We thank Katharina Haberland for image editing. &#160;Funding: The senior author received a KKF Grant from the Technische Universit&#228;t M&#252;nchen to set up a new research laboratory.

NOTE: Prior to implementation of the presented model, the corresponding animal protection laws must be consulted and permission must be obtained from the local authorities. In this work, all experiments were performed in conformity with the guiding principles for research involving animals and the German legislation on protection of animals. The experiments were approved by the local animal care committee.
1. Animal Preparation and Surgical Elevation of the Flap
<ol style="margin-left: 40px;">
	<li>Keep animals in single cages at room temperature of 22&#8211;24 &#176;C and at a relative humidity of 60&#8211;65% with a 12-hr day-and-night cycle. Allow mice free access to drinking water and standard laboratory chow. Always maintain sterile conditions during survival surgery.</li>
	<li>Anesthetize the mouse by intraperitoneal (i.p.) injection of 0.1 ml saline solution per 10 g bw containing 90 mg/kg bw ketamine hydrochloride and 25 mg/kg bw dihydroxylidinothiazine hydrochloride. Anesthesia is necessary for animal preparation, surgery and subsequent microscopy. Confirm proper anesthetization by checking the response to a painful stimulus. During the time of anesthesia, apply vet ointment on eyes to prevent dryness.</li>
	<li>After induction of sufficient anesthesia, depilate the back with an electric shaver. Hereafter, apply depilation cream to the shaved area to remove the remaining hair.</li>
	<li>During residence time of the depilation cream of ~7&#8211;10 min, prepare instruments, including skin forceps and micro forceps, scissors and micro scissors, suture material and a marker pen. Disinfect and prepare the titanium chamber by applying the two screws with the nut at the chamber&#8217;s base and by fixing self-adhesive foam (Fig. 1 A-C) around the window of the chamber&#8217;s counterpart to guarantee tightness of the chamber system.</li>
	<li>Remove all depilation cream from the back by washing it off with tepid water. Then dry and disinfect the skin with an alcohol-containing solution.</li>
	<li>Place the mouse in prone position and define the midline of the back. Grasp the skin centrally and lift it up to create a fold (Fig. 2A). By trans-illumination using any kind of light source, decide where to provisorily place the titanium frame (Fig. 2B). Make sure that the distal branches of the lateral thoracic artery (LTA) cranially and the deep circumflex iliac artery (DCIA) caudally course through the center of the chamber&#8217;s window (Fig. 2B-D). Once the positioning of the chamber is done, perforate both layers of the skin at the very bottom of the fold for later fixation of the titanium frame. Now remove the titanium frame, place the mouse in prone position and redefine the midline of the back if necessary.</li>
	<li>Outline the flap perpendicular to the spine starting at the midline with a width-to-length ratio of 15 x 11 mm to result in a laterally based and randomly perfused flap. Then outline an additional skin area of 2 mm extending to the contralateral side (Fig. 2C, D). This area is picked with the forceps and later sutured to the titanium frame without interfering with the visible flap area.</li>
	<li>Following final marking, incise the flap. In doing so, transect both the LTA and DCIA and elevate the flap (Fig. 2E). There is no need for coagulation or ligation of the transected vessels.</li>
	<li>Place the chamber&#8217;s screw through the previously made skin perforations (Fig. 2F) and fixate the skinfold both anteriorly and posteriorly within the chamber&#8217;s frame of the elevated flap to the backside part of the frame using the holes of the chamber&#8217;s frame and a 5-0 interrupted sutures (Fig. 2G).</li>
	<li>Suture the vertical limbs of the flap back to the surrounding skin by means of 5-0 interrupted sutures (Fig. 2H, I).</li>
	<li>Excise a hemi-circular area of skin lateral to the small skin strip of 2 mm, i.e., on the other side of the flap to observe the flap&#8217;s vasculature. This allows direct view through the observation window of the chamber that has a surface of 90 mm2.</li>
	<li>Remove the gelatin-like loose areolar tissue from the striated muscle using microsurgical instruments and optical magnifying lens or microscope in order to improve the image quality during microscopy. Try not to damage vessels within the muscular tissue layers.</li>
	<li>Finally, seal the chamber by mounting the counterpart of the frame that has previously been endued with self-adhering strips of foam anteriorly, cranially and posteriorly, bedew the flap with topical bisbenzimide and saline (Fig. 2J). Thereafter, apply the observation window with a cover glass using a snap ring. Try not to entrap any air blisters under the glass (Fig. 2K, L). The skin will adhere to the cover glass by adhesion forces.</li>
</ol>
2. Intravital Epifluorescence Microscopy
<ol style="margin-left: 40px;">
	<li>Wait 24 hr after surgical preparation for the first microscopic analysis for ideal optical quality. This analysis represents the baseline of microscopy and is repeated at day 3, 5, 7 and 10 after surgery. Each time, anesthetize the mouse as described in step 1.2. Place the animal in a lateral decubital position on a custom-made Plexiglas carrier. That way, the muscular tissue layers of the flap adhering to the cover glass are facing up-wards.</li>
	<li>Inject 0.05 ml of 5% green fluorescence dextran (molecular weight 150,000) and 0.05 ml 1% highly fluorescent Rhodamine family dye in a tail vein or the retro-bulbar venous plexus. The green fluorescent dextran stains the non-cellular components of the blood if applied intravenously (i.v.). The fluorescent Rhodamine stains leukocytes and platelets that can be distinguished from other cellular and non-cellular components. Finally, bisbenzimide stains nuclear components that emit more or less fluorescence according to the state of condensation.</li>
	<li>Subsequently, place the mouse under the microscope. Ensure the microscope includes a LED system &#8212; LED beam combinations for 470 &#38; 425 nm, 365 &#38; 490 nm, and 540 &#38; 580 nm as well as the corresponding filter sets (62 HE BFP/GFP/HcRed, range 1: 350&#8211;390 nm excitation wavelength, split 395 nm / 402&#8211;448 nm; range 2: 460&#8211;488 nm, split 495 nm / 500&#8211;557 nm; range 3: 567&#8211;602 nm, split 610 nm / 615 nm to infinite. 20 Rhodamine, range: 540&#8211;552 nm, split 560 nm, emission 575&#8211;640 nm).</li>
	<li>Record the stills and motion-pictures using a charge-coupled device video camera and save them on a computer hard disk for further off-line analysis.</li>
	<li>Use different objectives (2.5X, NA (numerical aperture) = 0.06 for panorama views of the chamber; 5X, NA = 0.16; 10X, NA = 0.32; 20X, NA = 0.50; 50X, NA = 0.55) for recordings of the regions of interest.</li>
	<li>Virtually, divide the flap surface that is visible through the chamber&#8217;s window into three areas of equal height, i.e., a proximal, a central, and a distal area most remote from the vascular inflow (Fig. 3F). For image acquisition, proceed the following way at each observation time point:
	<ol>
		<li>Scan the tissue image by image within the chamber&#8217;s window from proximal to distal using the 5X objective.</li>
		<li>In each area of the flap, select second- or third-order arterioles and their accompanying venules with easily identifiable branching patterns. Using the 5X, 10X, or 20X objective, make printouts of the arteriolovenular bundles in order to easily re-localize the bundles throughout the whole observation period.</li>
		<li>With the 20X objective and the 50X objective, further record 5&#8211;6 capillary fields and &#8220;apoptotic&#8221; fields per area of the flap respectively. All images recorded with the 10X and 20X objectives use the blue light (fluorescent dextran) and green light (Rhodamine) filter, whereas images recorded with the 5X and 50X objective use blue light (fluorescent dextran) and white light (Bisbenzimide) filter, respectively.</li>
	</ol>
	</li>
</ol>
3. Analysis of Recorded Data
NOTE: With the use of a computer-assisted image analysis system quantify all recorded parameters off-line as follows29.
<ol style="margin-left: 40px;">
	<li>Measurement of the area of non-perfused, respectively necrotic tissue (mm2).
	<ol>
		<li>Use the complete scanned chamber window image recorded with the 5X objective (from 2.6.1) and the fluorescent dextran to measure non-perfused respectively necrotic tissue. Use &#8220;AreaBo&#8221; function to measure the non-perfused dark area: Border the non-perfused area and calculate the area in mm2 by using the software&#8217;s Area measurement algorithms.</li>
	</ol>
	</li>
	<li>Measurement of microvascular diameter in arterioles, capillaries and venules (&#181;m).
	<ol>
		<li>Load video or pictures from the recorded AV-bundles or the capillary fields into the software. For best results use the stills or videos with the highest magnification where definite borders of the vessel are clearly visible.</li>
		<li>Use the &#8220;DiamPe&#8221; function of the software to make sure the measurements are performed perpendicular to the vessel&#8217;s wall. To do so mark two points on the vessel&#8217;s wall and with the third click mark the perpendicular diameter of the vessel. The software will perform the measurement in &#181;m.</li>
		<li>Repeat the measurements on the same vessels for all recordings at the same place. Measure multiple capillaries to accumulate average diameter.</li>
	</ol>
	</li>
	<li>Analysis of red blood cell (RBC) velocity in arterioles, capillaries and venules (mm/sec).
	<ol>
		<li>For arteriolar red blood cell (RBC) velocity (mm/s) measurements use the &#8220;VeloLSD&#8221; function of the software and choose the same vessels in the fluorescent dextran window for which the diameters have been measured.</li>
		<li>Analyze it with the computer-assisted image analysis system using the line shift method, which is on the basis of the measurement of the shift (mm) of an individual intravascular gray level pattern over time (sec).</li>
	</ol>
	</li>
	<li>Measurement of volumetric blood flow in vessels (pl/sec).
	<ol>
		<li>Calculate volumetric blood flow (pl/sec) in arterioles, venules and capillaries from RBC velocity and vessel cross-sectional area (&#960; * r2) according to the equation of Gross and Aroesty, that is, Q = V * &#960; * r2, assuming a cylindrical vessel shape30.</li>
	</ol>
	</li>
	<li>Measurement of functional capillary density of RBC-perfused capillaries (nutritive perfusion: cm/cm2).
	<ol>
		<li>Load a recorded fluorescent capillary field with 20X objective magnification into the software. Use the &#8220;DensLA&#8221; function to measure functional capillary density of RBC-perfused microvessels.</li>
		<li>Trace the perfused capillaries with the cursor and mark the perfused vessels. Do not mark non-perfused capillaries, arterioles or venules. The software will measure functional capillary density of the perfused capillaries in cm/cm2. Repeat the steps for the other recorded capillary fields.</li>
	</ol>
	</li>
	<li>Measurement of tortuosity of the microvessels (microvascular remodeling), early signs of angiogenesis such as bud and sprout-formation and newly formed microvessels.
	<ol>
		<li>Load recorded videos or frames of fluorescent capillary fields into the software. Identify gyrose vessels and use the &#8220;TorqIx&#8221; function of the software. Trace the definite vessel flow with the course and end with a right click. The software will draw a straight line for the direct way and will set it in relation with the traced path. 
		NOTE: Watch out for signs of angiogenesis such as buds, sprouts and newly formed capillaries, which usually emanate perpendicularly from the pre-existing capillaries. Measure the density of these newly formed vessels as described in step 3.5.</li>
	</ol>
	</li>
	<li>Identification of characteristics for apoptotic cell death, such as nuclear condensation, fragmentation and/or margination (cells/mm2).
	<ol>
		<li>Load Bisbenzimide-recorded videos (50X objective) into the software. Use the &#8220;DenseNA&#8221; function. Mark apoptotic cells according to the mentioned characteristics such as nuclear condensation, fragmentation and margination with a left click.</li>
		<li>After marking all characteristic cells throughout the video, click &#8220;N/A&#8220; in the software, which will automatically count all marked cells and divide it throughout the area. The result will be apoptotic cells/mm2.</li>
	</ol>
	</li>
	<li>Analysis of leukocyte adherence to the vascular endothelium representing inflammation. Intermittent adherence of leukocytes (rolling leukocytes; adherence &#60; 30 sec: number of rollers/mm2 endothelial surface) and firm adherence of leukocytes (sticking leukocytes; adherence &#62; 30 sec: number of stickers/mm2 endothelial surface).
	<ol>
		<li>Analyze the inflammatory response by counting the number of Rhodamine labeled leukocytes that adhered to the endothelial lining of post-capillary venules for a time period of &#8805;30 sec (&#8220;stickers&#8221;) and the intermittently adhering leukocytes (&#60;30 sec, &#8220;rollers&#8221;). Venular leukocyte adherence is expressed as cells per mm2 endothelial surface.</li>
	</ol>
	</li>
	<li>Measurement of intravascular and interstitial grey levels as a parameter for microvascular leakage (macromolecular extravasation E=E1/E2).
	<ol>
		<li>Assess macromolecular leakage as a parameter of microvascular permeability after an i.v. injection of a fluorescent dextran. Densitometrically determine multiple grey levels in the tissue directly adjacent to the capillary vessel wall (E1), as well as in the marginal cell-free plasma of the vessel (E2). Then calculate macromolecular extravasation (E) as the ratio of E1/E2 31.</li>
	</ol>
	</li>
</ol>
4. Postoperative Care
<ol style="margin-left: 40px;">
	<li>Return the animal in a separate cage to avoid the company of other animals until fully recovered. Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. Monitor animals daily for bleeding, local and systemic signs of infection and the position of the chamber.</li>
	<li>Evaluate the general state of the animal by observing motor activity, body weight, signs of pain, tolerance to the dressing and auto-mutilation.</li>
	<li>Keep the mice one per cage to avoid mutual manipulation of the chamber. At any sign of pain, apply Buprenorphine at a dose of 0.05&#8211;0.1 mg/kg bw, subcutaneously in 8 hr intervals.</li>
</ol>
5. Euthanasia and Explantation of the Skinfold Chamber
<ol style="margin-left: 40px;">
	<li>At day 10, sacrifice animals using an overdose of an anesthetic drug (150 mg/kg Pentobarbital).</li>
	<li>Remove the chamber and sample the flap tissue for immunohistochemical and molecular protein assays. Sample tissues for conventional histology (formalin) and quantifiable protein assays (liquid nitrogen or dry ice).</li>
</ol>

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F., Harder, Y., Menger, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-acetylcysteine+attenuates+leukocytic+inflammation+and+microvascular+perfusion+failure+in+critically+ischemic+random+pattern+flaps.">N-acetylcysteine attenuates leukocytic inflammation and microvascular perfusion failure in critically ischemic random pattern flaps.</a> Microvasc Res. 82, 28-34 (2011).</li><li>Rezaeian, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ghrelin+protects+musculocutaneous+tissue+from+ischemic+necrosis+by+improving+microvascular+perfusion.">Ghrelin protects musculocutaneous tissue from ischemic necrosis by improving microvascular perfusion.</a> Am J Physiol Heart Circ Physiol. 302, 603-610 (2012).</li><li>Rezaeian, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+preconditioning+with+Erythropoietin+reduces+ischemia-induced+skin+necrosis.">Long-term preconditioning with Erythropoietin reduces ischemia-induced skin necrosis.</a> Microcirculation. , (2013).</li><li>Harder, Y., 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=Heat+shock+preconditioning+reduces+ischemic+tissue+necrosis+by+heat+shock+protein+(HSP)-32-mediated+improvement+of+the+microcirculation+rather+than+induction+of+ischemic+tolerance.">Heat shock preconditioning reduces ischemic tissue necrosis by heat shock protein (HSP)-32-mediated improvement of the microcirculation rather than induction of ischemic tolerance.</a> Ann Surg. 242, 869-878 (2005).</li><li>Tobalem, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+shockwave-induced+capillary+recruitment+improves+survival+of+musculocutaneous+flaps.">Local shockwave-induced capillary recruitment improves survival of musculocutaneous flaps.</a> J Surg Res. 184, 1196-1204 (2013).</li><li>Lindenblatt, N., Calcagni, M., Contaldo, C., Menger, M. D., Giovanoli, P., Vollmar, B. <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+model+for+studying+the+revascularization+of+skin+grafts+in+vivo:+the+role+of+angiogenesis.">A new model for studying the revascularization of skin grafts in vivo: the role of angiogenesis.</a> Plast Reconstr Surg. 122, 169-1680 (2008).</li><li>Schweizer, R., 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=Morphology+and+hemodynamics+during+vascular+regeneration+in+critically+ischemic+murine+skin+studied+by+intravital+microscopy+techniques.">Morphology and hemodynamics during vascular regeneration in critically ischemic murine skin studied by intravital microscopy techniques.</a> Eur Surg Res. 47, 222-230 (2011).</li><li>Klyscz, T., J&#252;nger, M., Jung, F., Zeintl, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cap+image&#8212;a+new+kind+of+computer-assisted+video+image+analysis+system+for+dynamic+capillary+microscopy.">Cap image&#8212;a new kind of computer-assisted video image analysis system for dynamic capillary microscopy.</a> Biomed. Tech. 42, 168-1675 (1997).</li><li>Gross, J. F., Aroesty, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mathematical+models+of+capillary+flow:+a+critical+review.">Mathematical models of capillary flow: a critical review.</a> Biorheology. 9, 225-264 (1972).</li><li>Menger, M. D., Pelikan, S., Steiner, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+ischemiareperfusion+injury+in+striated+muscle:+significance+of+&#8216;reflow+paradox.">Microvascular ischemiareperfusion injury in striated muscle: significance of &#8216;reflow paradox.</a> Am J Physiol. 263 (6 part 2), 1901-1906 (1992).</li></ol>

In order to decrease ischemic complications and thereby improve the clinical outcome, more detailed knowledge of pathophysiologic processes in critically perfused flap tissue is required. The development of new animal models that mimic acute persistent ischemia is therefore mandatory. Accordingly, we were able to develop an easily reproducible and reliable model allowing for repetitive morphological, dynamic and functional real-time evaluation of various parameters of muscle and skin vasculature that can be correlated wi...

Coverage of exposed tendon, bone and implant material in reconstructive surgery relies on the use of flaps. A flap is a block of tissue that is transferred on its vascular pedicle that guarantees arterial inflow and venous outflow. Despite broad expertise and the availability of a variety of flaps to be transferred, ischemia-induced complications ranging from wound breakdown to total tissue loss are still encountered. Whereas conservative treatment and healing by secondary intention can be expected after minor tissue necrosis, significant flap necrosis usually requires surgical revision, including debridement, wound conditioning and secondary reconstruction. This increases morbidity, prolongs hospital stay and consequently leads to increased health care costs.
Flaps with an undefined pattern of vasculature or randomly perfused areas in the distal zone most remote from the arterial inflow are particularly prone to ischemic damage. Accordingly, numerous experimental and clinical studies have evaluated the development of necrosis in both, axial pattern flaps (defined blood supply) and random pattern flaps (undefined blood supply)1-3. The main findings are commonly based on macroscopic evaluation of the size of the necrotic area. In order to assess the causes and mechanisms of tissue necrosis more in detail, several studies focused on the analysis of microcirculation. Different techniques have been used to measure tissue perfusion, including the analysis of tissue oxygen tension using polarographic electrodes4-5, as well as the measurement of blood flow using laser Doppler flowmetry6-7, dye diffusion8, and microspheres9-10. These techniques, however, only allow for measuring indirect parameters of tissue perfusion and do not enable any morphological analysis of the microhemodynamic processes within an individual area of interest of a flap.
Sandison is known to be the first who has used a transparent chamber for prolonged in vivo studies, which he performed in rabbits11. In 1943 &#8212; approximately 20 years later &#8212; Algire was the first to adapt such a transparent chamber to be applicable in mice in order to study the behavior of micro-implants of tumor cells12. Due to the fact that mice are so-called loose skin animals and after some technical refinements over the following years, Lehr and co-workers were able to adapt such a dorsal skinfold chamber developing a smaller and lighter titanium chamber. This chamber enabled evaluation using intravital fluorescence microscopy, a technique that allows direct and repetitive visualization of a number of morphologic and microcirculatory features and their changes over time under different physiological and pathophysiological conditions, such as ischemia-reperfusion injury13.
In the investigation of perfusion of skin, muscle and bone flaps under normal and pathological conditions two trends occurred: First, the &#8220;acute&#8221; flap models that do not use the dorsal skinfold chamber such as the pedicled ear flap in the mouse14, the laterally based island skin flap in the hamster15 and the pedicled composite flap in the rat16. Second, the &#8220;chronic&#8221; flap model where the combination of a flap with a dorsal skinfold chamber permits repetitive microcirculatory analyses over several days with intravital fluorescence microscopy. It consists of a randomly perfused musculocutaneous flap that is integrated in the skinfold chamber of the mouse 17. Its width-to-length ratio was chosen that a situation of acute persistent ischemia consistently results in ~50% flap tissue necrosis 10 to 14 days after flap elevation. This reproducible extent of tissue necrosis allows further evaluation of both, protective (i.e., development of less necrosis) and detrimental factors (i.e., development of more necrosis) on flap pathophysiology. During the last years, several experimental publications demonstrating the effect of different pre-, peri- and post-conditioning procedures, including the administration of tissue-protective substances18-24 and the local application of physiologic stressors such as heat25 and shockwaves26, have emerged.
The quantitative analyses of necrosis, microvascular morphology and microcirculatory parameters can further be correlated to immunohistochemical analyses and protein assays. Different proteins and molecules including vascular endothelial growth factor (VEGF), nitric oxide synthases (NOS), nuclear factor kappa B (NF-&#954;B) and heat shock proteins (HSP-32: heme-oxygenase 1 (HO-1) and HSP-70) have been shown to play a role in tissue protection. Based on this chamber flap model, two modifications have been developed in order to analyze neovascularization and microcirculation during skin graft healing27 and angiogenic developments in a pedicled flap with axial pattern perfusion28. We present a reproducible and reliable model that includes an ischemically challenged musculocutaneous flap in the mouse skinfold chamber. This model allows visualization and quantification of the microcirculation and hemodynamics by intravital epi-fluorescence microscopy.

<table><tbody><tr><td>C57Bl/6 mice, 6-8 w, 20-22 g</td><td>Charles River</td><td></td><td></td></tr><tr><td>Depilation cream</td><td>Balea</td><td></td><td>Any depilation cream</td></tr><tr><td>Titanium chamber</td><td>Irola</td><td>160001</td><td>Halteblech M</td></tr><tr><td>Custom-made Plexiglass mounting frame</td><td></td><td></td><td>Frame to secure chamber to avoid chamber movement due to the animal&#039;s respiration.</td></tr><tr><td>Slotted cheese head screw</td><td>Screws and More</td><td>842210</td><td>DIN84 M2x10</td></tr><tr><td>Hexagon full nut</td><td>Screws and More</td><td>93422</td><td>DIN934 M2</td></tr><tr><td>Snap ring</td><td>Schaefer-Peters</td><td>472212</td><td>DIN472 J12x1,0</td></tr><tr><td>Cover glass</td><td>Volab</td><td></td><td>Custom-made cover glass, 11.8 mm in diameter.</td></tr><tr><td>Fixing foam</td><td>tesamoll</td><td>05559-100</td><td>tesamoll Standard I-Profile</td></tr><tr><td>Ketamine hydrochloride</td><td>Parke Davis</td><td></td><td>Ketavet&amp;reg;</td></tr><tr><td>Dihydroxylidinothiazine hydrochloride</td><td>Bayer</td><td></td><td>Rompun&amp;reg;</td></tr><tr><td>Buprenorphin</td><td>Essex Pharma</td><td></td><td>Temgesic&amp;reg;</td></tr><tr><td>Saline 0.9%</td><td></td><td></td><td></td></tr><tr><td>Desinfection alcohol</td><td></td><td></td><td></td></tr><tr><td>Vicryl 5-0</td><td>Ethicon</td><td>V 490 H</td><td></td></tr><tr><td>Ethilon 5-0</td><td>Ethicon</td><td>EH 7823 H</td><td></td></tr><tr><td>1 ml syringes</td><td></td><td></td><td></td></tr><tr><td>Surgical skin marker with flexible ruler</td><td>Purple surgical</td><td>PS3151</td><td>Any surgical skin marker and flexible ruler</td></tr><tr><td>Pointed scissors</td><td></td><td></td><td></td></tr><tr><td>Micro-Scissors</td><td></td><td></td><td></td></tr><tr><td>Normal scissors</td><td></td><td></td><td></td></tr><tr><td>2 clamps</td><td></td><td></td><td></td></tr><tr><td>Fine anatomic forceps</td><td></td><td></td><td></td></tr><tr><td>Micro-forceps</td><td></td><td></td><td></td></tr><tr><td>Hex nuter driver</td><td>wiha</td><td>1018</td><td></td></tr><tr><td>Screwdriver</td><td>wiha</td><td>685</td><td></td></tr><tr><td>Snap ring plier</td><td>Knipex</td><td>4411J1</td><td>12-25 mm</td></tr><tr><td>Wire cutter</td><td>Knipex</td><td>70 02 160</td><td>Wire cutter is used to cut screws short; 160 mm</td></tr><tr><td>Trans-illumination light</td><td>IKEA</td><td>501.632.02</td><td>LED light Jansj&amp;ouml;; any light&amp;nbsp;</td></tr><tr><td>Magnification glasses</td><td></td><td></td><td></td></tr><tr><td>Intravital microscope</td><td>Zeiss</td><td>490035-0001-000</td><td>Scope.A1.Axiotech</td></tr><tr><td>LED system</td><td>Zeiss</td><td>423052-9501-000</td><td>Colibri.2</td></tr><tr><td>LED module 365nm</td><td>Zeiss</td><td>423052-9011-000</td><td></td></tr><tr><td>LED module 470nm</td><td>Zeiss</td><td>423052-9052-000</td><td></td></tr><tr><td>LED module 540-580nm</td><td>Zeiss</td><td>423052-9121-000</td><td></td></tr><tr><td>Filter set 62 62 HE BFP + GFP + HcRed</td><td>Zeiss</td><td>489062-9901-000</td><td>Range 1: 350-390 nm excitation wavelength, split 395 nm / 402-448 nm; range 2: 460-488 nm, split 495 nm / 500-557 nm; range 3: 567-602 nm, split 610 nm / 615 nm to infinite.</td></tr><tr><td>Filter set 20 Rhodamine</td><td>Zeiss</td><td>485020-0000-000</td><td>540-552 nm, split 560 nm, emission 575-640 nm</td></tr><tr><td>2.5X objective NA=0.06</td><td>Zeiss</td><td>421020-9900-000</td><td>A-Plan 2.5X/0.06</td></tr><tr><td>5X objective NA=0.16</td><td>Zeiss</td><td>420330-9901-000</td><td>EC Plan-Neofluar 5X/0.16 M27</td></tr><tr><td>10X objetive NA=0.30</td><td>Zeiss</td><td>420340-9901-000</td><td>EC Plan-Neofluar 10X/0.30 M27</td></tr><tr><td>20X objective NA=0.50</td><td>Zeiss</td><td>420350-9900-000</td><td>EC Plan-Neofluar 20X/0.50 M27</td></tr><tr><td>50X objective NA=0.55</td><td>Zeiss</td><td>422472-9960-000</td><td>LD Epiplan-Neofluar 50X/0.55 DIC 27</td></tr><tr><td>ZEN imaging software</td><td>Zeiss</td><td></td><td>ZenPro 2012</td></tr><tr><td>CapImage</td><td>Dr. Zeintl</td><td></td><td></td></tr><tr><td>Fluorescein isothiocyanate-dextran</td><td>Sigma-Aldrich</td><td>45946</td><td></td></tr><tr><td>bisBenzimide H 33342 trihydrochloride</td><td>Sigma-Aldrich</td><td>B2261</td><td>Harmful if swallowed; causes severe skin burns and eye damage, may cause respiratory irritation.</td></tr><tr><td>Rhodamine 6G chloride</td><td>Invitrogen</td><td>R634</td><td>Harmful if swallowed; may cause genetic defects; may cause cancer; may damage fertility or the unborn child.</td></tr><tr><td>Pentobarbital</td><td>Merial</td><td></td><td>Narcoren&amp;reg;</td></tr></tbody></table>

ischemic tissue injury in the dorsal skinfold chamber of the mouse: a skin flap model to investigate acute persistent ischemia

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are still occurring, particularly in reconstructive flap surgery. Multiple experimental flap models have been developed to analyze underlying causes and mechanisms and to investigate treatment strategies to prevent ischemic complications. The limiting factor of most models is the lacking possibility to directly and repetitively visualize microvascular architecture and hemodynamics. The goal of the protocol was to present a well-established mouse model affiliating these before mentioned lacking elements. Harder et al. have developed a model of a musculocutaneous flap with a random perfusion pattern that undergoes acute persistent ischemia and results in ~50% necrosis after 10 days if kept untreated. With the aid of intravital epi-fluorescence microscopy, this chamber model allows repetitive visualization of morphology and hemodynamics in different regions of interest over time. Associated processes such as apoptosis, inflammation, microvascular leakage and angiogenesis can be investigated and correlated to immunohistochemical and molecular protein assays. To date, the model has proven feasibility and reproducibility in several published experimental studies investigating the effect of pre-, peri- and postconditioning of ischemically challenged tissue.

Necrosis
The main endpoint of this model&#160;&#8212; tissue necrosis following flap elevation (i.e., induction of acute persistent ischemia) &#8212; is repeatedly measured and illustrated macroscopically as shown in Figure 3 over a period of 10 days. Final demarcation of flap necrosis usually occurs between day 5 and 7 after surgery and is characterized by a red fringe, i.e., zone of vasodilation and microvascular remodeling, developing betw...

Watch this Scientific Journal Video about Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia at JoVE.com

Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia

The window of the murine dorsal skinfold chamber presented visualizes a zone of acute persistent ischemia of a musculocutaneous flap. Intravital epi-fluorescence microscopy permits for direct and repetitive assessment of the microvasculature and quantification of hemodynamics. Morphologic and hemodynamic results can further be correlated with histological and molecular analyses.

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are ...

ischemic-tissue-injury-dorsal-skinfold-chamber-mouse-skin-flap-model

Research

JoVE Journal

Medicine

16.7K Views.  Technische Universität München.  Het raam van de muizen dorsale huidplooi kamer gepresenteerd visualiseert een zone van acute aanhoudende ischemie van een musculocutane flap. Intravitale epi-fluorescentie microscopie vergunningen voor directe en repetitieve evaluatie van de microvasculatuur en kwantificering van hemodynamiek. Morfologische en hemodynamische resultaten kunnen verder worden gecorreleerd met histologische en moleculaire analyses. 

Video: Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia

A Murine Closed-chest Model of Myocardial Ischemia and Reperfusion

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ischemia and reperfusion. Immune response includes cytokine expression and release or secretion of endogenous ligands of innate immune receptors. Activation of innate immunity can potentially modulate infarct size. We have modified an existing murine closed-chest model using hanging weights which could be useful for studying myocardial pre- and postconditioning and the role of innate immunity in myocardial ischemia and reperfusion. This model allows animals to recover from surgical trauma before onset of myocardial ischemia.
Volatile anesthetics have been intensely studied and their preconditioning effect for the ischemic heart is well known. However, this protective effect precludes its use in open chest models of coronary artery ligation. Thus, another advantage could be the use of the well controllable volatile anesthetics for instrumentation in a chronic closed-chest model, since their preconditioning effect lasts up to 72 hours. Chronic heart diseases with intermittent ischemia and multiple hit models are other possible applications of this model.
For the chronic closed-chest model, intubated and ventilated mice undergo a lateral blunt thoracotomy via the 4th intercostal space. Following identification of the left anterior descending a ligature is passed underneath the vessel and both suture ends are threaded through an occluder. Then, both suture ends are passed through the chest wall, knotted to form a loop and left in the subcutaneous tissue. After chest closure and recovery for 5 days, mice are anesthetized again, chest skin is reopened and hanging weights are hooked up to the loop under ECG control.
At the end of the ischemia/reperfusion protocol, hearts can be stained with TTC for infarct size assessment or undergo perfusion fixation to allow morphometric studies in addition to histology and immunohistochemistry.

Surgical trauma induces an inflammatory response. Cytokines and endogenous ligands are known to modulate myocardial infarct size following ischemia and reperfusion. We present a modified closed-chest model of murine ischemia and reperfusion using hanging weights to minimize effects of thoracotomy.

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ...

Murine Model of Hindlimb Ischemia 

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide. Medical therapies for symptomatic relief are limited. Surgical or endovascular interventions are useful for some individuals, but long-term results are often disappointing. As a result, there is a need for developing new therapies to treat PAD. The murine hindlimb ischemia preparation is a model of PAD, and is useful for testing new therapies. When compared to other models of tissue ischemia such as coronary or cerebral artery ligation, femoral artery ligation provides for a simpler model of ischemic tissue. Other advantages of this model are the ease of access to the femoral artery and low mortality rate. 
In this video, we demonstrate the methodology for the murine model of unilateral hindimb ischemia. The specific materials and procedures for creating and evaluating the model will be described, including the assessment of limb perfusion by laser Doppler imaging. This protocol can also be utilized for the transplantation and non-invasive tracking of cells, which is demonstrated by Huang et al.1.

Murine Model of Hindlimb Ischemia

The surgical procedure for induction of unilateral hindlimb ischemia is demonstrated, with confirmation of ischemia by laser Doppler perfusion imaging.

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide.  Medical therapies for ...

Biology

In situ Transverse Rectus Abdominis Myocutaneous Flap: A Rat Model of Myocutaneous Ischemia Reperfusion Injury

Free tissue transfer is the gold standard of reconstructive surgery to repair complex defects not amenable to local options or those requiring composite tissue. Ischemia reperfusion injury (IRI) is a known cause of partial free flap failure and has no effective treatment. Establishing a laboratory model of this injury can prove costly both financially as larger mammals are conventionally used and in the expertise required by the technical difficulty of these procedures typically requires employing an experienced microsurgeon. This publication and video demonstrate the effective use of a model of IRI in rats which does not require microsurgical expertise. This procedure is an in situ model of a transverse abdominis myocutaneous (TRAM) flap where atraumatic clamps are utilized to reproduce the ischemia-reperfusion injury associated with this surgery. A laser Doppler Imaging (LDI) scanner is employed to assess flap perfusion and the image processing software, Image J to assess percentage area skin survival as a primary outcome measure of injury.

In situ Transverse Rectus Abdominis Myocutaneous Flap: A Rat Model of Myocutaneous Ischemia Reperfusion Injury

Free tissue transfer is widely employed in reconstructive surgery to restore form and function following oncological resection and trauma. Preconditioning this tissue prior to surgery may improve outcome. This article describes an in situ transverse rectus abdominis myocutaneous flap (TRAM) in rats as a means for testing preconditioning strategies.

Free tissue transfer is the gold standard of reconstructive surgery to repair complex defects not amenable to local options or those requiring composite ...

Demonstration of the Rat Ischemic Skin Wound Model

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and unrelieved pressure due to immobility. Animal models have been developed that attempt to mimic these conditions for the purpose of furthering our understanding of the complexity of chronic wounds. The model described herein is a rat ischemic skin flap model that permits a prolonged reduction of blood flow resulting in wounds that become ischemic and resemble a chronic wound phenotype (reduced vascularization, increased inflammation and delayed wound closure). It consists of a bipedicled dorsal flap with 2 ischemic wounds placed centrally and 2 non-ischemic wounds lateral to the flap as controls. A novel addition to this ischemic skin flap model is the placement of a silicone sheet beneath the flap that functions as a barrier and a splint to prevent revascularization and reduce contraction as the wounds heal. Despite the debate of using rats for wound healing studies due to their quite distinct anatomic and physiologic differences compared to humans (i.e., the presence of a panniculus carnosus muscle, short life-span, increased number of hair follicles, and their ability to heal infected wounds) the modifications employed in this model make it a valuable alternative to previously developed ischemic skin flap models.

The rat, due to its size, availability, and rather docile behavior, has been utilized as a research model for many years. The goal of this protocol is to utilize the rat as an ischemic skin wound healing model to provide valuable insight into the pathophysiology of chronic wounds.

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and ...

Methods for Acute and Subacute Murine Hindlimb Ischemia

Peripheral artery disease (PAD) is a leading cause of cardiovascular morbidity and mortality in developed countries, and animal models that reliably reproduce the human disease are necessary to develop new therapies for this disease. The mouse hindlimb ischemia model has been widely used for this purpose, but the standard practice of inducing acute limb ischemia by ligation of the femoral artery can result in substantial tissue necrosis, compromising investigators&#39; ability to study the vascular and skeletal muscle tissue responses to ischemia. An alternative approach to femoral artery ligation is the induction of gradual femoral artery occlusion through the use of ameroid constrictors. When placed around the femoral artery in the same or different locations as the sites of femoral artery ligation, these devices occlude the artery over 1 - 3 days, resulting in more gradual, subacute ischemia. This results in less substantial skeletal muscle tissue necrosis, which may more closely mimic the responses seen in human PAD. Because genetic background influences outcomes in both the acute and subacute ischemia models, consideration of the mouse strain being studied is important in choosing the best model. This paper describes the proper procedure and anatomical placement of ligatures or ameroid constrictors on the mouse femoral artery to induce subacute or acute hindlimb ischemia in the mouse.

Surgical induction of hindlimb ischemia in the mouse is useful to examine angiogenesis, however this is compromised in certain inbred mouse strains that display marked ischemia-induced tissue necrosis. Methods are described to induce subacute limb ischemia using ameroid constrictors to circumvent this problem through the induction of gradual arterial occlusion.

Peripheral artery disease (PAD) is a leading cause of cardiovascular morbidity and mortality in developed countries, and animal models that reliably ...

Induction of Ischemic Stroke and Ischemia-reperfusion in Mice Using the Middle Artery Occlusion Technique and Visualization of Infarct Area

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of stroke on tissue injury and to evaluate the effectiveness of therapeutic interventions, several experimental models in a variety of species were developed. They include complete global cerebral ischemia, incomplete global ischemia, focal cerebral ischemia, and multifocal cerebral ischemia. The model described in this protocol is based on the middle cerebral artery occlusion (MCAO) and is related to the focal ischemia category. This technique produces consistent focal ischemia in a strictly defined region of the hemisphere and is less invasive than other methods. The procedure described is performed on mice, given the availability of several genetic variants and the high number of tests standardized for mice to aid in the behavioral and neurodeficit evaluation.

We describe a mouse model of stroke induced by the occlusion of the middle cerebral artery using a silicone coated suture. The protocol can be applied to induce permanent occlusion or a temporary ischemia, followed by reperfusion.

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of ...

Inducing Ischemia-reperfusion Injury in the Mouse Ear Skin for Intravital Multiphoton Imaging of Immune Responses

Ischemia-reperfusion injury (IRI) occurs when there is transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent re-oxygenation of the tissues (reperfusion). In the skin, ischemia-reperfusion (IR) is the main contributing factor to the pathophysiology of pressure ulcers. While the cascade of events leading up to the inflammatory response has been well studied, the spatial and temporal responses of the different subsets of immune cells to an IR injury are not well understood. Existing models of IR using the clamping technique on the skin flank are highly invasive and unsuitable for studying immune responses to injury, while similar non-invasive magnet clamping studies in the skin flank are less-than-ideal for intravital imaging studies. In this protocol, we describe a robust model of non-invasive IR developed on mouse ear skin, where we aim to visualize in real-time the cellular response of immune cells after reperfusion via multiphoton intravital imaging (MP-IVM).

This protocol describes the induction of an ischemia-reperfusion (IR) model on mouse ear skin using magnet clamping. Using a custom-built intravital imaging model, we study in vivo inflammatory responses post-reperfusion. The rationale behind the development of this technique is to extend the understanding of how leukocytes respond to skin IR injury.

Ischemia-reperfusion injury (IRI) occurs when there is transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent ...

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective treatment. Appropriate in vitro and in vivo approaches are essential for the identification of new target molecules for drug treatments to improve the skin wound healing process. We identified the &#946;3 subunit of voltage-gated calcium channels (Cav&#946;3) as a potential target molecule to influence the wound healing in two independent assays, i.e., the in vitro scratch migration assay and the in vivo dorsal skinfold chamber model. Primary mouse embryonic fibroblasts (MEFs) acutely isolated from wild-type (WT) and Cav&#946;3-deficient mice (Cav&#946;3 KO) or fibroblasts acutely isolated from WT mice treated with siRNA to down-regulate the expression of the Cacnb3 gene, encoding Cav&#946;3, were used. A scratch was applied on a confluent cell monolayer and the gap closure was followed by taking microscopic images at defined time points until complete repopulation of the gap by the migrating cells. These images were analyzed, and the cell migration rate was determined for each condition. In an in vivo assay, we implanted a dorsal skinfold chamber on WT and Cav&#946;3 KO mice, applied a defined circular wound of 2 mm diameter, covered the wound with a glass coverslip to protect it from infections and desiccation, and monitored the macroscopic wound closure over time. Wound closure was significantly faster in Cacnb3-gene-deficient mice. Because the results of the in vivo and the in vitro assays correlate well, the in vitro assay may be useful for the high-throughput screening before validating the in vitro hits by the in vivo wound healing model. What we have shown here for wild-type and Cav&#946;3-deficient mice or cells might also be applicable for specific molecules other than Cav&#946;3.

Here, we present a protocol for an in vitro scratch assay using primary fibroblasts and for an in vivo skin wound healing assay in mice. Both assays are straightforward methods to assess in vitro and in vivo wound healing.

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective ...

The Superficial Inferior Epigastric Artery Axial Flap to Study Ischemic Preconditioning Effects in a Rat Model

Fasciocutaneous flaps (FCF) have become the gold standard for complex defect reconstruction in plastic and reconstructive surgery. This muscle-sparing technique allows transferring vascularized tissues to cover any large defect. FCF can be used as pedicled flaps or as free flaps; however, in the literature, failure rates for pedicled FCF and free FCF are above 5%, leaving room for improvement for these techniques and further knowledge expansion in this area. Ischemic preconditioning (I.P.) has been widely studied, but the mechanisms and the optimization of the I.P. regimen are yet to be determined. This phenomenon is indeed poorly explored in plastic and reconstructive surgery. Here, a surgical model is presented to study the I.P. regimen in a rat axial fasciocutaneous flap model, describing how to safely and reliably assess the effects of I.P. on flap survival. This article describes the complete surgical procedure, including suggestions to improve the reliability of this model. The objective is to provide researchers with a reproducible and reliable model to test various ischemic preconditioning regimens and assess their effects on flap survivability.

This protocol describes harvesting, suturing, and monitoring fasciocutaneous flaps in rats that allow for good visualization and manipulation of blood flow through the superficial inferior epigastric vessels by means of clamping and ligating the femoral vessels. This is critical for studies involving ischemic preconditioning.

Fasciocutaneous flaps (FCF) have become the gold standard for complex defect reconstruction in plastic and reconstructive surgery. This muscle-sparing ...

Immunology and Infection

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...

Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia

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