Name: performing colonoscopic-guided pinch biopsies in mice and evaluating subsequent tissue changes
Uploaded: 2021-02-05T12:30:00
Description: Watch this Scientific Journal Video about Performing Colonoscopic-Guided Pinch Biopsies in Mice and Evaluating Subsequent Tissue Changes at JoVE.com

This work was supported by grants from Crohn&#8217;s and Colitis Foundation (D.C.M) and New York Crohn&#8217;s Foundation (D.C.M. and A.J.D.).&#160;The authors thank Ms. Carmen Ferrara for assistance with creating the video accompaniment to this article.

All procedures described here were approved by the Institutional Animal Care and Use Committee of Weill Cornell Medicine.&#8221; To: &#8220;All procedures described here were approved by the Institutional Animal Care and Use Committees of Weill Cornell Medicine and Stony Brook University.
1. Colonoscopy and wound induction
<ol>
	<li>Pre-assemble the components of the endoscope by first inserting the 1.9 mm rigid bore endoscope into the sheath (Figure 1A-...

<ol>
	<li>Boal Carvalho, P., Cotter, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucosal+Healing+in+Ulcerative+Colitis:+A+Comprehensive+Review.">Mucosal Healing in Ulcerative Colitis: A Comprehensive Review.</a> Drugs. 77 (2), 159-173 (2017).</li><li>Chen, M. L., Sundrud, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+Networks+and+T-Cell+Subsets+in+Inflammatory+Bowel+Diseases.">Cytokine Networks and T-Cell Subsets in Inflammatory Bowel Diseases.</a> Inflammatory Bowel Diseases. 22 (5), 1157-1167 (2016).</li><li>Habtezion, A., Nguyen, L. P., Hadeiba, H., Butcher, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+Trafficking+to+the+Small+Intestine+and+Colon.">Leukocyte Trafficking to the Small Intestine and Colon.</a> Gastroenterology. 150 (2), 340-354 (2016).</li><li>Halfvarson, J., 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=Dynamics+of+the+human+gut+microbiome+in+inflammatory+bowel+disease.">Dynamics of the human gut microbiome in inflammatory bowel disease.</a> Nature Microbiology. 2, 17004 (2017).</li><li>Johansson, M. E., 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=Bacteria+penetrate+the+normally+impenetrable+inner+colon+mucus+layer+in+both+murine+colitis+models+and+patients+with+ulcerative+colitis.">Bacteria penetrate the normally impenetrable inner colon mucus layer in both murine colitis models and patients with ulcerative colitis.</a> Gut. 63 (2), 281-291 (2014).</li><li>Luissint, A. C., Parkos, C. A., Nusrat, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+and+the+Intestinal+Barrier:+Leukocyte-Epithelial+Cell+Interactions,+Cell+Junction+Remodeling,+and+Mucosal+Repair.">Inflammation and the Intestinal Barrier: Leukocyte-Epithelial Cell Interactions, Cell Junction Remodeling, and Mucosal Repair.</a> Gastroenterology. 151 (4), 616-632 (2016).</li><li>Pineton de Chambrun, G., Blanc, G., Peyrin-Biroulet, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+evidence+supporting+mucosal+healing+and+deep+remission+as+important+treatment+goals+for+inflammatory+bowel+disease.">Current evidence supporting mucosal healing and deep remission as important treatment goals for inflammatory bowel disease.</a> Expert Review of Gastroenterology &amp; Hepatology. 10 (8), 915-927 (2016).</li><li>Fung, K. Y., Putoczki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vivo+Models+of+Inflammatory+Bowel+Disease+and+Colitis-Associated+Cancer.">In Vivo Models of Inflammatory Bowel Disease and Colitis-Associated Cancer.</a> Methods in Molecular Biology. 1725, 3-13 (2018).</li><li>Jurjus, A. R., Khoury, N. N., Reimund, J. M. <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+inflammatory+bowel+disease.">Animal models of inflammatory bowel disease.</a> Journal of Pharmacological and Toxicological Methods. 50 (2), 81-92 (2004).</li><li>Mizoguchi, A., Takeuchi, T., Himuro, H., Okada, T., Mizoguchi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+engineered+mouse+models+for+studying+inflammatory+bowel+disease.">Genetically engineered mouse models for studying inflammatory bowel disease.</a> Journal of Pathology. 238 (2), 205-219 (2016).</li><li>Seno, H., 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=Efficient+colonic+mucosal+wound+repair+requires+Trem2+signaling.">Efficient colonic mucosal wound repair requires Trem2 signaling.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (1), 256-261 (2009).</li><li>Alam, 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=The+microenvironment+of+injured+murine+gut+elicits+a+local+pro-restitutive+microbiota.">The microenvironment of injured murine gut elicits a local pro-restitutive microbiota.</a> Nature Microbiology. 1, 15021 (2016).</li><li>Alam, 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=Redox+signaling+regulates+commensal-mediated+mucosal+homeostasis+and+restitution+and+requires+formyl+peptide+receptor+1.">Redox signaling regulates commensal-mediated mucosal homeostasis and restitution and requires formyl peptide receptor 1.</a> Mucosal Immunology. 7 (3), 645-655 (2014).</li><li>Kuhn, K. A., Manieri, N. A., Liu, T. C., Stappenbeck, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-6+stimulates+intestinal+epithelial+proliferation+and+repair+after+injury.">IL-6 stimulates intestinal epithelial proliferation and repair after injury.</a> PLoS One. 9 (12), 114195 (2014).</li><li>Leoni, G., 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=Annexin+A1,+formyl+peptide+receptor,+and+NOX1+orchestrate+epithelial+repair.">Annexin A1, formyl peptide receptor, and NOX1 orchestrate epithelial repair.</a> Journal of Clinical Investigation. 123 (1), 443-454 (2013).</li><li>Manieri, N. 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=Mucosally+transplanted+mesenchymal+stem+cells+stimulate+intestinal+healing+by+promoting+angiogenesis.">Mucosally transplanted mesenchymal stem cells stimulate intestinal healing by promoting angiogenesis.</a> Journal of Clinical Investigation. 125 (9), 3606-3618 (2015).</li><li>Miyoshi, H., Ajima, R., Luo, C. T., Yamaguchi, T. P., Stappenbeck, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt5a+potentiates+TGF-beta+signaling+to+promote+colonic+crypt+regeneration+after+tissue+injury.">Wnt5a potentiates TGF-beta signaling to promote colonic crypt regeneration after tissue injury.</a> Science. 338 (6103), 108-113 (2012).</li><li>Neurath, M. F., 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=Assessment+of+tumor+development+and+wound+healing+using+endoscopic+techniques+in+mice.">Assessment of tumor development and wound healing using endoscopic techniques in mice.</a> Gastroenterology. 139 (6), 1837-1843 (2010).</li><li>Montrose, D. C., 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=Colonoscopic-Guided+Pinch+Biopsies+in+Mice+as+a+Useful+Model+for+Evaluating+the+Roles+of+Host+and+Luminal+Factors+in+Colonic+Inflammation.">Colonoscopic-Guided Pinch Biopsies in Mice as a Useful Model for Evaluating the Roles of Host and Luminal Factors in Colonic Inflammation.</a> American Journal of Pathology. 188 (12), 2811-2825 (2018).</li><li>Bruckner, M., 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=Murine+endoscopy+for+in+vivo+multimodal+imaging+of+carcinogenesis+and+assessment+of+intestinal+wound+healing+and+inflammation.">Murine endoscopy for in vivo multimodal imaging of carcinogenesis and assessment of intestinal wound healing and inflammation.</a> Journal of Visualized Experiments. (90), (2014).</li></ol>

Ensuring consistent and accurate biopsies as well as measurements of wound size are of paramount importance when attempting to effectively evaluate the rate of wound closure in this model. Therefore, several measures should be taken to be confident that the procedures are being correctly carried out. First, the depth of the biopsy must not be too shallow or deep. If too shallow, there will not be a sufficient window to evaluate wound closure. Figure 2 demonstrates an optimal biopsy depth and...

The gastrointestinal (GI) tract is a complex organ system&#160;given its multiple functions, host cell types (e.g. epithelial, immune, stromal, etc.) as well as trillions of microbes. In light of this complexity, diseases of the GI tract often involve the interplay of all of these factors. For example, inflammatory bowel diseases (IBD) are associated with cycles of inflammation and remission in the GI tract, involving the activation of inflammatory cells, dysbiosis, and epithelial repair1,2,3,4,5,6,7. Having appropriate model systems to study IBD and other inflammatory conditions of the GI tract is critical for elucidating the pathogenesis of disease. Several models exist to study IBD pathogenesis including genetically engineered mice and the use of chemicals such as dextran sodium sulfate (DSS) in rodents8,9,10. Limitations of these models include an inability to precisely control the induction of inflammation as well as difficulties in evaluating wound healing. Alternative methods to mimic aspects of IBD pathogenesis could prove useful for developing therapies.
Colonoscopic-guided pinch biopsies in mice offer a useful model system to study the pathogenesis of the inflammatory response, wound healing, as well as host-microbe interactions in the colon. This approach was first used as an experimental tool in 2009, which demonstrated its utility for studying the acute inflammatory response and wound healing in the gut11. Subsequent studies utilized this technique to evaluate the roles of different signaling pathways as well as the gut microbiota, in colonic wound healing11,12,13,14,15,16,17,18. More recently, our group used this model to investigate the importance of sphingosine-1-phosphate signaling and bacteria in the acute response to colonic injury19. Although useful, carrying out colonoscopic-guided pinch biopsies in mice and evaluating subsequent tissue changes can be technically challenging. For instance, perforation of the bowel can occur upon induction of injury and ensuring consistent measurements of the wound bed through serial colonoscopies can be difficult. Additionally, orienting the colonic tissue properly to visualize the wound bed for histological or immunohistochemical analyses can be challenging. Although some information does exist regarding these methods18,20, a precise step-wise description of these techniques along will visual aids promises to enhance the reliability and broader utility of this model. Here, we present a detailed method to carry out colonoscopic-guided pinch biopsies in mice, track wound closure over time and prepare the tissue to enable histologic and molecular analyses of the wound bed. Creating a standard method to carry out these techniques can expand the use of this model to study previously uninvestigated mediators that are potentially important for GI inflammation and wound repair.

<table>
	<tbody>
		<tr>
			<td>Biopsy forceps, 3 Fr</td>
			<td>Karl Storz</td>
			<td>61071ZJ</td>
			<td></td>
		</tr>
		<tr>
			<td>Coloview Tower system</td>
			<td>Karl Storz</td>
			<td>contact company</td>
			<td></td>
		</tr>
		<tr>
			<td>Examination sheath, 9 Fr, Kit</td>
			<td>Karl Storz</td>
			<td>61029DK</td>
			<td></td>
		</tr>
		<tr>
			<td>Hopkins telescope, 0&#39;, 1.9 mm x 10 cm</td>
			<td>Karl Storz</td>
			<td>64301AA</td>
			<td></td>
		</tr>
		<tr>
			<td>isofluorane</td>
			<td>Covetrus</td>
			<td>2905</td>
			<td></td>
		</tr>
		<tr>
			<td>methylene blue</td>
			<td>Sigma-Aldrich</td>
			<td>M9140</td>
			<td></td>
		</tr>
		<tr>
			<td>micro iris scissors</td>
			<td>Integra</td>
			<td>18-1619</td>
			<td></td>
		</tr>
		<tr>
			<td>NIH ImageJ</td>
			<td>NIH</td>
			<td>N/A</td>
			<td>software available for free download from: https://imagej.nih.gov/ij/</td>
		</tr>
		<tr>
			<td>Pawfly MA-60 aquarium pump</td>
			<td>Amazon</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>scalpal with #10 blade</td>
			<td>Hill-Rom</td>
			<td>372610</td>
			<td></td>
		</tr>
	</tbody>
</table>

performing colonoscopic-guided pinch biopsies in mice and evaluating subsequent tissue changes

Understanding the tissue and cellular changes that occur in the acute injury response as well as during the wound healing process is of paramount importance when studying diseases of the gastrointestinal (GI) tract. The murine colonic pinch biopsy model is a useful tool to define these processes. Additionally, the interplay between gut luminal content (e.g., microbes) and the colon can be studied. However, wound induction and the ability to track wound closure over time in a reliable manner can be challenging. Moreover, tissue preparation and orientation must be carried out in a standardized way to optimally interrogate histologic and molecular changes. Here, we present a detailed method describing biopsy-induced injury and the monitoring of wound closure through repeat colonoscopies. An approach is described that ensures consistent and reproducible measurements of wound size, the ability to collect the wound bed for molecular analyses as well as visualize the wound bed upon sectioning of tissues. The ability to successfully carry out these techniques allows for studies of the acute injury response, wound healing and luminal-host interactions within the colon.

The small items (lens, sheath, biopsy forceps) needed to carry out biopsies are shown in Figure 1 along with indicators of proper assembly of these components. Figure 2 shows representative images of acceptable views of the wound bed in order to accurately quantify the size of the wound bed and closure rate of the wound. An example of an ex vivo view of the wound bed is shown in Figure 3A inclusive of indicators of the perimeter of ...

Watch this Scientific Journal Video about Performing Colonoscopic-Guided Pinch Biopsies in Mice and Evaluating Subsequent Tissue Changes at JoVE.com

Performing Colonoscopic-Guided Pinch Biopsies in Mice and Evaluating Subsequent Tissue Changes

Here, we provide a detailed description of the procedure to induce colonoscopic-guided pinch biopsies in mice and track wound closure in real time. Additionally, methods for the preparation of tissues for histological, immunohistochemical and molecular analyses of the wound bed are provided.

Understanding the tissue and cellular changes that occur in the acute injury response as well as during the wound healing process is of paramount ...

This method provides an ideal approach for studying colonic wound healing and the molecular and histologic changes that occur following injury. This method can also shed light on IBD pathogenesis. The main advantage of this technique is to precisely control the location of injury and the timing of the wound healing process.Individuals performing this method for the first time may have difficulty inserting the colonoscope into the mouse's colon and creating consistent wounds. Repetitively practicing these techniques should overcome these issues. Given the multiple coordinated steps and nuance involved with this procedure, visual demonstration of this method is critical.Before beginning the procedure, insert a 1.9 millimeter rigid bore endoscope into an endoscope sheath. And attach the assembled endoscope to the light source and video imaging device per the manufacturer's instructions. Use the provided tubing to attach the air pump to the gas valve on the left side of the sheath next to the working channel.Ensure that the working channel is in the open position and insert three French biopsy forceps through the working channel. Advance the forceps to the end of the sheath without protruding out of the sheath. Next, place the anesthetized mouse onto an endoscopic staging platform on its ventral side and confirm the appropriate level of sedation by lack of response to pedal reflex.After applying eye ointment, fill a three milliliter syringe with an attached rat gavage needle with room temperature PBS. And insert the needle approximately one centimeter into the mouse's anus. Gently infuse with PBS until the fecal material has been cleared.Several fecal pellets should exit the mouse along with the PBS that was infused. Insert the assembled endoscope 0.5 centimeters into the anus and advance the biopsy forceps into the cleared lumen of the rectum until the full jaws of the forceps are beyond the end of the sheath. Turn the forceps 90 degrees so that the jaws open in an east-west orientation and open the tips and advance the forceps approximately one centimeter, closing and retracting the forceps in one smooth, quick motion to harvest the biopsy.To avoid fully insufflating the colon while performing the biopsy, leave the right side of the gas valve open. Immediately after the biopsy, depress the foot pedal attached to the colonoscope recording device to initiate the video recording. And firmly press an index finger against the right side of the gas valve to completely cover the opening, forcing air into the endoscope and thus into the colon.Retract the forceps back out of the sheath and into the rectal lumen while in the closed position. And place the forceps against the rectal wall immediately above the wound until the base of the jaws is aligned with the top edge of the viewing field. Continue fully insufflating the colon until a clear view of the wound can be observed.To obtain the best measurements of the wound bed, be patient then place the colonoscope at a precise angle so that you get your best image of the wound bed. Open the video in an appropriate software program and advance the video to a frame showing a point in time at which the wound bed can be easily visualized. And at which the closed forceps are above the wound bed and against the rectal wall, and the wall is taut.At each time point, obtain a snapshot of this frame and code the file name to ensure that the measurements of the wound beds are carried out in a blinded manner. To quantify the size of the wound bed, open the images in ImageJ and use the Free Hand selections tool to draw a perimeter around the wound. Under Analyze, select Measure.And the value of that measurement will automatically populate in the Results window. When all of the measurements have been acquired, calculate the size of the wound on subsequent days relative to the size on day zero in a spreadsheet. At the appropriate experimental endpoint, open the skin and abdominal muscle layers to expose the body cavity of the experimental animal and place closed forceps under the colon.Gently lift the colon to release it from the underlying mesentery and cut the tissue at its midpoint and at the anus to collect it from the mouse. Use a 20 milliliter syringe filled with ice-cold PBS and equipped with a rat gavage needle to flush out the fecal contents. And place the cleared colon onto a piece of filter paper.Open the colon longitudinally, taking care that the mesenteric side is face down against the filter paper. And use a Pasteur pipette to cover the mucosa with 0.2%methylene blue. After a few seconds, drain off the excess stain and view the colon under a dissecting microscope to locate the wound bed.Use four-inch Micro Iris Scissors to cut around the edge of the bed, being careful not to cut into the muscle layer. And use fine point tweezers to transfer the dissected tissue into a tube for snap freezing and or storage. At the appropriate experimental endpoint, open the harvested colon longitudinally on a piece of filter paper, mesenteric side down.Gently cover the tissue with your fixative of choice. Cover the tissue with parafilm in a sealed container for 4 to 6 hours before storage in 70%ethanol. On the day of the processing, remove the parafilm and add 0.2%methylene blue to the tissue, as demonstrated.After draining off the excess stain, place the tissue under a dissecting microscope to locate the wound bed. Using a scalpel with a number 10 blade, cut directly through the center of the wound bed and continue cutting through the remainder of the colon in a straight line, such that the colon is cut in half lengthwise. For cryosectioning, embed the colon pieces such that the side that was cut by the scalpel is face down in a base mold half-filled with tissue freezing medium.Secure the tissue in place with fine tweezers and place the base mold onto a metal plate or thick aluminum foil on top of dry ice to harden the freezing medium. Once the bottom portion of the medium is frozen, fill the remaining volume of the base mold with freezing medium at room temperature. And return the mold to the dry ice until the entire tissue block is frozen.Then, transfer the mold to a 80 degrees Celsius freezer until sectioning. Here, representative images of acceptable views of the wound bed for an accurate quantification of the size of the wound bed and closure rate of the wound are shown. In this ex-vivo view of a wound bed, indicators of the perimeter of the wound bed and where to cut the tissue to enable visualization of the wound bed upon sectioning can be observed.In this representative image, an H and E stain section of a wound bed can be clearly observed. It is important to acquire instant images of the wound bed at all time points to ensure an accurate assessment of the wound healing rate. Following the pinch biopsy, different agents of interest can be injected into the wound bed to test their effects on the wound healing rate.

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Immunology and Infection

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 ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte function, in general these are limited in terms of the information they can provide. For example, most existing assays merely provide a percentage of how many granulocytes are activated following a single, fixed length incubation. Complicating matters, most assays focus on only one aspect of function due to limitations in detection technology. This report demonstrates a technique for simultaneous measurement of granulocyte phagocytosis of bacteria and oxidative burst. By measuring both of these functions at the same time, three unique phenotypes of activated granulocytes were identified: 1) Low Activation (minimal phagocytosis, no oxidative burst), 2) Moderate Activation (moderate phagocytosis, some oxidative burst, but no co-localization of the two functional events), and 3) High Activation (high phagocytosis, high oxidative burst, co-localization of phagocytosis and oxidative burst). A fourth population that consisted of inactivated granulocytes was also identified. Using assay incubations of 10, 20, and 40-min the effect of assay incubation duration on the redistribution of activated granulocyte phenotypes was assessed. A fourth incubation was completed on ice as a control. By using serial time incubations, the assay may be able to able to detect how a treatment spatially affects granulocyte function. All samples were measured using an image-based flow cytometer equipped with a quantitative imaging (QI) option, autosampler, and multiple lasers (488, 642, and 785 nm).

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

This method demonstrates a technique for assessing granulocyte function by simultaneously measuring phagocytosis of bacteria and oxidative burst. Image-based flow cytometry allowed for the identification of three distinct subsets of activated granulocytes that all differed in their relative functional capacity.

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte ...

Methods to Study Lipid Alterations in Neutrophils and the Subsequent Formation of Neutrophil Extracellular Traps

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of lipids. The function of lipids (e.g., in host-pathogen interactions or host entry) has been reported to play a crucial role in cellular processes. Here, we show a method to determine lipid composition, with a focus on the cholesterol level of primary blood-derived neutrophils, by HPTLC in comparison to high performance liquid chromatography (HPLC). The aim was to investigate the role of lipid/cholesterol alterations in the formation of neutrophil extracellular traps (NETs). NET release is known as a host defense mechanism to prevent pathogens from spreading within the host. Therefore, blood-derived human neutrophils were treated with methyl-&#946;-cyclodextrin (M&#946;CD) to induce lipid alterations in the cells. Using HPTLC and HPLC, we have shown that M&#946;CD treatment of the cells leads to lipid alterations associated with a significant reduction in the cholesterol content of the cell. At the same time, M&#946;CD treatment of the neutrophils led to the formation of NETs, as shown by immunofluorescence microscopy. In summary, here we present a detailed method to study lipid alterations in neutrophils and the formation of NETs.

Lipids are known to play an important role in cellular functions. Here, we describe a method to determine the lipid composition of neutrophils, with emphasis on the cholesterol level, by using both HPTLC and HPLC to gain a better understanding of the underlying mechanisms of neutrophil extracellular trap formation.

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of ...

Human Lung Dendritic Cells: Spatial Distribution and Phenotypic Identification in Endobronchial Biopsies Using Immunohistochemistry and Flow Cytometry

The lungs are constantly exposed to the external environment, which in addition to harmless particles, also contains pathogens, allergens, and toxins. In order to maintain tolerance or to induce an immune response, the immune system must appropriately handle inhaled antigens. Lung dendritic cells (DCs) are essential in maintaining a delicate balance to initiate immunity when required without causing collateral damage to the lungs due to an exaggerated inflammatory response. While there is a detailed understanding of the phenotype and function of immune cells such as DCs in human blood, the knowledge of these cells in less accessible tissues, such as the lungs, is much more limited, since studies of human lung tissue samples, especially from healthy individuals, are scarce. This work presents a strategy to generate detailed spatial and phenotypic characterization of lung tissue resident DCs in healthy humans that undergo a bronchoscopy for the sampling of endobronchial biopsies. Several small biopsies can be collected from each individual and can be subsequently embedded for ultrafine sectioning or enzymatically digested for advanced flow cytometric analysis. The outlined protocols have been optimized to yield maximum information from small tissue samples that, under steady-state conditions, contain only a low frequency of DCs. While the present work focuses on DCs, the methods described can directly be expanded to include other (immune) cells of interest found in mucosal lung tissue. Furthermore, the protocols are also directly applicable to samples obtained from patients suffering from pulmonary diseases where bronchoscopy is part of establishing the diagnosis, such as chronic obstructive pulmonary disease (COPD), sarcoidosis, or lung cancer.

Lung-resident immune cells, including dendritic cells (DCs) in humans, are critical for defense against inhaled pathogens and allergens. However, due to the scarcity of human lung tissue, studies are limited. This work presents protocols to process human mucosal endobronchial biopsies for studying lung DCs using immunohistochemistry and flow cytometry.

The lungs are constantly exposed to the external environment, which in addition to harmless particles, also contains pathogens, allergens, and toxins. In ...

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human diseases have been reported to be associated with four species of bacteria: Aeromonas dhakensis, Aeromonas hydrophila, Aeromonas veronii, and Aeromonas caviae. The model organism Caenorhabditis elegans is a bacterivore that provides an excellent infection model by which to study the bacterial pathogenesis of Aeromonas. Here, we introduce three different experiments to study Aeromonas infection using a C. elegans model, including survival, liquid toxicity, and muscle necrosis assays. The results of the three methods determining the virulence of Aeromonas were consistent. A. dhakensis was shown to be the most toxic among the 4 major Aeromonas species causing clinical infections. These methods are shown to be a convenient way to evaluate the toxicity among and within Aeromonas species and contribute to our understanding of the pathogenesis of Aeromonas infection.

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

Here, we introduce three different experiments to study Aeromonas infection in C. elegans. Using these convenient methods, it is easy to evaluate the toxicity among and within Aeromonas species.

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human ...

Live-Cell Fluorescence Microscopy to Investigate Subcellular Protein Localization and Cell Morphology Changes in Bacteria

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence microscopy as observations made at a population level may not truly reflect what occurs at a single cell level. Live-cell timelapse microscopy allows investigators to monitor the changes in cell division or cell morphology which provide valuable insights regarding subcellular localization of proteins and timing of gene expression, as it happens, to potentially aid in answering important biological questions. Here, we describe our protocol to monitor phenotypic changes in Bacillus subtilis and Staphylococcus aureus using a high-resolution deconvolution microscope. The objective of this report is to provide a simple and clear protocol that can be adopted by other investigators interested in conducting fluorescence microscopy experiments to study different biological processes in bacteria as well as other organisms.

This article provides a step-by-step guide to investigate protein subcellular localization dynamics and to monitor morphological changes using high-resolution fluorescence microscopy in Bacillus subtilis and Staphylococcus aureus.

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence ...

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of UTI in male and female mice have illustrated the procedures for bacterial inoculation and enumeration in urine and tissues. During an initial bladder infection in C57BL/6 mice, UPEC establish latent reservoirs inside bladder epithelial cells that persist following clearance of UPEC bacteriuria. This model builds on these studies to examine rUTI caused by the emergence of UPEC from within latent bladder reservoirs. The urogenital bacterium Gardnerella vaginalis is used as the trigger of rUTI in this model because it is frequently present in the urogenital tracts of women, especially in the context of vaginal dysbiosis that has been associated with UTI. In addition, a method for in situ bladder fixation followed by scanning electron microscopy (SEM) analysis of bladder tissue is also described, with potential application to other studies involving the bladder.

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy.

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of ...

Exploring the Therapeutic Potential of Du-Moxibustion in Ankylosing Spondylitis

Du-Moxibustion in a Mouse Model of Ankylosing Spondylitis

Ankylosing spondylitis (AS) is a progressively worsening and disabling form of arthritis that primarily affects the axial skeleton. This disease mainly involves the spine and the sacroiliac joint. Fusion of the spine and the sacroiliac joint may occur in the later stage of the disease, resulting in spinal stiffness and kyphosis, as well as difficulty in walking, which seriously affects the quality of work and daily living activities and imposes a heavy burden on the patient, the family, and society. Increasing attention has been paid to non-pharmacotherapy as an alternative therapy for AS. Moxibustion is an ancient therapeutic technique used in Traditional Chinese Medicine (TCM). Du-moxibustion therapy, a unique and innovative external treatment developed on the basis of ordinary moxibustion, has a definite therapeutic effect on AS. Du-moxibustion skillfully combines the compatible techniques of TCM to integrate meridians, acupoints, Chinese herbal medicine, and moxibustion. This paper describes the operation procedures and precautions to be taken during Du-moxibustion in experimental mice in detail to provide an experimental basis for the study of the mechanism of Du-moxibustion in the treatment of AS.

Author Spotlight: Evaluating Traditional Chinese Therapy for Ankylosing Spondylitis in Mice 

This paper describes in detail the operation procedures and precautions to be taken during Du-moxibustion in the treatment of ankylosing spondylitis in experimental mice.

Ankylosing spondylitis (AS) is a progressively worsening and disabling form of arthritis that primarily affects the axial skeleton. This disease mainly ...

Development of pHluorin2-Based Sensor for Glycosomal pH in &lt;i&gt;Trypanosoma brucei&lt;/i&gt;

Measuring Dynamic Glycosomal pH Changes in Living Trypanosoma brucei

Glucose metabolism is critical for the African trypanosome, Trypanosoma brucei, as an essential metabolic process and regulator of parasite development. Little is known about the cellular responses generated when environmental glucose levels change. In both bloodstream and procyclic form (insect stage) parasites, glycosomes house most of glycolysis. These organelles are rapidly acidified in response to glucose deprivation, which likely results in the allosteric regulation of glycolytic enzymes such as hexokinase. In previous work, localizing the chemical probe used to make pH measurements was challenging, limiting its utility in other applications.
This paper describes the development and use of parasites that express glycosomally localized pHluorin2, a heritable protein pH biosensor. pHluorin2 is a ratiometric pHluorin variant that displays a pH (acid)-dependent decrease in excitation at 395 nm while simultaneously yielding an increase in excitation at 475 nm. Transgenic parasites were generated by cloning the pHluorin2 open reading frame into the trypanosome expression vector pLEW100v5, enabling inducible protein expression in either lifecycle stage. Immunofluorescence was used to confirm the glycosomal localization of the pHluorin2 biosensor, comparing the localization of the biosensor to the glycosomal resident protein aldolase. The sensor responsiveness was calibrated at differing pH levels by incubating cells in a series of buffers that ranged in pH from 4 to 8, an approach we have previously used to calibrate a fluorescein-based pH sensor. We then measured pHluorin2 fluorescence at 405 nm and 488 nm using flow cytometry to determine glycosomal pH. We validated the performance of the live transgenic pHluorin2-expressing parasites, monitoring pH over time in response to glucose deprivation, a known trigger of glycosomal acidification in PF parasites. This tool has a range of potential applications, including potentially being used in high-throughput drug screening. Beyond glycosomal pH, the sensor could be adapted to other organelles or used in other trypanosomatids to understand pH dynamics&#160;in the live cell setting.

Author Spotlight: Advancements in Glycosomal pH Monitoring in &lt;i&gt;Trypanosoma brucei&lt;/i&gt; Using pHluorin2 Biosensor

We describe a method to study how pH responds to environmental cues in the glycosomes of the bloodstream form of African trypanosomes. This approach involves a pH-sensitive heritable protein sensor in combination with flow cytometry to measure pH dynamics, both as a time-course assay and in a high-throughput screen format.

Glucose metabolism is critical for the African trypanosome, Trypanosoma brucei, as an essential metabolic process and regulator of parasite development. ...