Name: the mouse cremaster muscle preparation for intravital imaging of the microcirculation
Uploaded: 2011-06-10T12:00:00
Duration: 7 min 19 s
Description: Watch this Scientific Journal Video about The Mouse Cremaster Muscle Preparation for Intravital Imaging of the Microcirculation at JoVE.com

Research in the authors' laboratory is supported by the National Institutes of Health grants R37-HL041026, R01-HL086483 and R01-HL056786 (SSS) and by F32-HL097463 and T32-AR048523 (PB) from the United States Public Health Service.

1. Mouse board, muscle pedestal, body wedge and superfusion solution
<ol>
 <li>Mouse board: A transparent rectangular Plexiglas board (6" wide X 8" long X 3/16" thick) is made to fit on the stage of the microscope. The "mouse board" is where the anesthetized mouse is secured during surgical preparation of the cremaster muscle. </li>
 <li>Muscle pedestal: A transparent silastic rubber pedestal is prepared from Sylgard 184, which is mixed according to the manufacturer&deg;s instructions. A convenient mold is a 15-mm thick x 50-mm diameter diameter disposable Petri dish. The Sylgard block is cut to the desired shape with a razor blade (see Figure 1C.). A thin layer of clear waterproof silicone adhesive is used to secure the Sylgard block to the mouse board. Helpful tips: After pouring the Sylgard into the Petri dish, degassing in a vacuum chamber for an hour removes air bubbles and improves clarity. Following degassing, curing time for Sylgard is shortened by placing the dish in a laboratory oven at 50 &deg;C for several hours.</li>
 <li>Body wedge and heating platform: A wedge (2" X 4" X 15&deg;) positioned underneath the mouse and against the pedestal tilts the body forward which facilitates extending the cremaster muscle over the pedestal from its origin. Incorporating an aluminum platform onto the surface provides conductive heat. The platform is heated by resistors connected to a DC power supply (Figure 1A). Calibrating the platform temperature to &tilde;40 &deg;C maintains esophageal temperature at &tilde;37 &deg;C. Otherwise, radiant heat from a lamp is used.</li>
 <li>Pins. To secure the cremaster muscle on the pedestal, pins are prepared from 0.15 mm insect pins that have been bent into an "L" shape.</li>
 <li>Physiological salt solution (PSS). Preparations are superfused (irrigated) continuously with bicarbonate-buffered PSS prepared in ultrapure (18.2 M&Omega;) H2O. Stock solutions are prepared at 20X final working concentration and sterilized through a 0.2 &mu;m filter. Stock solutions remain good for several weeks when stored at 4&deg;C. Preparing salts separately from the bicarbonate and mixing them with dilution on the day of the experiment prevents the precipitation of calcium bicarbonate. The stock salt solution is comprised of (in mmol/L): 2638 NaCl, 94 KCl, 40 MgS04, 23.4 CaCl2. The stock bicarbonate is 360 NaHCO3. On the day of an experiment, respective solutions are brought to a final volume (typically 2 L for a given experiment) in a volumetric flask and placed in a water bath at &tilde;37&deg;C. Equilibrating the PSS with 5% CO2 / 95% N2 for &tilde;15 minutes using a gas disperser adjusts the pH to &tilde;7.4 and prevents precipitation. The PSS is bubbled continuously with 5% CO2 / 95% N2 throughout experiments. The final working solution composition (in mmol/L) is: 132 NaCl, 4.7 KCl, 1.2 MgS04, 2 CaCl2, 18 NaHCO3.</li>
</ol>
2. Anesthesia and preparation for surgery
<ol>
 <li>Following approval from the Institutional Animal Care and Use Committee, male mice at least 12 weeks old are used. The mouse is anesthetized with pentobarbital sodium (60 mg/kg) via intraperitoneal (i.p.) injection. A restraining tube proves useful for securing the mouse during the initial injection. Throughout surgical procedures and experimental protocols, anesthesia is maintained by supplements (10-20% of initial injection, i.p.) as needed (every 30-60 minutes; indicated by withdrawal response to toe or tail pinch). Tip: Diluting the pentobarbital 10 mg/ml in sterile saline before injection reduces the possibility of overdose.</li>
 <li>Anesthesia compromises temperature regulation so the mouse must be kept warm immediately after the first injection. Placing the mouse in a metal carrier (ventilated aluminum basket) on top of a heating plate (calibrated to &tilde;37 &deg;C) works well. Alternatively, a heat lamp can be used taking care to position it at an appropriate distance from the mouse. The mouse should be monitored every 5-10 minutes until the appropriate level of anesthesia is achieved (lack of withdrawal to toe or tail pinch). A supplementary dose may be required after 15-20 minutes. It is best to be patient and proceed with caution to avoid overdose, especially with obese or aged animals. </li>
 <li>Hair is removed from the lower abdomen, lower back, scrotum and legs by carefully shaving respective regions. Particular attention should be taken to avoid trauma to the scrotum and testes, which will otherwise injure the cremaster muscle. Remove loose hair with a fresh alcohol swab. </li>
 <li>If the bladder is full it will feel like a small grape through the abdominal wall. Empty the bladder using gentle pressure to prevent the mouse from urinating on the cremaster preparation during experiments. A Kimwipe is an effect sponge to collect the urine. </li>
 <li>Position the mouse on its back, lying on the wedge with its legs straddling the pedestal. A silk suture (4-0 or 5-0) tied to each foot provides a tether for securing the mouse with its crotch against the pedestal. A piece of tape placed loosely across the chest and secured to the wedge maintains overall body position. Surgical procedures are performed while viewing through a stereomicroscope using microdissection scissors and angled forceps.</li>
</ol>
3. Surgical preparation of the open cremaster muscle
<ol>
 <li>A pin is placed through the apex of the scrotal sac (either the left or right side) and secured in the pedestal to place tension on the skin. Superfusion with PSS (34-35 &deg;C) is initiated over the surgical field and maintained throughout dissection to keep the exposed tissue warm and moist. A wick (made from Kimwipe) positioned with one end next to the scrotal sac and the other next to a vacuum line removes the effluent PSS. A bead of silicone sealant adhered to the Plexiglas board and completely encircling the wedge and pedestal serves as "moat" to collect any PSS that is not aspirated, serving as an effective precaution to prevent PSS leakage onto the microscope.</li>
 <li>An incision is made along the ventral surface of the scrotal sac. If the testicle has been retracted by the cremaster muscle into the abdominal cavity, gentle pressure on the lower abdomen directs the testicle into the sac. The surface of the cremaster muscle overlying the testicle should be identified at this time. Connective tissue between the scrotal skin and the cremaster muscle is carefully removed to free the cremaster muscle from surrounding tissue. </li>
 <li>The scrotal skin is gently retracted behind the proximal edge of the pedestal and secured on each side with a pin. The exterior surface of the cremaster muscle surface is then cleared of connective tissue. Continuous superfusion during dissection hydrates the connective tissue, facilitating visibility and removal. </li>
 <li>A pin through the apex of the cremaster muscle is used to place it under longitudinal tension. A longitudinal incision is made through the ventral surface of the muscle with great care taken to minimize disruption of the vascular supply. Blood flow dynamics near the periphery of the preparation are altered by this tissue damage14. Therefore, to minimize these affects on data collection, blood vessels near the center of the preparation are used for imaging and physiological studies.</li>
 <li>The cremaster muscle is connected by a thin ligament to the epididymis underneath the testicle. Reflecting the testicle to one side exposes this ligament, which is carefully separated from the epididymis. Near the apex of the cremaster muscle is small artery and vein which connect it to the epididymis. Occluding these vessels between forceps and pulling them apart minimizes bleeding. The testicle, epididymis, testicular artery and vein are ligated proximally (4-0 or 5-0 silk suture), severed and discarded (orchiectomy) along with the associated inguinal fat pad. Alternatively, the testicle can be gently pushed back into the abdominal cavity and retained with a piece of cotton or Kimwipe. The orchiectomy serves to avoid possible trauma to the tissue when pushing the testes back into the abdominal cavity. We have not found noticeable differences in the quality of the cremaster microcirculation preparation (see Section 3.8) using either procedure7,15. </li>
 <li>The external surface of the cremaster muscle is cleared of remaining connective tissue and then spread radially on the surface of the pedestal. The edges are secured with pins (see Section 1.4) in 5-6 places to create a flat sheet of striated muscle with intact microcirculation. </li>
 <li>The completed preparation is transferred to the stage of an intravital microscope, superfused continuously (3-5 ml/min, 34 &deg;C) and equilibrated for 30 minutes. </li>
 <li>Integrity of the cremaster preparation is evaluated according to several criteria. Spontaneous vasomotor tone with minimal surgical trauma is indicated by brisk flow in arterioles and lack of adherent leukocytes in venules. The most sensitive index of a responsive preparation is constriction of arterioles to raising the superfusion solution oxygen content for 5-10 minutes. This is done by equilibrating it with 21% O2 (vs. 5% O2; see Section 1.5; all with 5% CO2, balance N2). The presence of leukocytes in arterioles, the absence of red blood cells flowing through capillaries, and/or leukocytes accumulating in venules and surrounding tissue are signs of tissue damage and inflammation. </li>
</ol>
4. Intravital imaging of the cremaster muscle microcirculation
<ol>
 <li>Intravital imaging is performed on a custom microscope based upon an Olympus MVX10 Stereo Zoom platform. The MVX10 microscope body is mounted to a MVX10 hybrid stand equipped with transillumination (halogen lamp) for brightfield (K&ouml;hler) imaging and with epi-illumination (mercury lamp) for fluorescence imaging. Using epi-illumination, GCaMP2 is excited at 472/30 nm with emission collected at 520/35 nm. Images are acquired through a MV PLAPO 2XC lens (numerical aperture = 0.50; Olympus) using an XR/Mega-10 intensified digital camera (Stanford Photonics, Inc.; SPI) via Piper Control Software (SPI) on a personal computer. With this system, the observed field of view (FOV) can be varied from 553 &mu;m X 442 &mu;m to 22 mm X 18 mm.</li>
 <li>Upon completion of intravital imaging experiments, the anesthetized mouse is euthanized with overdose of pentobarbital followed by cervical dislocation.</li>
</ol>
5. Representative Results
<img src="/files/ftp_upload/2874/2874fig1.jpg" alt="Figure 1" /> 
Figure 1. Mouse board for intravital imaging of the mouse cremaster preparation. A) Body wedge with aluminum platform (insulated with yellow plastic) viewed from the bottom. Heating resistors secured to the underside of the platform provide conducted heat. B) The heating platform rests on a plastic wedge. C) The body wedge is placed on a Plexiglas board for surgery and subsequent transfer to the stage of the intravital microscope. Sylgard pedestal is indicated with red arrow. A bead of waterproof silicone surrounds the entire preparation to contain any solution that may leak during the intravital procedure, preventing it from dripping onto the microscope.
<img src="/files/ftp_upload/2874/2874fig2.jpg" alt="Figure 2" /> 
Figure 2. Custom MacroZoom microscope for widefield imaging. A) MVX10 microscope body (Olympus) with XR/Mega-10 ICCD camera (Stanford Photonics) mounted on trinocular port. B) Close-up view of the microscope body. (a) zoom control (0.63 to 6.3X); (b) filter wheel; (c) image doubler (NA = 0.50 with 25X optical magnification). C) Substage condenser for brightfield (K&ouml;hler) illumination (Condenser NA = 0.55, Working Distance = 27 mm). 
<img src="/files/ftp_upload/2874/2874fig3.jpg" alt="Figure 3" /> 
Figure 3. Completed mouse cremaster preparation. Anesthetized mouse in supine position on warm plastic-coated aluminum platform (yellow). Body position is secured with tape. The cremaster muscle is spread radially on the transparent silastic rubber pedestal and pinned at the edges. Superfusion solution is introduced at the proximal end through a plastic dripper (white arrow). A vacuum line (white arrowhead) removes the solution via a Kimwipe wick. Two micropipettes are shown positioned with their tips in the tissue. A reference electrode (silver wire) is secured at the lower edge of the cremaster muscle.
<img src="/files/ftp_upload/2874/2874fig4.jpg" alt="Figure 4" /> 
Figure 4. Illustrating the magnification range for visualizing arteriolar networks expressing GCaMP2 in endothelium. A) Fluorescent and B) brightfield image taken at an optical magnification of 3.2X for a total magnification = 42X on the video monitor. [Field of view (FOV) = 4,375 x 3,470 &mu;m]. Scale bar = 500 &mu;m. Working at this magnification facilitates placement of micropipettes at desired locations. C) Fluorescent and D) brightfield image taken at optical magnification of 6.4X for total magnification = 83X (FOV = 2,200 x 1,759 &mu;m). Scale bar = 200 &mu;m. E) Fluorescent and F) brightfield image taken at optical magnification of 12.6X for total magnification = 165X (FOV = 1,100 x 885 &mu;m). Scale bar = 100 &mu;m. G) With image doubler, optical magnification = 25.2X. Scale bar = 50 &mu;m.

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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Observation+of+Skeletal+Muscle+Blood+Vessels+(Rat+Cremaster).">Direct Observation of Skeletal Muscle Blood Vessels (Rat Cremaster).</a> J. Physiol. 172, 123-137 (1964).</li><li>Baez, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+open+cremaster+muscle+preparation+for+the+study+of+blood+vessels+by+in+vivo+microscopy.">An open cremaster muscle preparation for the study of blood vessels by in vivo microscopy.</a> Microvasc. Res. 5, 384-394 (1973).</li><li>Bohlen, H. G., Gore, R. W., Hutchins, P. 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C., &amp;amp, S. e. g. a. l., S, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vasomotor+control+in+arterioles+of+the+mouse+cremaster+muscle.">Vasomotor control in arterioles of the mouse cremaster muscle.</a> FASEB J. 14, 197-207 (2000).</li><li>Figueroa, X. F., Paul, D. L., Simon, A. M., Goodenough, D. A., Day, K. H., Damon, D. N., Duling, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+role+of+connexin40+in+the+propagation+of+electrically+activated+vasodilation+in+mouse+cremasteric+arterioles+in+vivo.">Central role of connexin40 in the propagation of electrically activated vasodilation in mouse cremasteric arterioles in vivo.</a> Circ. Res. 92, 793-800 (2003).</li><li>Wolfle, S. E., Schmidt, V. J., Hoepfl, B., Gebert, A., Alcolea, S., Gros, D., de Wit, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connexin45+cannot+replace+the+function+of+connexin40+in+conducting+endothelium-dependent+dilations+along+arterioles.">Connexin45 cannot replace the function of connexin40 in conducting endothelium-dependent dilations along arterioles.</a> Circ. Res. 101, 292-1299 (2007).</li><li>Milkau, M., Kohler, R., de Wit, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crucial+importance+of+the+endothelial+K++channel+SK3+and+connexin40+in+arteriolar+dilations+during+skeletal+muscle+contraction.">Crucial importance of the endothelial K+ channel SK3 and connexin40 in arteriolar dilations during skeletal muscle contraction.</a> FASEB J. 24, 3572-3579 (2010).</li><li>Bagher, P., Duan, D., Segal, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+impaired+neurovascular+transmission+in+a+murine+model+of+Duchenne+Muscular+Dystrophy.">Evidence for impaired neurovascular transmission in a murine model of Duchenne Muscular Dystrophy.</a> J. Appl. Physiol. 110, 601-610 (2011).</li><li>Tallini, Y. N., Brekke, J. F., Shui, B., Doran, R., Hwang, S. M., Nakai, J., Salama, G., Segal, S. S., Kotlikoff, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propagated+endothelial+Ca2++waves+and+arteriolar+dilation+in+vivo:+measurements+in+Cx40BAC-GCaMP2+transgenic+mice.">Propagated endothelial Ca2+ waves and arteriolar dilation in vivo: measurements in Cx40BAC-GCaMP2 transgenic mice.</a> Circ. Res. 101, 1300-1309 (2007).</li><li>Grant, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+denervation+on+skeletal+muscle+blood+vessels+(rat+cremaster).">The effects of denervation on skeletal muscle blood vessels (rat cremaster).</a> J. Anat. 100, 305-316 (1966).</li><li>Proctor, K. G., Busija, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationships+among+arteriolar,+regional,+and+whole+organ+blood+flow+in+cremaster+muscle.">Relationships among arteriolar, regional, and whole organ blood flow in cremaster muscle.</a> Am. J. Physiol. 249, H34-H41 (1985).</li><li>Bagher, P., Segal, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+blood+flow+in+the+microcirculation:+Role+of+conducted+vasodilation.">Regulation of blood flow in the microcirculation: Role of conducted vasodilation.</a> Acta Physiol. , (2011).</li><li>Hill, M. A., Simpson, B. E., Meininger, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+cremaster+muscle+hemodynamics+due+to+disruption+of+the+deferential+feed+vessels.">Altered cremaster muscle hemodynamics due to disruption of the deferential feed vessels.</a> Microvasc. Res. 39, 349-363 (1990).</li></ol>

All procedures and protocols involving animals were approved by the Animal Care and Use Committee of the University of Missouri and performed in accord with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. 
Production of this video-article has been sponsored by Stanford Photonics.

Here we describe the open cremaster muscle preparation in the mouse for observing the microcirculation in vivo. This 
procedure is modeled after the "open" cremaster preparation first described in the rat4. With practice the entire surgical procedure can be completed in 
less than 1 hour. The versatility of this preparation allows for a variety of experimental manipulations and is readily adapted to hamsters as well 
as rats, enabling a variety of experimental models to be studied in similar fashion. The preparation is limited to male animals and measurements 
should be made towards the center of the tissue, avoiding damaged regions near the edges of the muscle14. It should also be recognized that, while 
pressure and flow distributions are altered when interconnecting vessels of the cremaster muscle are cut16, microvessels remain responsive and suitable 
for reproducible data collection. The primary limitation to visualizing the microcirculation in the cremaster muscle is the buildup of connective tissue 
as animals mature and increase in size, particularly in rats but also in hamsters. Further, as animals get fat, it becomes more difficult to control 
anesthesia because pentobarbital is lipophilic and can be absorbed in adipose tissue. The best way to proceed is by being patient while ensuring that 
the animal&deg;s body temperature is maintained at ~37 &deg;C while exposed tissue is irrigated continuously with PSS.

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent or device</th>
			<th>Company</th>
			<th>Catalogue number</th>
			<th>Comments</th>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher</td>
			<td>S642-212</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma</td>
			<td>P9541</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Sigma</td>
			<td>M2643</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma</td>
			<td>C1016</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Fisher</td>
			<td>S233</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nembutal Sodium Solution</td>
			<td>Lundbeck</td>
			<td>NDC 67386-501-55</td>
			<td>Also referred to as sodium pentobarbital</td>
		</tr>
		<tr>
			<td>Temperature Controller</td>
			<td>Warner Instruments</td>
			<td>TC-344B</td>
			<td>Alternate: adjustable 12V DC power supply</td>
		</tr>
		<tr>
			<td>Series 20 platform heater kit RH-2</td>
			<td>Warner Instruments</td>
			<td>64-0274</td>
			<td>Requires custom-built aluminum plate</td>
		</tr>
		<tr>
			<td>Mega-10 Camera</td>
			<td>Stanford Photonics</td>
			<td>XR/Mega-10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MVX10</td>
			<td>Olympus</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MV PLAPO 2XC lens</td>
			<td>Olympus</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MVX10 hybrid stand</td>
			<td>Leeds</td>
			<td>LBX-Hybrid</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Piper Control Software</td>
			<td>Stanford Photonics</td>
			<td>&nbsp;</td>
			<td>Program for Mega-10 camera</td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>Nikon</td>
			<td>SMZ645</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Waterproof Silicone Sealant (Clear)</td>
			<td>General Electric</td>
			<td>47970-72643-LW5000</td>
			<td>Other clear silicone sealants also work</td>
		</tr>
		<tr>
			<td>60 x 15mm Petri Dish</td>
			<td>Fisher</td>
			<td>08-757-13A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sylgard</td>
			<td>Dow Corning</td>
			<td>184</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microdissection Scissors</td>
			<td>Fine Science Tools</td>
			<td>15003-08</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont Forceps</td>
			<td>Fine Science Tools</td>
			<td>#5/45</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>GP Millipore Express PLUS Membrane</td>
			<td>Millipore</td>
			<td>SCGPT05RE</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Minutiens Insect Pins</td>
			<td>Austerlitz</td>
			<td>M size 0.15 mm</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Compact Pet Trimmer</td>
			<td>Wahl Clipper Corp.</td>
			<td>Model 9966</td>
			<td>Clean after each use</td>
		</tr>
	</tbody>
</table>

the mouse cremaster muscle preparation for intravital imaging of the microcirculation

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic by-products. This balance is achieved by the movement of blood through the microcirculation, which encompasses the smallest branches of the vascular supply throughout all tissues and organs. Arterioles branch from arteries to form networks that control the distribution and magnitude of oxygenated blood flowing into the multitude of capillaries intimately associated with parenchymal cells. Capillaries provide a large surface area for diffusional exchange between tissue cells and the blood supply. Venules collect capillary effluent and converge as they return deoxygenated blood towards the heart. To observe these processes in real time requires an experimental approach for visualizing and manipulating the living microcirculation.
The cremaster muscle of rats was first used as a model for studying inflammation using histology and electron microscopy post mortem1,2. 
The first in vivo report of the exposed intact rat cremaster muscle investigated microvascular responses to vasoactive drugs using reflected light3. However 
curvature of the muscle and lack of focused illumination limited the usefulness of this preparation. The major breakthrough entailed opening the muscle, 
detaching it from the testicle and spreading it radially as a flat sheet for transillumination under a compound microscope4. While shown to be a valuable 
preparation to study the physiology of the microcirculation in rats5 and hamsters6, the cremaster muscle in mice7 has proven particularly useful in dissecting 
cellular pathways involved in regulating microvascular function8-11 and real-time imaging of intercellular signaling12. 
The cremaster muscle is derived from the internal oblique and transverse abdominus muscles as the testes descend through the inguinal canal13. 
It serves to support (Greek: cremaster = suspender) and maintain temperature of the testes. As described here, the cremaster muscle is prepared as a thin flat sheet 
for outstanding optical resolution. With the mouse maintained at a stable body temperature and plane of anesthesia, surgical preparation involves freeing the muscle 
from surrounding tissue and the testes, spreading it onto transparent pedestal of silastic rubber and securing the edges with insect pins while irrigating it 
continuously with physiological salt solution. The present protocol utilizes transgenic mice expressing GCaMP2 in arteriolar endothelial cells. GCaMP2 is a 
genetically encoded fluorescent calcium indicator molecule12. Widefield imaging and an intensified charge-coupled device camera enable in vivo study of 
calcium signaling in the arteriolar endothelium.

Watch this Scientific Journal Video about The Mouse Cremaster Muscle Preparation for Intravital Imaging of the Microcirculation at JoVE.com

The Mouse Cremaster Muscle Preparation for Intravital Imaging of the Microcirculation

A tissue preparation is described for visualization and experimental manipulation of the living microcirculation. In anesthetized male mice, the thin, highly vascularized cremaster muscle is prepared for intravital microscopy to study microvascular networks including arterioles, capillaries and venules. This preparation is readily adapted for rats and hamsters.

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic ...

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Research

JoVE Journal

Biology

Microiontophoresis and Micromanipulation for Intravital Fluorescence Imaging of the Microcirculation

Microiontophoresis entails passage of current through a micropipette tip to deliver a solute at a designated site within an experimental preparation. Microiontophoresis can simulate synaptic transmission1 by delivering neurotransmitters and neuropeptides onto neurons reproducibly2. Negligible volume (fluid) displacement avoids mechanical disturbance to the experimental preparation. Adapting these techniques to the microcirculation3 has enabled mechanisms of vasodilation and vasoconstriction to be studied at the microscopic level in vivo4,5. A key advantage of such localized delivery is enabling vasomotor responses to be studied at defined sites within a microvascular network without evoking systemic or reflexive changes in blood pressure and tissue blood flow, thereby revealing intrinsic properties of microvessels.
A limitation of microiontophoresis is that the precise concentration of agent delivered to the site of interest is difficult to ascertain6. Nevertheless, its release from the micropipette tip is proportional to the intensity and duration of the ejection current2,7, such that reproducible stimulus-response relationships can be readily determined under defined experimental conditions (described below). Additional factors affecting microiontophoretic delivery include solute concentration and its ionization in solution. The internal diameter of the micropipette tip should be &tilde; 1 &mu;m or less to minimize diffusional 'leak', which can be counteracted with a retaining current. Thus an outward (positive) current is used to eject a cation and a negative current used to retain it within the micropipette.
Fabrication of micropipettes is facilitated with sophisticated electronic pullers8. Micropipettes are pulled from glass capillary tubes containing a filament that 'wicks' solution into the tip of the micropipette when filled from the back end ("backfilled"). This is done by inserting a microcapillary tube connected to a syringe containing the solution of interest and ejecting the solution into the lumen of the micropipette. Micromanipulators enable desired placement of micropipettes within the experimental preparation. Micromanipulators mounted on a movable base can be positioned around the preparation according to the topography of microvascular networks (developed below). 
The present protocol demonstrates microiontophoresis of acetylcholine (ACh+ Cl-) onto an arteriole of the mouse cremaster muscle preparation (See associated protocol: JoVE ID#2874) to produce endothelium-dependent vasodilation. Stimulus delivery is synchronized with digitized image acquisition using an electronic trigger. The use of Cx40BAC-GCaMP2 transgenic mice9 enables visualization of intracellular calcium responses underlying vasodilation in arteriolar endothelial cells in the living microcirculation.

Microiontophoresis entails movement of ions from a micropipette in response to a difference in electrical potential between the inside and outside of the micropipette. Biologically active molecules are thereby delivered in proportion to electrical current. We illustrate acetylcholine microiontophoresis in conjunction with micromanipulation to study endothelium-dependent vasodilation in the microcirculation.

Microiontophoresis entails passage of current through a micropipette tip to deliver a solute at a designated site within an experimental  preparation. ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo cell-free layer width and its spatiotemporal variations can provide a comprehensive understanding of hemodynamics in microcirculation. In this study, we used an intravital microscopic system coupled with a high-speed video camera to quantify the cell-free layer widths in arterioles in vivo. The cremaster muscle of Sprague-Dawley rats was surgically exteriorized to visualize the blood flow. A custom-built imaging script was also developed to automate the image processing and analysis of the cell-free layer width. This approach enables the quantification of spatiotemporal variations more consistently than previous manual measurements. The accuracy of the measurement, however, partly depends on the use of a blue filter and the selection of an appropriate thresholding algorithm. Specifically, we evaluated the contrast and quality of images acquired with and without the use of a blue filter. In addition, we compared five different image histogram-based thresholding algorithms (Otsu, minimum, intermode, iterative selection, and fuzzy entropic thresholding) and illustrated the differences in their determination of the cell-free layer width.

This study demonstrates the surgical preparation of the rat cremaster muscle for the visualization of the in vivo cell-free layer. Considerable factors affecting the accuracy of the cell-free layer width measurement are discussed in this study.

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo ...

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle composition. Various time-consuming methods have traditionally been used to study fiber populations in many fields of research. However, recently developed immunohistochemical methods based on myosin heavy chain protein expression provide a quick alternative to identify multiple fiber types in a single section. Here, we present a rapid, reliable and reproducible protocol for improved staining quality, allowing automatic acquisition of whole cross sections and automatic quantification of fiber populations with ImageJ. For this purpose, embedded skeletal muscles are cut in cross sections, stained using myosin heavy chains antibodies with secondary fluorescent antibodies and DAPI for cell nuclei staining. Whole cross sections are then scanned automatically using a slide scanner to obtain high-resolution composite pictures of the entire specimen. Fiber population analyses are subsequently performed to quantify slow, intermediate and fast fibers using an automated macro for ImageJ. We have previously shown that this method can identify fiber populations reliably to a degree of &#177;4%. In addition, this method reduces inter-user variability and time per analyses significantly using the open source platform ImageJ.

Here, we present a protocol for rapid muscle fiber analyses, which allows improved staining quality, and thereby automatic acquisition and quantification of fiber populations using the freely available software ImageJ.

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is regulated spatially and temporally. The use of a fluorescent gene under the control of an immunity-related promoter in combination with an automated fluorescence microscopy is a simple way to understand spatiotemporal regulation of plant immunity. In contrast to the root tissues that have been used for a number of various intravital fluorescent imaging experiments, there exist few fluorescent live-imaging examples for the leaf tissues that encounter an array of airborne microbial infections. Therefore, we developed a simple method to mount leaves of Arabidopsis thaliana plants for live-cell imaging over an extended period of time. We used transgenic Arabidopsis plants expressing the yellow fluorescent protein (YFP) genefused to the nuclear localization signal (NLS) under the control of the promoter of a defense-related marker gene, Pathogenesis-Related 1 (PR1). We infiltrated a transgenic leaf with Pseudomonas syringae pv. tomato DC3000 (avrRpt2) strain (Pst_a2) and performed in vivo time-lapse imaging of the YFP signal for a total of 40 h using an automated fluorescence stereomicroscope. This method can be utilized not only for studies on plant immune responses but also for analyses of various developmental events and environmental responses occurring in leaf tissues.

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune responses.

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is ...

Sample Preparation for Metabolic Profiling using MALDI Mass Spectrometry Imaging

Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to investigate the biological activities during development and diseases. Metabolomics approaches&#160;are widely employed in the study of cancer, nutrition/diet, diabetes, and other physiological and pathological conditions involving metabolic processes. An advantageous tool that aids in metabolomic profiling advocated in this paper is matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI). Its ability to detect metabolites in situ without labeling, structural modifications, or other specialized reagents, such as those used in immunostaining, makes MALDI MSI a unique tool in advancing methodologies relevant in the field of metabolomics. An appropriate sample preparation process is critical to yield optimal results and will be the focus of this paper.

The goal of this protocol is to provide detailed guidance on the sample preparation when planning for experiments using MALDI MSI to maximize metabolic and molecular detection in biological samples.

Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to ...

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...