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

Summary

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.

Abstract

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.

Introduction

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 model^5,6. 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.

Protocol

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.

1. Anesthesia
   NOTE: Before injection of the MMF-mix anesthesia, it is recommended to first anesthetize with 5% isoflurane until the mouse is sleeping.
   1. 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.
   2. Fill a 1 mL syringe with MMF-mix and inject a final volume of 100 µL s.c. using a 30 G needle (after anesthesia with 5% isoflurane).
   3. 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 µL) of the MMF-mix.
   4. 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 analgesia.
2. Femoral artery ligation (FAL)
   NOTE: Use sterile surgical instruments and sutures. Disinfect the skin with disinfectant before opening and after closing.
   1. Place the anesthetized mouse on a heating plate (35-37 °C) to maintain physiological body temperature. Apply eye cream (dexpanthenol) to each eye to prevent drying of the eyes under anesthesia.
   2. Transfer the anesthetized mouse to a Laser Doppler imaging (LDI) chamber before and after FAL to check blood flow and perfusion (Figure 1A-C).
   3. 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 vessels close to the skin.
   4. 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.
   5. 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.
   6. 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.
   7. 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.

2. Tissue preparation for multiphoton intravital imaging

1. 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.
   1. 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).
   2. Prepare the antibody solution for i.v. injection: CD45-PE and CD41-FITC (20 µg/mouse) at a final volume of 100 µL.
   3. 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).
   4. Inject the antibodies prior to imaging (at least 15 min before) and after finishing the tissue preparation.
2. 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.
   1. 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).
   2. 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).
   3. To ensure a stable body temperature of 37 °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).
   4. 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).
   5. 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).
   6. 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).
   7. 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).
   8. 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).

3. Intravital multiphoton microscopy

1. 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.
   1. Remove the saline from the two prepared pockets and replace it with ultrasonic gel to cover the collateral vessels (Figure 2C).
   2. Inject the antibody solution through the tail vein catheter. 15 min after antibody injection, transfer the pad with the fixed mouse into the warmed multiphoton imaging chamber (Figure 2F).
   3. After completion of the image acquisition, euthanize the mouse by cervical dislocation under deep narcosis.
2. 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.
   1. 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).
   2. Launch the acquisition software (Inspector software). Set the wavelength to 800 nm and open the microscope shutter (Figure 3A ii).
   3. In the Measurement Wizard dialog, choose the Instrument Mode (Single beam) and in Measurement Mode (3D-scan Time lapse) (Figure 3A i).
   4. 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).
   5. 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.
   6. 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).
   7. 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).
   8. 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).
   9. 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 µm) position in the axial direction (Figure 3A vii).
   10. Set the step size to 2 µm. 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.
   11. If the microscope is equipped with live-drift correction⁹, start the drift correction software (e.g., VivoFollow) and follow the steps described below.
       1. 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.
       2. 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.
       3. Go to the xyz Stage Z dialog window and enlarge the Scan Range by 200 µm on both ends: in Start, enter (200 µm) and in End, enter (-240 µm), the range will automatically be set (-440 µm). Keep the step size (2 µm) as shown in Figure 3A xi.
       4. Open the VivoFollow Frontend dialog and click the Motion Profile box (Figure 3A xii).
       5. 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).
       6. 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 µm that was added to the first and last z positions (here, 200 µm) and click OK.
       7. 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.
       8. Stop imaging acquisition after ~35 min when another cycle of anesthesia re-injection is required. Inject half a dose of the MMF-mix (50 µl s.c) and re-start imaging acquisition by pressing the green arrowhead again (step 3.2.11.5).
       9. Repeat this procedure until the experiment is finished.

Results

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...

Discussion

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...

Materials

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