Name: Effects of Exposure of Formaldehyde to a Rat Model of Atopic Dermatitis Induced by Neonatal Capsaicin Treatment (Video) | JoVE Journal
Uploaded: 2017-09-27T12:30:00.000+00:00
Duration: 6 min 47 s
Description: Learn to induce atopic dermatitis-like symptoms in rats using neonatal capsaicin treatment and assess formaldehyde exposure effects. This method helps researchers study pathophysiological mechanisms of skin inflammation.

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5 minutes

This research was supported by the Public Welfare &#38; Safety research program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2014M3C8A5030612), and supported by The Chung Yang, Cha Young Sun, M.D. and Jang Hi Joo Yeu Sa Memorial Fund.

All methods described here have been approved by the International Animal Care and Use Committee (IACUC) of Korea University College of Medicine. &#160;6 weeks after birth, experimenters can induce enthansasia and &#160;collect various samples such as the skin and serum of rats.
1. Induction of AD by Neonatal Capsaicin Treatment
<ol>
	<li>To prepare 5 mg/mL capsaicin solution, add 50 mg of capsaicin to 1 mL of Tween 80 and 1 mL of 100% ethanol in a 15 mL conical tube.</li>
	<li>Add 8 mL of normal saline to the conical tube. Shake the tube vigorously until no separated layers are visible</li>
	<li>Inject the capsaicin solution (10 &#181;L/g) into the subcutaneous tissue, on the midline and backside of the neck of a neonatal Sprague-Dawley rat.
	<ol>
		<li>Deliver the capsaicin between 12 h to 48 h after birth (CRITICAL). 
		NOTE: If the injection is delayed by more than a few days, dermatitis will not develop or it will persist only for few weeks. If the injection is too early, respiratory distress can occur due to severe pain.</li>
		<li>To prevent neonatal rats from respiratory arrest due to the capsaicin injection, push the chest of neonates to encourage breathing or keep the neonates in an oxygen chamber until the respiration becomes normal. &#160;Since capsaicin-induced pain &#160;can discourage breathing, adding general anesthesia&#160;could&#160;possibly worsen breathing difficulty. Thus, experimenters should not use anesthesics to increase survival of rats.</li>
	</ol>
	</li>
</ol>
2. Evaluation of Pruritus and Dermatitis in a Rat Model of AD
<ol>
	<li>Measurement of pruritus by counting spontaneous scratching behavior
	<ol>
		<li>Prepare plastic chambers (20 cm x 30 cm x 20 cm) equipped with a mirror to allow for full coverage for viewing and holes to allow the rats to breathe.</li>
		<li>Set up and adjust a digital video camera properly to record the mirror views and front views of the rats simultaneously.</li>
		<li>Place rats in the plastic chamber and cover the ceiling of the chamber with a heavy object to prevent from opening.</li>
		<li>Record the spontaneous behaviors of rats for 1 h, play back the video clips, and count the number of scratching behaviors.</li>
	</ol>
	</li>
	<li>Assessment of skin lesion using a scoring system
	<ol>
		<li>Place the rat in the induction chamber.</li>
		<li>Turn the oxygen flowmeter to 2 L/min flow rate and adjust the isoflurane vaporizer to 3% for anesthesia.</li>
		<li>Identify the extent and severity of dermatitis in the ears and other parts of a rat.</li>
		<li>Define the unit size for the extent of skin lesions as 0.25 cm2 and the severity of skin lesions by following Table 1: severity index; degree of dermatitis score by multiplying the extent and severity (see dermatitis score in Figure 1B).</li>
	</ol>
	</li>
</ol>
3. Exposure of Gaseous FA to Animals
<ol>
	<li>Preparation of the inhalation chamber
	<ol>
		<li>Prepare an acrylic glass box (55 cm x 40 cm x 35 cm) that has two outlets on opposite sides.</li>
		<li>CAUTION. For circulation of FA gas, place the outlet higher than the inlet and make the size of the outlet larger than that of the inlet.</li>
		<li>CAUTION. For the measurement of internal gas concentration, on another side of the outlets, drill three small passages, which can be closed.</li>
		<li>Connect tubes to both outlets. Place an outlet tube to circulation hood or outside of the building for emission of the gas, and connect the other outlet tube to a medical oxygen regulator.</li>
		<li>Place a conventional rat cage inside the acrylic glass box.</li>
		<li>Put rats in the cage from one week after birth for 5 weeks (2 h/day and 5 days/week).</li>
		<li>Seal the acrylic glass box as tightly as possible to prevent gas leakage. 
		NOTE: For tight sealing, the chamber lid should contain rubber. Apply cling film to wrap the chamber for tight sealing.</li>
	</ol>
	</li>
	<li>Generation of gaseous FA by using a medical oxygen regulator
	<ol>
		<li>Dissolve 5 g of FA into 1 L of water.</li>
		<li>Put the 0.5% FA solution in the humidifier bottle of the medical oxygen regulator.</li>
		<li>After connecting the inlet tube to the regulator, turn on the regulator and set the flow rate to 5 L/min.</li>
		<li>Adjust the solution concentration and the flow rate to determine the concentration of gas in the chamber.</li>
	</ol>
	</li>
</ol>

<ol><li>Geha, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Allergy+and+hypersensitivity.+Nature+versus+nurture+in+allergy+and+hypersensitivity%5BTitle%5D)+AND+%22Curr+Opin+Immunol%22%5BJournal%5D)">Allergy and hypersensitivity. Nature versus nurture in allergy and hypersensitivity.</a> Curr Opin Immunol. 15 (6), 603-608 (2003).</li>
<li>Novak, N., Bieber, T., Leung, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immune+mechanisms+leading+to+atopic+dermatitis%5BTitle%5D)+AND+%22The+J+Allergy+Clin+Immunol%22%5BJournal%5D)">Immune mechanisms leading to atopic dermatitis.</a> The J Allergy Clin Immunol. 112 (Suppl 6), S128-S139 (2003).</li>
<li>Back, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chronically+relapsing+pruritic+dermatitis+in+the+rats+treated+as+neonate+with+capsaicin%3B+a+potential+rat+model+of+human+atopic+dermatitis%5BTitle%5D)+AND+%22J+Dermatol+Sci%22%5BJournal%5D)">Chronically relapsing pruritic dermatitis in the rats treated as neonate with capsaicin; a potential rat model of human atopic dermatitis.</a> J Dermatol Sci. 67 (2), 111-119 (2012).</li>
<li>Jin, H., He, R., Oyoshi, M., Geha, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Animal+models+of+atopic+dermatitis%5BTitle%5D)+AND+%22J+Invest+Dermatol%22%5BJournal%5D)">Animal models of atopic dermatitis.</a> J Invest Dermatol. 129 (1), 31-40 (2009).</li>
<li>Han, R. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Formaldehyde-Induced+Aggravation+of+Pruritus+and+Dermatitis+Is+Associated+with+the+Elevated+Expression+of+Th1+Cytokines+in+a+Rat+Model+of+Atopic+Dermatitis%5BTitle%5D)+AND+%22PLOS+ONE%22%5BJournal%5D)">Formaldehyde-Induced Aggravation of Pruritus and Dermatitis Is Associated with the Elevated Expression of Th1 Cytokines in a Rat Model of Atopic Dermatitis.</a> PLOS ONE. 11 (12), e0168466(2016).</li>
<li>Xu, B., Aoyama, K., Takeuchi, M., Matsushita, T., Takeuchi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expression+of+cytokine+mRNAs+in+mice+cutaneously+exposed+to+formaldehyde%5BTitle%5D)+AND+%22Immunol+Lett%22%5BJournal%5D)">Expression of cytokine mRNAs in mice cutaneously exposed to formaldehyde.</a> Immunol Lett. 84 (1), 49-55 (2002).</li>
<li>Kim, J. Y., Jeong, M. S., Park, K. Y., Seo, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aggravation+of+atopic+dermatitis-like+symptoms+by+consecutive+low+concentration+of+formaldehyde+exposure+in+NC%2FNga+mice%5BTitle%5D)+AND+%22Exp+Dermatol%22%5BJournal%5D)">Aggravation of atopic dermatitis-like symptoms by consecutive low concentration of formaldehyde exposure in NC/Nga mice.</a> Exp Dermatol. 22 (3), 219-221 (2013).</li>
<li>Laske, N., Niggemann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Does+the+severity+of+atopic+dermatitis+correlate+with+serum+IgE+levels%3F%5BTitle%5D)+AND+%22Pediatr+Allergy+Immunol%22%5BJournal%5D)">Does the severity of atopic dermatitis correlate with serum IgE levels?</a> Pediatr Allergy Immunol. 15 (1), 86-88 (2004).</li>
<li>Fujii, Y., Takeuchi, H., Sakuma, S., Sengoku, T., Takakura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+a+2%2C4-dinitrochlorobenzene-induced+chronic+dermatitis+model+in+rats%5BTitle%5D)+AND+%22Skin+Pharmacol+Physiol%22%5BJournal%5D)">Characterization of a 2,4-dinitrochlorobenzene-induced chronic dermatitis model in rats.</a> Skin Pharmacol Physiol. 22 (5), 240-247 (2009).</li>
<li>Sugimoto, T., Xiao, C., Ichikawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neonatal+primary+neuronal+death+induced+by+capsaicin+and+axotomy+involves+an+apoptotic+mechanism%5BTitle%5D)+AND+%22Brain+Res%22%5BJournal%5D)">Neonatal primary neuronal death induced by capsaicin and axotomy involves an apoptotic mechanism.</a> Brain Res. 807 (1-2), 147-154 (1998).</li>
<li>Saito, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+skin+inflammation+induced+by+repeated+exposure+of+toluene%2C+xylene%2C+and+formaldehyde+in+mice%5BTitle%5D)+AND+%22Environ+Toxicol%22%5BJournal%5D)">Characterization of skin inflammation induced by repeated exposure of toluene, xylene, and formaldehyde in mice.</a> Environ Toxicol. 26 (3), 224-232 (2011).</li>
<li>Jung, W. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Formaldehyde+exposure+induces+airway+inflammation+by+increasing+eosinophil+infiltrations+through+the+regulation+of+reactive+oxygen+species+production%5BTitle%5D)+AND+%22Environ+Toixcol+Pharmacol%22%5BJournal%5D)">Formaldehyde exposure induces airway inflammation by increasing eosinophil infiltrations through the regulation of reactive oxygen species production.</a> Environ Toixcol Pharmacol. 24 (2), 174-182 (2007).</li>
</ol>

The authors have nothing to disclose.

The protocol described here for the induction of AD-like symptoms in rats can be adapted for studying AD pathophysiology and used as a screening tool for drug test. The instrument for the exposure of gaseous FA to capsaicin-treated rats can be applied to evaluate the effects of exposure to various gases on diverse animal models.
The neonatal capsaicin-treated AD model relies on a more simple procedure than other atopic models such as the DNCB treatment model9 and epicut...

To interrogate the effects of formaldehyde (FA) inhalation on atopic dermatitis (AD) progression, we established a protocol for AD induction and developed a conventional circulation chamber for chronic exposure of FA.
AD is a chronic inflammatory skin disease attributed to the complex interactions between genetic predisposition and environmental triggers1,2. A proper animal model is required to study AD in the preclinical state. This is most important when elucidating the pathogenesis of AD under the influence of environmental factors. Moreover, to better investigate the effects of the environmental triggers on the AD progression, it is essential to separate the individual environmental factors and quantify the degree of exposure.
We have demonstrated a novel rat model of AD induced by neonatal capsaicin treatment. This AD model3 has some merits over other AD models such as the AD models induced by epicutaneous sensitization, the genetically-engineered AD models, and the spontaneous mouse models of AD4. First, this model develops AD-like symptoms at the age of 3 weeks, which corresponds to infant AD and is earlier than other spontaneous mouse models of AD such as the NC/Nga mouse. In some cases, unlike the genetically-engineered AD models, the symptoms of the AD models subside and relapse spontaneously over a 20-week period. Moreover, the induction process of AD-like symptoms in the model is easier than that of the epicutaneous sensitization model, which is more similar to the allergic contact dermatitis in the perspective of the induction process.
We have introduced the novel protocol for FA exposure to an animal model by using an acrylic glass chamber and a conventional medical oxygen regulator5 to study the effects of FA inhalation on the development of AD in infantile and adolescent periods. The regulator has allowed us to expose the animals to a constant concentration of gas by adjusting the flow rates and the solution concentration. Previous studies have reported several ways of exposure of FA to animal models. Xu et al. described the exposure method in which FA solution was directly applied to the skin6. Another recent study addressed the protocol of FA gas exposure using a specialized volatile solvents generator, model 49127. Compared to methods of previous studies, the new method is more easily equipped and can be applied for other solutions in addition to FA.

<table><tbody><tr><td>Medical Oxygen Regulator</td><td>Yuyao Jiahua Medical</td><td></td><td>manufacturer will not affect results</td></tr><tr><td>Tube</td><td></td><td></td><td>manufacturer will not affect results</td></tr><tr><td>Acrylic Chamber</td><td>Taejin Acryl</td><td></td><td>manufacturer will not affect results</td></tr><tr><td>Plastic Chamber</td><td>Taejin Acryl</td><td></td><td>manufacturer will not affect results</td></tr><tr><td>Paraformaldehyde, powder</td><td>Sigma</td><td>158127</td><td>manufacturer will not affect results</td></tr><tr><td>Wrap filme</td><td>Uniwrap</td><td></td><td>manufacturer will not affect results</td></tr><tr><td>Capsaicin</td><td>Sigma</td><td>M2028</td><td>manufacturer will not affect results</td></tr><tr><td>Tween 80</td><td>Sigma</td><td>P4780</td><td>manufacturer will not affect results</td></tr><tr><td>Digital Video Camera</td><td>Sony</td><td>HDR-CX380y</td><td>manufacturer will not affect results</td></tr><tr><td>Isoflurane</td><td>JW pharmaceutical</td><td></td><td>manufacturer will not affect results</td></tr></tbody></table>

effects of exposure of formaldehyde to a rat model of atopic dermatitis induced by neonatal capsaicin treatment

Effects of Exposure of Formaldehyde to a Rat Model of Atopic Dermatitis Induced by Neonatal Capsaicin Treatment

Atopic dermatitis is chronically relapsing pruritic eczema and prevails around the world especially in developed countries. Complex interactions between genetic and environmental factors are known to play an important role in the pathophysiology of atopic dermatitis. However, we still lack a detailed picture of the pathogenesis of this disease. Thus, it is of importance to develop appropriate animal models for elucidating the progression of atopic dermatitis. Moreover, investigating the effect of environmental factors such as air pollutants on atopic dermatitis expands understanding of the disease. Here, we describe a method for inducing atopic dermatitis in rats with neonatal capsaicin treatment and a protocol for exposure of a constant concentration of formaldehyde to rats to reveal effects on the development of atopic dermatitis in infantile and adolescent periods. These protocols have been successfully applied to several experiments and can be used for other substances.

The AD-like skin inflammation of the capsaicin-treated AD rat model at 7 weeks of age is presented in Figure 1A. Robust scratching behaviors of the capsaicin-treated AD rat model is shown in Video 1. Neonatal capsaicin treatment elicited dermatitis and aggravated scratching behaviors in rats 3 weeks of age, and AD-like symptoms persisted for several weeks (Figure 1B, C). Though the degree was les...

We describe here methods for neonatal capsaicin treatment to induce atopic dermatitis in rats and exposure of vaporized formaldehyde to investigate the effects of formaldehyde inhalation on atopic dermatitis.

Atopic dermatitis is chronically relapsing pruritic eczema and prevails around the world especially in developed countries. Complex interactions between ...

The overall goal of this procedure is to induce atopic dermatitis-like symptoms by neonatal capsaicin treatment. And to measure the effect of formaldehyde on atopic dermatis-like symptoms. This method can help answer a key question related to the pathophysiological of atopic dermatitis in the dermatological field.The major advantage of this technique are the induction of atopic dermatitis-like symptoms and the establishment of an important exposure system with the increments. To prepare five milligrams per milliliter capsaicin solution add one milliliter of Tween 80, one milliliter of 100%ethanol and 50 milligrams of capsaicin to a 15 milliliter conical tube. Then add eight milliliters of normal saline to the conical tube.Cap and shake the tube vigorously until no separated layers are visible. It is critical to deliver the capsaicin between 12 to 48 hours after birth. Inject the capsaicin solution into the subcutaneous tissue on the midline and backside of the neck of a neonatal Sprague Dawley Rat.To prevent respiratory arrest due to the capsaicin injection place the neonates in an oxygen chamber until respiration becomes normal. If necessary push the chest of neonates to encourage breathing. Begin by preparing plastic chambers equipped with a mirror to allow for full coverage for viewing and holes to allow the rats to breath.Place the rats in the plastic chamber and cover the ceiling of the chamber with a heavy object to prevent the lid from opening. Then set up a digital video camera to simultaneously record both the mirror views and front views of the rats. Then record the spontaneous behaviors of the rats for one hour.Play back the video clips and count the number of scratching behaviors such as those exhibited by the rat on the right. And the rat in the center. To score the lesions identify the extent and severity of dermatitis in the ears and other parts of an anesthetized rat.Define the unit size for the extent of skin lesions as 0.25 centimeters squared. And the severity of skin lesions using this table. Prepare an acrylic glass box that has two connectors on opposite sides.For circulation of formaldehyde gas the venting outlet should be placed higher than the gas intake outlet. Connect tubes to both connectors. Run the venting outlet tube to the circulation hood or outside of the building for emission of the gas.And connect the other inlet tube to a medical oxygen regulator. Place a conventional rat cage inside the acrylic glass box. Put one week old rats in the cage and then seal the acrylic glass box as tightly as possible to prevent gas leakage.Apply cling film to wrap the chamber and close the chamber with a rubberized lid for tight sealing. Add 0.5%formaldehyde solution to the humidifier bottle connected to the medical oxygen regulator. Don't forget that working in formaldehyde can be extremely hazardous.And precaution such as masks and gloves should always be taken by performing this procedure. Next turn on the regulator and set the flow rate to five liters per minute. Expose the rats to formaldehyde gas for a two hour period.Shown here are representative photographs of skin inflammation in the capsaicin-treated rat. The chronological development of dermatitis of the capsaicin-treated rats is shown here. This image shoes the chronological development of scratching behaviors in the capsaicin-treated rats.This is a representative image of relapsing skin lesions of 16 week old capsaicin-treated rats. Serum IgE levels are significantly elevated in capsaicin-treated animals. Significant elevation of IL-4 mRNA expression is also observed in capsaicin-treated animals.Significant elevation of IL-13 mRNA expression in capsaicin-treated animals is shown here. These representative photographs show atopic dermatitis after the fifth week of treatment using 1.2 parts per million. And 0.8 parts per million formaldehyde compared to controlled treatment with air.Exposure to 1.2 parts per million formaldehyde but not 0.8 parts per million formaldehyde significantly exacerbated pruritis in the capsaicin-treated atopic dermatitis model. Similar results were observed when dermatitis was measured. Increased serum IgE levels were observed in the capsaicin-treated AD rat model treated with 1.2 parts per million formaldehyde exposure.Following this procedure other materials like gylcol solution can be used to answer additional questions related to the effects of particular matter on atopic dermatitis. After these developments this technique paved the way for researchers in the field of dermatology to explore the role of environmental factors in the pathogenesis of atopic dermatitis.

effects-exposure-formaldehyde-to-rat-model-atopic-dermatitis-induced

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Learn to induce atopic dermatitis-like symptoms in rats using neonatal capsaicin treatment and assess formaldehyde exposure effects. This method helps researchers study pathophysiological mechanisms of skin inflammation.

Effects of Exposure of Formaldehyde to a Rat Model of Atopic Dermatitis Induced by Neonatal Capsaicin Treatment

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