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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.
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.
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 arising from ischemia lead to energy metabolism disorders at the cellular level5. For instance, impaired mitochondrial function activates multiple signaling pathways, initiating cellular death mechanisms6. Although rapid reperfusion enables the restoration of aerobic metabolism, the restoration of blood flow to an ischemic region often causes extensive tissue injury due to reactive oxygen species (ROS) production7, calcium overload8, endothelial dysfunction9, and inflammatory responses10.
Many experimental animal models employing temporary vascular occlusion of the superior mesenteric artery have been established to elucidate the molecular mechanisms during ischemia-reperfusion (I/R) injury11. The use of such models in combination with intravital microscopy (IVM) is fundamental for gaining insights into the underlying cellular interactions and disease progression11. Gut barrier dysfunction is observed shortly after ischemia12, with the epithelium losing its integrity13, resulting in bacterial translocation to extraintestinal sites such as the mesenteric lymph nodes, spleen, liver, and kidney12,14. Reperfusion further aggravates the mucosal injury15. An inflammatory response in the arterioles is manifested by the rolling and firm adhesion of leukocytes on the endothelial cell layer, which is dependent on molecules such as L-selectin16, P-selectin (CD62P)17, platelet GPIIb/IIIa, and fibrinogen18. Interestingly, leukocyte adhesion to mesenteric venules is influenced by the presence of gut microbiota19 and Toll-like receptor 4 signaling10. I/R injury also triggers neutrophil extracellular trap (NET) formation10. Therapeutic targeting of the extracellular DNA, originating from activated leukocytes such as neutrophils, improves the outcome of intestinal I/R injury20. In the liver, I/R injury is ignited by resident microphages21, and microRNA inhibition notably attenuates apoptosis22. Of note, I/R injury in the mesentery results in gut dysbiosis, which is reversible and resolved after revascularization23.
The endothelium is also influenced by the damaging effects of I/R injury, with ROS playing a pivotal role in the process24. Leukocyte adhesion to the endothelial cell layer, endothelial damage, and alterations of nitric oxide (NO) metabolism are observed9. However, the molecular pathways involved have not yet been fully explored.
Protease-activated receptor 1 (PAR1, F2r), is a membrane receptor expressed by a variety of cells, including endothelial cells, and is activated during inflammatory conditions25. Its activation occurs when serine proteases cleave the receptor at the N-terminus, exposing the tethered ligand that, in turn, activates the receptor by binding to the second extracellular loop of the molecule26. The endothelial protein C receptor (EPCR)-PAR1 signaling pathway impacts endothelial NO homeostasis and facilitates the restoration of blood flow in peripheral ischemia27.
In this study, I/R injury was applied to a genetic mouse model characterized by endothelial cell-specific PAR1 deficiency to visualize leukocyte rolling, adhesion, and NET formation. The combination of a genetic mouse model and intravital microscopy after I/R microsurgery provides valuable insights by highlighting the specific cell types involved in the disease progression. Applying I/R injury to genetic mouse models is instrumental in identifying potential therapeutic targets that can be used to improve the course of the disease. Moreover, the significance of endothelial PAR1 in NETosis was shown.
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+ (F2RΔEC) exhibited reduced F2r (PAR1) expression on endothelial cells, and flox-F2r x VE-Cadherin Cre- (WT) express normal endothelial F2r (PAR1) levels. The details of the reagents and the equipment used in this study are listed in the Table of Materials.
1. Preparation for the surgery
2. Animal preparation
3. Surgical procedure
4. Intravital high-speed video epifluorescence microscopy
5. Liver endothelial cell (LEC) isolation and quantitative RT-PCR
To study the endothelial cell-dependent effects on leukocyte rolling and adhesion as well as on NET formation, initially, the efficacy of the VE-Cadherin Cre inducible F2r-deficiency was examined in endothelial cells. Magnetic cell sorting was employed to isolate hepatic endothelial cells. Subsequently, RNA was isolated, followed by quantitative real-time PCR (qRT-PCR). The F2rΔEC mice, in which the Cre recombinase expressed under the control of the VE-Cadherin promoter is absent, showed...
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 hours23,31, influencing the inflammatory outcome32 and mortality
The authors disclose no conflict of interest.
C.R. acknowledges funding from the Forschungsinitiative Rheinland-Pfalz and ReALity (project MORE), the BMBF Cluster4Future CurATime (project MicrobAIome; 03ZU1202CA), the Wilhelm Sander-Stiftung (Nr. 2022.131.1), and the Deutsche Zentren der Gesundheitsforschung (DZG) Innovation Fund "Microbiome" (81X2210129). C.R. is a scientist at the German Center for Cardiovascular Research (DZHK). C.R. is a member of the Center for Translational Vascular Biology (CTVB), the Research Center for Immunotherapy (FZI), and the Potentialbereich EXPOHEALTH at the Johannes Gutenberg-University Mainz. C.R. is a member of the DFG Research Unit 5644 INFINITE (RE 3450/15-1). C.R. was awarded a Fellowship from the Gutenberg Research College at the Johannes Gutenberg-University Mainz. K.K. is supported by the DZHK "Promotion of Women Scientists" Excellence Programme and is a member of Young DZHK. Z.G and O.D. are PhD students at the Mainz Research School of Translational Biomedicine (TransMed).
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 |
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