

  28. Glycopyrrolate solution (see Recipes)

  29. Myricetin solution (see Recipes)

  30. Myricetin-loaded lipid nanoparticles (M-LNPs) (see Recipes)

  31. Starch solution (see Recipes)

  32. Iodine solution (see Recipes)

Equipment

  1. Pipets (20, 200, and 1,000 µl) (Gilson, catalog numbers: FA10003M, FA10005M, and FA10006M, respectively)

  2. Electric pharynx

  3. Pioneer analytical balance, capacity 320 g (Ohaus, model: PX323E)

  4. Cages for stabilizing between the time of anesthetic injection and complete anesthesia

  5. Paintbrushes to apply iodine and starch solutions on the hind footpads of mice

  6. Digital camera (Canon, model: EOS 800D)

  7. Mouse restrainers were prepared by making holes in the plastic wall of conical tubes and the lid using an electric pharynx (Figure 1)

Software

  1. ImageJ (NIH, http://rsb.info.nih.gov/ij/) for image-processing and determination of the sweating area %

  2. SigmaPlot 10.0 Windows version (IBM Co.) for drawing graphs

  3. SPSS Statistics version 23.0 software (IBM Co.) for the statistical analyzes

Procedure

Male mice (5 mice per cage) were housed at a temperature of 21 ± 1°C and a humidity of 55 ± 10% under controlled laboratory conditions and maintained with food and water ad libitum. Mice were tested during the light phase of a 12-h light cycle (lights on at 6:00 am and off at 6:00 pm).


  1. Anesthesia

    1. Six-week-old Institute of Cancer Research (ICR) mice, weighing 25-35 g, were purchased from Koatec Co. (Pyeongtaek, Korea). After arrival, the mice were housed in an animal facility for 7 days for acclimatization.

    2. The mice were anesthetized by intraperitoneal injection of anesthesia solution (8.5 ml·kg−1; Recipe 2). All procedures for the sweat assay were terminated within 1 h before the mice woke up.

    3. The mice were placed on a 37°C-heating pad in an empty cage for 5 min without any stimulus (e.g., noise and light) for stabilization of anesthesia.

    4. The anesthetized mice were carefully transported into a mouse restrainer (Equipment 7) and covered using a screw-lid.


  2. Drug administration

    1. The drug solutions (pilocarpine, glycopyrrolate, BoNT/A, myricetin, and M-LNP, see the Recipes section) were prepared and warmed to 37°C immediately before administration. If needed, the drug solutions were sterilized by passing through a 0.2-μm syringe filter before use.

    2. Administration of the drug solution after stabilization of anesthesia

      1. Application on hind footpad skin (Figure 1A)

        1. The drug solution was pipetted onto the hind footpad skin. Precautions were taken not to over-apply the solution.

        2. A mouse-containing restrainer was placed on the heating pad for 30 min to allow sufficient time for absorption and action of the agents.

      2. Subcutaneous injection (Figure 1B)

        1. A needle-equipped syringe was loaded with drug solution. Care was taken not to inject the drug solution over 20 µl to avoid excessive bulging of the skin.

        2. To remove the air in the syringe column, the piston was pressed while keeping the syringe with the needle-side up.

        3. The needle was slowly inserted into the right hind footpad skin as close to the surface as possible, while maintaining an almost parallel angle between the needle and the skin surface to avoid subcutaneous structural damage and bleeding. Thin needles (17 gauge) were used to minimize skin damage, and fresh needles were used every time to prevent infection.

        4. The drug solution was injected and the needle was removed. If saliva secretion stimulants such as pilocarpine hydrochloride were used, saliva overflow was eliminated using a wipe to prevent suffocation.

        5. Any overflow was wiped off.

        6. The mouse-containing restrainer was placed down on the heating pad for 10 min to allow sufficient time for action of the agents.



    Figure 1. Administration of the drugs in the sweat assay after anesthesia. A. Procedures for the application of the drug solutions on the skin of the right hind footpads of mice. B. Procedures for the subcutaneous injection of the drug solutions into the right hind footpads of mice.


  3. Iodine-starch test (Figure 2)

    1. After administration of the drugs (pilocarpine, glycopyrrolate, BoNT/A, myricetin, and M-LNP), the remainder of the solution was wiped off the footpad skin, the skin washed with warm PBS, and the liquid wiped off again.

    2. Using a paintbrush, the iodine solution was applied to both the left and right hind footpads and allowed to dry for 1 min. The paintbrush used was wider than the horizontal width of the hind footpads, which enabled painting of enough iodine solution at once. Further, the paintbrush was soaked in iodine solution before use to avoid the formation of iodine crystals resulting from ethanol-drying. To avoid the formation of spots with iodine crystals, which can influence the values of the resulting sweating area %, application was done only once.

    3. To block sweat evaporation from the skin surface, a coat of starch-oil solution was applied once on the footpad skin by brushing at 37°C. A different paintbrush was used for this step. To avoid starch aggregation during the application, the starch solution was stirred.

    4. Immediately after coating with the starch solution, photographs of the hind footpad were acquired under fixed conditions of distance, magnification, and exposure. Atmospheric exposure of the starch-covered hind footpads for longer than 5 min can affect the unwanted color change due to moisture; thus, we suggest 5 min as the time limit for determining the sweating area (%).

    5. Description of the test: The iodine-in-ethanol solution was first applied over the footpad skin and the ethanol was then evaporated for 1 min. Next, the starch-in-oil solution was applied over the footpad skin covered with iodine. The sweat is captured between the skin and the oil layer since the moisture cannot diffuse out through the hydrophobic oil layer. In turn, dissolution into the captured sweat makes the brownish iodine react with the starch and turns a blue-black color as a result of ionization to polyiodide ions. In addition, the concentrations of starch and iodine in the starch solution and iodine solution, respectively, are high enough to induce the blue-black color change if the mice sweat.

      Note: The number of solution applications, once each, is more important than the volume. Therefore, the color can be changed to blue-black by each serial application of the iodine and starch solutions only once, without any risk of a lack of color change.



    Figure 2. Iodine-starch test in the sweat assay. A. Procedures of the test. B. Illustration of an expected mechanism for the test.

Data analysis

  1. Image-processing and sweating area % determination

    1. Area selection (Figure 3A): The images of the hind footpad skin of mice were individually processed in ImageJ and only the footpad area was cropped.

    2. Modifying the images (Figure 3A): “Image > Type > RGB stack”, the cropped real color images of the footpad skin area were converted into grayscale.

    3. Auto local threshold (Figure 3B): The grayscale images of the blue stack were adjusted by selecting “Image > Adjust > Auto Local Threshold” using the Phansalkar method. Radius: 15; Parameter 1 and 2: 0; check “White objects on black background”; uncheck “Stack.” Black pixels in the adjusted images indicate the sweating spot on the hind footpad skin. As shown in Figure 3C, the Phansalkar method is the best way to select the sweating spots among all the modules available the Auto Local Threshold.

      Note: Do not touch any initial settings for the Auto Local Threshold except the Phansalkar method selection.

    4. Determination of the sweating area % (Figure 3D): The measurements were set by selecting “Analyze > Set Measurements” and checking “‘Area fraction” before the measurement. The area fraction values of the black pixels in the adjusted images were measured and recorded by either pressing the “Ctrl + M” keys on a keyboard or clicking “Analyze > Measure.” The sweating area % was calculated by subtracting the area fraction from 100%.



    Figure 3. ImageJ-based image-processing and measurement of the sweating area %. A. Area-selection of the mouse footpad skin in the obtained image and conversion of the selected image into grayscale (the blue stack among red, green, and blue stacks) using RGB stack. B. Adjustment of the blue stack-based grayscale image using the Auto Local Threshold, according to the Phansalkar method, for extracting the sweating area. C. The adjusted images using all the modules for the Auto Local Threshold, including the Bernsen, Contrast, Mean, Median, MidGrey, Niblack, Otsu, Phansalkar, and Sauvola methods. D. Establishing the method for the measurement of the Area fraction that is used for determining the sweating area % values.


  2. Assessment of the reliability of image-processing

    All the sweating area % values of each group were determined from the footpad skin images obtained at different predetermined times (0, 1, 2, 3, 4, and 5 min) after the iodine-starch test using ImageJ (Figure 4A and 4B). The reliability of the image-processing for determining the sweating area % values of each group was assessed using the coefficient of determination (R2) of the linear-fitting curves, as shown in Figure 4C. Reliable data: R2 ≥ 0.9; unreliable data: R2 < 0.9. Unreliable datasets were excluded when determining the sweating area % values of a certain group.



    Figure 4. Reliability assessment of the image-processing procedures for determining the sweating area %. A. Procedures for the representative image-processing of the right hind footpad skin that is not administered with any drug (non-treatment) for 5 min after the initiation of the iodine-starch test. B. Area fraction values (%Area) determined 0, 1, 2, 3, 4, and 5 min after the procedures for the representative image-processing. C. The linear-fitting curves for the sweating area % values of non-treatment, negative control (Pilocarpine) and positive control (Glycopyrrolate) groups. Data are expressed as the mean ± SD (standard deviation; n = 5).


  3. Statistical analysis and plotting the graph for the sweating area %

    All data from the sweat assay are reported as the average and standard deviation (SD) of at least five independent mouse experiments. Statistical analyses were performed using a Student’s t-test with SPSS Statistics. The bar graph including the average and SD of the sweating area % value for each mouse group was generated using SigmaPlot 10.0 (Figure 5).



    Figure 5. Representative sweating area % values of the mouse hind-footpad skin 5 min after the iodine-starch test. Sweating area % values (%Area of black pixel fraction) on (A) right hind footpads, directly treated (by either subcutaneous injection or applied on the skin), and (B) left hind footpads, not treated with drug. Groups: no treatment, PBS (0.8 ml/kg), pilocarpine HCl (2.5 mg/kg, negative control), glycopyrrolate bromide (0.25 mg/kg, positive control), BoNT/A (0.8 U/kg, positive control), myricetin (M; 0.8 mg/kg), and M-LNP (myricetin-loaded lipid nanoparticle; 0.8 ml/kg). Data are expressed as the mean ± SD (n = 5; Student’s t-test; ns, non-significant; *, P < 0.05; **, P < 0.01).


  4. Assessment of the focal anti-perspiratory effect of drugs

    Sweating area % values for left hind footpads, to which drugs were not administered, are represented as separate graphs to assess the circulatory-system or topical efficacy of the drugs. Herein, the sweating area % reduction in both the right and left footpads indicates the circulatory-system anti-perspiration efficacy of the drug, whereas that only in the right footpad indicates the topical efficacy.

Recipes

  1. Rumpun solution

    Rumpun (xylazine hydrochloride, 23.32 mg/ml) diluted with PBS to 20 mg/ml xylazine hydrochloride

  2. Anesthesia solution

    Mixture of Alfaxan and Rumpun solution at a volumetric ratio of 16:1

  3. Pilocarpine solution

    Pilocarpine hydrochloride diluted with PBS to 3.125 mg/ml

  4. Glycopyrrolate solution

    Glycopyrrolate bromide diluted with PBS to 312.5 μg/ml

  5. Myricetin solution

    5% (w/v) myricetin solution (in DMSO) diluted with PBS to 1 mg/ml immediately before administration to prevent crystal formation

  6. Myricetin-loaded lipid nanoparticles (M-LNPs)

    M-LNPs prepared using the method previously reported by our group (Ban et al., 2020); concentration of myricetin in the M-LNP system: 1 mg/ml

  7. Starch solution

    10% (w/v) starch dispersed in castor oil and vortexed immediately before use to prevent sedimentation

  8. Iodine solution

    3.5% (w/v) iodine dissolved in ethanol

Acknowledgments

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (No. 2020R1A2C2101964, 2017R1A6A1A03015642, and 2018R1D1A1B07050508). This protocol was derived from Ban et al. (2020).

Competing interests

There are no competing interests to be declared.

Ethics

This study was approved by the Ethics Committee of Sungkyunkwan University (approval no. SKKUIACUC2018-04-11-2). All mice included in this study were housed in a facility at Sungkyunkwan University, and all experimental protocols were approved by the Animal Care and Use Committee of Sungkyunkwan University.

References

  1. Ban, C., Park, J. B., Cho, S., Kim, H. R., Kim, Y. J., Choi, Y. J., Chung, W. J. and Kweon, D. H. (2020). Reduction of focal sweating by lipid nanoparticle-delivered myricetin. Sci Rep 10(1): 13132.
  2. Chia, K. Y. and Tey, H. L. (2013). Approach to hypohidrosis. J Eur Acad Dermatol Venereol 27(7): 799-804.
  3. Colin, J. J. and Gaultier de Claubry, H. F. (1814). Mémoire sur les combinassions de l'iode avec les substances végétales et animaux. Ann Chim Phys 90: 87-110.
  4. Fealey, R. D., Low, P. A. and Thomas, J. E. (1989). Thermoregulatory sweating abnormalities in diabetes mellitus. Mayo Clin Proc 64(6): 617-628.
  5. Haider, A. and Solish, N. (2005). Focal hyperhidrosis: diagnosis and management. CMAJ 172(1): 69-75.
  6. Liu, Y., Sebastian, B., Liu, B., Zhang, Y., Fissel, J. A., Pan, B., Polydefkis, M. and Farah, M. H. (2017). Sensory and autonomic function and structure in footpads of a diabetic mouse model. Sci Rep 7: 41401.
  7. Madhu, S., Evans, H. A., Doan-Nguyen, V. V., Labram, J. G., Wu, G., Chabinyc, M. L., Seshadri, R. and Wudl, F. (2016). Infinite Polyiodide Chains in the Pyrroloperylene-Iodine Complex: Insights into the Starch-Iodine and Perylene-Iodine Complexes. Angew Chem Int Ed Engl 55(28): 8032-8035.
  8. Minor, V. (1928). Ein neues Verfahren zu der klinischen Untersuchung der Schweißabsonderung. Dtsch Z Nervenheilkd 101(1): 302-308.
  9. Nejsum, L. N., Kwon, T. H., Jensen, U. B., Fumagalli, O., Frokiaer, J., Krane, C. M., Menon, A. G., King, L. S., Agre, P. C. and Nielsen, S. (2002). Functional requirement of aquaporin-5 in plasma membranes of sweat glands. Proc Natl Acad Sci U S A 99(1): 511-516.
  10. Provitera, V., Nolano, M., Caporaso, G., Stancanelli, A., Santoro, L. and Kennedy, W. R. (2010). Evaluation of sudomotor function in diabetes using the dynamic sweat test. Neurology 74(1): 50-56.
  11. Quattrini, C., Jeziorska, M., Tavakoli, M., Begum, P., Boulton, A. J. and Malik, R. A. (2008). The Neuropad test: a visual indicator test for human diabetic neuropathy. Diabetologia 51(6): 1046-1050.
  12. Solish, N., Bertucci, V., Dansereau, A., Hong, H. C., Lynde, C., Lupin, M., Smith, K. C., Storwick, G. and Canadian Hyperhidrosis Advisory, C. (2007). A comprehensive approach to the recognition, diagnosis, and severity-based treatment of focal hyperhidrosis: recommendations of the Canadian Hyperhidrosis Advisory Committee. Dermatol Surg 33(8): 908-923.
  13. Strutton, D. R., Kowalski, J. W., Glaser, D. A. and Stang, P. E. (2004). US prevalence of hyperhidrosis and impact on individuals with axillary hyperhidrosis: results from a national survey. J Am Acad Dermatol 51(2): 241-248.

简介

[摘要]在临床试验之前,体内汗液定量测定是开发局灶多汗症治疗药物所需要的; ħ H但是,在体内测定法,特别是小鼠模型,是罕见的。即使我Ñ汗试验使用小鼠,出汗是定量itat的ED通过手动计数出汗斑点,这可以向由于任意判定各种错误的数目。在这项研究中,我们开发了一个鼠标汗水试验模型和孔定量方法达荷兰国际集团出汗,除去可能出现的错误的数量。使用碘的淀粉在蓖麻油覆盖后测试anesthet的脚掌皮肤美化版小鼠导致出汗区域被染成蓝色-黑色。麻醉和治疗后用药物(毛果芸香碱,格隆溴铵,肉毒杆菌神经毒素,杨梅黄酮,杨梅黄酮和加载的脂质纳米颗粒小号),足垫皮肤的剩余区域从所获取的足垫消除图像使用ImageJ。从脚踏板图像中提取的蓝光像素使用Phansalkar方法自动调整,其中蓝色区域的百分比是根据整个后脚踏板皮肤区域确定的,最终指示出汗区域的百分比。使用该小鼠模型和用于汗水测定分析小号,明确二fference对照组和止汗剂给药组之间相对于所述出汗面积%,没有观察到错误。总之,该测定法可用作筛选潜在止汗药的临床前工具。



图形摘要:

小鼠模型汗试验和目标孔定量的概述吨的通货膨胀的焦点汗水荷兰国际集团区域


[背景]人口众多 -近3%的25岁的人- 64年-经验焦点多汗症,特发性异常,其特征在于超-出汗集中在身体的某些区域,例如,手掌,脚掌,面部,和腋下(海德尔。等人,2005)。它会干扰日常活动;因此,这种情况造成了巨大的情感,心理,社会和职业负担(Strutton等,2004),甚至导致了社交焦虑症(Solish等,2007)。在用于治疗局灶性多汗症的药物或药物的开发过程中,评估出汗的合理分析方法对于评估过度出汗症状是必要的。临床实践和研究中使用的几种测试不够灵敏,无法检测出药物引起的细微的汗液变化(Provitera等,2010)。体温调节性汗液测试可刺激整个身体的汗液,包括中央和周围的成分,但不会使出汗的病变部位局部化(Fealey等,1989)。简单的比色程序(例如Neuropad测试)可评估出汗异常,但未提供详细分析(Quattrini等人,2008)。此外,大量的临床前临床数据对于在临床试验之前验证止汗离子的功效是必不可少的。^ h H但是,研究,特别是利用小鼠模型中,并不多见。

碘-淀粉试验是淀粉和碘,它立即产生之间的公知的化学反应š一个强烈的蓝色-黑色。该测试首次在1814年进行了描述(Colin等,1814),并在1928年首次用作医学测试,以显示sudomotor功能(出汗或出汗)(Minor,1928年)。聚碘络合的淀粉形式无限多碘均聚物的直链淀粉螺旋和产生蓝黑色,而非离子化碘溶解在非极性溶剂和个体碘化物(I - )不与淀粉反应(马杜等人,2016) 。三碘化物(I 3 - )离子是最简单的多碘,其是黄色〜褐色与无淀粉水性条件。另外,三碘化物-淀粉复合物的蓝黑色非常深,当碘的浓度在20°C时低至2×10 -5 M时,也可以目视检测到。因此,碘-淀粉试验已被用来作为良好-建立的诊断工具,以评估运动不足(少汗)和过度活动性(多汗症)出汗(正大等人,2013年)。

即使在小鼠汗试验,因为出汗斑点的数量,归因于碘可能由于任意判定发生各种错误α-淀粉反应,被手动计数,以孔定量泰特发汗得分(Nejsum等人,2002;刘等人。,2017) 。对于数据分析,使用ImageJ,由全国学院提供一个免费的软件图像处理小号卫生(NIH),可以被用来消除可能的任意判断和错误。在此协议中,我们生成了一个小鼠汗液检测模型,并将ImageJ用于图像分析(Ban等人,2020年)。对麻醉小鼠的后足垫皮肤出汗面积染成蓝色-使用黑色ñ碘型淀粉测试。蓝色-黑色像素从得到的足垫图像中提取,使用自动局部阈值功能在ImageJ的调整,并测量到的面积率(出汗面积%)。使用本方案,相对于出汗面积%,观察到对照和给予止汗剂的小鼠之间有明显的差异,没有错误。此外,是否预期止汗利用任一循环-系统或局灶性止汗功效也可使用此协议评估。

关键字:汗液分析, 小鼠模型, 汗液定量, 鼠后足底汗, 非任意判断的定量



材料和试剂


移液管尖端,20微升,200微升,和1 ,000微升(吉尔森,目录号:F161671,F1739311,和F161451,分别地)
0.2微米注射器式过滤器
带盖锥形管,50毫升(SPL Life Sciences,目录号:50050)
200 µl微量管和1.5 ml微量管(Sigma-Aldrich,目录号:Z374873; Eppendorf,目录号:EP0030125150)
注射器用消毒针,17号(韩国疫苗)
无菌注射器过滤器(Sartorius,货号:17764-ACK)
普通消毒注射器,1毫升和50毫升(韩国疫苗)
小鼠(6周龄研究所癌症研究,体重25 - 35克; Koatec有限公司平泽,韩国)
杨梅素(Sigma-Aldrich,目录号:M6760)
三硬脂酸甘油酯(Sigma-Aldrich,目录号:T5016)
甘油三辛酸酯(Sigma-Aldrich,目录号:T9126)
Brij S100(Sigma-Aldrich,目录号:466387)
Pluronic P-123(Sigma-Aldrich,目录号:435465)
盐酸毛果芸香碱(毛果芸香碱)(东京化学工业,目录号:P0434)
溴乙酸格隆溴铵(东京工业化学,目录号:G0392)
Neuronox 100U(A型肉毒神经毒素,BoNT / A)(Medytox )
Alfaxalone溶液,10 mg · ml -1 (Alfaxan )(Alfaxan Multidose,Jurox动物保健)
盐酸赛拉嗪溶液,23.32 mg · ml -1 (Rumpun ,Bayer)
淀粉(Millipore,目录号:101252)
蓖麻油(Sigma-Aldrich,目录号:259853)
碘(Sigma-Aldrich,目录号:207772)
乙醇(Sigma-Aldrich,目录号:02891)
二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:276855)
HyClone磷酸盐缓冲盐水,pH 7.4(PBS)(GE Healthcare,目录号:SH30256.01)
Rumpun解决方案(请参阅食谱)
麻醉溶液(请参阅食谱)
毛果芸香碱溶液(请参阅食谱)
格隆溴铵溶液(请参阅配方)
杨梅素溶液(请参阅食谱)
装有杨梅素的脂质纳米颗粒(M-LNP)(请参阅食谱)
淀粉解决方案(请参阅食谱)
碘溶液(请参阅食谱)


设备


吸管小号(20,200,和1000微升)(吉尔森,目录号:FA10003M,FA10005M,和FA10006M,分别地)
电动咽
先锋分析天平,容量320克(Ohaus,型号:PX323E)
在麻醉剂注射和完全麻醉之间保持稳定的笼子
在老鼠的后脚掌上涂抹碘和淀粉溶液的画笔
数码相机(佳能,型号:EOS 800D)
通过制备小鼠限制器形成孔我Ñ锥形管和盖使用电动咽的塑料壁(图1)


软件


ImageJ(NIH,http: //rsb.info.nih.gov/ij/ ),用于图像处理和确定出汗面积%
SigmaPlot 10.0 Windows版本(IBM Co.),用于绘制图形
用于统计分析的SPSS Statistics 23.0版软件(IBM Co.)


程序


雄性小鼠(5只小鼠每笼)圈养在温度21±1 ℃,和一个55±10%的控制的实验室条件下的湿度和食物和水保持自由采食。在12小时光照周期的光照阶段对小鼠进行了测试(上午6:00点亮,下午6:00熄灭)。


                麻醉
癌症研究(ICR)小鼠,体重25六周龄研究所- 35克,自购Koatec有限公司(平泽,韩国)。到任后,小鼠被安置在一个动物设施7天以适应环境。
小鼠瓦特ERE麻醉由麻醉溶液的腹膜内注射(8.5 ml的·千克-1 ;配方2)。用于汗水测定所有程序均在1个小时内终止是前小鼠瓦特Ó柯达。
将小鼠放置于37℃,加热垫在一个空的保持架5分钟而没有任何刺激物(例如,噪声和光)为stabiliz的通货膨胀麻醉。
麻醉小鼠瓦特ERE小心运送到小鼠限制器(设备7 ),并使用螺旋盖覆盖。


                药物管理
该药物溶液(毛果芸香碱,格隆溴铵,的BoNT / A,杨梅黄酮,和M-LNP,见配方小号部)瓦特ERE制备,并温热至37℃ ,立即给药之前。如果需要,药物溶液通过一个0.2通过灭菌-微米注射器过滤器在使用前。
管理的药液麻醉稳定后
                 在后脚掌皮肤上的应用(图1A)
                              将药物溶液吸移到后足垫皮肤上。采取了预防措施以免过度使用该溶液。
                             将包含小鼠的限制器放在加热垫上30分钟,以留出足够的时间吸收和作用药物。
                皮下注射(图1B)
                              甲针装有注射器装有药物溶液。小心不要过度注入20内的药液微升,以避免过多的鼓胀的皮肤。
                             为了除去注射器色谱柱中的空气,在保持注射器针头朝上的同时,对活塞施压。
                           针缓慢插入右后肢足垫皮肤为接近表面成为可能,同时保持针和皮肤表面之间的大致平行的角度,以避免皮下structur人损伤和出血。使用细针(17号针头)以最大程度地减少对皮肤的伤害,并且每次都使用新鲜的针来防止感染。
                           钍é药物溶液注入并在取出针。如果使用唾液分泌刺激剂(如盐酸匹洛卡品),可使用抹布消除唾液溢出以防止窒息。
                             任何溢出都被清除了。
                           将包含小鼠的限制器放到加热垫上10分钟,以留出足够的时间来使药剂起作用。






图1.麻醉后在汗液测定中施用这些药物。A.程序的药液对皮肤应用的小鼠的右后足垫。B.程序的皮下注射药物溶液进入了小鼠的右后足垫。


                我odine -淀粉试验(图2)
后的药物(在施用毛果芸香碱,格隆溴铵,的BoNT / A,杨梅黄酮,和M-LNP ),其余的的溶液擦去足垫皮肤,皮肤用温暖PBS,并在液体再擦去。
使用画笔,碘溶液施加到两个所述左,右后足垫并使其干燥1分钟。所使用的画笔比后脚垫的水平宽度宽,从而可以立即绘制足够的碘溶液。另外,在使用前将画笔浸入碘溶液中,以避免乙醇干燥导致形成碘晶体。为了避免与碘晶体斑点,这可以影响的值的形成的结果ING汗水荷兰国际集团面积%,应用程序是只进行一次。
为了阻止汗液从皮肤表面蒸发,通过在37°C的刷子刷洗一次,将淀粉-油溶液的涂层涂在脚垫皮肤上。此步骤使用了另一种画笔。为了避免在施用过程中淀粉聚集,搅拌淀粉溶液。
用淀粉溶液涂布后,立即在固定的距离,放大倍率和曝光条件下获取后脚垫的照片。淀粉覆盖的后脚掌在大气中暴露超过5分钟可能会由于水分而影响不必要的颜色变化;吨HUS,我们建议5分钟作为用于确定出汗面积(%)的时间限制。
描述的测试:Ť他碘在乙醇溶液首先施加在足垫皮肤,然后蒸发乙醇1分钟。接下来,将油包淀粉溶液涂在覆盖有碘的脚垫皮肤上。汗水被捕获之间的皮肤和油层,因为湿气不能透过扩散出来的疏水油层。反过来,溶解到所捕获的汗水化妆š的褐色碘与淀粉反应并变成一个蓝黑色颜色作为结果电离到多碘离子。此外,淀粉在淀粉溶液和碘溶液浓度和碘,分别是足够高到引起如果蓝黑颜色变化的小鼠汗水。
注意:每个解决方案应用程序的数量比数量更重要。因此,色彩可以由每个序列申请改为蓝黑色的ication碘和淀粉溶液只有一次,没有任何风险缺乏颜色变化。






图2.汗液分析中的碘-tarch试验。一。测试程序。B.测试的预期机制的图示。


数据分析


图像处理和出汗面积%测定
                 区域选择(图3A):在ImageJ中分别处理了小鼠后脚掌皮肤的图像,仅裁剪了脚掌区域。
                修改所述图像(图3A):“图像>类型> RGB栈”,将裁剪的实际彩色图像的足垫皮肤区域被转换成克一个yscale。
                 自动局部阈值(图3B):在GR一个蓝色叠层的yscale图像通过调整选择“图像>调整>汽车局部阈值”使用Phansalkar方法。半径:15;参数1和2:0;选中“黑色背景上的白色物体”;取消选中“堆栈” 。调整后的图像中的黑色像素表示后脚掌皮肤上的出汗点。如图3C所示,该Phansalkar方法是选择出汗斑点的最佳方式中的所有可用的自动局部阈值的模块。
注意:不要触摸任何初始设置小号的自动局部阈值除Phansalkar方法选择。


                出汗面积百分比的确定(图3D):通过选择“分析>设置测量值”并在测量前检查“面积分数”来设置测量值。黑色像素的调整后的图像面积率的值分别为米通过按按“Ctrl + M”键的键盘上或点击“分析>测量easured并记录。通过从100%中减去面积分数来计算出汗面积%。






图3.基于ImageJ的图像处理和出汗面积%的测量。A.区域选择所获得的图像在小鼠足垫皮肤和转换所选图像的成克的一个使用RGB堆yscale(红色,绿色,和蓝色的堆叠蓝色栈)。B.蓝色基于堆栈的GR的调整一个yscale使用自动局部阈值,图像根据所述Phansalkar方法,用于提取出汗区域。C.使用“自动局部阈值”的所有模块(包括Bernsen,Contrast,Mean,Median,MidGrey ,Niblack ,Otsu,Phansalkar和Sauvola方法)调整后的图像。D.建立用于确定出汗面积%值的面积分数的测量方法。


评估图像处理的可靠性
各组的所有出汗面积%值确定从所述后在不同的预定时间获得的足垫皮肤图像(0,1,2,3,4,和5分钟)碘-淀粉使用ImageJ(图4A和4B)测试。所述图像处理用于确定每个组的出汗面积%值的可靠性进行评估使用的决定系数(- [R 2的线性拟合曲线的),如图4C所示。可靠的数据:- [R 2 ≥ 0.9; 数据不可靠:R 2 <0.9。在确定特定组的出汗面积%值时,将不可靠的数据集排除在外。






图4.确定出汗面积%的图像处理程序的可靠性评估。A.在开始进行碘-淀粉测试后5分钟内未使用任何药物(未治疗)的右后足垫皮肤的代表性图像处理程序。B.在代表性图像处理的程序之后的0、1、2、3、4和5分钟确定面积分数值(%Area)。C.非治疗组,阴性对照组(毛果芸香碱)和阳性对照组(甘草次酸盐)的出汗面积%值的线性拟合曲线。数据表示为平均值±SD(标准偏差;n = 5)。


统计分析并绘制出汗面积的图表%
来自汗液测定法的所有数据均报告为至少五个独立小鼠实验的平均值和标准偏差(SD)。使用带有SPSS Statistics的Student t检验进行统计分析。使用SigmaPlot 10.0生成包括每个小鼠组出汗面积%值的平均值和SD的条形图(图5)。






图5.碘-淀粉测试后5分钟,小鼠后足垫皮肤的代表性出汗面积%值。上出汗面积%的值(黑象素部分的面积%)(A)右后肢足垫,直接进行处理(由任一皮下注入离子或施加在皮肤上),和(B)左后肢足垫,没有吨用药物处理。组:不治疗,PBS(0.8 ml ·kg -1 ),盐酸匹洛卡品(2.5 mg ·kg -1 ,阴性对照),格隆溴铵溴化物(0.25 mg ·kg -1 ,阳性对照),BoNT / A(0.8 U ·kg -1 ,阳性对照),杨梅素(M; 0.8 mg ·kg -1 )和M-LNP(杨梅素负载的脂质纳米颗粒; 0.8 ml ·kg -1 )。数据表示为平均值±SD(n = 5; Student's t检验; ns,无显着性; *,P <0.05; **,P <0.01)。


药物对局部止汗作用的评估
出汗面积%值左后肢足垫,向其中不施用药物,被表示为单独的图形,以评估循环系统的系统或局部效力的药物。此处,右脚垫和左脚垫上的出汗面积%减少表明该药物的循环系统抗汗功效,而仅右脚垫上的汗液面积%表明局部功效。


菜谱


Rumpun解决方案
用PBS稀释的Rumpun (盐酸赛拉嗪23.32 mg · ml -1 )稀释至20 mg · ml -1赛拉嗪盐酸盐


麻醉液
体积比为16:1的Alfaxan和Rumpun溶液的混合物


毛果芸香碱溶液
PBS将盐酸匹罗卡品稀释至3.125 mg· ml -1


格隆溴铵溶液
用PBS稀释的溴化格隆溴铵至312.5μg· ml -1


杨梅素溶液
给药前立即用PBS将5%(w / v)的杨梅素溶液(在DMSO中)稀释至1 mg· ml -1 ,以防止形成晶体


杨梅素脂质脂质纳米粒(M-LNPs)
用我们小组先前报道的方法制备的M-LNPs (Ban et al。,2020); M-LNP系统中杨梅素的浓度:1 mg ·ml -1


淀粉溶液
10%(w / v)淀粉分散在蓖麻油中并在使用前立即涡旋以防止沉淀


碘溶液
溶于乙醇的3.5%(w / v)碘


致谢


这项工作是由韩国国家研究基金会(NRF)的支持授予由韩国政府(MSIT)(第2020R1A2C2101964,2017R1A6A1A03015642资助,并2018R1D1A1B07050508)。该协议源自(Ban et al。,2020)。


利益争夺


没有要宣布的利益冲突。


伦理


这项研究是经Ë thics Ç韩国成均馆大学(批准文号SKKUIACUC2018-04-11-2)的ommittee。这项研究中包括的所有小鼠均被安置在成均馆大学的设施中,所有实验方案均已得到成均馆大学动物保护和使用委员会的批准。


参考


Ban,C.,Park,JB,Cho,S.,Kim,HR,Kim,YJ,Choi,YJ,Chung,WJ和Kweon ,DH(2020)。通过脂质纳米颗粒递送的杨梅素减少局部出汗。科学代表10(1):13132。
Chia,KY和Tey ,HL(2013)。多汗症的方法。J Eur Acad Dermatol Venereol 27(7):799-804。
约翰·科林(Colin)和HF·高迪埃·德·克劳布里(Gaultier de Claubry)(1814)。蜜饯和动漫的蜜饯大事记。Ann Chim Phys 90:87-110。
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引用:Ban, C. and Kweon, D. (2021). Objective Quantitation of Focal Sweating Areas Using a Mouse Sweat-assay Model. Bio-protocol 11(11): e4047. DOI: 10.21769/BioProtoc.4047.
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