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.
<ol>
	<li>Adequately anesthetize a mouse using sodium pentobarbital (50 mg/kg i.p.). Make sure that mice do not respond to a hindlimb toe pinch. 
	NOTE: Conduct the procedure of immobilization between 10 a.m. and 7 p.m. to minimize the possible effects on the feeding activity of mice.</li>
	<li>Apply surgical tapes to the bilateral hindlimbs of the mouse laid in a supine position with the knee joints extended and ankle joints plantar-flexed.</li>
	<li>Place an aluminum wire (see Table of Materials) on the trunk at L4-5 spine level and coil the wire in a spiral configuration around the hindlimbs with 5 mm gaps between each turn of the spiral layer (Figure 1A). Make sure not to coil the wire too tightly and avoid disturbing the local blood flow.</li>
	<li>To minimize the possibility of escape from wiring, immobilize the hip joints at the position of 90&#176; abduction by manually adjusting the configuration of aluminum wire.</li>
	<li>Return the mice to their original cages. 3 h later, make sure that they recover from anesthesia and access to food and water as usual.</li>
	<li>House 3 - 6 immobilized mice per cage as before immobilization.</li>
</ol>
2. Measurement of intramuscular pressure of mouse gastrocnemius muscles
NOTE: Several different weights of cylindrical units (36 g, 66 g, and 200 g) were tested in the pressure-monitoring experiments combined with LCC. This measurement was conducted separately from the experiments to analyze muscle inflammation and atrophy (see step 3 - 5 for more details) i.e., the mice subjected to pressure measurement were not used for histological analyses.
<ol>
	<li>Because pressure measurement involves more invasive procedures (e.g., skin incision and needle insertion) as compared to hindlimb wiring and LCC, use a mixture of three anesthetic agents (medetomidine 0.75 mg/kg, midazolam 4.0 mg/kg, and butorphanol 5.0 mg/kg, i.p.). Make sure that mice do not respond to the hindlimb toe pinch.</li>
	<li>Lay the mouse in a prone position, make a 2-mm incision with a scalpel on the posterior calf after depilating with an electric shaver and semi-sterilizing the skin surface with 70% ethanol- and Chlorhexidine-soaked absorbent cotton.</li>
	<li>Insert a 20 G indwelling needle into the gastrocnemius muscle at an obtuse angle (150&#176; &#8211; 170&#176;) to the skin surface.</li>
	<li>Using the plastic sheath of the needle as a guide, place a sensor of the blood pressure telemeter (see Table of Materials) in the mid-belly of gastrocnemius muscle, and then remove the sheath from the muscle.</li>
	<li>After suturing the skin with 4-0 nylon suture, apply LCC with several different weights of cylindrical units to the calf in the mice (see step 3 for more details), and monitor the intramuscular pressure using software for biological signal analysis (see Table of Materials).</li>
	<li>Return the mice to their original cages. 3 h later, make sure that they recover from anesthesia/analgesia&#160;and have access to food and water as usual.</li>
</ol>
3. Local cyclical compression (LCC) on mouse calves
<ol>
	<li>Except for the intramuscular pressure measurement and euthanizing (i.e., cervical dislocation), use sodium pentobarbital (50 mg/kg i.p.) for anesthesia.</li>
	<li>Disengage the mouse from hindlimb wiring and lay it in a prone position with the knee joints extended and the ankle joints plantar-flexed so that the calves faced upward. Do not fix the mouse hindlimbs on the stage.</li>
	<li>Apply LCC to the calf by vertically moving a cylindrical weight unit (Figure 1B) covered with a cushion pad (Figure 1C) at 1 Hz for 30 min per day, 7 days.</li>
	<li>After each bout of daily LCC, re-wire the mouse hindlimbs.</li>
</ol>
4. Immunohistochemical analysis of gastrocnemius
<ol>
	<li>Euthanize the mouse by cervical dislocation under adequate anesthesia/analgesia by intraperitoneal injection of a mixture of three anesthetic agents (medetomidine 0.75 mg/kg, midazolam 4.0 mg/kg, and butorphanol 5.0 mg/kg).</li>
	<li>After depilating the posterior calf surface, make a skin incision, and dissect gastrocnemius muscles by separating from tibio-fibular bone using a surgical scissor and quickly freeze them in an optimal cutting temperature compound solution.</li>
	<li>Using a cryostat, prepare cryo-section samples of gastrocnemius muscles on glass slides. Store the samples in a -80 &#176;C freezer until analysis.</li>
	<li>Take out the gastrocnemius cryo-section samples to be analyzed from the freezer and dehydrate them by air drying at room temperature.</li>
	<li>Use a liquid blocker pen to draw an area that includes all the cryo-sections on the slide. The circle will prevent the solutions from flowing off the slide.</li>
	<li>Avoid drying of the samples by placing the slides in a tray in which a moist environment is created with water-soaked paper cloth.</li>
	<li>Apply 100 &#181;L of blocking buffer (phosphate-buffered saline (PBS) containing 0.25% casein, carrier protein, and 0.015 M sodium azide) for 30 min at room temperature.</li>
	<li>Rinse the slides twice by incubating with PBS-T (PBS containing 0.1% polyoxyethylene sorbitan monolaurate (see Table of Materials) for 5 min.</li>
	<li>Apply 100 &#181;L of primary antibody diluted with PBS on each sample, cover the tray with a lid, and incubate overnight at room temperature.</li>
	<li>Wash 3 times with PBS-T (5 min for each wash).</li>
	<li>Apply 100 &#181;L of secondary antibody diluted with PBS on each sample and incubate for 1 h at room temperature. 
	NOTE: For anti-laminin staining, use Alexa Fluor 568-conjugated secondary antibody. For anti-F4/80, anti-MCP-1, and anti-TNF-&#945;, use Alexa Fluor 568- or 488-conjugated secondary antibody.</li>
	<li>Wash 3 times with PBS-T (5 min for each wash).</li>
	<li>Apply 100 &#181;L of DAPI solution diluted with PBS-T on each sample and incubate for 3 min at room temperature.</li>
	<li>Wash 3 times with PBS-T (5 min for each).</li>
	<li>Mount the samples with mounting medium and cover them with coverslips.</li>
</ol>
5. Histo-morphometric analysis of gastrocnemius
<ol>
	<li>Place the sample slides on a fluorescence microscope (see Table of Materials) and view the samples using a 20&#215; objective with appropriate filters (DAPI-B, 360/40 nm for excitation and 460/50 nm for emission; GFP-B, 470/40 nm for excitation and 535/50 nm for emission; FRITC, 540/25 nm for excitation and 605/55 nm for emission.</li>
	<li>Using the software for image analysis (see Table of Materials), measure the cross-sectional area (CSA) of each myofiber, and count the number of F4/80-, MCP-1-, and TNF-&#945;-positive cells. 
	NOTE: Determine CSA of each myofiber by tracing the internal margin of the basement membrane visualized with anti-laminin-2 immunostaining.</li>
</ol>

<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. D., Hangg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intramuscular+pressure+during+walking:+An+experimental+study+using+the+wick+catheter+technique.">Intramuscular pressure during walking: An experimental study using the wick catheter technique.</a> Clinical Orthopaedics Related Research. 145, 292-299 (1979).</li><li>Lee, I., 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=Effect+of+physical+inactivity+on+major+non-communicable+diseases+worldwide:+an+analysis+of+burden+of+disease+and+life+expectancy.">Effect of physical inactivity on major non-communicable diseases worldwide: an analysis of burden of disease and life expectancy.</a> Lancet. 380, 219-229 (2012).</li></ol>

The authors declare that there are no competing interests associated with the manuscript.

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 ...

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

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5.5K Views.  National Rehabilitation Center for Persons with Disabilities. 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.

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

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. ...

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 ...

The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine via a series of lymphatic vessels and nodes that converge at the superior mesenteric lymph duct. Cannulation of the mesenteric lymph duct thus enables the collection of mesenteric lymph flowing from the intestine. Mesenteric lymph consists of a cellular fraction of immune cells (99% lymphocytes), aqueous fraction (fluid, peptides and proteins such as cytokines and gut hormones) and lipoprotein fraction (lipids, lipophilic molecules and apo-proteins). The mesenteric lymph duct cannulation model can therefore be used to measure the concentration and rate of transport of a range of factors from the intestine via the lymphatic system. Changes to these factors in response to different challenges (e.g., diets, antigens, drugs) and in disease (e.g., inflammatory bowel disease, HIV, diabetes) can also be determined. An area of expanding interest is the role of lymphatic transport in the absorption of orally administered lipophilic drugs and prodrugs that associate with intestinal lipid absorption pathways. Here we describe, in detail, a mesenteric lymph duct cannulated rat model which enables evaluation of the rate and extent of lipid and drug transport via the lymphatic system for several hours following intestinal delivery. The method is easily adaptable to the measurement of other parameters in lymph. We provide detailed descriptions of the difficulties that may be encountered when establishing this complex surgical method, as well as representative data from failed and successful experiments to provide instruction on how to confirm experimental success and interpret the data obtained.

Here we describe a technique to cannulate the mesenteric lymph duct in rats which enables quantification of lipid and drug transport via the lymphatic system following intestinal delivery. The technique can be adapted to assess mesenteric lymph concentrations and/or transport of fluid, immune cells, peptides, proteins and lipophilic molecules.

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine ...

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and virus-infected or malignantly transformed cells due to a set of germline encoded inhibitory and activating receptors. Upon virus or tumor cell recognition a variety of different NK cell functions are initiated including cytotoxicity against the target cell as well as cytokine and chemokine production leading to the activation of other immune cells. It has been demonstrated that accurate NK cell functions are crucial for the treatment outcome of different virus infections and malignant diseases. Here a simple and reliable method is described to analyze different NK cell functions using a flow cytometry-based assay. NK cell functions can be evaluated not only for the whole NK cell population, but also for different NK cell subsets. This technique enables scientists to easily study NK cell functions in healthy donors or patients in order to reveal their impact on different malignancies and to further discover new therapeutic strategies.

A simple and reliable method is described here to analyze a set of NK cell functions such as degranulation, cytokine and chemokine production within different NK cell subsets.

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and ...

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 ...