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 arteriog....

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 ....

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