Name: effects of exposure of formaldehyde to a rat model of atopic dermatitis induced by neonatal capsaicin treatment
Uploaded: 2017-09-27T12:30:00
Description: 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

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 n...

<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 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 width="776">
	<tbody>
		<tr height="21">
			<td height="21" style="width:231px;height:21px;">Medical Oxygen Regulator</td>
			<td style="width:159px;">Yuyao Jiahua Medical</td>
			<td style="width:129px;"></td>
			<td style="width:257px;">manufacturer will not affect results</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:231px;height:21px;">Tube</td>
			<td style="width:159px;"></td>
			<td style="width:129px;"></td>
			<td>manufacturer will not affect results</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:231px;height:21px;">Acrylic Chamber</td>
			<td style="width:159px;">Taejin Acryl</td>
			<td style="width:129px;"></td>
			<td>manufacturer will not affect results</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:231px;height:21px;">Plastic Chamber</td>
			<td style="width:159px;">Taejin Acryl</td>
			<td style="width:129px;"></td>
			<td>manufacturer will not affect results</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:231px;height:21px;">Paraformaldehyde, powder</td>
			<td style="width:159px;">Sigma</td>
			<td align="right">158127</td>
			<td>manufacturer will not affect results</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:231px;height:21px;">Wrap filme</td>
			<td style="width:159px;">Uniwrap</td>
			<td></td>
			<td>manufacturer will not affect results</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:231px;height:21px;">Capsaicin</td>
			<td style="width:159px;">Sigma</td>
			<td>M2028</td>
			<td>manufacturer will not affect results</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:231px;height:21px;">Tween80</td>
			<td style="width:159px;">Sigma</td>
			<td>P4780</td>
			<td>manufacturer will not affect results</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:231px;height:21px;">Digital Video Camera</td>
			<td style="width:159px;">Sony</td>
			<td>HDR-CX380y</td>
			<td>manufacturer will not affect results</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:231px;height:21px;">Isoflurane</td>
			<td style="width:159px;">JW pharmaceutical</td>
			<td style="width:129px;"></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 ...

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

Research

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Immunology and Infection

Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient

Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with causing food- and water-borne disease outbreaks. The definitive host is the feline species where sexual replication occurs resulting in the development of the highly infectious and environmentally resistant oocyst. Infection occurs via ingestion of tissue cysts from contaminated meat or oocysts from soil or water. Infection is typically asymptomatic in healthy individuals, but results in a life-long latent infection that can reactivate causing toxoplasmic encephalitis and death if the individual becomes immunocompromised. Meat contaminated with T. gondii cysts have been the primary source of infection in Europe and the United States, but recent changes in animal management and husbandry practices and improved food handling and processing procedures have significantly reduced the prevalence of T. gondii cysts in meat1, 2. Nonetheless, seroprevalence in humans remains relatively high suggesting that exposure from oocyst contaminated soil or water is likely. Indeed, waterborne outbreaks of toxoplasmosis have been reported worldwide supporting the theory exposure to the environmental oocyst form poses a significant health risk3-5. To date, research on understanding the prevalence of T. gondii oocysts in the water and environment are limited due to the lack of tools to detect oocysts in the environment 5, 6. This is primarily due to the lack of efficient purification protocols for obtaining large numbers of highly purified T gondii oocysts from infected cats for research purposes. This study describes the development of a modified CsCl method that easily purifies T. gondii oocysts from feces of infected cats that are suitable for molecular biological and tissue culture manipulation7.

Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient

This study describes the development of a modified CsCl method that easily purifies T. gondii oocysts from feces of infected cats that are suitable for molecular biological and tissue culture manipulation

Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with ...

Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis

Tissue transglutaminase 2 (tTG2) is an intestinal digestive enzyme which deamidates already partially digested dietary gluten e.g. gliadin peptides. In genetically predisposed individuals, tTG2 triggers autoimmune responses that are characterized by the production of tTG2 antibodies and their direct deposition into small intestinal wall 1,2. The presence of such antibodies constitutes one of the major hallmarks of the celiac disease (CD). Epidermal transglutaminase (eTG) is another member of the transglutaminase family that can also function as an autoantigen in a small minority of CD patients. In these relatively rare cases, eTG triggers an autoimmune reaction (a skin rash) clinically known as dermatitis herpetiformis (DH). Although the exact mechanism of CD and DH pathogenesis is not well understood, it is known that tTG2 and eTG share antigenic epitopes that can be recognized by serum antibodies from both CD and DH patients 3,4.
In this study, the confocal microscopy examination of biopsy samples from skin lesions of two rhesus macaques (Macaca mulatta) with dermatitis (Table 1, Fig. 1 and 2) was used to study the affected tissues. In one animal (EM96) a spectral overlap of IgA and tTG2 antibodies (Fig. 3) was demonstrated. The presence of double-positive tTG2+IgA+ cells was focused in the deep epidermis, around the dermal papillae. This is consistent with lesions described in DH patients 3. When EM96 was placed on a gluten-free diet, the dermatitis, as well as tTG2+IgA+ deposits disappeared and were no longer detectable (Figs. 1-3). Dermatitis reappeared however, based on re-introduction of dietary gluten in EM96 (not shown). In other macaques including animal with unrelated dermatitis, the tTG2+IgA+ deposits were not detected. Gluten-free diet-dependent remission of dermatitis in EM96 together with presence of tTG2+IgA+ cells in its skin suggest an autoimmune, DH-like mechanism for the development of this condition. This is the first report of DH-like dermatitis in any non-human primate.

Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis

Dermatitis herpetiformis (DH) is a chronic inflammatory condition characterized by an autoimmune reaction between IgA and epidermal transglutaminase (eTG). DH develops in a very small portion of gluten-sensitive and/or celiac patients. The results of this study indicate that DH can also develop in a rhesus monkey host with symptoms of idiopatic dermatitis.

Tissue transglutaminase 2 (tTG2) is an intestinal digestive enzyme which deamidates already partially digested dietary gluten e.g. gliadin peptides. In ...

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity 1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection 3. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop 4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.
	Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.
	The principle and protocol of this methodology have been described in the literature 7,8. Additionally, alternate methodologies to isolate primary microglia are well described 9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia 12. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture 9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.
	It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions. This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5. Mast cell granule component and the allergic mediator &#946;-hexosaminidase, which is released linearly in tandem with histamine from mast cells6, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1. Originally published by Naal et al.1, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.
Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using &#949;280 = 4,200 L/M/cm)12. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.

Mast cell degranulation, the release of allergic mediators, is important in allergy, asthma, and parasite defense. Here we demonstrate techniques1 for assessing effects of drugs and toxicants on degranulation, methodology recently utilized to exhibit the powerful inhibitory effect of antibacterial agent triclosan2.

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

A Controlled Mouse Model for Neonatal Polymicrobial Sepsis

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse polymicrobial sepsis can be induced by injecting cecal slurry intraperitoneally into day of life 7 mice and monitoring them for the following week. Presented here are the detailed steps necessary for the implementation of this neonatal sepsis model. This includes making a homogeneous cecal slurry stock, diluting it to a weight- and litter-adjusted dose, an outline of the monitoring schedule, and a definition of observed health categories used to define humane endpoints. The generation of a homogeneous cecal slurry stock from pooled donors allows for the administration into many litters over time, reducing the variation between donors, and preventing the use of potentially toxic glycerol. The monitoring strategy used allows for the anticipation of survival outcome and the identification of mice that would later progress to death, allowing for an earlier identification of the humane endpoint. Two main behavioral features are used to define the health scores, namely, the ability of the neonatal mice to right themselves when placed on their back and their level of mobility. These criteria could potentially be applied to address humane endpoints in other studies of neonatal disease in mice, as long as a pilot study is performed to confirm accuracy. In conclusion, this approach provides a standardized method to model newborn sepsis in mice, while providing resources to assess animal welfare used to define early humane endpoints for challenged animals.

This protocol provides the necessary steps to establish and evaluate neonatal sepsis in 7-day-old mice.

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse ...

Humanized NOG Mice for Intravaginal HIV Exposure and Treatment of HIV Infection

Humanized mice provide a sophisticated platform to study human immunodeficiency virus (HIV) virology and to test antiviral drugs. This protocol describes the establishment of a human immune system in adult NOG mice. Here, we explain all the practical steps from isolation of umbilical cord blood derived human CD34+ cells and their subsequent intravenous transplantation into the mice, to the manipulation of the model through HIV infection, combination antiretroviral therapy (cART), and blood sampling. Approximately 75,000 hCD34+ cells are injected intravenously into the mice and the level of human chimerism, also known as humanization, in the peripheral blood is estimated longitudinally for months by flow cytometry. A total of 75,000 hCD34+ cells yields 20%&#8211;50% human CD45+ cells in the peripheral blood. The mice are susceptible to intravaginal infection with HIV and blood can be sampled once weekly for analysis, and twice monthly for extended periods. This protocol describes an assay for quantification of plasma viral load using droplet digital PCR (ddPCR). We show how the mice can be effectively treated with a standard-of-care cART regimen in the diet. The delivery of cART in the form of regular mouse chow is a significant refinement of the experimental model. This model can be used for preclinical analysis of both systemic and topical pre-exposure prophylaxis compounds as well as for testing of novel treatments and HIV cure strategies.

We have developed a protocol for the generation and evaluation of a humanized and human immunodeficiency virus-infected NOG mouse model based on stem cell transplant, intravaginal human immunodeficiency virus exposure, and droplet digital PCR RNA quantification.

Humanized mice provide a sophisticated platform to study human immunodeficiency virus (HIV) virology and to test antiviral drugs. This protocol describes ...

A Neonatal Imaging Model of Gram-Negative Bacterial Sepsis

Neonates are at an increased risk of bacterial sepsis due to the unique immune profile they display in the first months of life. We have established a protocol for studying the pathogenesis of E. coli O1:K1:H7, a serotype responsible for high mortality rates in neonates. Our method utilizes intravital imaging of neonatal pups at different time points during the progression of infection. This imaging, paralleled by measurement of bacteria in the blood, inflammatory profiling, and tissue histopathology, signifies a rigorous approach to understanding infection dynamics during sepsis. In the current report, we model two infectious inoculums for comparison of bacterial burdens and severity of disease. We find that subscapular infection leads to disseminated infection by 10 h post-infection. By 24 h, infection of luminescent E. coli was abundant in the blood, lungs, and other peripheral tissues. Expression of inflammatory cytokines in the lungs is significant at 24 h, and this is followed by cellular infiltration and evidence of tissue damage that increases with infectious dose. Intravital imaging does have some limitations. This includes a luminescent signal threshold and some complications that can arise with neonates during anesthesia. Despite some limitations, we find that our infection model offers an insight for understanding longitudinal infection dynamics during neonatal murine sepsis, that has not been thoroughly examined to date. We expect this model can also be adapted to study other critical bacterial infections during early life.

Infection of neonatal mice with bioluminescent E. coli O1:K1:H7 results in a septic infection with significant pulmonary inflammation and lung pathology. Here, we describe procedures to model and further study neonatal sepsis using longitudinal intravital imaging in parallel with enumeration of systemic bacterial burdens, inflammatory profiling, and lung histopathology.

Neonates are at an increased risk of bacterial sepsis due to the unique immune profile they display in the first months of life. We have established a ...