This work was funded by the National Institutes of Health 1R21HL092370-01 (GG), 1R01 HL095764-01 (GG); R01HL082694 (JW); American Heart Association, Founders affiliate (0855943D, GG); Stony Wold - Herbert Fund, New York (SHP).

1. Preparation
<ol>
 <li>Prepare the following solutions and tubes (Table 1) as follows:
 <ol>
 <li>Hanks solution, no calcium, magnesium or indicator with Penicillin (100 U/ml) / Streptomycin (100 mg/ml).</li>
 <li>Phosphate buffered saline (PBS), 1x, no calcium, no magnesium.</li>
 <li>Ethanol, 70 %, make 500 ml.</li>
 <li>Buffered formaldehyde, 7-10 % with PBS, make 500 ml.</li>
 <li>Anaesthesia solutions:
 <ol>
 <li>Avertin. Carefully add 5 ml of 2-Methyl-2-butanol to 5 g of 2,2,2-Tribromoethanol. Once dissolved, keep stock solution at room temperature in the dark. Take 0.25 ml of the stock solution and dilute it with 10 ml of 1xPBS in glass bottom bottle. Wrap the bottle with aluminium foil, place at 37 &deg;C until dissolved and then aliquot into 5 ml polypropylene tubes and keep in refrigerator. Warm one aliquot on the morning of the experiment.</li>
 <li>Barbiturate. Dilute stock solution with PBS to give 2.6% barbiturate solution. Place 3-4 ml aliquots in polypropylene tubes.</li>
 </ol>
 </li>
 </ol>
 </li>
 <li>Organize three work-areas (Figure 1).</li>
 <li>
 <ol>
 <li>Area A: Arrange the following items: study form to record study identification number, date, body weight, weights of right heart, left heart and septum; precision scale (0-50 g at 0.01 g precision) to determine body weight; micro scale to determine heart weights at 0.001 g precision; weigh boats; anaesthesia solutions (clearly marked); small dewar with liquid nitrogen; insulated container with ice; tubes and 24-well plate to collect blood and tissue specimens (Table 1); surgical instruments (Table 2, Figure 2); and solutions to clean the instruments (Table 1).</li>
 <li>Area B: Arrange the following items: dissecting microscope; right heart catheter connected to a pressure control unit that is connected to an amplifier and laptop computer. The catheter is placed into a beaker containing water. Arrange surgical instruments (Table 2), pieces of suture cut to optimal length and placed into a weigh boat; tape (autoclave tape is optimal) cut to optimal length and width and lined up on microscope or lab bench for easy access; cotton swabs and hair remover solution. Calibrate the catheter at the beginning of the study.</li>
 <li>Area C: Arrange the following items: magnifier lens; tracheal cannula; 1 ml syringes; suture cut to optimal length and placed into a weigh boat; small dewar with liquid nitrogen; insulated container with ice; tubes and 24-well plate to collect BAL and tissue specimens (Table 1); surgical instruments (Table 2); solutions to perform BAL and to clean the tracheal cannula and the instruments (Table 1).</li>
 </ol>
 </li>
</ol>
2. Right Heart Catheterization
<ol>
 <li>Weigh mouse using a precision scale by gently placing mouse into a weigh boat. Make sure to handle the animal gently and quietly. Record weight. The described protocol works best for mice that are 20 g or more because the diameter of the jugular vein has to accommodate the pressure catheter; it is possible to catheterize smaller mice (17 g or more) as long as the vein is large enough.</li>
 <li>Anaesthetize the mouse by injection with Avertin depending on the body weight (10 &mu;l/g), e.g. for 20 g give 200 &mu;l, for 25 g give 250 &mu;l, for 30 g give 300 &mu;l. The exact injection volume needs to be adjusted depending on the mouse strain. Make sure to handle the animal gently and quietly because stress can alter the response to the anaesthetic.</li>
 <li>Once the mouse is sedated, place on double-pleated tissue paper. Note the ID number on the paper. Gently rub hair removal lotion into the ventral aspect of the neck to clear the surgical field. Place the mouse back onto the paper and wait for 1-2 min.</li>
 <li>Remove hair from the ventral aspect of the neck by gently stroking the hair against the direction of the hair growth with cotton swabs.</li>
 <li>Carefully determine the sufficient depth of anaesthesia by checking for the absence of the toe reflex: the mouse does not react to pinching of its toes.</li>
 <li>Working quickly and relaxed, the remainder of the right heart catheterization should not take more than 10 min, optimally 3-6 min. It is important that the tissue does not dry out because the catheter needs to glide into the vein and move inside of the vein on a layer of moisture. Consistent timing of the procedure is important for the generation of data that are comparable between groups.</li>
 <li>Place mouse back on tissue paper and transfer with tissue paper onto styrofoam board. Fix paws and head into place using autoclave tape.</li>
 <li>Make incision of skin from mandibles to sternum (breast-bone).</li>
 <li>Carefully dissect the thyroid grand upwards towards the mandibles to expose the right jugular vein and trachea.</li>
 <li>Place styrofoam board holding the mouse underneath the dissection microscope with the focus plane already in place and the lens at approximately 0.8x magnification (total magnification [lens x eye piece] is approximately 8x.</li>
 <li>Carefully microdissect the right jugular vein by removing surrounding connective tissue with surgical forceps.</li>
 <li>Place two pieces of suture on top of the right jugular vein. Tie the suture closest to the mandibles to close off blood flow in the vein to a tiny trickle, place the suture that is closest to the sternum / breast-bone at a loose knot around the vein. One can use the tiny trickle of blood to later find the hole in the vein, should this become necessary.</li>
 <li>Breathe and relax (loosen the muscles in the neck and jaw) to keep your eyes on the vein through the eyepiece. This will ensure that your hands and fingers will move securely, smoothly and relaxed. While holding the vein with forceps at the side closest to the mandibles, cut a hole into the vein between the two sutures using micro-scissors operated by the other hand. Put down the micro-scissors, and keeping that hand relaxed, feel for the catheter. Gently retrieve the catheter holding it between thumb and first finger (Figure 3). Insert the catheter into the hole of the vein.</li>
 <li>Gently advance the catheter into the right heart (Figure 3). Observe the marks at the catheter that give an approximate index of how far the catheter needs to be inserted and observe the monitor. Once the pressure curves are displayed, gently tighten the lower suture around the catheter. Because the catheter has tensile strength that gives it a coil, and because the breathing and heart beat of the mouse add motion, additionally affix the catheter using tape.</li>
 <li>Record pressure measurements for 2 min for later analysis (Figure 4).</li>
 <li>Open the suture holding the catheter, then gently retrieve catheter. Wipe the part behind the pressure transducer, place back into the beaker with water. Do not touch the pressure transducer.</li>
 <li>Transfer the mouse on top of the tissue paper to area A for euthanasia, collection of blood and spleen tissue. Clean surgical instruments.</li>
 <li>Start the procedure with the next animal. When the timing permits, inject the anaesthetic while waiting to record the pressure curve (2.15).</li>
</ol>
3. Collection of Blood and Spleen Samples
<ol>
 <li>Inject barbiturate solution, 400 &mu;l per mouse and wait for 1-2 min. This is routinely performed in our laboratory to avoid the possibility that the mice inadvertently recover from avertin-anaesthesia while being bled. Appropriate standardization of the experiment is achieved by injecting the same dose of barbiturate solution per mouse, and by waiting the same amount of time prior to harvest of blood. If permitted by the Institutional Animal Care and Use Committee and as long as the animals are carefully monitored for the depth of anaesthesia prior to exsanguination, this step can be omitted.</li>
 <li>Make incision into the abdomen. Open the caudal vein (vena cava) and collect blood using a transfer pipette.</li>
 <li>Remove the spleen, or a piece of the spleen into the Eppendorf tube and snap freeze in liquid nitrogen, or into the 24 well plate into a well containing Hanks buffer for later preparation of single cells.</li>
 <li>Transfer the mouse on top of the tissue paper to work-area C for collection of BAL, lung draining lymph node, lung, and heart tissues. Clean surgical instruments.</li>
</ol>
4. Collection of Lung and Heart Samples
<ol>
 <li>Dissect the trachea free of muscle and connective tissue. Placing forceps under the trachea, pull suture through. Gently generate sufficient stretch to cut a hole. Maintaining the gentle stretch, insert the tracheal cannula using a slightly turning motion, and suture cannula into place. For the insertion of the tracheal cannula, make sure that the trachea is moist, if necessary add a drop of Hanks solution. Throughout the procedure, breathe, relax neck and jaw muscles, to keep hand movements secure and smooth.</li>
 <li>Dissect open the rib cage by carefully removing the breast-bone starting from the abdominal end. Do not disturb the contents of the thorax because this will make it very hard to find the lymph node.</li>
 <li>Perform bronchoalveolar lavage (BAL) by gently inserting 1 ml of Hanks buffer, gently turn the syringe and retrieve BAL fluid. Repeat 3 times, close tube and place on ice. Throughout the procedure, keep eyes on the end of the tracheal cannula that connects to the syringe. Glance to thorax to observe the inflation and deflation of the lungs.</li>
 <li>Harvest lung lymph node by holding rib cage wall with one set of forceps, finding and retrieving the lymph node with another forceps. Place lymph node into 24 well plate. It is important to know where to search: starting from the neck, find the entrance of the rib cage from the right side of the mouse and look above the spine (Figure 5).</li>
 <li>Harvest heart and place on tissue paper. Isolate the right heart by carefully dissecting alongside the septum. Transfer to work-area A to weigh, record the weight and place into eppendorf tubes to snap freeze in liquid nitrogen.</li>
 <li>Place suture around the left lung lobe to close the main bronchus. Dissect the left lung lobe free, place into eppendorf tube and snap freeze in liquid nitrogen, or place into a well of a 24 well plate containing Hanks solution for later isolation of single cell suspensions.</li>
 <li>Insert approx. 0.5 ml buffered formaldehyde solution through the tracheal cannula into the remaining lung lobes. After inflation, dissect the lung lobes out of the thorax and place into 50 ml tube containing formaldehyde solution. For an alternative study design, the lungs can be inflated with optimal cutting media (OCT, diluted 1:4 with PBS) and frozen in a mold containing undiluted OCT for later preparation of frozen sections. Another study design might necessitate obtaining snap frozen lung tissue and single cell suspensions from the lungs. In that case, no inflation is necessary: dissect the remaining lung lobes; retrieve them from the thorax and place into the 24 well plate into a well containing Hanks buffer.</li>
 <li>Remove tracheal cannula, clean by rinsing with Hanks buffer, clean surgical instruments.</li>
</ol>
5. Analysis of Pressure Curves Generated from the Right Heart Catheter
The recorded right ventricular pressure data are analyzed using LabChart 7 software with no knowledge of the group identity of each recording. More than 20 curves are selected randomly and the difference between maximum and minimum ventricular pressure for each curve measured (&Delta;P). The average &Delta;P is calculated to give the right ventricular systolic pressure.

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No conflicts of interest declared.

The experimental flow outlined here allows for rapid and simultaneous measurement of right ventricular systolic pressure and harvest of samples for the analysis of the responses in the lungs, heart and the immune system in mice. The procedure combines heart physiology measurements, micro-dissection and subsequent tissue harvest for live cell studies, histological analysis, or omics-analysis of the tissues. The complete procedure takes less than 20 min per mouse. Because of the work-area-organized workflow, 2-3 animals ca...

<table border="1">
 <tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagents</td>
 </tr>
 <tr>
 <td>2-Methyl-2-butanol </td>
 <td>Sigma-Aldrich </td>
 <td>152463 </td>
 <td></td>
 </tr>
 <tr>
 <td>2,2,2-Tribromoethanol </td>
 <td>Sigma-Aldrich </td>
 <td>T48402 </td>
 <td></td>
 </tr>
 <tr>
 <td>disinfectant soap (Coverage Spray TB plus Steris)</td>
 <td>Fisher Scientific</td>
 <td>1629-08</td>
 <td></td>
 </tr>
 <tr>
 <td>Ethyl Alcohol, 200 Proof, Absolute, Anhydrous ACS/USP Grade </td>
 <td>PHARMCO-AAPER</td>
 <td>111000200</td>
 <td>Dilute to 70 % with distilled water</td>
 </tr>
 <tr>
 <td>Formaldehyde solution </td>
 <td>Sigma-Aldrich</td>
 <td>F1635-500ML</td>
 <td>Dilute to a 7-10 % formaldehyde concentration at a PBS concentration of 1x using PBS stock solution and water</td>
 </tr>
 <tr>
 <td>Hanks solution, no calcium, magnesium </td>
 <td>Fisher Scientific </td>
 <td>21-022-CV</td>
 <td></td>
 </tr>
 <tr>
 <td>O.C.T</td>
 <td>Tissue-Tek</td>
 <td>4583</td>
 <td></td>
 </tr>
 <tr>
 <td>Penicillin (10,000 U/ml) / Streptomycin (10,000 mg/ml) solution</td>
 <td>Thermo Scientific</td>
 <td>SV30010</td>
 <td></td>
 </tr>
 <tr>
 <td>Phosphate buffered saline (PBS), no calcium, no magnesium, 1x and 10x solutions </td>
 <td>Fisher Scientific </td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium pentobarbital 26% </td>
 <td>Fort Dodge Animal Health </td>
 <td>NDC 0856-0471-01 </td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Labware </td>
 </tr>
 <tr>
 <td>Plates 12, 24, 96 well </td>
 <td>Falcon</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Transfer Pipet </td>
 <td>Fisher Scientific </td>
 <td>13-711-9BM </td>
 <td></td>
 </tr>
 <tr>
 <td>Tube, EDTA coated </td>
 <td>Sarstedt </td>
 <td>2013-08 </td>
 <td></td>
 </tr>
 <tr>
 <td>Tubes 0.65 ml and 1.7 ml micro-centrifuge </td>
 <td>VWR</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Tubes 12 x 75 mm polypropylene </td>
 <td>Fisher Scientific</td>
 <td>14-956-1D</td>
 <td></td>
 </tr>
 <tr>
 <td>Tubes, various sizes, polypropylene</td>
 <td>Fisher Scientific</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Instruments </td>
 </tr>
 <tr>
 <td>Forceps, Dumon #5 Fine</td>
 <td>Fine Science Tools </td>
 <td>11254-20 </td>
 <td></td>
 </tr>
 <tr>
 <td>Forceps, extra fine graefe -0.5 mm tips curved</td>
 <td>Fine Science Tools </td>
 <td>11152-10</td>
 <td></td>
 </tr>
 <tr>
 <td>Forceps, extra fine graefe -0.5 mm tips straight</td>
 <td>Fine Science Tools </td>
 <td>11150-10</td>
 <td></td>
 </tr>
 <tr>
 <td>Cannula 18 ga, 19 ga</td>
 <td>BD</td>
 <td>Precision Glide Needles</td>
 <td>Cut to optimal length, blunted and outside rasped to create a rough outside surface.</td>
 </tr>
 <tr>
 <td>Scissors,
 Dissector scissors-slim blades 9 cm</td>
 <td>Fine Science Tools </td>
 <td>14081-09 </td>
 <td></td>
 </tr>
 <tr>
 <td>Suture for BAL, braided silk suture, 4-0 </td>
 <td>Fine Science Tools </td>
 <td>SP116 </td>
 <td></td>
 </tr>
 <tr>
 <td>Suture for right heart catheterization, braided silk suture, 6-0</td>
 <td>Teleflex medical</td>
 <td>18020-60 </td>
 <td></td>
 </tr>
 <tr>
 <td>Syringe, 1 ml</td>
 <td>BD</td>
 <td>309659</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment </td>
 </tr>
 <tr>
 <td>Amplifier, PowerLab 4/30</td>
 <td>ADInstrument</td>
 <td>Model ML866</td>
 <td></td>
 </tr>
 <tr>
 <td>Catheter, pressure F1.4</td>
 <td>Millar Instruments, Inc</td>
 <td>840-6719 </td>
 <td></td>
 </tr>
 <tr>
 <td>Dissecting Microscope</td>
 <td>Variscope</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Forceps, 
 Vannas spring scissors-2 mm blades </td>
 <td>Fine Science Tools </td>
 <td>15000-00 </td>
 <td></td>
 </tr>
 <tr>
 <td>Halogen Illuminated Desk Magnifier</td>
 <td>Fisher Scientific</td>
 <td>11-990-56</td>
 <td></td>
 </tr>
 <tr>
 <td>Laptop computer</td>
 <td>Asus</td>
 <td>Model number A52F i5 processor; 15 inch</td>
 <td></td>
 </tr>
 <tr>
 <td>Light Source </td>
 <td>Amscope</td>
 <td>HL-250-A</td>
 <td></td>
 </tr>
 <tr>
 <td>Pressure Control Unit </td>
 <td>Millar Instruments, Inc </td>
 <td>PCU-2000</td>
 <td></td>
 </tr>
 <tr>
 <td>Software, Labchart-Pro V.7</td>
 <td>AD Instruments</td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

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.

The primary outcome for obtaining right heart pressure curves is achieved by the correct position of the right heart catheter. The shape of the pressure time curves is critical because the correct placement of the catheter inside of the right ventricle will result in pressure plateaus (Figure 4). Spiky curves, instead, indicate a catheter that is moved by the breathing or heart beat motion against the wall of the right ventricle. To detect potential problems with the stage of survival of the anima...

Watch this Scientific Journal Video about Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice at JoVE.com

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

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 ...

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25.1K 조회수.  New York University School of Medicine, Tuxedo. 동시에 오른쪽 심장 기능, 폐 염증 및 면역 반응을 조사하기 위해 구체적이고 빠른 프로토콜은 학습 도구로 설명되어 있습니다. 비디오 및 그림은 큰 규모의 연구에 작은에 사용할 수에 적용 할 수 있습니다 조직 팀 접근 방식에서 생리학 및 microdissection 기술을 설명합니다.

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

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

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 ...

Induction of Maternal Immune Activation in Mice at Mid-gestation Stage with Viral Mimic Poly(I:C)

Maternal immune activation (MIA) model is increasingly well appreciated as a rodent model for the environmental risk factor of various psychiatric disorders. Numerous studies have demonstrated that MIA model is able to show face, construct, and predictive validity that are relevant to autism and schizophrenia. To model MIA, investigators often use viral mimic polyinosinic:polycytidylic acid (poly(I:C)) to activate the immune system in pregnant rodents. Generally, the offspring from immune activated dam exhibit behavioral abnormalities and physiological alterations that are associated with autism and schizophrenia. However, poly(I:C) injection with different dosages and at different time points could lead to different outcomes by perturbing brain development at different stages. Here we provide a detailed method of inducing MIA by intraperitoneal (i.p.) injection of 20 mg/kg poly(I:C) at mid-gestational embryonic 12.5 days (E12.5). This method has been shown to induce acute inflammatory response in the maternal-placental-fetal axis, which ultimately results in the brain perturbations and behavioral phenotypes that are associated with autism and schizophrenia.

Induction of Maternal Immune Activation in Mice at Mid-gestation Stage with Viral Mimic PolyI:C

Maternal immune activation (MIA) is a model for an environmental risk factor of autism and schizophrenia. The goal of this article is to provide a step-by-step procedure of how to induce MIA in the pregnant mice in order to enhance the reproducibility of this model.

Maternal immune activation (MIA) model is increasingly well appreciated as a rodent model for the environmental risk factor of various psychiatric ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy 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 ...

Application of Consistent Massage-Like Perturbations on Mouse Calves and Monitoring the Resulting Intramuscular Pressure Changes

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of massage on skeletal muscles, the molecular mechanisms behind are poorly understood. We have recently developed a simple device to apply local cyclical compression (LCC), which can generate intramuscular pressure waves with varying amplitudes. Using this device, we have demonstrated that LCC modulates inflammatory responses of macrophages in situ and alleviates immobilization-induced muscle atrophy. Here, we describe protocols for the optimization and application of LCC as a massage-like intervention against immobilization-induced inflammation and atrophy of skeletal muscles of mouse hindlimbs. The protocol that we have developed can be useful for investigating the mechanism underlying beneficial effects of physical exercise and massage. Our experimental system provides a prototype of the analytical approach to elucidate the mechanical regulation of muscle homeostasis, although further development needs to be made for more comprehensive studies.

Here we describe the protocols for applying defined mechanical loads to mouse calves and for monitoring the concomitant intramuscular pressure changes. The experimental systems that we have developed can be useful for investigating the mechanism behind the beneficial effects of physical exercise and massage.

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of ...

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 ...

Monitoring Neutrophil Elastase and Cathepsin G Activity in Human Sputum Samples

Proteases are regulators of countless physiological processes and the precise investigation of their activities remains an intriguing biomedical challenge. Among the ~600 proteases encoded by the human genome, neutrophil serine proteases (NSPs) are thoroughly investigated for their involvement in the onset and progression of inflammatory conditions including respiratory diseases. Uniquely, secreted NSPs not only diffuse within extracellular fluids but also localize to plasma membranes. During neutrophil extracellular trap (NETs) formation, NSPs become an integral part of the secreted chromatin. Such complex behavior renders the understanding of NSPs pathophysiology a challenging task. Here, detailed protocols are shown to visualize, quantify and discriminate free and membrane-bound neutrophil elastase (NE) and cathepsin G (CG) activities in sputum samples. NE and CG are NSPs whose activities have pleiotropic roles in the pathogenesis of cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD): they promote tissue remodeling, regulate downstream immune responses and correlate with lung disease severity. The protocols show how to separate fluid and cellular fraction, as well as the isolation of neutrophils from human sputum for enzymatic activity quantification via small-molecule F&#246;rster resonance energy transfer-based (FRET) reporters. To gather specific insights into the relative role of NE and CG activities, a FRET readout can be measured by different technologies: i) in vitro plate reader measurements allow for high-throughput and bulk detection of protease activity; ii) confocal microscopy spatiotemporally resolves membrane-bound activity at the cell surface; iii) small-molecule FRET flow cytometry enables for the rapid evaluation of anti-inflammatory treatments via single-cell protease activity quantification and phenotyping. The implementation of such methods opens the doors to explore NSPs pathobiology and their potential as biomarkers of disease severity for CF and COPD. Given their standardization potential, their robust readout and simplicity of transfer, the described techniques are immediately shareable for implementation across research and diagnostic laboratories.

The protocols herein described provide a guide to visualize and quantify the activity of neutrophil proteases in human sputum. The applications of such analysis span from the evaluation of anti-inflammatory treatments, to biomarker validation, drug screening and large cohort clinical studies.

Proteases are regulators of countless physiological processes and the precise investigation of their activities remains an intriguing biomedical ...

Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms include irregular fever, lymphadenopathy, and significantly increased lymphocytes in peripheral blood. The pathogenic mechanism of IM is still unclear; there is no effective treatment method for it, with mainly symptomatic therapies being available. The main question in EBV immunobiology is why only a small subset of infected individuals shows severe clinical symptoms and even develop EBV-associated malignancies, whilemost individuals are asymptomatic for life with the virus.
B cells are first involved in IM because EBV receptors are presented on their surface. Natural killer (NK) cells are cytotoxic innate lymphocytes that are important for killing EBV-infected cells. The proportion of CD4+ T cells decreases while that of CD8+ T cells expands dramatically during acute EBV infection, and the persistence of CD8+ T cells is important for lifelong control of IM. Those immune cells play important roles in IM, and their functions need to be identified separately. For this purpose, monocytes are separated first from peripheral blood mononuclear cells (PBMCs) of IM individuals using CD14 microbeads, a column, and a magnetic separator.
The remaining PBMCs are stained with peridinin-chlorophyll-protein (PerCP)/Cyanine 5.5 anti-CD3, allophycocyanin (APC)/Cyanine 7 anti-CD4, phycoerythrin (PE) anti-CD8, fluorescein isothiocyanate (FITC) anti-CD19, APC anti-CD56, and APC anti-CD16 antibodies to sort CD4+ T cells, CD8+ T cells, B cells, and NK cells using a flow cytometer. Furthermore, transcriptome sequencing of five subpopulations was performed to explore their functions and pathogenic mechanisms in IM.

We describe a method combining immunomagnetic beads and fluorescence-activated cell sorting to isolate and analyze defined immune cell subpopulations of peripheral blood mononuclear cells (monocytes, CD4+ T cells, CD8+ T cells, B cells, and natural killer cells). Using this method, magnetic and fluorescently labeled cells can be purified and analyzed.

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms ...

Induction of Periodontitis via a Combination of Ligature and Lipopolysaccharide Injection in a Rat Model

Periodontitis (PD) is a highly prevalent, chronic immune-inflammatory disease of the periodontium, that results in a loss of gingival soft tissue, periodontal ligament, cementum, and alveolar bone. In this study, a simple method of PD induction in rats is described. We provide detailed instructions for placement of the ligature model around the first maxillary molars (M1) and a combination of injections of lipopolysaccharide (LPS), derived from Porphyromonas gingivalis at the mesio-palatal side of the M1. The induction of periodontitis was maintained for 14 days, promoting the accumulation of bacteria biofilm and inflammation. To validate the animal model, IL-1&#946;, a key inflammatory mediator, was determined by an immunoassay in the gingival crevicular fluid (GCF), and alveolar bone loss was calculated using cone beam computed tomography (CBCT). This technique was effective in promoting gingiva recession, alveolar bone loss, and an increase in IL-1&#946; levels in the GCF at the end of the experimental procedure after 14 days. This method was effective in inducing PD, thus being able to be used in studies on disease progression mechanisms and future possible treatments.

Induction of Periodontitis via a Combination of Ligature and Lipopolysaccharide Injection in a Rat Model

In this study, a rat model of induction of periodontitis is presented via a combination of retentive ligature and repetitive injections of lipopolysaccharide derived from Porphyromonas gingivalis, over 14 days around the first maxillary molars. The ligation and LPS injection techniques were effective in inducing peridontitis, resulting in alveolar bone loss and inflammation.