Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury (IRI) of the Rat Cremaster Muscle

Summary

Digital intravital epifluorescence microscopy of postcapillary venules in the cremasteric microcirculation is a convenient method to gain insights into leukocyte-endothelial interaction in vivo in ischemia-reperfusion injury (IRI) of striated muscle tissue. We here provide a detailed protocol to safely perform the technique and discuss its applications and limitations.

Abstract

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, intestinal ischemia as well as following transplant and cardiovascular surgery.¹ Reperfusion of previously ischemic tissue, while essential for the prevention of irreversible tissue injury, elicits excessive inflammation of the affected tissue. Adjacent to the production of reactive oxygen species, activation of the complement system and increased microvascular permeability, the activation of leukocytes is one of the principle actors in the pathological cascade of inflammatory tissue damage during reperfusion.^{2, 3} Leukocyte activation is a multistep process consisting of rolling, firm adhesion and transmigration and is mediated by a complex interaction between adhesion molecules in response to chemoattractants such as complement factors, chemokines, or platelet-activating factor.⁴

While leukocyte rolling in postcapillary venules is predominantly mediated by the interaction of selectins⁵ with their counter ligands, firm adhesion of leukocytes to the endothelium is selectin-controlled via binding to intercellular adhesion molecules (ICAM) and vascular cellular adhesion molecules (VCAM).^{6, 7}

Gold standard for the in vivo observation of leukocyte-endothelial interaction is the technique of intravital microscopy, first described in 1968.⁸

Though various models of IRI (ischemia-reperfusion injury) have been described for various organs, ^9-12 only few are suitable for direct visualization of leukocyte recruitment in the microvascular bed on a high level of image quality.⁸

We here promote the digital intravital epifluorescence microscopy of the postcapillary venule in the cremasteric microcirculation of the rat ¹³ as a convenient method to qualitatively and quantitatively analyze leukocyte recruitment for IRI-research in striated muscle tissue and provide a detailed manual for accomplishing the technique. We further illustrate common pitfalls and provide useful tips which should enable the reader to truly appreciate, and safely perform the method.

In a step by step protocol we depict how to get started with respiration controlled anesthesia under sufficient monitoring to keep the animal firmly anesthetized for longer periods of time. We then describe the cremasteric preparation as a thin flat sheet for outstanding optical resolution and provide a protocol for leukocyte imaging in IRI that has been well established in our laboratories.

Protocol

1. Anesthesia and Monitoring

1. Appropriate national and institutional ethics should be in place before performing animal experiments. Following approval from the ethics committee anesthetize male Sprague Dawley rats with a body weight from 120 - 180 g. Deliver 2 - 3 vol% isoflurane to a Plexiglas box via isoflurane vaporizer and place the rat inside.
2. As soon as the appropriate level of anesthesia is achieved (lack of reaction to toe or tail pinch) the rat is weighted and shaved on the ventral cervical area.
3. Place the rat in dorsal recumbency on a heating pad to maintain body temperature at 37 °C and apply isoflurane at 2 vol% usin....

Discussion

Leukocyte-endothelial interaction, production of reactive oxygen species and activation of the complement system are the key features of IRI-induced tissue dysfunction.²⁶ The microcirculation of the affected tissue is considered as the integral site for the inflammatory onset. Apart from ex vivo experiments such as flow chamber assays^{27, 28} it is mandatory to provide well-established models of intravital imaging to further evaluate the in vivo relevance. Though IRI has been implica.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Name of the equipment:	Company:	Catalogue No.:	Comments:
Forene 100% (V/V)	Abbot	B506	API isoflurane
Terylene Suture	Serag Weissner	OC108000
Portex Fine Bore Polythene Tubing	Smiths Medical	800/100/100	0.28 mm inner Diameter
0,9% saline solution	Fresinus Kabi	808771
Change-A-tip deluxe cautery kit	Bovie Medical	DEL1
Abbocath -T 14G	Venisystems	G713 - A01	used as lens tube
Servo Ventilator 900C	Maquet		used as animal ventialtor
Logical pressure transducer	Smiths Medical	MX1960
Sirecust 404 Monitor	Siemens
ABL 700 Benchtop Analyzer	Radiometer		for blood gas measurement
Heating pad	Effenberger	8319
Aluminum stage	Alfun	AW7022
Surgical microscope OPMI 6-SDFC	Carl Zeiss
Microsurgical instruments lab set	S&T	767
Biemer vessel clip	Diener	64.562
Applying forceps	Diener	64.568	for Biemer vessel clip
Rhodamine 6G	Sigma-Aldrich	R4127
Vaseline white DAB	Winthrop	2726853
Cover glasses			32x32 mm
Intravital setup
Zeis Axio Scope A-1 MAT	Carl Zeis	490036	epifluorescence microscope
470 nm LED	Carl Zeis	423052	fluorescence light source
Colibri 2 System	Carl Zeis	423052
W Plan-Apochromat 20x/1,0 DIC	Carl Zeis	421452	water immersion objective
AxioCam MRm Rev. 3 FireWire	Carl Zeis	426509	high resolution digital camera
Axio vision LE software	Carl Zeis	410130	use for offline analysis