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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allergy+and+hypersensitivity.+Nature+versus+nurture+in+allergy+and+hypersensitivity.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+mechanisms+leading+to+atopic+dermatitis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronically+relapsing+pruritic+dermatitis+in+the+rats+treated+as+neonate+with+capsaicin;+a+potential+rat+model+of+human+atopic+dermatitis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+atopic+dermatitis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formaldehyde-Induced+Aggravation+of+Pruritus+and+Dermatitis+Is+Associated+with+the+Elevated+Expression+of+Th1+Cytokines+in+a+Rat+Model+of+Atopic+Dermatitis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+cytokine+mRNAs+in+mice+cutaneously+exposed+to+formaldehyde.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aggravation+of+atopic+dermatitis-like+symptoms+by+consecutive+low+concentration+of+formaldehyde+exposure+in+NC/Nga+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+the+severity+of+atopic+dermatitis+correlate+with+serum+IgE+levels?.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+2,4-dinitrochlorobenzene-induced+chronic+dermatitis+model+in+rats.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+primary+neuronal+death+induced+by+capsaicin+and+axotomy+involves+an+apoptotic+mechanism.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+skin+inflammation+induced+by+repeated+exposure+of+toluene,+xylene,+and+formaldehyde+in+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formaldehyde+exposure+induces+airway+inflammation+by+increasing+eosinophil+infiltrations+through+the+regulation+of+reactive+oxygen+species+production.">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

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...

Watch this Scientific Journal Video about Effects of Exposure of Formaldehyde to a Rat Model of Atopic Dermatitis Induced by Neonatal Capsaicin Treatment at JoVE.com

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

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 ...

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

Research

JoVE Journal

Immunology and Infection

9.5K Views.  Korea University. We beschrijven hier methoden voor de behandeling van neonatale capsaïcine ertoe atopische dermatitis bij ratten en blootstelling van gevaporiseerde formaldehyde te onderzoeken van de effecten van formaldehyde inhalatie op atopische dermatitis.

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

Chronic Post-Ischemia Pain Model for Complex Regional Pain Syndrome Type-I in Rats

Complex regional pain syndrome type-I (CRPS-I) is a neurological disease that causes severe pain among patients and remains an unresolved medical condition. However, the underlying mechanisms of CRPS-I have yet to be revealed. It is known that ischemia/reperfusion is one of the leading factors that causes CRPS-I. By means of prolonged ischemia and reperfusion of the hind limb, the rat chronic post-ischemia pain (CPIP) model has been established to mimic CRPS-I. The CPIP model has become a well-recognized animal model for studying the mechanisms of CRPS-I. This protocol describes the detailed procedures involved in the establishment of the rat model of CPIP, including anesthesia, followed by ischemia/reperfusion of the hind limb. Characteristics of the rat CPIP model are further evaluated by measuring the mechanical and thermal hypersensitivities of the hind limb as well as the nocifensive responses to acute capsaicin injection. The rat CPIP model exhibits several CRPS-I-like manifestations, including hind limb edema and hyperemia in the early stage after establishment, persistent thermal and mechanical hypersensitivities, and increased nocifensive responses to acute capsaicin injection. These characteristics render it a suitable animal model for further investigation of the mechanisms involved in CRPS-I.

Provided here is a protocol that details steps to establish an animal model of chronic post-ischemia pain (CPIP). This is a well-recognized model mimicking human complex regional pain syndrome type-I. Mechanical and thermal hypersensitivities are further evaluated, as well as capsaicin-induced nocifensive behaviors observed in the CPIP rat model.

Complex regional pain syndrome type-I (CRPS-I) is a neurological disease that causes severe pain among patients and remains an unresolved medical ...

Behavior

Separation of Rat Epidermis and Dermis with Thermolysin to Detect Site-Specific Inflammatory mRNA and Protein

Easy-to-use and inexpensive techniques are needed to determine the site-specific production of inflammatory mediators and neurotrophins during skin injury, inflammation, and/or sensitization. The goal of this study is to describe an epidermal-dermal separation protocol using thermolysin, a proteinase that is active at 4 &deg;C. To illustrate this procedure, Sprague Dawley rats are anesthetized, and right hind paws are injected with carrageenan. Six and twelve hours after injection, rats with inflammation and na&iuml;ve rats are euthanized, and a piece of hind paw, glabrous skin is placed in cold Dulbecco's Modified Eagle Medium. The epidermis is then separated at the basement membrane from the dermis by thermolysin in PBS with calcium chloride. Next, the dermis is secured by microdissection forceps, and the epidermis is gently teased away. Toluidine blue staining of tissue sections show that the epidermis is separated cleanly from the dermis at the basement membrane. All keratinocyte cell layers remain intact, and the epidermal rete ridges along with indentations from dermal papillae are clearly observed. Qualitative and real-time RT-PCR is used to determine nerve growth factor and interleukin-6 expression levels. Western blotting and immunohistochemistry are finally performed to detect amounts of nerve growth factor. This report illustrates that cold thermolysin digestion is an effective method to separate epidermis from dermis for evaluation of mRNA and protein alterations during inflammation.

Presented here is a protocol for the separation of epidermis from dermis to evaluate inflammatory mediator production. Following inflammation, rat hind paw epidermis is separated from the dermis by thermolysin at 4 &#176;C. The epidermis is then used for mRNA analysis by RT-PCR and protein evaluation by western blot and immunohistochemistry.

Easy-to-use and inexpensive techniques are needed to determine the site-specific production of inflammatory mediators and neurotrophins during skin ...

Cheek Injection Model for Simultaneous Measurement of Pain and Itch-related Behaviors

Itch was defined as &#34;an unpleasant cutaneous sensation that provokes a desire to scratch&#34;&#160;by Rothman in 1941. In mouse models, scratch bouts are typically counted to evaluate itch induced by pruritogens. However, previous reports have shown that algesic substances also induce scratching behaviors in a mouse neck injection model, which is the most common test used for scratching behaviors. This finding makes it difficult to study itch in mice. &#160;In contrast, capsaicin, a common algogen, reduced scratching behaviors in some neck injection experiments. Therefore, the effect of pain on scratching behaviors remains unclear. It is thus necessary to develop a method to concurrently investigate itch and pain sensation using behavioral tests. Here, a cheek injection model is introduced which can be used to simultaneously measure pain- and itch-related behaviors. In this model, pruritogens induce scratching behaviors while algesic substances induce wiping behaviors. Using this model, lysophosphatidic acid (LPA), an itch mediator found in cholestatic patients with itch, is shown to exclusively induce itch but not pain. However, in mouse models, LPA has been reported to be both a pruritogen and an algogen. Investigation into the effects of LPA in a mouse cheek injection model showed that LPA only induced scratching, but not wiping behaviors. This indicates that LPA acts as a pruritogen similarly in mice and humans, and demonstrates the utility of a cheek injection model for itch research.

Typically, the mouse neck injection model is used for evaluate pruritogen-induced scratch behaviors. However, the model provides information only on itch, not pain. Here, a cheek injection model is introduced in mice which can be used to simultaneously measure pain and itch-related behaviors.

Itch was defined as &#34;an unpleasant cutaneous sensation that provokes a desire to scratch&#34;&#160;by Rothman in 1941. In mouse models, scratch bouts ...

Neuroscience

Contact Hypersensitivity as a Murine Model of Allergic Contact Dermatitis

Contact hypersensitivity (CHS) is an experimental model of allergic contact dermatitis (ACD) that can be studied in mice. This study aims to present an objective laboratory method that may help to study the CHS reaction in mice, which can be measured and quantified by various tests. To induce CHS, on day "0", mice were sensitized on a previously shaved spot by abdominal skin painting with the hapten 2,4,6-trinitrochlorobenzene (TNCB) in an acetone-ethanol mixture, whereas negative control mice were sham sensitized with vehicle alone-acetone-ethanol mixture. On day "4", the baseline ear thickness was measured with a micrometer prior to the elicitation of CHS (challenge) by painting both ears with diluted TNCB both in the test and control groups. After 24 h, the ear swelling was measured with a micrometer. CHS is an example of a T cell-mediated immune response that causes swelling in inflamed tissue, peaking 24 h after the skin challenge with the same hapten. An increase in ear edema correlated with augmented ear weight, myeloperoxidase (MPO) activity, pro-inflammatory cytokine concentration in the ear extracts, increased thickening of the edematous dermis in the histological examination, and ear vascular permeability. There was also an increase in the concentration of TNP-specific IgG1 antibodies in the sera of the test group when compared with the control mice. Additionally, CHS can be successfully transferred with the CHS-effector cells obtained from donors previously sensitized with TNCB. The CHS-effector cells were administered intravenously into na&#239;ve recipient mice, which were subsequently challenged with the same diluted hapten. Ear swelling was measured with a micrometer 24 h later.

Contact hypersensitivity (CHS) is a murine experimental model of allergic contact dermatitis (ACD). CHS is based on sensitization with reactive hapten by painting the shaved skin of the chest and abdomen, with a subsequent ear skin challenge with a diluted hapten, causing a swelling reaction that is assessed in various ways.

Contact hypersensitivity (CHS) is an experimental model of allergic contact dermatitis (ACD) that can be studied in mice. This study aims to present an ...

A Mouse Ear Model for Allergic Contact Dermatitis Evaluation

Skin is the human body&#39;s first line of defense and one of the most exposed organs to environmental chemicals. Allergic contact dermatitis (ACD) is a common skin disease that manifests as a local rash, redness, and skin lesions. The occurrence and development of ACD are influenced by both genetic and environmental factors. Although many scholars have constructed a series of models of ACD in recent years, the experimental protocols of these models are all different, which makes it difficult for readers to establish them well. Therefore, a stable and efficient animal model is of great significance to further study the pathogenesis of atopic dermatitis. In this study, we detail a modeling method using 1-fluoro-2,4-dinitrobenzene (DNFB) to induce ACD-like symptoms in the ears of mice and describe several methods for assessing the severity of dermatitis during modeling. This experimental protocol has been successfully applied in some experiments and has a certain promotional role in the field of ACD research.

Here, we describe the methods for inducing allergic contact dermatitis in mouse ears by 1-fluoro-2,4-dinitrobenzene (DNFB) and how to evaluate the severity of allergic contact dermatitis.

Skin is the human body&#39;s first line of defense and one of the most exposed organs to environmental chemicals. Allergic contact dermatitis (ACD) is a ...

Ex Vivo Release of Calcitonin Gene-Related Peptide from the Trigeminovascular System in Rodents

Calcitonin gene-related peptide (CGRP) was first discovered in the 1980s as a splice variant from the calcitonin gene. Since its discovery, its role in migraine pathophysiology has been well established, first by its potent vasodilator properties and subsequently by its presence and function as a neurotransmitter in the sensory trigeminovascular system. The migraine-provoking ability of CGRP gave support to the pharma industry to develop monoclonal antibodies and antagonists inhibiting the effect of CGRP. A new treatment paradigm has proven effective in the prophylactic treatment of migraine. One of the useful tools to further understand migraine mechanisms is the ex vivo model of CGRP release from the trigeminovascular system. It is a relatively simple method that can be used with various pharmacological tools to achieve know-how to further develop new effective migraine treatments. The present protocol describes a CGRP release model and the technique to quantify the effect of pharmacological agents on the amount of CGRP released from the trigeminovascular system in rodents. A procedure describing the experimental approach from euthanasia to the measurement of protein levels is provided. The essential isolation of the trigeminal ganglion and the trigeminal nucleus caudalis from both mice and rats and the preparation of rat dura mater are described in detail. Furthermore, representative results from both species (rats and mice) are presented. The technique is a key tool to investigate the molecular mechanisms involved in migraine pathophysiology by using various pharmacological compounds and genetically modified animals.

Ex Vivo Release of Calcitonin Gene-Related Peptide from the Trigeminovascular System in Rodents

The present protocol describes the ex vivo calcitonin gene-related peptide (CGRP) release model and the strategy to quantify the effect of pharmacological agents on the amount of CGRP released from the trigeminovascular system in rodents.

Calcitonin gene-related peptide (CGRP) was first discovered in the 1980s as a splice variant from the calcitonin gene. Since its discovery, its role in ...

Creating a Preterm Rat Model for Early Postnatal Pain Research

A Preterm Rat Model for Pain Studies

This research delves into the consequences of consistent pinprick stimulation on preterm offspring to ascertain its long-term implications for pain sensitivity. The primary objective of this protocol was to investigate the impact of neonatal pinprick stimuli on the pain threshold in the later stages of life using a preterm rat model. By establishing this model, we aim to advance the research on understanding and managing early postnatal pain associated with prematurity. The findings of this study indicate that while the baseline thresholds to mechanical stimuli remained unaffected, there was a notable increase in mechanical hypersensitivity following complete Freund's adjuvant (CFA) injection in adult rats. Interestingly, compared with male rats, female rats demonstrated heightened inflammatory hypersensitivity. Notably, maternal behavior, the weight of the litters, and the growth trajectory of the offspring remained unchanged by the stimulation. The manifestation of altered nociceptive responses in adulthood after neonatal painful stimuli could be indicative of changes in sensory processing and the functioning of glucocorticoid receptors. However, further research is needed to understand the underlying mechanisms involved and to develop interventions for the consequences of prematurity and neonatal pain in adults.

Here, we present a concise protocol for creating a preterm rat model, facilitating research on early postnatal pain management. The method involves performing a cesarean section three days before the expected birth, extracting preterm rat pups via hysterectomy, and integrating them with the biological offspring of a surrogate dam.

This research delves into the consequences of consistent pinprick stimulation on preterm offspring to ascertain its long-term implications for pain ...

Multicolor Immunofluorescence of the Nasal Tissue in an Allergic Rhinitis Rat Model

Immunofluorescent Labeling in Nasal Mucosa Tissue Sections of Allergic Rhinitis Rats via Multicolor Immunoassay

Allergic rhinitis (AR) is a chronic, non-infectious inflammatory disease of the nasal mucosa, primarily mediated by specific immunoglobulin E (IgE), affecting approximately 10%-20% of the world's population. While immunofluorescence (IF) staining has long been a standard technique for detecting disease-specific protein expression, conventional IF techniques are limited in their ability to detect the expression levels of three or more proteins in the same sample. Consequently, multicolor IF techniques have been developed in recent years, which allow the simultaneous labeling of multiple targets in cells or tissues.
This protocol provides a comprehensive overview of the process for establishing a rat model of AR, obtaining nasal mucosal samples, and the technical procedures for multicolor immunofluorescence. All rats in the AR group exhibited typical symptoms such as sneezing, a runny nose, and an itchy nose, with behavioral observations scoring &#8805;5 points. Hematoxylin and eosin (H&amp;E) staining revealed increased inflammatory cell counts and disrupted nasal mucosal integrity in the AR group. Multicolor immunofluorescence (mIF) demonstrated increased expression of ROR&#947;t and TICAM-1, while Foxp3 expression decreased in the nasal mucosa tissue of AR rats.

Author Spotlight: Advancing Allergic Rhinitis Research with Multicolor Immunofluorescence

This protocol describes a multicolor immunofluorescence technique for evaluating the rat model of allergic rhinitis.

Allergic rhinitis (AR) is a chronic, non-infectious inflammatory disease of the nasal mucosa, primarily mediated by specific immunoglobulin E (IgE), ...

Medicine

Advanced Glycation End-Products Sensitize Human Sensory-Like Neuron Cells to Capsaicin-Induced Calcium Influx

Increased collagen-derived advanced glycation end products (AGEs) are consistently linked to painful diseases, including osteoarthritis, diabetic neuropathy, and neurodegenerative disorders. Human sensory-like neurons differentiated from the SH-SY5Y cell line gain pro-nociceptive functions when exposed to AGEs by releasing substance P and upregulating the transient receptor potential vanilloid 1 (TRPV1) expression. Here, we investigated whether this receptor was functionally active and whether the glycation process sensitizes sensory neurons to capsaicin excitation. Sensory-like neuron cells were obtained from the differentiation of SH-SY5Y cells with all-trans-retinoic acid and brain-derived neurotrophic factor. Incubation with glycated collagen extracellular matrix (ECM-GC) simulated a pro-nociceptive stimulus. Control cells were incubated with a non-glycated extracellular collagen matrix (ECM-NC). Fluo-8 Calcium Flux Assay Kit was used to assess calcium influx, which was stimulated by capsaicin. The results show that glycation increases calcium influx compared with cells treated with normal collagen, suggesting that sensory-like neurons express functional TRPV1 channels and that glycation increases capsaicin excitation. These data indicate AGEs hypersensitive sensory-like neuron&#160;cells, triggering pro-nociceptive signaling. Together, our results suggest that we established a functional model responsive to capsaicin that can be useful for screening candidates for managing painful conditions.

Increased collagen-derived advanced glycation end products (AGEs) are consistently linked to painful diseases. Here, we investigated whether glycation sensitizes sensory neurons to capsaicin excitation.

Increased collagen-derived advanced glycation end products (AGEs) are consistently linked to painful diseases, including osteoarthritis, diabetic ...

Establishing a Compound Acne Model in Rats Using Oleic Acid and C. acnes

A Rat Model of Compound Acne

Acne vulgaris is a prevalent chronic skin condition characterized by the presence of comedones, papules, and pustules on the face, neck, and chest. To simulate the inflammation of acne vulgaris, this protocol details an approach to establish a compound acne rodent model by inducing acne inflammation in rat ears using oleic acid and Cutibacterium acnes (C. acnes).&#160;Rats were randomly divided into four groups: the normal control group (NC), ears treated with oleic acid group (OA), ears treated with C. acnes group (C. acnes), ears treated with oleic acid and C. acnes (OA + C. acnes). To mimic excessive sebum production, oleic acid was smeared on the ears of rats in OA and OA + C. acnes groups for 25 days.
From days 21 to 25, C. acnes suspension was injected intradermally into the ears of rats in the C. acnes and OA + C. acnes groups to aggravate the acne inflammation. Ear thickness was measured weekly as a gauge of inflammation severity. Gross observation, hematoxylin and eosin staining, and immunohistochemistry (IHC) were conducted and the results showed that the ears of the OA group and the OA + C. acnes group were thickened and indurated, accompanied by erythema and the presence of comedones. Additionally, papules were observed in C. acnes and OA + C. acnes groups. The histopathology exhibited hyperkeratinization and expanded infundibulum of the hair follicles in OA and OA + C. acnes groups. Infiltration of inflammatory cells and abscesses were found in the dermis of C. acnes and OA + C. acnes groups. The IHC results confirmed increased levels of tumor necrosis factor (TNF)-&#945; in the dermis of C. acnes and OA + C. acnes groups. All the above results collectively indicated the successful establishment of the compound acne rodent model.

Author Spotlight: Development and Evaluation of a Compound Acne Rodent Model Using C. acnes and Oleic Acid

This protocol outlines the procedure for inducing acne inflammation in rat skin with oleic acid and Cutibacterium acnes.

Acne vulgaris is a prevalent chronic skin condition characterized by the presence of comedones, papules, and pustules on the face, neck, and chest. To ...

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

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