Name: application of consistent massage-like perturbations on mouse calves and monitoring the resulting intramuscular pressure changes
Uploaded: 2019-09-20T13:00:00
Description: Watch this Scientific Journal Video about Application of Consistent Massage-Like Perturbations on Mouse Calves and Monitoring the Resulting Intramuscular Pressure Changes at JoVE.com

We thank K. Nakanishi, K. Hamamoto, N. Kume, and K. Tsurumi for their consistent support throughout the project. This work was in part supported by Intramural Research Fund from the Japanese Ministry of Health, Labour and Welfare; Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science; MEXT-Supported Program for the Strategic Research Foundation at Private Universities, 2015-2019 from the Japanese Ministry of Education, Culture, Sports, Science and Technology (S1511017).

All animal experiments were conducted under the approval by the Institutional Animal Care and Use Committee of the National Rehabilitation Center for Persons with Disabilities.
1. Immobilization of the mouse bilateral hindlimbs
NOTE: Male C57BL/6 mice were used for experiments at the age of 11 - 12 weeks after acclimation for at least 7 days.
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	<li>Adequately anesthetize a mouse using sodium pentobarbital (50 mg/kg i.p.). Make sure that mice do not respond to ...

<ol>
	<li>Furlan, A. D., Imamura, M., Dryden, T., Irvin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Massage+for+low+back+pain:+an+updated+systematic+review+within+the+framework+of+the+Cochrane+Back+Review+Group.">Massage for low back pain: an updated systematic review within the framework of the Cochrane Back Review Group.</a> Spine. 34 (16), 1669-1684 (2009).</li><li>Robertson, A., Watt, J. M., Galloway, S. D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+leg+massage+on+recovery+from+high+intensity+cycling+exercise.">Effects of leg massage on recovery from high intensity cycling exercise.</a> British Journal of Sports Medicine. 38 (2), 173-176 (2004).</li><li>Waters-Banker, C., Butterfield, T. A., Dupont-Versteegden, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunomodulatory+effects+of+massage+on+nonperturbed+skeletal+muscle+in+rats.">Immunomodulatory effects of massage on nonperturbed skeletal muscle in rats.</a> Journal of Applied Physiology. 116 (2), 164-175 (2014).</li><li>Haas, 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=Massage+timing+affects+postexercise+muscle+recovery+and+inflammation+in+a+rabbit+model.">Massage timing affects postexercise muscle recovery and inflammation in a rabbit model.</a> Medicine &amp; Science in Sports &amp; Exercise. 45 (6), 1105-1112 (2013).</li><li>Crane, J. D., 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=Massage+therapy+attenuates+inflammatory+signaling+after+exercise-induced+muscle+damage.">Massage therapy attenuates inflammatory signaling after exercise-induced muscle damage.</a> Science Translational Medicine. 4 (119), 119ra113 (2012).</li><li>Bove, G. M., Harris, M. Y., Zhao, H., Barbe, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manual+therapy+as+an+effective+treatment+for+fibrosis+in+a+rat+model+of+upper+extremity+overuse+injury.">Manual therapy as an effective treatment for fibrosis in a rat model of upper extremity overuse injury.</a> Journal of the Neurological Sciences. 361, 168-180 (2016).</li><li>Andrzejewski, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+skeletal+muscle+expression+of+VEGF+induced+by+massage+and+exercise.">Increased skeletal muscle expression of VEGF induced by massage and exercise.</a> Folia Histochemica et Cytobiologica. 53 (2), 145-151 (2015).</li><li>Mantovani Junior, N., 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=Effects+of+massage+as+a+recuperative+technique+on+autonomic+modulation+of+heart+rate+and+cardiorespiratory+parameters:+a+study+protocol+for+a+randomized+clinical+trial.">Effects of massage as a recuperative technique on autonomic modulation of heart rate and cardiorespiratory parameters: a study protocol for a randomized clinical trial.</a> Trials. 19 (1), 459 (2018).</li><li>Zeng, H., Butterfield, S., Agarwal, F., Haq, T., Zhao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+engineering+approach+for+quantitative+analysis+of+the+lengthwise+strokes+in+massage+therapies.">An engineering approach for quantitative analysis of the lengthwise strokes in massage therapies.</a> Journal of Medical Devices. 2 (4), (2008).</li><li>Wang, Q., 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=A+mechatronic+system+for+quantitative+application+and+assessment+of+massage-like+actions+in+small+animals.">A mechatronic system for quantitative application and assessment of massage-like actions in small animals.</a> Annals of Biomedical Engineering. 42 (1), 36-49 (2014).</li><li>Saitou, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+cyclical+compression+modulates+macrophage+function+in+situ+and+alleviates+immobilization-induced+muscle+atrophy.">Local cyclical compression modulates macrophage function in situ and alleviates immobilization-induced muscle atrophy.</a> Clinical Science. 132 (19), 2147-2161 (2018).</li><li>Onda, 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=A+New+mouse+model+of+skeletal+muscle+atrophy+using+spiral+wire+immobilization.">A New mouse model of skeletal muscle atrophy using spiral wire immobilization.</a> Muscle Nerve. 54 (4), 788-791 (2016).</li><li>Luster, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokines--chemotactic+cytokines+that+mediate+inflammation.">Chemokines--chemotactic cytokines that mediate inflammation.</a> The New England Journal of Medicine. 338, 436-445 (1998).</li><li>Reid, M. B., Li, Y. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+necrosis+factor-&#945;+and+muscle+wasting:+a+cellular+perspective.">Tumor necrosis factor-&#945; and muscle wasting: a cellular perspective.</a> Respiratory Research. 2 (5), 269-272 (2001).</li><li>Baumann, J. U., Sutherland, M. 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We have described a method for applying a massage-like mechanical stimulus, which has anti-inflammatory effects. Our system has following advantages even when compared with those reported previously. First, previous studies did not quantitatively define the mechanical forces applied2 or defined their magnitudes based on the measurement at the body surface, but not inside the tissues10. In contrast, we measured intramuscular pressure using a blood pressure telemeter. Second,...

Massage is generally recognized to be beneficial for both pain relief and improvement of the physical performance among competitive athletes and non-athletes alike1,2. In fact, previous studies have shown that massage suppresses local inflammation3 and prompts recovery from the post-exercise muscle damage4,5. Molecular mechanisms underlying the beneficial effects of massage remain largely unknown.
One of the difficulties with the mechanistic investigation on massage relates to the reproducibility of experimental techniques by which massage-like interventions are tested. In previous studies, experimental procedures that mimic massage mostly involve the application of physical interventions using practitioners&rsquo; body parts, such as palms and fingers6,7,8. This makes it is difficult to precisely reproduce their magnitude, frequency, duration, and mode.
Many devices have been developed to apply defined mechanical loads to the target tissues. For example, Zeng et al. have developed a pneumatic system for the length-wise mechanical loading to rats&rsquo; hindlimbs9 and Wang et al. have developed a mechatronic device that can apply massage-like mechanical loads to hindlimbs of rats and rabbits with real-time feedback control10. Compared to them, our local cyclical compression (LCC) system is much simpler, demanding far less cost for construction. Nonetheless, we can reproduce the intramuscular pressure changes that are generated during the mild muscle contraction. Using this device, we have successfully demonstrated that the massage-like mechanical interventions modulate local interstitial fluid dynamics and alleviate immobilization-induced muscle atrophy11.
Here, we describe the details of our device and the protocol, which may help explore the molecular mechanisms behind the positive effects of exercises and massages. The schematics of the protocol is presented as Supplementary Figure 1.

<table>
<tbody>
<tr>
<td>Aluminum wire</td>
<td>DAISO JAPAN</td>
<td>B028</td>
<td>An aluminum wire is used to avoid escaping restriction by the wire</td>
</tr>
<tr>
<td>Blood pressure telemeter</td>
<td>Millar</td>
<td>SPR-671</td>
<td>A blood pressure telemeter is used to mesure intramuscular pressure.</td>
</tr>
<tr>
<td>DAPI</td>
<td>Thermo Fisher Scientific</td>
<td>D1306</td>
<td>DAPI is a fluorescent probe which is commonly used to stain DNA for fluorescent microscopy.</td>
</tr>
<tr>
<td>Goat anti-rabbit Alexa Fluor 488 (Dilution ratio, 1:500)</td>
<td>Invitrogen </td>
<td>A11034</td>
<td>Antibody for immunohistochemical staining.</td>
</tr>
<tr>
<td>Goat anti-rat Alexa Fluor 568 (Dilution ratio, 1:500))</td>
<td>Invitrogen </td>
<td>A11077</td>
<td>Antibody for immunohistochemical staining.</td>
</tr>
<tr>
<td>ImageJ</td>
<td>NIH</td>
<td>N/A</td>
<td>Analysis software for image</td>
</tr>
<tr>
<td>LabChart8</td>
<td>ADInstrumens</td>
<td>&nbsp;</td>
<td>Analysis software for acquiring biological signals.</td>
</tr>
<tr>
<td>Prolong gold</td>
<td>Thermo Fisher Scientific</td>
<td>P36930</td>
<td>Prolong gold is for mounting stained samples.</td>
</tr>
<tr>
<td>Protein Block Serum-Free</td>
<td>Dako</td>
<td>X090930-2</td>
<td>For blocking non-specific background staining in immunohistochemical procedures.</td>
</tr>
<tr>
<td>Rat monoclonal anti-laminin-2 antibody (Dilution ratio, 1:1000)</td>
<td>Sigma Aldrich</td>
<td>L0663</td>
<td>Antibody for immunohistochemical staining.</td>
</tr>
<tr>
<td>Rat monoclonal anti-F4/80 antibody (Dilution ratio, 1:500)</td>
<td>Abcam</td>
<td>ab6640</td>
<td>Antibody for immunohistochemical staining.</td>
</tr>
<tr>
<td>Rabbit polyclonal anti-MCP-1 antibody (Dilution ratio, 1:1000)</td>
<td>Abcam</td>
<td>ab25124</td>
<td>Antibody for immunohistochemical staining.</td>
</tr>
<tr>
<td>Rabbit polyclonal anti-TNF-&alpha; antibody (Dilution ratio, 1:1000)</td>
<td>Abcam</td>
<td>ab66579</td>
<td>Antibody for immunohistochemical staining.</td>
</tr>
<tr>
<td>Surgical tape</td>
<td>3M Japan</td>
<td>1530EP-0</td>
<td>Surgical tape is used to restrict joint movement.</td>
</tr>
</tbody>
</table>

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.

Consistent with our previous observations12, the CSA of gastrocnemius myofibers were significantly decreased by hindlimb immobilization (Figure 2A,B). Furthermore, our immunofluorescence staining analysis revealed that cells expressing MCP-1 and TNF-&#945;, both of which play key roles in regulating inflammatory processes13,14, significantly increased in gastrocnemius muscle tissues of immobil...

Watch this Scientific Journal Video about Application of Consistent Massage-Like Perturbations on Mouse Calves and Monitoring the Resulting Intramuscular Pressure Changes at JoVE.com

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

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 ...

Our protocol can be useful in the systems and mechanism. And the right information shows the effect of physical stress and massage. Our local cyclic completion system is much more simple.The conventional methods aren't required for local insertion. Apply surgical tape to the bilateral hind limbs in the supine position with the knee joints extended and the ankle joints plantar flexed. Place an aluminum wire on the trunk at the L4-5 spine level, and coil the wire in a spiral configuration around the hind limbs with five millimeter gaps between each turn of the spiral.To minimize the possibility of escape from the wiring, manually adjust the wire configuration to immobilize the hip joints at a 90 degree position of abduction. Then return the mouse to it's home cage with monitoring until full recovery. To measure the intramuscular gastrocnemius muscle pressure place the anesthetized mouse in the prone position and shave, depilate, and semi-sterilize the skin of the posterior calf.Use a scalpel to make a two millimeter incision in the disinfected skin. And insert a 20 gauge indwelling needle into the gastrocnemius muscle at a 150 to 170 degree angle to the skin surface. Next, using the plastic sheath of the needle as a guide, place a blood pressure telemeter sensor in the mid belly of gastrocnemius muscle.Enclose the skin with a 4-O nylon suture. Then, use several different cylindrical unit weights to apply local cylindrical compression to the calf muscle for one minute. And monitor the intramuscular pressure using the appropriate software for biological signal analysis.For local cyclical compression treatment disengage the anesthetized mouse from the hind limb wiring. And place the animal in the prone position. Adjust the hind limbs so that the knee joints are extended and the ankle joints are plantar flexed with the calves facing upward.And vertically move a cylindrical weight unit, covered with a cushioned pad, over the calf muscle at one hertz for 30 minutes per day for seven days. After each daily bout of compression rewire the mouse hind limbs before returning the animal to its home cage. At the end of the experiment depilate the posterior calf surface and make a skin incision to allow separation of the gastrocnemius muscles from the tibiofibular bone.Quickly freeze the harvested muscle tissue in an optimal cutting temperature compound solution. And use a cryostat to obtain sections of the gastrocnemius muscles on glass slides. For immunohistochemical labeling thaw and dehydrate the sections by air drying at room temperature before using a hydrophobic pen to draw a ring around the tissue sections on each slide.Next, place the slides in humidity chamber. And add 100 microliters of blocking buffer to each side. After 30 minutes at room temperature rinse the slides with two 5 minute washes in PBS-T, before labeling the sections with 100 microliters of the primary antibody of interest overnight at room temperature.The next morning wash the slides 3 times in PBS-T for 5 minutes per wash. And label the samples with an appropriate secondary antibody for 1 hour at room temperature, protected from light. At the end of the incubation, wash the slides 3 times in fresh PBS-T as demonstrated.And stain the nuclei with 100 microliters of DAPI for three minutes at room temperature in the dark. Then, wash the slide three more times. And mount the samples with a compatible mounting medium and cover slips.For histomorphometric analysis, place the slides on the stage of a fluorescence microscope and view the samples under a 20-X subjective in the appropriate filters. Then, in the image analysis software measure the cross sectional area of each myofiber and count the number of cells expressing the experimental markers of interest. The cross sectional area of gastrocnemius myofiber are significantly decreased by hind limb immobilization.Further, immunofluorescent staining analysis reveals that cells expressing MCP-1 and TNF-alpha are significantly increased in the gastrocnemius muscle tissues of immobilized hind limbs. Together, with the increase in cells positively stained with F4/80, hind limb immobilization appears to instigate calf muscle atrophy, involving local inflammatory responses, including macrophage accumulation. Among the several different local cyclical compression magnitudes tested by changing the weight of the cylindrical unit, the compression corresponding to 50 milliliters of mercury intermuscular pressure waves, appears to most efficiently alleviate the immobilization induced decrease in myofiber cross sectional area and to increase in the macrophage accumulation in gastrocnemius muscles.A 66 gram local cyclical compression, applied at 1 hertz for 30 minutes per day for 7 days, significantly alleviates the observed immobilization induced decreases in the myofiber cross sectional area of the gastrocnemius muscles. More, local cyclical compression partially tempers immobilization induced decreases in the contracting force of the triceps surae muscles. In addition, local cyclical compression tempers the increase in F4/80 positive, TNF-alpha positive, and MCP-1 one positive cells in the gastrocnemius muscle tissue of immobilized hind limbs.Our approach will provide a scientific basis for muscle rise in the patient and our potential strategy for preventive procedure. The rat's found the limited of physical inactivity. Using our system, we have started assessing the most patients that's muscles lack mechanic in the patient.Moderate local industrial way to do it makes a perfect immobilization induced muscle atrophy.

application-consistent-massage-like-perturbations-on-mouse-calves

Research

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Immunology and Infection

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all gynecologic malignancies among US women 1,2,3. Serous tumors are the most widespread forms of ovarian cancer and 4,5 the Tg-MISIIR-TAg transgenic represents the only mouse model that spontaneously develops this type of tumors. Tg-MISIIR-TAg mice express SV40 transforming region under control of the Mullerian Inhibitory Substance type II Receptor (MISIIR) gene promoter 6. Additional transgenic lines have been identified that express the SV40 TAg transgene, but do not develop ovarian tumors. Non-tumor prone mice exhibit typical lifespan for C57Bl/6 mice and are fertile. These mice can be used as syngeneic allograft recipients for tumor cells isolated from Tg-MISIIR-TAg-DR26 mice.
Objective: Although tumor imaging is possible 7, early detection of deep tumors is challenging in small living animals. To enable preclinical studies in an immunologically intact animal model for serous ovarian cancer, we describe a syngeneic mouse model for this type of ovarian cancer that permits in vivo imaging, studies of the tumor microenvironment and tumor immune responses.
Methods: We first derived a TAg+ mouse cancer cell line (MOV1) from a spontaneous ovarian tumor harvested in a 26 week-old DR26 Tg-MISIIR-TAg female. Then, we stably transduced MOV1 cells with TurboFP635 Lentivirus mammalian vector that encodes Katushka, a far-red mutant of the red fluorescent protein from sea anemone Entacmaea quadricolor with excitation/emission maxima at 588/635 nm 8,9,10. We orthotopically implanted MOV1Kat in the ovary 11,12,13,14 of non-tumor prone Tg-MISIIR-TAg female mice. Tumor progression was followed by in vivo optical imaging and tumor microenvironment was analyzed by immunohistochemistry.
Results: Orthotopically implanted MOV1Kat cells developed serous ovarian tumors. MOV1Kat tumors could be visualized by in vivo imaging up to three weeks after implantation (fig. 1) and were infiltrated with leukocytes, as observed in human ovarian cancers 15 (fig. 2).
Conclusions: We describe an orthotopic model of ovarian cancer suitable for in vivo imaging of early tumors due to the high pH-stability and photostability of Katushka in deep tissues. We propose the use of this novel syngeneic model of serous ovarian cancer for in vivo imaging studies and monitoring of tumor immune responses and immunotherapies.

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

To study in vivo tumor growth and tumor microenvironment, we used a syngeneic and orthotopic mouse model of ovarian cancer in immunocompetent animals. We transduced a mouse tumor cell line (MOV1) with Katushka fluorescent protein (MOV1KAT) and here we show its orthotopic implantation in ovary and in vivo imaging.

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all ...

Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. In the vasculature hypertension is associated with endothelial dysfunction and increased inflammation leading to atherosclerosis and various disease states such as chronic kidney disease2, stroke3 and heart failure4. An initial step in vascular inflammation leading to atherogenesis is the adhesion cascade which involves the rolling, tethering, adherence and subsequent transmigration of leukocytes through the endothelium. Recruitment and accumulation of leukocytes to the endothelium is mediated by an upregulation of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intracellular cell adhesion molecule-1 (ICAM-1) and E-selectin as well as increases in cytokine and chemokine release and an upregulation of reactive oxygen species5. In vitro methods such as static adhesion assays help to determine mechanisms involved in cell-to-cell adhesion as well as the analysis of cell adhesion molecules. Methods employed in previous in vitro studies have demonstrated that acute increases in pressure on the endothelium can lead to monocyte adhesion, an upregulation of adhesion molecules and inflammatory markers6 however, similar to many in vitro assays, these findings have not been performed in real time under physiological flow conditions, nor with whole blood. Therefore, in vivo assays are increasingly utilised in animal models to demonstrate vascular inflammation and plaque development. Intravital microscopy is now widely used to assess leukocyte adhesion, rolling, migration and transmigration7-9. When combining the effects of pressure on leukocyte to endothelial adhesion the in vivo studies are less extensive. One such study examines the real time effects of flow and shear on arterial growth and remodelling but inflammatory markers were only assessed via immunohistochemistry10. Here we present a model for recording leukocyte adhesion in real time in intact pressurised blood vessels using whole blood perfusion. The methodology is a modification of an ex vivo vessel chamber perfusion model9 which enables real-time analysis of leukocyte -endothelial adhesive interactions in intact vessels. Our modification enables the manipulation of the intraluminal pressure up to 200 mmHg allowing for study not only under physiological flow conditions but also pressure conditions. While pressure myography systems have been previously demonstrated to observe vessel wall and lumen diameter11 as well as vessel contraction this is the first time demonstrating leukocyte-endothelial interactions in real time. Here we demonstrate the technique using carotid arteries harvested from rats and cannulated to a custom-made flow chamber coupled to a fluorescent microscope. The vessel chamber is equipped with a large bottom coverglass allowing a large diameter objective lens with short working distance to image the vessel. Furthermore, selected agonist and/or antagonists can be utilized to further investigate the mechanisms controlling cell adhesion. Advantages of this method over intravital microscopy include no involvement of invasive surgery and therefore a higher throughput can be obtained. This method also enables the use of localised inhibitor treatment to the desired vessel whereas intravital only enables systemic inhibitor treatment.

This is a method to visualise leukocyte adhesion to the endothelium in harvested pressurised vessels. The technique enables studying vascular adhesion under shear flow with differing intraluminal pressures up to 200 mmHg thus mimic-ing the pathophysiological conditions of high blood pressure.

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. ...

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&amp;#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin synthesis. They also supply the host with a considerable amount of nutrients, making this ecosystem an essential metabolic organ. In the context of increasing evidence of the link between the gut flora and the metabolic syndrome, understanding the metabolic interaction between the host and its gut microbiota is becoming an important challenge of modern biology.1-4
 Colonization (also referred to as normalization process) designates the establishment of micro-organisms in a former germ-free animal. While it is a natural process occurring at birth, it is also used in adult germ-free animals to control the gut floral ecosystem and further determine its impact on the host metabolism. A common procedure to control the colonization process is to use the gavage method with a single or a mixture of micro-organisms. This method results in a very quick colonization and presents the disadvantage of being extremely stressful5. It is therefore useful to minimize the stress and to obtain a slower colonization process to observe gradually the impact of bacterial establishment on the host metabolism.
 In this manuscript, we describe a procedure to assess the modification of hepatic metabolism during a gradual colonization process using a non-destructive metabolic profiling technique. We propose to monitor gut microbial colonization by assessing the gut microbial metabolic activity reflected by the urinary excretion of microbial co-metabolites by 1H NMR-based metabolic profiling. This allows an appreciation of the stability of gut microbial activity beyond the stable establishment of the gut microbial ecosystem usually assessed by monitoring fecal bacteria by DGGE (denaturing gradient gel electrophoresis).6 The colonization takes place in a conventional open environment and is initiated by a dirty litter soiled by conventional animals, which will serve as controls. Rodents being coprophagous animals, this ensures a homogenous colonization as previously described.7
 Hepatic metabolic profiling is measured directly from an intact liver biopsy using 1H High Resolution Magic Angle Spinning NMR spectroscopy. This semi-quantitative technique offers a quick way to assess, without damaging the cell structure, the major metabolites such as triglycerides, glucose and glycogen in order to further estimate the complex interaction between the colonization process and the hepatic metabolism7-10. This method can also be applied to any tissue biopsy11,12.

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

A progressive colonization procedure is described to further assess its impact on the host hepatic metabolism. Colonization is monitored non invasively by evaluating the urinary excretion of microbial co-metabolites by NMR-based metabolic profiling while hepatic metabolism is assessed by High Resolution Magic Angle Spinning (HR MAS) NMR profiling of intact biopsy.

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin ...

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

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 ...

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on the amber suppression technology that was originally developed by Peter Schultz and colleagues1. An amber mutation is first introduced at a specific codon of the gene encoding the protein of interest. The amber mutant is then expressed in E. coli together with genes encoding an amber suppressor tRNA and an amino acyl-tRNA synthetase derived from Methanococcus jannaschii. Using this system, the photo activatable amino acid analog p-benzoylphenylalanine (Bpa) is incorporated at the amber codon. Cells are then irradiated with ultraviolet light to covalently link the Bpa residue to proteins that are located within 3-8 &#197;. Photocrosslinking is performed in combination with pulse-chase labeling and immunoprecipitation of the protein of interest in order to monitor changes in protein-protein interactions that occur over a time scale of seconds to minutes. We optimized the procedure to study the assembly of a bacterial virulence factor that consists of two independent domains, a domain that is integrated into the outer membrane and a domain that is translocated into the extracellular space, but the method can be used to study many different assembly processes and biological pathways in both prokaryotic and eukaryotic cells. In principle interacting factors and even specific residues of interacting factors that bind to a protein of interest can be identified by mass spectrometry.

This article illustrates the use of pulse-chase radio labeling in combination with site-specific photocrosslinking to monitor interactions between a protein of interest and other factors in E. coli. Unlike traditional chemical cross-linking methods, this approach generates high resolution &#8220;snapshots&#8221; of an ordered assembly pathway in a living cell.

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on ...

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability.
Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay.
A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated peripheral blood.

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into possible MOA, morphologic changes associated with antibiotic exposure can be visualized using scanning electron microscopy (SEM). Integrating SEM imaging with traditional kill curves may improve our insight into drug action and advance the drug development process. To test this premise, kill curves and SEM studies were conducted using drugs with known but different MOA (vancomycin and metronidazole). C. difficile cells (R20291) were grown with or without the presence of antibiotic for up to 48 h. Throughout the 48 h interval, cells were collected at multiple time points to determine antibiotic efficacy and for imaging on the SEM. Consistent with previous reports, vancomycin and metronidazole had significant bactericidal activity following 24 h of treatment as measured by colony-forming unit (CFU) counting. Using SEM imaging we determined that metronidazole had significant effects on cell length (&#62; 50% reduction in cell length for each antibiotic; P&#60; 0.05) compared to controls and vancomycin. While the phenotypic response to drug treatment has not been documented previously in this manner, they are consistent with the drug&#39;s MOA demonstrating the versatility and reliability of the imaging and measurements and the application of this technique for other experimental compounds.

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Antibiotic efficacy is most commonly determined by conducting killing kinetic studies and measuring colony forming units (CFUs). By integrating scanning electron microscopy (SEM) with these standard methods, we can distinguish the pharmacological effects of treatment between different antibiotics.

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...