Visualization of Neutrophil Extracellular Traps in Mesenteric Venules After Mesenteric Ischemia-Reperfusion Injury via Intravital Microscopy

Summary

This protocol presents a method for inducing mesenteric ischemia-reperfusion injury and visualizing neutrophil extracellular traps (NETs) in mesenteric venules using intravital microscopy. Blood flow in the superior mesenteric artery was restricted for 1 h, followed by reperfusion, allowing direct quantification of leukocyte adhesion and NETosis.

Abstract

Intestinal ischemia-reperfusion (I/R) injury is an acute condition characterized by tissue damage resulting from restricted blood flow to the mesenteric vessels, leading to both local and systemic pathologies with a poor prognosis. Both ischemia and reperfusion trigger a series of cellular and molecular responses, with inflammatory cells serving as key regulators of the pathology. These interactions with the ischemic endothelium are mediated by multiple adhesion receptors. Several animal models have been established to mimic this pathology and investigate the involved molecular pathways. In this study, a microsurgical model of I/R injury is combined with intravital microscopy to visualize leukocyte rolling, adhesion, and neutrophil extracellular trap (NET) formation. This model is applied to transgenic mice deficient in endothelial PAR1 (F2r) to assess the impact of PAR1 on leukocyte rolling and NET formation 1 h after ischemia and immediately following reperfusion. In vivo, Acridine Orange leukocyte staining was employed, and NETs were visualized using a nucleic acid stain. Interestingly, reduced leukocyte adhesion and NET formation were observed in mice lacking the endothelial PAR1 receptor. This model enables the in vivo analysis of key regulators involved in I/R injury.

Introduction

Acute mesenteric ischemia (AMI) is a rare pathology¹ with a high mortality risk², characterized by reduced blood flow in the mesentery due to thrombi in the celiac axis, or the superior or inferior mesenteric artery. In 60%-70% of the cases, acute occlusions of the superior mesenteric artery due to thrombosis or embolization are responsible for acute bowel ischemia³. In contrast, in 5-15% of cases, mesenteric venous thrombosis accounts for mesenteric ischemia involving the superior mesenteric vein and, more rarely, the inferior mesenteric vein⁴. The hypoxia and malnutrition....

Protocol

All procedures involving mice were approved by the local committee on the legislation of animals (Landesuntersuchungsamt Rheinland-Pfalz, Koblenz, Germany; G21-1-041). 6-8 week-old male and female B6.FVB-F2rtm1a(EUCOMM) Tg/Cdh5-cre)7Mlia/Tarc mice were used for the study. The endothelial cell-specific regulation of F2r (PAR1) occurs via the VE-Cadherin Cre promoter²⁸. In this study, flox-F2r x VE-Cadherin Cre⁺ (F2R^ΔEC) exhibited reduced

Discussion

The mesenteric I/R injury combined with intravital microscopy was applied to a genetic mouse model of F2r (PAR1) deficiency in endothelial cells for the in vivo analysis of leukocytes and NETs after 1 h of ischemia. The mesenteric I/R injury model is frequently used in rodents with both ischemia and reperfusion times varying from minutes to several hours^23,31, influencing the inflammatory outcome³² and mortality

Materials

Name	Company	Catalog Number	Comments
Acridine orange	Sigma-Aldrich	A-6014
96-well plate Multiply PCR-Platten	Sarstedt	721977202.00
Anaesthesia Systems for Rodents	GROPPLER medizintechnik	Uni Vet -Porta T-8
Aqua ad injectabilia IVM	B Braun Melsungen	53402101
black dough	Staedtler		modelling clay
CD146 Microbeads, mouse	Miltenyi Biotec	130-092-007
cellSens	Olympus	Olympus cellSense Dimension Desktop 2.3	software
charge-coupled device camera	Hamamatsu Photonics	ORCA-R2	camera
cotton swabs	Böttger	09-119-9100
Curved Hemostat 125mm/5"	Indigo Instruments	22466
Depilatory cream	Balea	not applicable
Dorbene Vet	zoetis
Dumont Forceps	Fine Science Tools	11251-35
Ethanol p.a. 99,5 %	Roth	5504.20
Extra Narrow Scissors – Straight/Sharp-Sharp/10.5	FST	14088-10
Fentanyl	Janssen-Cilag GmBH
Forceps "Dumont #5" (Inox, tip 0.1mm, length 11 cm)	FST	11251-20
GentleMACS C Tubes	Miltenyi Biotec	130-093-236
Graphpadprism	Graphpad	Prism 10	software
isoflurane	Piramal	4150097146757.00
iTaq UniversalSYBR Green Supermix 10x5ml	BioRad	1725125.00
Leukofix medical tape	BSNMedical	0213600
Liver Dissociation Kit, mouse	Miltenyi Biotec	130-105-807
LS-Separation columns MACS	Miltenyi Biotec	130-042-401
MACS Smart Strainer 100 µm	Miltenyi Biotec	130-098-463
Microseal B Seal Seals	Biorad	MSB1001
Midazolam	hameln	5918501.00
NaCl	Braun	2350748
Needle holder	FST	12001-13
Nonabsorbable Braided Silk Suture, Size: 7/0, 91 Meters	Fine Science Tools	18020-70
Olympus BX51WI fluorescence microscope	Olympus		microscope
PCR for Tube Strips	Sarstedt	72985002.00
PCR Tube Lids	Sarstedt	65989002.00
Plyethylene Catheter	Smith Medical Deutschland GmbH	800/100/100
Prolene (8648G), Polypropylen 6-0, Nahtmaterial	Johnson & Johnson Medical GmbH	8695H
Relia Prep RNA Miniprep Systems	Promega	Z6112
Reverse Tanscription Kit (High Capacity cDNA)	Applied Biosystems	4368813.00
School scale	KERN & SOHN GmbH	EMB 500-1
Spring Scissors - Angled to Side	FST	15006-09
Stereo microscope	Leica	M50
Straight Hemostats 135mm/5.5"	Indigo Instruments	22468
Syringe 1 mL	Braun	9166017V
SYTOX Orange	Thermo Fisher Scientific	S11368
Temperature Controller TC-1000	FMI Föhr Medical Instruments GmbH	not applicable
Tissue Forceps - 1x2 Teeth	FST	18057-14
vascular clamp	Fine Science Tools	18055-02
Vetiva mini	WAHL	1584-0480