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

Summary

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.

Abstract

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.

Introduction

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 triggers^1,2. 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.

Protocol

All methods described here have been approved by the International Animal Care and Use Committee (IACUC) of Korea University College of Medicine.  6 weeks after birth, experimenters can induce enthansasia and  collect various samples such as the skin and serum of rats.

1. Induction of AD by Neonatal Capsaicin Treatment

1. 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.
2. Add 8 mL of normal saline to the conical tube. Shake the tube vigorously until no separated layers are visible
3. Inject the capsaicin solution (10 µL/g) into the subcutaneous tissue, on the midline and backside of the neck of a neonatal Sprague-Dawley rat.
   1. 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.
   2. 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.  Since capsaicin-induced pain  can discourage breathing, adding general anesthesia could possibly worsen breathing difficulty. Thus, experimenters should not use anesthesics to increase survival of rats.

2. Evaluation of Pruritus and Dermatitis in a Rat Model of AD

1. Measurement of pruritus by counting spontaneous scratching behavior
   1. 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.
   2. Set up and adjust a digital video camera properly to record the mirror views and front views of the rats simultaneously.
   3. Place rats in the plastic chamber and cover the ceiling of the chamber with a heavy object to prevent from opening.
   4. Record the spontaneous behaviors of rats for 1 h, play back the video clips, and count the number of scratching behaviors.
2. Assessment of skin lesion using a scoring system
   1. Place the rat in the induction chamber.
   2. Turn the oxygen flowmeter to 2 L/min flow rate and adjust the isoflurane vaporizer to 3% for anesthesia.
   3. Identify the extent and severity of dermatitis in the ears and other parts of a rat.
   4. 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).

3. Exposure of Gaseous FA to Animals

1. Preparation of the inhalation chamber
   1. Prepare an acrylic glass box (55 cm x 40 cm x 35 cm) that has two outlets on opposite sides.
   2. 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.
   3. CAUTION. For the measurement of internal gas concentration, on another side of the outlets, drill three small passages, which can be closed.
   4. 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.
   5. Place a conventional rat cage inside the acrylic glass box.
   6. Put rats in the cage from one week after birth for 5 weeks (2 h/day and 5 days/week).
   7. 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.
2. Generation of gaseous FA by using a medical oxygen regulator
   1. Dissolve 5 g of FA into 1 L of water.
   2. Put the 0.5% FA solution in the humidifier bottle of the medical oxygen regulator.
   3. After connecting the inlet tube to the regulator, turn on the regulator and set the flow rate to 5 L/min.
   4. Adjust the solution concentration and the flow rate to determine the concentration of gas in the chamber.

Results

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...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Medical Oxygen Regulator	Yuyao Jiahua Medical		manufacturer will not affect results
Tube			manufacturer will not affect results
Acrylic Chamber	Taejin Acryl		manufacturer will not affect results
Plastic Chamber	Taejin Acryl		manufacturer will not affect results
Paraformaldehyde, powder	Sigma	158127	manufacturer will not affect results
Wrap filme	Uniwrap		manufacturer will not affect results
Capsaicin	Sigma	M2028	manufacturer will not affect results
Tween 80	Sigma	P4780	manufacturer will not affect results
Digital Video Camera	Sony	HDR-CX380y	manufacturer will not affect results
Isoflurane	JW pharmaceutical		manufacturer will not affect results