The study was funded by the Deutsche Forschungsgemeinschaft SFB 914 (HI-A/SM, project Z01). We thank Dr. Susanne Stutte for reading the manuscript.

This study was approved by the Bavarian Animal Care and Use Committee (approved by ethical code: ROB-55.2Vet-2532.Vet_02-17-99); these experiments were carried out in strict accordance with the German animal legislation guidelines.
1. Animals and femoral artery ligation (FAL)
NOTE: To induce sterile inflammation and arteriogenesis, 8-10 weeks old male C57BL/6J mice were used. None of the mice died or suffered from wound infection or wound healing disturbance during or post-FAL or sham surgery, respectively.
<ol>
	<li>Anesthesia 
	NOTE: Before injection of the MMF-mix anesthesia, it is recommended to first anesthetize with 5% isoflurane until the mouse is sleeping.
	<ol>
		<li>Weigh the mouse and prepare MMF mix with a dose of midazolam 5.0 mg/kg, medetomidine 0.5 mg/kg, and fentanyl 0.05 mg/kg.</li>
		<li>Fill a 1 mL syringe with MMF-mix and inject a final volume of 100 &#181;L s.c. using a 30 G needle (after anesthesia with 5% isoflurane).</li>
		<li>Place the mouse on a warm pad and wait until the mouse is completely anesthetized. Set the timer to 35 min. After this time, inject s.c. half of the dose (50 &#181;L) of the MMF-mix.</li>
		<li>Check the depth of anesthesia by testing the pedal and interdigital reflexes (by firm toe pinch) of the mouse. 
		NOTE: An absence of response indicates an adequate depth of&#160;analgesia.</li>
	</ol>
	</li>
	<li>Femoral artery ligation (FAL) 
	NOTE: Use sterile surgical instruments and sutures. Disinfect the skin with disinfectant before opening and after closing.
	<ol>
		<li>Place the anesthetized mouse on a heating plate (35-37 &#176;C) to maintain physiological body temperature. Apply eye cream (dexpanthenol) to each eye to prevent drying of the eyes under anesthesia.</li>
		<li>Transfer the anesthetized mouse to a Laser Doppler imaging (LDI) chamber before and after FAL to check blood flow and perfusion (Figure 1A-C).</li>
		<li>Remove hair, disinfect the skin, and make a cut to access the femoral artery. Under a surgical microscope, ligate the right femoral artery of the mouse hind limb using a braided silk suture (7-0) and perform a sham operation on the left femoral artery to serve as an internal control as previously described6 (Figure 1D-K). 
		NOTE: The incision in the skin was made with small scissors to avoid direct injury to the blood&#160;vessels close to the skin.</li>
		<li>Close the skin using a braided silk suture (6-0). Administer buprenorphine (0.1 mg/kg) s.c. every 12 h, starting 10 min before antagonizing the anesthesia for pain relief. Observe the animal for signals of pain, i.e., reduced grooming activity, reluctance to move, or hunched posture for 24 h post-surgery.</li>
		<li>Inject (s.c.) a combination of naloxone (1.2 mg/kg), flumazenile (0.5 mg/kg), and atipamezole (2.5 mg/kg) to antagonize the anesthesia after finishing the FAL and closing the skin.</li>
		<li>After surgery, keep the mouse on the warm pad and continuously monitor the animal until it can maintain an upright posture and start walking normally around the cage.</li>
		<li>When the mouse is fully awake, place it back in the cage together with the other mice and return the cage to the animal housing room.</li>
	</ol>
	</li>
</ol>
2. Tissue preparation for multiphoton intravital imaging
<ol>
	<li>Tail vein injection 
	NOTE: The animal needs to be under anesthesia before starting the preparation for intravital imaging. Before introducing the needle of the vein catheter, it is recommended to apply a skin disinfectant on the tail. This will also help to visualize the vessels.
	<ol>
		<li>Prepare a tail vein injection catheter using a 2 x 30 G needle, 10 cm of a fine bore polythene tubing (0.28 mm internal diameter and 0.61 mm outer diameter), a 1 mL syringe, and a histoacryl glue to fix the catheter on the mouse tail for further i.v. injections during imaging (Figure 2A,B and Figure 2F).</li>
		<li>Prepare the antibody solution for i.v. injection: CD45-PE and CD41-FITC (20 &#181;g/mouse) at a final volume of 100 &#181;L.</li>
		<li>Check the tail for the laterally located tail veins and dam the vein to identify the tail vein. Disinfect the tail, insert the vein catheter, and fix it using tissue histoacryl glue (Figure 2B).</li>
		<li>Inject the antibodies prior to imaging (at least 15 min before) and after finishing the tissue preparation.</li>
	</ol>
	</li>
	<li>Tissue preparation 
	NOTE: Always use a warm pad and administer a drop of eye cream (dexpanthenol) in each eye before tissue preparation. Use sterile surgical instruments and sutures. Disinfect the skin with disinfectant before open and after closing.
	<ol>
		<li>Place the anesthetized mouse on a stable and portable pad. Fix the mouse in a supine position with a 4 point fixation on the pad using adhesive tape (Figure 2B).</li>
		<li>Mold two pieces of modeling clay and place the pieces under the upper hind limb of the mouse to ensure a plane position of the adductor muscle (Figure 2B, indicated by red arrows).</li>
		<li>To ensure a stable body temperature of 37 &#176;C of the mouse, place the pad with the mouse on a heating plate under a surgical microscope with a 0.64-4.0-fold zoom (Figure 1L).</li>
		<li>Remove the hair from both legs, disinfect the skin, and cut the skin in a circle around the scar of the earlier femoral artery ligation of the right hind limb (Figure 1M).</li>
		<li>Remove the skin and fat layer from the top of the leg (Figure 1N,O), and pull aside the remaining skin using sutures to create a pocket around the adductor muscle (Figure 1P,Q).</li>
		<li>Remove subcutaneous fat to get a clear vision of the adductor muscle and the profound artery/vein as well as the collateral vessels (Figure 1Q,R).</li>
		<li>Carefully start removing the superficial muscle layer on top of the collateral vessels by carefully dissecting it out with fine forceps (Figure 1R). 
		NOTE: The collateral artery and the collateral vein are clearly visible and ready for imaging (Figure 1S).</li>
		<li>Fill the prepared pocket with saline to avoid drying of the tissue and continue with the same procedure for the sham-operated left hind limb. Before imaging, add ultrasonic gel in both pockets, which prevents drying and serves as an immersion medium for optical coupling with the objective (Figure 2C and Figure 2F).</li>
	</ol>
	</li>
</ol>
3. Intravital multiphoton microscopy
<ol>
	<li>Preparation 
	NOTE: Keep a syringe with ultrasonic gel inside the incubator chamber of the microscope to maintain a warm temperature for refilling the pockets during long-term imaging.
	<ol>
		<li>Remove the saline from the two prepared pockets and replace it with ultrasonic gel to cover the collateral vessels (Figure 2C).</li>
		<li>Inject the antibody solution through the tail vein catheter.&#160;15 min after antibody injection, transfer the pad with the fixed mouse into the warmed multiphoton imaging chamber (Figure 2F).</li>
		<li>After completion of the image acquisition, euthanize the mouse by cervical dislocation under deep narcosis.</li>
	</ol>
	</li>
	<li>Multiphoton microscopy image acquisition 
	NOTE: Turn on the microscope 30 min before imaging to allow for laser stabilization. For the best optical stability of the microscope, it is recommended to keep the microscope chamber permanently heated.
	<ol>
		<li>Turn on the key of the Ti:Sapphire laser box, the electronic interfaces boxes, the heating unit of the incubation chamber, the fluorescent lamp, and the computer (Figure 2D,E).</li>
		<li>Launch the acquisition software (Inspector software). Set the wavelength to 800 nm and open the microscope shutter (Figure 3A ii).</li>
		<li>In the Measurement Wizard dialog, choose the Instrument Mode (Single beam) and in Measurement Mode (3D-scan Time lapse) (Figure 3A i).</li>
		<li>Transfer the anesthetized mouse into the pre-warmed incubation chamber of the microscope. Position the area with the ultrasonic gel (step 2.2.8) directly in contact with the objective front lens (Figure 2F).</li>
		<li>Open the epifluorescence microscope shutter, choose an adequate filter/dichroic setting for FITC visualization (4) to find the area of interest by following the blood flow under epifluorescence illumination. After bringing the area of interest into the center field of view, close the epifluorescence microscope shutter.</li>
		<li>In the xy-Scanner dialog, set the following parameters: Image size (554x554), Pixel (512x512), Frequency (400), Line average (1) (Figure 3A iii). Adjust the laser power (5%) (Figure 3A iv), and set the photomultiplier (PMT) gain for the channels green (66), red (66), and blue (63) (Figure 3A v).</li>
		<li>Select anadequate objective for intravital imaging and drift correction settings (see details in 3.2.11). 
		NOTE: Here, the selected objective was the Nikon LWD 16x (Figure 3A vi).</li>
		<li>Define the image stack range by starting the preview acquisition mode by pressing the red button on the top right corner of the Measurement Wizard dialog window (Figure 3A i).</li>
		<li>Define the area and structure of interest by focusing to obtain a good image. Then, while observing the screen, change focus by moving the objective up until the image disappears, and set it as zero (first= 0). Change focus in the opposite direction by moving the objective down until the image disappears from the screen again, and set it as the last (end= 40 &#181;m) position in the axial direction (Figure 3A vii).</li>
		<li>Set the step size to 2 &#181;m. Click the red button on the top right corner of the Measurement Wizard dialog to stop the preview scanning. 
		NOTE: The number of images will be calculated and set automatically (21 images), as indicated in Figure 3A vii.</li>
		<li>If the microscope is equipped with live-drift correction9, start the drift correction software (e.g., VivoFollow) and follow the steps described below.
		<ol>
			<li>In the Measurement Wizard dialog, choose the Python axis (Python Ax) (Figure 3A viii) as the first axis device; DO NOT activate the auto save mode. Check the auto save (AS) box only on the time axis (Time Time). Press the Python icon on the Available Devices window (Figure 3A ix) to open the Python dialog window.</li>
			<li>In the Python dialog Axis settings, enter in From (0 zero) and To (-40). Enter Number of steps (21) as indicated in Figure 3A x.</li>
			<li>Go to the xyz Stage Z dialog window and enlarge the Scan Range by 200 &#181;m on both ends: in Start, enter (200 &#181;m) and in End, enter (-240 &#181;m), the range will automatically be set (-440 &#181;m). Keep the step size (2 &#181;m) as shown in Figure 3A xi.</li>
			<li>Open the VivoFollow Frontend dialog and click the Motion Profile box (Figure 3A xii).</li>
			<li>Start image acquisition by pressing the green arrowhead in the top left corner of the Measurement Wizard dialog of the Inspector Software (Figure 3A i).</li>
			<li>If live-drift correction is used, look for a dialog for the drift correction configuration to appear as shown in Figure 3A xiii. Set the acquisition channel that will be used as an immobile landmark reference (for this protocol, choose channel 2, where the vascular tracer signal is to be recorded). Enter the maximal correction offset in &#181;m that was added to the first and last z positions (here, 200 &#181;m) and click OK.</li>
			<li>Monitor the current drift offset in x, y, and z in real time during the image acquisition in the VivoFollow fronted dialog window Figure 3A xiv.</li>
			<li>Stop imaging acquisition after ~35 min when another cycle of anesthesia re-injection is required. Inject half a dose of the MMF-mix (50 &#181;l s.c) and re-start imaging acquisition by pressing the green arrowhead again (step 3.2.11.5).</li>
			<li>Repeat this procedure until the experiment is finished.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol>
	<li>The top 10 causes of death. World Health Organization Available from: <a target="_blank" href="https://www.who.int/news-room/fact-sheets/detail/the-top-10-causes-of-death">https://www.who.int/news-room/fact-sheets/detail/the-top-10-causes-of-death</a> (2020)</li><li>Deindl, E., Schaper, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+art+of+arteriogenesis.">The art of arteriogenesis.</a> Cell Biochemistry and Biophysics. 43 (1), 1-15 (2005).</li><li>Lasch, 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=Extracellular+RNA+released+due+to+shear+stress+controls+natural+bypass+growth+by+mediating+mechanotransduction+in+mice.">Extracellular RNA released due to shear stress controls natural bypass growth by mediating mechanotransduction in mice.</a> Blood. 134 (17), 1469-1479 (2019).</li><li>Kumaraswami, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+effective+flow+Ccytometry-based+method+for+identification+and+quantification+of+tissue+infiltrated+leukocyte+subpopulations+in+a+mouse+model+of+peripheral+arterial+disease.">A simple and effective flow Ccytometry-based method for identification and quantification of tissue infiltrated leukocyte subpopulations in a mouse model of peripheral arterial disease.</a> International Journal of Molecular Sciences. 21 (10), 3593 (2020).</li><li>Padgett, M. E., McCord, T. J., McClung, J. M., Kontos, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+acute+and+subacute+murine+hindlimb+ischemia.">Methods for acute and subacute murine hindlimb ischemia.</a> Journal of Visualized Experiments: JoVE. (112), e54166 (2016).</li><li>Limbourg, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+postnatal+arteriogenesis+and+angiogenesis+in+a+mouse+model+of+hind-limb+ischemia.">Evaluation of postnatal arteriogenesis and angiogenesis in a mouse model of hind-limb ischemia.</a> Nature Protocols. 4 (12), 1737 (2009).</li><li>Wagner, 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=Intravital+microscopy+of+monocyte+homing+and+tumor-related+angiogenesis+in+a+murine+model+of+peripheral+arterial+disease.">Intravital microscopy of monocyte homing and tumor-related angiogenesis in a murine model of peripheral arterial disease.</a> Journal of Visualized Experiments: JoVE. (126), e56290 (2017).</li><li>Ishikawa-Ankerhold, H. C., Ankerhold, R., Drummen, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+fluorescence+microscopy+techniques--FRAP,+FLIP,+FLAP,+FRET+and+FLIM.">Advanced fluorescence microscopy techniques--FRAP, FLIP, FLAP, FRET and FLIM.</a> Molecules. 17 (4), 4047-4132 (2012).</li><li>Vladymyrov, M., Abe, J., Moalli, F., Stein, J. V., Ariga, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+tissue+offset+correction+system+for+intravital+multiphoton+microscopy.">Real-time tissue offset correction system for intravital multiphoton microscopy.</a> Journal of Immunological Methods. 438, 35-41 (2016).</li><li>Chandraratne, S., 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=Critical+role+of+platelet+glycoprotein+ibalpha+in+arterial+remodeling.">Critical role of platelet glycoprotein ibalpha in arterial remodeling.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 35 (3), 589-597 (2015).</li></ol>

The authors have no conflicts of interest to declare.

The described method of multiphoton in vivo analysis of leukocyte recruitment represents an addition to commonly used tools for leukocyte recruitment studies such as (immuno-) histological or FACS analyses. With this imaging method, it is possible to visualize in greater detail the dynamic processes in leukocyte adherence and extravasation during arteriogenesis10. Despite the added value of this method, the offered protocol includes some critical steps and limitations. See Table 1...

Despite intensive research interest during recent years, cardiovascular diseases, e.g., ischemic heart disease and stroke, are still the leading global cause of death1. Current treatments for these diseases are highly invasive therapies such as percutaneous transluminal angioplasty, percutaneous transluminal coronary angioplasty, or bypass surgery2. Therefore, the development of alternative, non-invasive therapeutic options is urgently needed. The body can create natural bypasses around a stenosed or occluded vessel to redirect the interrupted blood flow to the distal part of the stenosis. This process is called arteriogenesis2. Many recent studies have shown that increased fluid shear impacts leukocyte recruitment, which plays an important role during arteriogenesis3. The main current options to analyze the recruitment of leukocytes during arteriogenesis are ex vivo (immuno-) histological analyses or fluorescence-activated cell sorting (FACS) methodology4. To enable the assessment of leukocyte dynamics during arteriogenesis, this paper presents an intravital imaging protocol with multiphoton microscopy.
Leukocytes are the major blood cells recruited during the process of arteriogenesis3. This protocol uses multiphoton imaging to show the crawling of adherent leukocytes labeled with injected anti-CD45-PE antibodies in collateral arteries, 24 h after the induction of arteriogenesis by femoral artery ligation (FAL) employing a murine hindlimb ischemia model5,6. Alternatively, immune cells can be labeled ex vivo and carefully injected into mice, as shown in studies on angiogenesis using intravital microscopy7. The blood flow inside vessels and arteries can be visualized by CD41-FITC (to label platelets), dextran-FITC (plasma), or by the second harmonic generation (SHG), which visualizes collagen type 1 present in the basement membrane of some part of the vascular tree. SHG is a unique free labeling effect of the multiphoton excitation. Multiphoton imaging allows long-term cell tracking without harming the tissue and activating the cells by laser power excitation. Multiphoton microscopy is the imaging method of choice for visualizing fluorescently labeled cells and structures in living animals due to its ability to excite the fluorophores deeper into the tissue/organs with minimal phototoxicity8.
The use of tuned infrared lasers with pulses delivered within femtosecond intervals excites the fluorochrome only at the focal plane, with no excitation above and below the focal plane8. Thus, multiphoton microscopy allows high spatio-temporal resolution, less photo-damage, and increased tissue penetration imaging for studying dynamic biological events inside the organs. It is an ideal microscopy tool for live-animal imaging. However, multiphoton and any other art of intravital microscopy is limited by tissue motion due to heartbeat, respiration, peristaltic movements, muscle tonality, and other physiological functions, which disturbs imaging acquisition and analysis. As these movements impair temporal and spatial resolution and sometimes even prohibit subsequent analysis, they must be addressed appropriately to enable accurate data analysis and interpretation. Several strategies have been developed to lower or prevent artifacts from tissue motion. This protocol applies an in situ drift correction software called VivoFollow9 to correct tissue drift during image acquisition. This approach provides the required image stabilization, enabling long-term imaging and cell-tracking analysis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.0 mL Syringe</td>
			<td>BD Biosciences, San Jose, CA, USA</td>
			<td>309628</td>
			<td>syringe for injection</td>
		</tr>
		<tr>
			<td>3M Durapore Surgical Tape 1538-0</td>
			<td>3M, St. Paul, MN, USA</td>
			<td>1538-0</td>
			<td>fixation tape</td>
		</tr>
		<tr>
			<td>Atipamezole</td>
			<td>Zoetis, Berlin, Germany</td>
			<td></td>
			<td>antagonize anesthesia</td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Reckitt Benckiser Healthcare, Slough, UK</td>
			<td></td>
			<td>antagonize anesthesia</td>
		</tr>
		<tr>
			<td>C57/B6J mouse</td>
			<td>Charles River, Sulzfeld, Germany</td>
			<td></td>
			<td>used animals for surgery/imaging</td>
		</tr>
		<tr>
			<td>CD41-FITC ab</td>
			<td>Biolegend</td>
			<td>133904</td>
			<td>Platelet labeling in vivo</td>
		</tr>
		<tr>
			<td>CD45-PE ab</td>
			<td>Biolegend</td>
			<td>368510</td>
			<td>Leukocytes labelling in vivo</td>
		</tr>
		<tr>
			<td>Disinfectant Cutasept</td>
			<td>Carl Roth GmbH, Karlsruhe, Deutschland</td>
			<td>AK64.2</td>
			<td>Disinfection</td>
		</tr>
		<tr>
			<td>Eye cream (Bepanthen)</td>
			<td>Bayer Vital GmbH</td>
			<td>5g</td>
			<td></td>
		</tr>
		<tr>
			<td>Fentanyl</td>
			<td>CuraMED Pharma, Karlsruhe, Germany</td>
			<td></td>
			<td>anesthesia</td>
		</tr>
		<tr>
			<td>Flumazenile</td>
			<td>Inresa Arzneimittel GmbH, Freiburg, Deutschland</td>
			<td></td>
			<td>antagonize anesthesia</td>
		</tr>
		<tr>
			<td>Fine bore polythene tubing</td>
			<td>Smiths medical</td>
			<td>Lot 278316</td>
			<td>0.28 mm ID and 0.61 mm OD, tubing for the vein catheter</td>
		</tr>
		<tr>
			<td>Histoacryl flexible</td>
			<td>BRAUM</td>
			<td>1050052</td>
			<td>tissue glue</td>
		</tr>
		<tr>
			<td>Imaris software</td>
			<td>Oxford Instruments</td>
			<td>version 9.2</td>
			<td>Used for cell tracking, cell speed analysis, 3D projection</td>
		</tr>
		<tr>
			<td>Laser Doppler Imaging instrument</td>
			<td>Moor LDI 5061 and Moor Software Version 3.01, Moor Instruments, Remagen, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LEICA KL300 LED</td>
			<td>Leica, Solms, Germany</td>
			<td></td>
			<td>light for microscope</td>
		</tr>
		<tr>
			<td>Leica M60</td>
			<td>Leica, Solms, Germany</td>
			<td></td>
			<td>microscope for surgery</td>
		</tr>
		<tr>
			<td>LEICA MC120 HD</td>
			<td>Leica, Solms, Germany</td>
			<td></td>
			<td>camera for microscope</td>
		</tr>
		<tr>
			<td>Medetomidine</td>
			<td>Pfizer Pharma, Berlin, Germany</td>
			<td></td>
			<td>anesthesia</td>
		</tr>
		<tr>
			<td>Midazolam</td>
			<td>Ratiopharm GmbH, Ulm, Germany</td>
			<td></td>
			<td>anesthesia</td>
		</tr>
		<tr>
			<td>Multiphoton microscope</td>
			<td>Lavision</td>
			<td>TRIMScope II</td>
			<td>WL 820 nm</td>
		</tr>
		<tr>
			<td>NaCl 0.9%</td>
			<td>Braun, Melsungen, Deutschland</td>
			<td>3570310</td>
			<td>saline for pocket</td>
		</tr>
		<tr>
			<td>Naloxone</td>
			<td>Inresa Arzneimittel GmbH, Freiburg, Deutschland</td>
			<td></td>
			<td>antagonize anesthesia</td>
		</tr>
		<tr>
			<td>Needle 30 G</td>
			<td>BD Biosciences, San Jose, CA, USA</td>
			<td>305128</td>
			<td>needle for i.v. catheter</td>
		</tr>
		<tr>
			<td>Silk braided suture (0/7)</td>
			<td>Pearsalls Ltd., Taunton, UK</td>
			<td>SUT-S&#160;103</td>
			<td>suture for femoral artery ligation</td>
		</tr>
		<tr>
			<td>Ultrason Gel</td>
			<td>SONOSID-ASID BONZ 250 mL</td>
			<td>782012</td>
			<td>gel for imaging</td>
		</tr>
		<tr>
			<td>Vicryl 6.0 suture</td>
			<td>Vicryl, Johnson&#38;Johnson, New Brunswick, NJ, USA</td>
			<td>NW-2347</td>
			<td>suture to build pocket</td>
		</tr>
		<tr>
			<td>VivoFollow drift correction software</td>
			<td>Developed by Mykhailo Vladymyrov</td>
			<td></td>
			<td>Reference 9</td>
		</tr>
	</tbody>
</table>

multiphoton intravital imaging for monitoring leukocyte recruitment during arteriogenesis in a murine hindlimb model

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for analyzing collateral arteries and leukocytes in arteriogenesis is ex vivo (immuno-) histological methodology. However, this technique does not allow the measurement of dynamic processes such as blood flow, shear stress, cell-cell interactions, and particle velocity. This paper presents a protocol to monitor in vivo processes in growing collateral arteries during arteriogenesis utilizing intravital imaging. The method described here is a reliable tool for dynamics measurement and offers a high-contrast analysis with minimal photo-cytotoxicity, provided by multiphoton excitation microscopy. Prior to analyzing growing collateral arteries, arteriogenesis was induced in the adductor muscle of mice by unilateral ligation of the femoral artery.
After the ligation, the preexisting collateral arteries started to grow due to increased shear stress. Twenty-four hours after surgery, the skin and subcutaneous fat above the collateral arteries were removed, constructing a pocket for further analyses. To visualize blood flow and immune cells during in vivo imaging, CD41-fluorescein isothiocyanate (FITC) (platelets) and CD45-phycoerythrin (PE) (leukocytes) antibodies were injected intravenously (i.v.) via a catheter placed in the tail vein of a mouse. This article introduces intravital multiphoton imaging as an alternative or in vivo complementation to the commonly used static ex vivo (immuno-) histological analyses to study processes relevant for arteriogenesis. In summary, this paper describes a novel and dynamic in vivo method to investigate immune cell trafficking, blood flow, and shear stress in a hindlimb model of arteriogenesis, which enhances evaluation possibilities notably.

Multiphoton microscopy offers a high spatio-temporal resolution for leukocyte tracking, wherein cell migration steps and speed can be tracked and monitored (Figure 4A,B). However, the physiological motion of the sample poses a challenge, especially for long-term intravital microscopy image acquisitions. Therefore, a good tissue preparation and holders to fix the tissue and tools, such as real-time imaging correction for tissue drift, are required for successful and stable im...

Watch this Scientific Journal Video about Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model at JoVE.com

Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model

The recruitment of leukocytes and platelets constitutes an essential component necessary for the effective growth of collateral arteries during arteriogenesis. Multiphoton microscopy is an efficient tool for tracking cell dynamics with high spatio-temporal resolution in vivo and less photo-toxicity to study leukocyte recruitment and extravasation during arteriogenesis.

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for ...

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Research

JoVE Journal

Medicine

1.3K Views.  Ludwig-Maximilians-Universität München. The recruitment of leukocytes and platelets constitutes an essential component necessary for the effective growth of collateral arteries during arteriogenesis. Multiphoton microscopy is an efficient tool for tracking cell dynamics with high spatio-temporal resolution in vivo and less photo-toxicity to study leukocyte recruitment and extravasation during arteriogenesis.

Video: Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology rely on the injection of a suspension of glioma cells into the brain parenchyma following incision of the dura-mater. Whereas the cells have to be injected superficially to be accessible to intravital two-photon microscopy, superficial injections fail to recapitulate the physiopathological conditions. Indeed, escaping through the injection tract most tumor cells reach the extra-dural space where they expand abnormally fast in absence of mechanical constraints from the parenchyma.
Our improvements consist not only in focally implanting a glioma spheroid rather than injecting a suspension of glioma cells in the superficial layers of the cerebral cortex but also in clogging the injection site by a cross-linked dextran gel hemi-bead that is glued to the surrounding parenchyma and sealed to dura-mater with cyanoacrylate. Altogether these measures enforce the physiological expansion and infiltration of the tumor cells inside the brain parenchyma. Craniotomy was finally closed with a glass window cemented to the skull to allow chronic imaging over weeks in absence of scar tissue development.
Taking advantage of fluorescent transgenic animals grafted with fluorescent tumor cells we have shown that the dynamics of interactions occurring between glioma cells, neurons (e.g. Thy1-CFP mice) and vasculature (highlighted by an intravenous injection of a fluorescent dye) can be visualized by intravital two-photon microscopy during the progression of the disease.
The possibility to image a tumor at microscopic resolution in a minimally compromised cerebral environment represents an improvement of current GBM animal models which should benefit the field of neuro-oncology and drug testing.

We have established a cortical orthotopic glioblastoma model in mice for intravital two-photon microscopy that recapitulates the biophysical constraints normally at play during the growth of the tumor. A chronic glass window replacing the skull above the tumor enables the follow-up of the tumor progression over time by two-photon microscopy.

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology ...

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or subvert anti-tumor immune responses in order to survive and flourish. Tumors take advantage of a number of different mechanisms of immune &#8220;escape,&#8221; including the recruitment of tolerogenic DC, immunosuppressive regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSC) that inhibit cytotoxic anti-tumor responses. Conversely, anti-tumor effector immune cells can slow the growth and expansion of malignancies: immunostimulatory dendritic cells, natural killer cells which harbor innate anti-tumor immunity, and cytotoxic T cells all can participate in tumor suppression. The balance between pro- and anti-tumor leukocytes ultimately determines the behavior and fate of transformed cells; a multitude of human clinical studies have borne this out. Thus, detailed analysis of leukocyte subsets within the tumor microenvironment has become increasingly important. Here, we describe a method for analyzing infiltrating leukocyte subsets present in the tumor microenvironment in a mouse tumor model. Mouse B16 melanoma tumor cells were inoculated subcutaneously in C57BL/6 mice. At a specified time, tumors and surrounding skin were resected en bloc and processed into single cell suspensions, which were then stained for multi-color flow cytometry. Using a variety of leukocyte subset markers, we were able to compare the relative percentages of infiltrating leukocyte subsets between control and chemerin-expressing tumors. Investigators may use such a tool to study the immune presence in the tumor microenvironment and when combined with traditional caliper size measurements of tumor growth, will potentially allow them to elucidate the impact of changes in immune composition on tumor growth. Such a technique can be applied to any tumor model in which the tumor and its microenvironment can be resected and processed.

This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses.

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or ...

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 ...

Combined Intravital Microscopy and Contrast-enhanced Ultrasonography of the Mouse Hindlimb to Study Insulin-induced Vasodilation and Muscle Perfusion

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate postprandial delivery of nutrients and hormones to insulin-sensitive tissues. We here describe a technique for combining intravital microscopy (IVM) and contrast-enhanced ultrasonography (CEUS) of the adductor compartment of the mouse hindlimb to simultaneously visualize muscle resistance arteries and perfusion of the microcirculation in vivo. Simultaneously assessing insulin's effect at multiple levels of the vascular tree is important to study relationships between insulin's multiple vasoactive effects and muscle perfusion. Experiments in this study were performed in mice. First, the tail vein cannula is inserted for the infusion of anesthesia, vasoactive compounds and ultrasound contrast agent (lipid-encapsulated microbubbles). Second, a small incision is made in the groin area to expose the arterial tree of the adductor muscle compartment. The ultrasound probe is then positioned at the contralateral upper hindlimb to view the muscles in cross-section. To assess baseline parameters, the arterial diameter is assessed and microbubbles are subsequently infused at a constant rate to estimate muscle blood flow and microvascular blood volume (MBV). When applied before and during a hyperinsulinemic-euglycemic clamp, combined IVM and CEUS allow assessment of insulin-induced changes of arterial diameter, microvascular muscle perfusion and whole-body insulin sensitivity. Moreover, the temporal relationship between responses of the microcirculation and the resistance arteries to insulin can be quantified. It is also possible to follow-up the mice longitudinally in time, making it a valuable tool to study changes in vascular and whole-body insulin sensitivity.

Insulin-induced vasodilation regulates muscle perfusion and increases the microvascular surface area (microvascular recruitment) available for solute exchange between blood and tissue interstitium. Combined intravital microscopy and contrast-enhanced ultrasonography is presented to simultaneously assess insulin&#39;s action at the larger vessels and the microcirculation in vivo.

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate ...

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 ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

Intravital Microscopy of Monocyte Homing and Tumor-Related Angiogenesis in a Murine Model of Peripheral Arterial Disease

The therapeutic goal for peripheral arterial disease and ischemic heart disease is to increase blood flow to ischemic areas caused by hemodynamic stenosis. Vascular surgery is a viable option in selected cases, but for patients without indications for surgery such as progression to rest pain, critical limb ischemia, or major disruptions to life or work, there are few possibilities for mitigating their disease. Cell therapy via monocyte-enhanced perfusion through the stimulation of collateral formation is one of a few non-invasive options.
Our group examines arteriogenesis after monocyte transplantation into mice using the hindlimb ischemia model. Previously, we have demonstrated improvement in hindlimb perfusion using tetanus-stimulated syngeneic monocyte transplantation. In addition to the effects on the collateral formation, tumor growth could be affected by this therapy as well. To investigate these effects, we use a basement membrane-like matrix mouse model by injecting the extracellular matrix of the Engelbreth-Holm-Swarm sarcoma into the flank of the mouse, after occlusion of the femoral artery.
After the artificial tumor studies, we use intravital microscopy to study in vivo tumor-angiogenesis and monocyte homing within collateral arteries. Previous studies have described the histological examination of animal models, which presupposes subsequent analysis to post-mortem artifacts. Our approach visualizes monocyte homing to areas of collateralization in real time sequences, is easy to perform, and investigates the process of arteriogenesis and tumor angiogenesis in vivo.

Monocytes are important mediators of arteriogenesis in the context of peripheral arterial disease. Using a basement membrane-like matrix and intravital microscopy, this protocol investigates monocyte homing and tumor-related angiogenesis after monocyte injection in the femoral artery ligation murine model.

The therapeutic goal for peripheral arterial disease and ischemic heart disease is to increase blood flow to ischemic areas caused by hemodynamic ...

Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard therapy, a considerable number of CLI patients are not suited for either surgical or endovascular revascularization. Angiogenic therapies are emerging as an option for these patients but are currently still under investigation. Before application in humans, those therapies must be tested in animal models and its mechanisms must be clearly understood. An animal model of hindlimb ischemia (HLI) has been developed by the ligation and excision of the distal external iliac and femoral arteries and veins in mice. A comprehensive panel of tests was assembled to assess the effects of ischemia and putative angiogenic therapies at functional, histologic and molecular levels. Laser Doppler was used for the flow measurement and functional assessment of perfusion. Tissue response was evaluated by the analysis of capillary density after staining with the anti-CD31 antibody on histological sections of gastrocnemius muscle and by measurement of collateral vessel density after diaphonization. Expression of angiogenic genes was quantified by RT-PCR targeting selected angiogenic factors exclusively in endothelial cells (ECs) after laser capture microdissection from mice gastrocnemius muscles. These methods were sensitive in identifying differences between ischemic and non-ischemic limbs and between treated and non-treated limbs. This protocol provides a reproducible model of CLI and a framework for testing angiogenic therapies.

Here, a critical hindlimb ischemia experimental model is presented followed by a battery of functional, histologic and molecular tests&#160;to assess the effectiveness of angiogenic therapies.&#8203;

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard ...