Leukocyte Infiltration of Cremaster Muscle in Mice Assessed by Intravital Microscopy

10.3791/60509 ◽  
2020 ◽  
Author(s):  
Simon Alexander Kranig ◽  
Trim Lajqi ◽  
Raphaela Tschada ◽  
Maylis Braun ◽  
Navina Kuss ◽  
...  
Keyword(s):  
Intravital Microscopy ◽  
Leukocyte Infiltration ◽  
Cremaster Muscle

Rock Inhibitor Fasudil Reduces Leukocyte-Endothelium Interactions in the Microvasculature of a Sickle Cell Mouse Model of Allergic Inflammation

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 961-961
Author(s):  
Athos Rodrigues Moraes ◽  
Hanan Chweih ◽  
Nicola Conran ◽  
Kleber Yotsumoto Fertrin ◽  
Fernando Ferreira Costa ◽  
...  
Keyword(s):  
Sickle Cell ◽  
Intravital Microscopy ◽  
Kinase Inhibitors ◽  
Rho Kinase ◽  
Allergic Inflammation ◽  
Cremaster Muscle ◽  
Kinase Signaling ◽  
Cell Disease ◽  
Rho Kinase Inhibitors ◽  
Pre Treatment

Abstract Introduction: Sickle cell disease (SCD) vaso-occlusion process involves different cell types, such as red blood cells,activated endothelial cells, platelets and leukocytes. Endothelialdysfunction contributes to the vaso-occlusion process and leadsto inflammation. Data suggest that Rho-kinase signaling may regulate numerous aspects of the inflammatory process. Alterations in the Rho-A/Rho-kinase signaling pathway modulate pathophysiological aspects of the sickle cell disease such as priapism, and these enzymes are involved in increased reactive oxygen species generation and altered sickle cell cytoskeletal phosphorylation. In addition, Rho kinase inhibitors were able to reduce endothelial activation and consequent eosinophil adhesion in vitro and reduced allergic inflammation in the lungs of SCD mice. However, the involvement of Rho/Rho-kinase signaling pathways in the mechanism underlying systemic vascular occlusion in SCD remains unclear. This study aimed to determine whether Rho/Rho-kinase pathways are involved in the mechanism of microvascular SCD vaso-occlusion. We investigated the effect of fasudil, a specific inhibitor of Rho-kinase, in the initial steps of the leukocyte transmigration process in a model of allergic inflammation using intravital microscopy of the cremaster muscle in SCD mice. Methods: Experimental groups consisted of male homozygous Tim Townes transgenic sickle cell mice and C57BL/6JuniB control mice. SCD and control mice were actively sensitized with a subcutaneous injection of 100 μg of ovalbumin (OVA) mixed with 1.6 mg Al(OH)3 in 0.9% NaCl (Day zero). On day 7, mice received a second injection of 100 μg of OVA. On day 14, mice were subcutaneously challenged with eotaxin (100 ng/per dose) with or without pre-treatment with intraperitoneal fasudil (10 mg/kg) 1 hour before eotaxin. Four hours later, the animals were surgically prepared for intravital microscopy of the microvasculature of the cremaster muscle. Results: Intravital microscopy showed that eotaxin challenge in OVA-sensitized animals increased rolling and adhesion of leukocytes to the endothelium, and accumulation of leukocytes outside the vessels was observed. However, this increase is significantly higher in SCD mice compared to control animals (Rolling: 23±1.9 and 15±1.2 leukocyte min-1; Adhesion: 16.3±0.9 and 11.7±0.5 leukocyte adhered 100µm-1; Extravasation: 3.88±0.3 and 2.4±0.2 leukocyte perx100x50 µm2, p<0.05, respectively). The leukocyte rolling flow was similarly inhibited by fasudil treatment in control animals by 50% and in SCD mice by 42%. Eotaxin-induced firm adhesion after fasudil was also reduced by 57% in control animals and 63% in SCD mice. Notably, pre-treatment with fasudil caused a greater decrease in leukocyte extravasation in SCD mice (44%) than in control animals (16%), p<0.0001. Conclusion: Our data show that inhibition of Rho-kinase decreased endothelial-leukocyte interaction. These findings suggest that Rho-kinase inhibitors may have therapeutic benefits in the vaso-occlusive process in SCD, limiting the extravasation of leukocytes, and reducing vascular inflammation. Disclosures Conran: Bayer AG: Research Funding. Fertrin: Alexion Pharmaceuticals: Consultancy.

Ischemic preconditioning attenuates postischemic leukocyte adhesion and emigration

1996 ◽  
Vol 271 (5) ◽  
pp. H2052-H2059 ◽  
Author(s):  
T. Akimitsu ◽  
D. C. Gute ◽  
R. J. Korthuis
Keyword(s):  
Adenosine Deaminase ◽  
Topical Application ◽  
Ischemic Preconditioning ◽  
Intravital Microscopy ◽  
Leukocyte Adhesion ◽  
Inhibitory Effect ◽  
Cremaster Muscle ◽  
Beneficial Effects ◽  
Reperfusion Period

Intravital microscopy was used to determine whether ischemic preconditioning (IPC; 5 min ischemia and 10 min reperfusion) would attenuate leukocyte adhesion and emigration induced by subsequent prolonged ischemia (60 min) and reperfusion (60 min) (I/R) in murine cremaster muscle and whether adenosine produced during IPC and/or reperfusion contributed to these beneficial effects. I/R elicited a marked increase in the number of adherent and emigrated leukocytes compared with the nonischemic control muscles, an effect that was largely prevented by IPC. Superfusion of the cremaster with adenosine deaminase only during IPC or only during 60-min reperfusion attenuated the inhibitory effect of IPC on postischemic leukocyte adhesion and emigration. However, the beneficial effects of IPC were mimicked in cremaster muscles preconditioned with adenosine (topical application for 10 min beginning 20 min before the onset of prolonged ischemia). Similar results were obtained in experiments in which adenosine was topically applied to the cremaster only during the 60-min reperfusion period. Our findings suggest that the ability of IPC to attenuate postischemic leukocyte adhesion and emigration may be mediated by adenosine released during IPC and during reperfusion after prolonged ischemia.

Arteriolar tone is determined by activity of ATP-sensitive potassium channels

1993 ◽  
Vol 265 (5) ◽  
pp. H1797-H1803 ◽  
Author(s):  
W. F. Jackson
Keyword(s):  
Potassium Channels ◽  
Intravital Microscopy ◽  
Cheek Pouch ◽  
Cremaster Muscle ◽  
Adrenergic Agonists ◽  
Beta Adrenergic Agonists ◽  
Muscle Concentration ◽  
Arteriolar Tone ◽  
Arteriolar Vasodilation

The role of ATP-sensitive potassium channels (KATP) in determining resting arteriolar tone and vasodilator reactivity was assessed in superfused, hamster microcirculatory beds studied via intravital microscopy. Under resting conditions, the selective KATP blocker, glibenclamide, produced concentration-dependent vasoconstriction in both the cheek pouch and the cremaster muscle. Concentration-related constriction of cheek pouch arterioles was also observed with tetrapentylammonium, although this agent appeared to have toxic effects on the microcirculation. Glibenclamide (2 microM) abolished arteriolar vasodilation to cromakalim and pinacidil over a concentration range (10 nM-1 microM) in which these agents are selective KATP agonists and also significantly inhibited adenosine-, carbacyclin-, and isoproterenol-induced vasodilation. In contrast, responses to other vasodilators were not significantly affected [methacholine, forskolin, and dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP)] or only slightly depressed (sodium nitroprusside). Thus the activity of KATP determines, in part, resting arteriolar tone in the hamster. Furthermore, vasodilators like adenosine, beta-adrenergic agonists, and prostacyclin appear to act through these ion channels by a mechanism that may not involve cAMP.

Prostaglandins do not mediate arteriolar oxygen reactivity

1986 ◽  
Vol 250 (6) ◽  
pp. H1102-H1108 ◽  
Author(s):  
W. F. Jackson
Keyword(s):  
Arachidonic Acid ◽  
Phospholipase A2 ◽  
Intravital Microscopy ◽  
Cheek Pouch ◽  
Cyclooxygenase Inhibitor ◽  
Cremaster Muscle ◽  
Hamster Cheek Pouch ◽  
Nonspecific Effects ◽  
Oxygen Reactivity ◽  
Phospholipase A2 Inhibitors

The hypothesis that prostaglandins mediate arteriolar O2 reactivity was tested by assessing the effects of cyclooxygenase and phospholipase A2 inhibitors on the O2 responses of arterioles in superfused hamster cheek pouch and hamster and rat cremaster muscle preparations by use of intravital microscopy. Superfusion of these three preparations with the cyclooxygenase inhibitor indomethacin (50 microM) completely inhibited the response of the vessels to exogenous arachidonic acid but had no effect on the arteriolar constriction induced by elevation of superfusion solution PO2 from 15 to 150 mmHg. Similar results were obtained in the hamster cheek pouch with another cyclooxygenase inhibitor, meclofenamate, or when indomethacin (5-50 mg/kg) was administered systemically. Dexamethasone (12.7 microM) and quinacrine (10 microM), two reported inhibitors of phospholipase A2, also had no significant effect on arteriolar O2 reactivity in the cheek pouch. At 50 microM, quinacrine significantly depressed arteriolar reactivity to O2, adenosine, methacholine, and phenylephrine, suggesting nonspecific effects. These data do not support the hypothesis that prostaglandins mediate arteriolar O2 reactivity.

scholarly journals Sensing of Vascular Permeability in Inflamed Vessel of Live Animal

2018 ◽  
Vol 2018 ◽  
pp. 1-6 ◽  
Author(s):  
Sang A Park ◽  
Soi Jeong ◽  
Young Ho Choe ◽  
Young-Min Hyun
Keyword(s):  
Vascular Permeability ◽  
Real Time ◽  
Intravital Microscopy ◽  
Early Stage ◽  
Neutrophil Migration ◽  
Cremaster Muscle ◽  
Live Animal ◽  
Two Photon ◽  
Inflammatory State ◽  
Permeability Increase

Increase in vascular permeability is a conclusive response in the progress of inflammation. Under controlled conditions, leukocytes are known to migrate across the vascular barriers to the sites of inflammation without severe vascular rupture. However, when inflammatory state becomes excessive, the leakage of blood components may occur and can be lethal. Basically, vascular permeability can be analyzed based on the intensity of blood outflow. To evaluate the amount and rate of leakage in live mice, we performed cremaster muscle exteriorization to visualize blood flow and neutrophil migration. Using two-photon intravital microscopy of the exteriorized cremaster muscle venules, we found that vascular barrier function is transiently and locally disrupted in the early stage of inflammatory condition induced by N-formylmethionyl-leucyl-phenylalanine (fMLP). Measurement of the concentration of intravenously (i.v.) injected Texas Red dextran inside and outside the vessels resulted in clear visualization of real-time increases in transient and local vascular permeability increase in real-time manner. We successfully demonstrated repeated leakage from a target site on a blood vessel in association with increasing severity of inflammation. Therefore, compared to other methods, two-photon intravital microscopy more accurately visualizes and quantifies vascular permeability even in a small part of blood vessels in live animals in real time.

Velocity differences between L- and P-selectin-dependent neutrophil rolling in venules of mouse cremaster muscle in vivo

1996 ◽  
Vol 271 (6) ◽  
pp. H2740-H2747 ◽  
Author(s):  
U. Jung ◽  
D. C. Bullard ◽  
T. F. Tedder ◽  
K. Ley
Keyword(s):  
Intravital Microscopy ◽  
Surgical Trauma ◽  
Leukocyte Rolling ◽  
Cremaster Muscle ◽  
Wild Type ◽  
Rolling Velocity ◽  
Physiological Conditions ◽  
Whole Mount ◽  
Deficient Mice

After surgical trauma, leukocyte rolling is initially normal in L-selectin-deficient mice and reduced at later times, whereas leukocyte rolling is initially absent in P-selectin-deficient mice but induced later. Here, we examined the possibility that P- and L-selectin support rolling at different characteristic velocities using intravital microscopy of venules of the exteriorized cremaster muscle venules of wild type (WT) and P- and L-selectin-deficient mice. At > 50 min after exteriorization, rolling in P-selectin-deficient mice occurred at significantly higher velocities (129 +/- 89 microns/s) than in WT mice (49 +/- 23 microns/s). Rolling velocity distribution in L-selectin-deficient mice was similar to WT mice immediately after exteriorization. Histological examination of Giemsa-stained whole-mount preparations in cremaster muscle venules revealed that the majority of rolling cells (approximately 90% in all genotypes) were granulocytes. We conclude that P-selectin mediates leukocyte rolling at velocities < 50 microns/s, whereas L-selectin sustains more rapid rolling. Under physiological conditions, P- and L-selectin synergize to support rolling at velocities between 20 and 70 microns/s as seen in WT mice.

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