This research was supported by grants from NIH K01HL133520 (PS) and K12HD055882 (PS). The authors thank Dr. Joanna Floros for the assistance with ozone exposure experiments.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Penn State University.
1. Assessment of the Estrous Cycle Stage
<ol>
	<li>Properly restrain a female C57BL/6 mouse (8&#8211;9 weeks old) using the one-handed mouse restraint technique described in Machholz et al.10.</li>
	<li>Fill the sterile plastic pipette with 10 &#956;L of ultra-pure water.</li>
	<li>Introduce the tip of plastic pipette into the vagina.</li>
	<li>Gently flush the liquid 4&#8211;5 times to collect the sample.</li>
	<li>Place the final flush containing vaginal fluid on a glass slide.</li>
	<li>Observe the unstained vaginal flush under a light microscope with a 20x objective. 
	NOTE: Animals that do not show regular cycles due to pseudopregnancy or other causes need to be excluded from the experiment. It is recommended to perform daily vaginal secretions for at least three consecutive cycles to confirm cyclicity.</li>
</ol>
2. Exposure to Ozone
<ol>
	<li>Place a maximum of 4 mice in two 1.2 L glass containers with wire mesh lids, and water ad libitum.</li>
	<li>Put one glass container in the ozone chamber and the other in the filtered air exposure chamber.</li>
	<li>Adjust the ozone concentration to 2 ppm and monitor ozone levels regularly. 
	NOTE: The ozone apparatus delivers a regulated airflow (&#62;30 air changes/h) with controlled temperature (25 &#176;C) and relative humidity (50%). The system generates ozone by an electrical discharge ozonizer, which is monitored and controlled by an ultraviolet ozone analyzer and mass flow controllers, as described previously11.</li>
	<li>Remove glass containers after 3 h of ozone/filtered air exposure. Return animals to cage with bedding, food, and water ad libitum.</li>
</ol>
3. Lung Collection
<ol>
	<li>4 h after exposure, anesthetize animals with an intraperitoneal injection of a ketamine/xylazine cocktail (90 mg/kg ketamine, 10 mg/kg xylazine). 
	NOTE: To confirm a proper level of anesthesia, check the mouse for a pedal reflex (firm toe pinch) and adjust the anesthetic as needed.</li>
	<li>Dampen the mouse skin with 70% ethanol.</li>
	<li>Make a 2 cm midline incision using an operating scissor and surgical tweezers to expose the vena cava.</li>
	<li>Sacrifice mice by transection of the vena cava and aorta. If needed, insert a 21 G gauge needle into the vena cava above the renal veins to collect blood prior to exsanguination. Alternatively, collect blood via heart puncture following standard protocols.</li>
	<li>Use a surgical scissor to cut open the abdominal cavity and remove skin/upper muscle, moving upwards toward the ribs.</li>
	<li>Use a surgical scissor to puncture the diaphragm. 
	NOTE: Lungs will collapse away from the diaphragm.</li>
	<li>Cut away the ribcage using a surgical scissor to expose the heart and lungs.</li>
	<li>Using forceps, take a 1.5 mL RNase-free microcentrifuge tube and submerge it in liquid nitrogen to fill the tube. 
	NOTE: Use goggles and protective gloves to handle liquid nitrogen.</li>
	<li>Remove the lungs, place them into the RNase-free microcentrifuge 1.5 mL tubes filled with liquid nitrogen to snap-freeze the tissue, and wait a few seconds until the liquid evaporates.</li>
	<li>Close the tube lid and store the tissue at -80 &#176;C until use.</li>
</ol>
4. RNA Preparation
<ol>
	<li>Pulverize whole lungs using a stainless-steel tissue pulverizer. 
	NOTE: The tissue pulverizer needs to be placed in liquid nitrogen prior to use. Clean the pulverizer after each use with RNAse solution.</li>
	<li>Split the pulverized lungs and place into two 1.5 mL tubes (half lung each).</li>
	<li>Add 500 &#181;L of guanidinium thiocyanate per sample tube and mix.&#160; Homogenize each sample using 18 G, 21 G, and 23 G needles, respectively. 
	NOTE: Samples can be spiked with 5.6&#8201; x&#8201; 108&#160;copies of a small RNA spike-in control (from a different species) before proceeding with the extraction.</li>
	<li>Add 500 &#181;L of ethanol to each sample and vortex for 15 s.</li>
	<li>Load the mixture in a spin column in a collection tube and centrifuge at 12,000 x g for 1 min. Discard the flow-through.</li>
	<li>For DNase I treatment (in-column);
	<ol>
		<li>Add 400 &#181;L of RNA Wash Buffer and centrifuge at 12,000 x g for 1 min.</li>
		<li>In an RNase-free tube, add 5 &#181;L of DNase I and 75 &#181;L of 1x DNA digestion buffer and mix. Add the mix directly to the column matrix.</li>
		<li>Incubate at room temperature for 15 min.</li>
	</ol>
	</li>
	<li>Add 400 &#181;L of RNA prewash solution to the column and centrifuge at 12,000 x g for 1 min. Discard the flow-through and repeat this step.</li>
	<li>Add 700 &#181;L of RNA wash buffer to the column and centrifuge at 12,000 x g for 1 min. Discard the flow-through.</li>
	<li>Centrifuge at 12,000 x g for 2 min to remove remaining buffer. Transfer the column into an RNase-free tube.</li>
	<li>To elute RNA, add 35 &#181;L of DNase/RNase-free water directly to the column matrix and centrifuge at 12,000 x g for 1.5 min.</li>
	<li>Measure total RNA concentration (260 nm) and purity using a spectrophotometer. Follow instructions to perform RNA quantification in a 1.5 &#181;L sample aliquot. Blank the instrument with DNase/RNase-free water used for elution. 
	NOTE: A 260/280 ratio of ~2.0 is generally accepted as &#8220;pure&#8221; for RNA. Typical RNA concentration usually ranges between 750 and 2,500 ng/&#181;L.</li>
	<li>Store at -80 &#176;C.</li>
</ol>
5. miRNA Profiling
<ol>
	<li>To retro-transcribe small RNAs, use 200 ng of total RNA.
	<ol>
		<li>Prepare the reverse-transcription reaction mix on ice (total volume per reaction is 20 &#181;L). For each reaction, add 4 &#181;L of 5x buffer, 2 &#181;L of 10x nucleotides mix, 2 &#181;L of reverse transcriptase, and 2 &#181;L of RNase-free water. Mix all the components and aliquot in 600 &#181;L RNAse-free plastic tubes (10 &#181;L of mix per reaction). 
		NOTE: The reverse-transcription master mix contains all components required for first-strand cDNA synthesis except template RNA. 
		NOTE: Calculate excess volume (10%) when preparing the master mix.</li>
		<li>Add the template RNA (200 ng in 10 &#181;L) to each tube containing reverse-transcription master mix. Mix, centrifuge for 15 s at 1,000 x g, and store them on ice until placing in thermocycler or dry block.</li>
		<li>Incubate for 60 min at 37 &#176;C.</li>
		<li>Incubate for 5 min at 95 &#176;C and place the tubes on ice.</li>
		<li>Dilute the cDNA by adding 200 &#181;L of RNase-free water to each 20 &#181;L reverse-transcription reaction.</li>
	</ol>
	</li>
	<li>Perform real-time PCR using the Mouse Inflammatory Response and Autoimmunity miRNA PCR Array.
	<ol>
		<li>Prepare a reaction mix (total volume 1100 &#181;L): For each reaction, add 550 &#181;L of 2x PCR master mix, 110 &#181;L of 10x universal primer mix, 340 &#181;L of RNase-free water, and 340 &#181;L of template cDNA (diluted reaction from step 5.1.5).</li>
		<li>Add 10 &#181;L of reaction mix to each well of the pre-loaded miRNA PCR Array using a multichannel pipettor.</li>
		<li>Seal the miRNA PCR Array plate with optical adhesive film.</li>
		<li>Centrifuge the plate for 1 min at 1,000 x g at room temperature to remove bubbles.</li>
		<li>Program the real-time cycler, PCR initial activation step for 15 min at 95 &#176;C, 3-step cycling containing denaturation for 15 s at 94 &#176;C, annealing for 30 s at 55 &#176;C, and extension for 30 s at 70 &#176;C for 40 cycles number. 
		NOTE: Follow manufacturer&#8217;s cycling conditions instructions to set up the real-time cycler. Perform the dissociation curve step built into the real-time cycler software.</li>
		<li>Perform data analysis.</li>
	</ol>
	</li>
</ol>
6. Data Analysis
<ol>
	<li>Extract Ct values from the real time PCR software for each sample into an analysis software. 
	NOTE: A Ct value of 34 is considered as cutoff. If samples contain spike-in control (e.g., cel-mir-39), normalize Ct values to the spike in control for each sample. Threshold values may need to be manually set. Baseline values are automatically set.</li>
	<li>Normalize Ct values to the average Ct of six miRNA housekeeping controls: SNORD61, SNORD68, SNORD72, SNORD95, SNORD96A, RNU6-2, using the following equation: 
	&#916;Ct = (Ct_Target &#8722; Ct_housekeeping)</li>
	<li>For fold change calculations, calculate &#916;&#916;Ct values using a specific sample as the control, using the relative expression equation12: 
	miRNA relative expression: 
	2-&#8710;&#8710;Ct, where - &#8710;&#8710;Ct = -[&#8710;Ct test - &#8710;Ct control] 
	NOTE: A fold change of 200 is considered as cutoff.</li>
	<li>Export fold change expression values to perform statistical analysis on R using the limma package in Bioconductor8.</li>
	<li>Correct for multiple comparisons using the Benjamini-Hochberg method13. 
	NOTE: &#160;A copy of the R script is available at: <a href="http://psilveyra.github.io/silveyralab/">http://psilveyra.github.io/silveyralab/</a>. Datasets and analyzed data are also available at the Gene Expression Omnibus under number GSE111667,&#160;https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE111667.</li>
</ol>
7. Data Analysis: Functional Analysis Software
<ol>
	<li>Arrange the dataset. Include miRNAs with their respective expression log ratios and p-values. See Table 1 for specific dataset formatting.</li>
	<li>Open functional analysis software (version 01-10).</li>
	<li>Upload the dataset using the following format: File format: &#8220;Flexible Format&#8221;, Contains Column Header: &#8220;Yes&#8221;, Select Identifier type: &#8220;miRBase (mature)&#8221;, Array platform used for experiments: Choose the array platform. 
	NOTE: Accepted files formats are .txt (tab delimited text files), .xls (excel files), and .diff (cuffdiff files).</li>
	<li>Select &#8220;Infer Observations&#8221; and verify that the experimental group labeling is correct.</li>
	<li>Go to &#8220;Dataset Summary&#8221; to revise the total amount of mapped and unmapped miRNAs.</li>
	<li>Click on the &#8220;new&#8221; button at the top left of program. Select &#8220;New MicroRNA Target Filter&#8221; and upload a microRNA dataset.
	<ol>
		<li>Set Source to: TarBase, Ingenuity Experts Findings, miRecords.</li>
		<li>Set Confidence to: Experimentally observed or high (predicted).</li>
		<li>Select &#8220;Add Columns&#8221; to include a variety of biological information about the targets such as species, diseases, tissues, pathways and more.</li>
		<li>To focus on targets that have changed expression in the experiment, select &#8220;Add/ replace mRNA dataset&#8221;. Use &#8220;Expression Pairing&#8221; to locate microRNAs with the same or different expression levels. 
		NOTE: The filter analysis will provide microRNA names and symbols, mRNA targets, the source that describes the target relationship, and the confidence level of the predicted relationship (Figure 2).</li>
	</ol>
	</li>
	<li>Click &#8220;Add to My Pathway&#8221; to send the filtered dataset to a pathway canvas and explore more biological relationships.</li>
	<li>Use Path Designer to create a publication-quality model of microRNA effects.</li>
	<li>An alternate option is to create a core analysis.
	<ol>
		<li>For core analysis type, select &#8220;Expression Analysis&#8221;.</li>
		<li>For measurement type, select &#8220;Expr Log Ratio&#8221;.</li>
		<li>Analysis Filter Summary: consider only molecules and/or relationships where: (species = mouse) AND (confidence = experimentally observed) AND (data sources = &#8220;Ingenuity Expert Findings&#8221;, &#8220;Ingenuity ExpertAssist Findings&#8221;, &#8220;miRecords&#8221;, &#8220;TarBase&#8221;, OR &#8220;TargetScan Human&#8221;).</li>
		<li>Select a cutoff of p-value = 0.05.</li>
		<li>Run the analysis. 
		NOTE: The report will include: canonical pathways, upstream regulators analysis, diseases and functions, regulator effects, networks, molecules and more.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing interests.

MicroRNA profiling is an advantageous technique for both disease diagnosis and mechanistic research. In this manuscript, we defined a protocol to evaluate the expression of miRNAs that are predicted to regulate inflammatory genes in the lungs of female mice exposed to ozone in different estrous cycle stages. Methods for the determination of the estrous cycle, such as the visual detection method, have been described16. However, these rely on one-time measurements, and therefore are unreliable. To a...

microRNAs (miRNAs) are short (19 to 25 nucleotides), naturally occurring, non-coding RNA molecules.Sequences of miRNAs are evolutionary conserved across species, suggesting the importance of miRNAs in regulating physiological functions1. microRNA expression profiling has been proven to be helpful for identifying miRNAs that are important in the regulation of a variety of processes, including the immune response, cell differentiation, developmental processes, and apoptosis2. More recently, miRNAs have been recognized for their potential use in disease diagnostics and therapeutics. For researchers studying mechanisms of gene regulation, measuring miRNA expression can enlighten systems-level models of regulatory processes, especially when miRNA information is merged with mRNA profiling and other genome-scale data3. On the other hand, miRNAs have also been shown to be more stable than mRNAs in a range of specimen types and are also measurable with greater sensitivity than proteins4. This has led to considerable interest in the development of miRNAs as biomarkers for diverse molecular diagnostic applications, including lung diseases.
In the lung, miRNAs play important roles in developmental processes and the maintenance of homeostasis. Moreover, their abnormal expression has been associated with the development and progression of various pulmonary diseases5. Inflammatory lung disease induced by air pollution has demonstrated greater severity and poorer prognosis in females, indicating that hormones and the estrous cycle can regulate lung innate immunity and miRNA expression in response to environmental challenges6. In this protocol, we use ozone exposure, which is a major component of air pollution, to induce a form of lung inflammation in female mice that occurs in the absence of adaptive immunity. By using ozone, we are inducing the development of airway hyperresponsiveness that is associated with airway epithelial cell damage and an increase in neutrophils and inflammatory mediators in proximal airways7. Currently, there are not well-described protocols to characterize and analyze miRNAs across the estrous cycle in ozone-exposed mice. 
Below, we describe a simple method to identify estrous cycle stages and miRNA expression in lung tissue of female mice exposed to ozone. We also address effective bioinformatics approaches to miRNA discovery and target identification, with an emphasis on computational biology. We analyze the microarray data using limma, an R/Bioconductor software that provides an integrated solution for analyzing data from gene expression experiments8. Analysis of PCR array data from limma has an advantage in terms of power over t-test based procedures when using small number of arrays/samples to compare expression. To comprehend the biological context of miRNA expression results, we then used the functional analysis software. In order to understand the mechanisms regulating transcriptional changes and to predict likely outcomes, the software combines miRNA-expression datasets and knowledge from the literature9. This is an advantage when compared with software that just look for statistical enrichment in overlapping to sets of miRNAs.

<table>
	<tbody>
		<tr>
			<td>C57BL/6J mice</td>
			<td>The Jackson Laboratory</td>
			<td>000664</td>
			<td>8 weeks old</td>
		</tr>
		<tr>
			<td>UltraPure Water</td>
			<td>Thermo Fisher Scientific</td>
			<td>10813012</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile plastic pipette</td>
			<td>Fisher Scientific</td>
			<td>13-711-25</td>
			<td>Capacity: 1.7mL</td>
		</tr>
		<tr>
			<td>Frosted Microscope Slides</td>
			<td>Thermo Fisher Scientific</td>
			<td>2951TS</td>
			<td></td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td>Microscope World</td>
			<td>MW3-H5</td>
			<td>10X and 20X objective</td>
		</tr>
		<tr>
			<td>Ketathesia- Ketamine HCl Injection USP</td>
			<td>Henry Schein Animal Health</td>
			<td>55853</td>
			<td>90 mg/kg. Controlled drug.</td>
		</tr>
		<tr>
			<td>Xylazine Sterile Solution</td>
			<td>Lloyd Laboratories</td>
			<td>139-236</td>
			<td>10mg/kg. Controlled Drug.</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher Scientific</td>
			<td>BP2818100</td>
			<td>Dilute to 70% ethanol with water.</td>
		</tr>
		<tr>
			<td>21G gauge needle</td>
			<td>BD Biosciences</td>
			<td>305165</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Fisher Scientific</td>
			<td>329654</td>
			<td>1mL</td>
		</tr>
		<tr>
			<td>Operating Scissors</td>
			<td>World Precision Instruments</td>
			<td>501221, 504613</td>
			<td>14cm, Sharp/Blunt, Curved and 9 cm, Straight, Fine Sharp Tip</td>
		</tr>
		<tr>
			<td>Tweezer Kit</td>
			<td>World Precision Instruments</td>
			<td>504616</td>
			<td></td>
		</tr>
		<tr>
			<td>-80 &#730;C freezer</td>
			<td>Forma</td>
			<td>7240</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrum&#160;Bessman Tissue Pulverizers</td>
			<td>Fisher Scientific</td>
			<td>08-418-1</td>
			<td>Capacity: 10 to 50mg</td>
		</tr>
		<tr>
			<td>RNase-free Microfuge Tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM12400</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td>TRIzol Reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>15596026</td>
			<td></td>
		</tr>
		<tr>
			<td>Direct-zol RNA MiniPrep Plus</td>
			<td>Zymo Research</td>
			<td>R2071</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop</td>
			<td>Thermo Fisher Scientific</td>
			<td>ND-ONE-W</td>
			<td></td>
		</tr>
		<tr>
			<td>miScript II RT kit</td>
			<td>Qiagen</td>
			<td>218161</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Inflammatory Response &#38; Autoimmunity miRNA PCR Array</td>
			<td>Qiagen</td>
			<td>MIMM-105Z</td>
			<td></td>
		</tr>
		<tr>
			<td>Thin-walled, DNase-free, RNase-free PCR tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM12225</td>
			<td>for 20 &#956;l reactions</td>
		</tr>
		<tr>
			<td>miRNeasy Serum/Plasma Spike-in Control</td>
			<td>Qiagen</td>
			<td>219610</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microsoft Corporation</td>
			<td></td>
			<td>https://office.microsoft.com/excel/</td>
		</tr>
		<tr>
			<td>Ingenuity Pathway Analysis</td>
			<td>Qiagen</td>
			<td></td>
			<td>https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis/</td>
		</tr>
		<tr>
			<td>R Software</td>
			<td>The R Foundation</td>
			<td></td>
			<td>https://www.r-project.org/</td>
		</tr>
		<tr>
			<td>Thermal cycler or chilling/heating block</td>
			<td>General Lab Supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>General Lab Supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Real-time PCR cycler</td>
			<td>General Lab Supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipettor</td>
			<td>General Lab Supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RNA wash buffer</td>
			<td>Zymo Research</td>
			<td>R1003-3-48</td>
			<td>48 mL</td>
		</tr>
		<tr>
			<td>DNA digestion buffer</td>
			<td>Zymo Research</td>
			<td>E1010-1-4</td>
			<td>4 mL</td>
		</tr>
		<tr>
			<td>RNA pre-wash buffer</td>
			<td>Zymo Research</td>
			<td>R1020-2-25</td>
			<td>25 mL</td>
		</tr>
		<tr>
			<td>Ultraviolet ozone analyzer</td>
			<td>Teledyne API</td>
			<td>Model T400</td>
			<td>http://www.teledyne-api.com/products/oxygen-compound-instruments/t400</td>
		</tr>
		<tr>
			<td>Mass flow controllers</td>
			<td>Sierra Instruments Inc</td>
			<td>Flobox 951/954</td>
			<td>http://www.sierrainstruments.com/products/954p.html</td>
		</tr>
	</tbody>
</table>

lung microrna profiling across the estrous cycle in ozone-exposed mice

MicroRNA (miRNA) profiling has become of interest to researchers working in various research areas of biology and medicine. Current studies show a promising future of using miRNAs in the diagnosis and care of lung diseases. Here, we define a protocol for miRNA profiling to measure the relative abundance of a group of miRNAs predicted to regulate inflammatory genes in the lung tissue from of an ozone-induced airway inflammation mouse model. Because it has been shown that circulating sex hormone levels can affect the regulation of lung innate immunity in females, the purpose of this method is to describe an inflammatory miRNA profiling protocol in female mice, taking into consideration the estrous cycle stage of each animal at the time of ozone exposure. We also address applicable bioinformatics approaches to miRNA discovery and target identification methods using limma, an R/Bioconductor software, and functional analysis software to understand the biological context and pathways associated with differential miRNA expression.

The different cell types observed in smears are used to identify the mouse estrous cycle stage (Figure 1). These are identified by cell morphology. During proestrus, cells are almost exclusively clusters of round-shaped, well-formed nucleated epithelial cells (Figure 1A). When the mouse is in the estrus stage, cells are cornified squamous epithelial cells, present in densely packed clusters (Figure 1B</strong...

Watch this Scientific Journal Video about Lung microRNA Profiling Across the Estrous Cycle in Ozone-exposed Mice at JoVE.com

Lung microRNA Profiling Across the Estrous Cycle in Ozone-exposed Mice

Here we describe a method to assess lung expression of miRNAs that are predicted to regulate inflammatory genes using mice exposed to ozone or filtered air at different stages of the estrous cycle.

MicroRNA (miRNA) profiling has become of interest to researchers working in various research areas of biology and medicine. Current studies show a ...

lung-microrna-profiling-across-the-estrous-cycle-in-ozone-exposed-mice

Research

JoVE Journal

Immunology and Infection

6.0K Views.  Pennsylvania State University College of Medicine. Here we describe a method to assess lung expression of miRNAs that are predicted to regulate inflammatory genes using mice exposed to ozone or filtered air at different stages of the estrous cycle.

Video: Lung microRNA Profiling Across the Estrous Cycle in Ozone-exposed Mice

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse models allow us to study how diseases unfold, often providing a good replica of the same processes occurring in humans. For some autoimmune diseases, like type 1A diabetes, there are models (the NOD mouse) that spontaneously develop a disease similar to the human counterpart. For many other autoimmune diseases, however, the model needs to be induced experimentally. A common approach in this regard is to inject the mouse with a dominant antigen derived from the organ being studied. For example, investigators interested in autoimmune thyroiditis inject mice with thyroglobulin2, and those interested in myasthenia gravis inject them with the acetylcholine receptor3. If the autoantigen for a particular autoimmune disease is not known, investigators inject a crude protein extract from the organ targeted by the autoimmune reaction. For autoimmune hypophysitis, the pathogenic autoantigen(s) remain to be identified4, and thus a crude pituitary protein preparation is used. In this video article we demonstrate how to induce experimental autoimmune hypophysitis in SJL mice.

This video shows how to induce autoimmune hypophysitis in SJL mice and how to assess its severity by histopathology.

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is a common modifier of heart and lung function, by elaborating cellular infiltration, production of cytokines and growth factors, and by initiating remodeling processes 1.
Compared to the left ventricle, the right ventricle is a low-pressure pump that operates in a relatively narrow zone of pressure changes. Increased pulmonary artery pressures are associated with increased pressure in the lung vascular bed and pulmonary hypertension 2. Pulmonary hypertension is often associated with inflammatory lung diseases, for example chronic obstructive pulmonary disease, or autoimmune diseases 3. Because pulmonary hypertension confers a bad prognosis for quality of life and life expectancy, much research is directed towards understanding the mechanisms that might be targets for pharmaceutical intervention 4. The main challenge for the development of effective management tools for pulmonary hypertension remains the complexity of the simultaneous understanding of molecular and cellular changes in the right heart, the lungs and the immune system.
Here, we present a procedural workflow for the rapid and precise measurement of pressure changes in the right heart of mice and the simultaneous harvest of samples from heart, lungs and immune tissues. The method is based on the direct catheterization of the right ventricle via the jugular vein in close-chested mice, first developed in the late 1990s as surrogate measure of pressures in the pulmonary artery5-13. The organized team-approach facilitates a very rapid right heart catheterization technique. This makes it possible to perform the measurements in mice that spontaneously breathe room air. The organization of the work-flow in distinct work-areas reduces time delay and opens the possibility to simultaneously perform physiology experiments and harvest immune, heart and lung tissues.
The procedural workflow outlined here can be adapted for a wide variety of laboratory settings and study designs, from small, targeted experiments, to large drug screening assays. The simultaneous acquisition of cardiac physiology data that can be expanded to include echocardiography5,14-17 and harvest of heart, lung and immune tissues reduces the number of animals needed to obtain data that move the scientific knowledge basis forward. The procedural workflow presented here also provides an ideal basis for gaining knowledge of the networks that link immune, lung and heart function. The same principles outlined here can be adapted to study other or additional organs as needed.

A specific and rapid protocol to simultaneously investigate right heart function, lung inflammation, and the immune response is described as a learning tool. Video and figures describe physiology and microdissection techniques in an organized team-approach that is adaptable to be used for small to large sized studies.

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is ...

Measuring Local Anaphylaxis in Mice

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. Exposure to an allergen in a sensitized individual can result in clinical symptoms that vary from minor erythema to life threatening anaphylaxis. In the laboratory, various animal models have been developed to understand the mechanisms driving allergic responses. Herein, we describe a detailed method for measuring changes in vascular permeability to quantify localized allergic responses. The local anaphylaxis assay was first reported in the 1920s, and has been adapted from the technique published by Kojima et al. in 20071. In this assay, mice sensitized to OVA are challenged in the left ear with vehicle and in the right ear with OVA. This is followed by an intravenous injection of Evans Blue dye. Ten min after injecting Evans Blue, the animal is euthanized and the dye that has extravasated into the ears is extracted overnight in formamide. The absorbance of the extracted dye is then quantified with a spectrophotometer. This method reliably results in a visual and quantifiable manifestation of a local allergic response.

Allergic responses, characterized by the activation of mast cells and basophils, are driven by the cross-linking of IgE and release of proinflammatory mediators. A quantitative assessment of allergic responses can be achieved by using Evans Blue dye to monitor changes in vascular permeability after allergen challenge.

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. ...

MicroRNA-based Regulation of Picornavirus Tropism

Cell-specific restriction of viral replication without concomitant attenuation can benefit vaccine development, gene therapy, oncolytic virotherapy, and understanding the biological properties of viruses. There are several mechanisms for regulating viral tropism, however they tend to be virus class specific and many result in virus attenuation. Additionally, many viruses, including picornaviruses, exhibit size constraints that do not allow for incorporation of large amounts of foreign genetic material required for some targeting methods. MicroRNAs are short, non-coding RNAs that regulate gene expression in eukaryotic cells by binding complementary target sequences in messenger RNAs, preventing their translation or accelerating their degradation. Different cells exhibit distinct microRNA signatures and many microRNAs serve as biomarkers. These differential expression patterns can be exploited for restricting gene expression in cells that express specific microRNAs while maintaining expression in cells that do not. In regards to regulating viral tropism, sequences complementary to specific microRNAs are incorporated into the viral genome, generally in the 3' non-coding regions, targeting them for destruction in the presence of the cognate microRNAs thus preventing viral gene expression and/or replication. MicroRNA-targeting is a technique that theoretically can be applied to all viral vectors without altering the potency of the virus in the absence of the corresponding microRNAs. Here we describe experimental methods associated with generating a microRNA-targeted picornavirus and evaluating the efficacy and specificity of that targeting in vitro. This protocol is designed for a rapidly replicating virus with a lytic replication cycle, however, modification of the time points analyzed and the specific virus titration readouts used will aid in the adaptation of this protocol to many different viruses.

We describe here a method for regulating picornavirus tropism by incorporating sequences complementary to specific microRNAs into the viral genome. This protocol can be adapted to all different classes of viruses with modifications based upon the length and nature of their life cycle.

Cell-specific restriction of viral replication without concomitant attenuation can benefit vaccine development, gene therapy, oncolytic virotherapy, and ...

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-&#945;), and interleukin 1 alpha (IL-1&#945;) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances.

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

Detection of MicroRNA Expression in the Kidneys of Immunoglobulin A Nephropathic Mice

Immunoglobulin A (IgA) nephropathy is a type of primary glomerulonephritis characterized by the abnormal deposition of IgA, leading to the end-stage renal failure. In recent years, the involvement of microRNAs (miRNAs) has been reported in the pathogenesis of IgA nephropathy. However, there is no established method for profiling miRNAs in IgA nephropathy using small animal models. Therefore, we developed a reliable method for analyzing miRNA in the kidney of an IgA mouse model (HIGA mouse). The goal of this protocol is to detect the altered expression levels of miRNAs in the kidneys of HIGA mice when compared with the levels in kidneys of control mice. In brief, this method consists of four steps: 1) obtaining kidney samples from HIGA mice; 2) purifying total RNA from kidney samples; 3) synthesizing complementary DNA from total RNA; and 4) quantitative reverse transcription polymerase chain reaction (qRT-PCR) of miRNAs. Using this method, we successfully detected the expression levels of several miRNAs (miR-155-5p, miR-146a-5p, and miR-21-5p) in the kidneys of HIGA mice. This new method can be applied to other studies profiling miRNAs in IgA nephropathy.

microRNAs are involved in the pathogenesis of IgA nephropathy. We have developed a reliable method for detecting microRNA expression levels in the kidneys of an IgA nephropathy mouse model (HIGA mice). This new method will facilitate to check for miRNAs involvement in IgA nephropathy.

Immunoglobulin A (IgA) nephropathy is a type of primary glomerulonephritis characterized by the abnormal deposition of IgA, leading to the end-stage renal ...

A DNA/Ki67-Based Flow Cytometry Assay for Cell Cycle Analysis of Antigen-Specific CD8 T Cells in Vaccinated Mice

The cell cycle of antigen-specific T cells in vivo has been examined by using a few methods, all of which possess some limitations. Bromodeoxyuridine (BrdU) marks cells that are in or recently completed S-phase, and carboxyfluorescein succinimidyl ester (CFSE) detects daughter cells after division. However, these dyes do not allow identification of the cell cycle phase at the time of analysis. An alternative approach is to exploit Ki67, a marker that is highly expressed by cells in all phases of the cell cycle except the quiescent phase G0. Unfortunately, Ki67 does not allow further differentiation as it does not separate cells in S-phase that are committed to mitosis from those in G1 that can remain in this phase, proceed into cycling, or move into G0.
Here, we describe a flow cytometric method for capturing a &#34;snapshot&#34; of T cells in different cell cycle phases in mouse secondary lymphoid organs. The method combines Ki67 and DNA staining with major histocompatibility complex (MHC)-peptide-multimer staining and an innovative gating strategy, allowing us to successfully differentiate between antigen-specific CD8 T cells in G0, in G1 and in S-G2/M phases of the cell cycle in the spleen&#160;and draining lymph nodes of mice after vaccination with viral vectors carrying the model antigen gag of human immunodeficiency virus (HIV)-1.
Critical steps of the method were the choice of the DNA dye and the gating strategy to increase the assay sensitivity and to include highly activated/proliferating antigen-specific T cells that would have been missed by current criteria of analysis. The DNA dye, Hoechst 33342, enabled us to obtain a high-quality discrimination&#160;of the G0/G1 and G2/M DNA peaks, while preserving membrane and intracellular staining. The method has great potential to increase knowledge about T cell response in vivo and to improve immuno-monitoring analysis.

Clonal expansion is a key feature of antigen-specific T cell response. However, the cell cycle of antigen-responding T cells has been poorly investigated, partly because of technical limitations. We describe a flow cytometric method to analyze clonally expanding antigen-specific CD8 T cells in spleen and lymph nodes of vaccinated mice.

The cell cycle of antigen-specific T cells in vivo has been examined by using a few methods, all of which possess some limitations. Bromodeoxyuridine ...

Lung microRNA Profiling Across the Estrous Cycle in Ozone-exposed Mice

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Rozdziały w tym wideo

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice

Measuring Local Anaphylaxis in Mice

MicroRNA-based Regulation of Picornavirus Tropism

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Detection of MicroRNA Expression in the Kidneys of Immunoglobulin A Nephropathic Mice

A DNA/Ki67-Based Flow Cytometry Assay for Cell Cycle Analysis of Antigen-Specific CD8 T Cells in Vaccinated Mice