Late Effects of TNF-α-Induced Inflammation on the Microcirculation of Cremaster Muscle Flaps under Intravital Microscopy

2002 ◽  
Vol 18 (1) ◽  
pp. 037-046 ◽  
Author(s):  
Kagan Ozer ◽  
Gokhan Adanali ◽  
Maria Siemionow
Keyword(s):  
Intravital Microscopy ◽  
Late Effects ◽  
Cremaster Muscle ◽  
Muscle Flaps ◽  
Tnf Α

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.

A Novel Selectin Antagonist, GMI-1070, Prevents Vaso-Occlusion in Sickle Cell Mice by Inhibiting Leukocyte Adhesion and Activation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2245-2245
Author(s):  
Jungshan Chang ◽  
John Patton ◽  
Arun Sarkar ◽  
John L. Magnani ◽  
Paul S. Frenette
Keyword(s):  
Sickle Cell ◽  
Intravital Microscopy ◽  
Competitive Inhibition ◽  
Leukocyte Adhesion ◽  
Fold Increase ◽  
Surgical Trauma ◽  
Mouse Bone Marrow ◽  
Leukocyte Rolling ◽  
Tnf Α ◽  
Adherent Leukocytes

Abstract Previous studies using intravital microscopy in a sickle cell disease (SCD) mouse model (Berkeley) suggest that adherent leukocytes (WBCs) play a key role in vaso-occlusion by capturing circulating erythrocytes (RBCs) in venules. In addition, mice deficient in both P-and E-selectins are protected from vaso-occlusion (VOC) induced by surgical trauma and TNF-α stimulation, suggesting that targeting selectins or their ligands represents a potentially useful strategy. Selectins bind to specific sialylated and fucosylated carbohydrate structures presented by glycoprotein or glycolipid ligands. Here, we tested the effect of novel small glycomimetic selectin inhibitors, GMI-1070 and GMI-1077, on leukocyte behavior and sickle cell VOC. Berkeley SCD mouse bone marrow was transplantated into lethally irradiated C57BL/6 animals to generate age- and gender-matched genetically identical cohorts of SCD mice. Fully engrafted male SCD mice were treated with TNF-α and prepared for intravital microscopy examination of the cremaster muscle 90 min later. GMI-1070, GMI-1077 (both 20 mg/kg) or vehicle (PBS) were administered immediately prior to cytokine stimulation (t=0 min), and an additional dose was given at t=70min. Another group of mice was injected with antibodies against P-and E-selectins (PES, 1 mg/kg) as positive control. Several post-capillary and collecting venules were examined between t= 90min and t= 150min. Antibody blockade of endothelial selectins completely ablated leukocyte rolling, whereas GMI-1070 and GMI-1077 significantly increased the rolling flux fractions (PBS: 5.0±1.2 GMI-1070: 10.6±1.3%%; GMI-1077: 9.9±1.0%; p< 0.001). Furthermore GMI-1070 and GMI-1077 significantly reduced the recruitment of adherent leukocytes (914±172 and 1433±119 cells/mm2, respectively) compared to sickle mice injected with PBS control (2400±392 cells/mm2, p< 0.001). Although the reduction in leukocyte adhesion was not as marked as with anti-P and E-selectins (61±25 cells/mm2, p< 0.001), GMI-1070, in particular, dramatically inhibited the capture of sickle RBCs by adherent leukocytes (PBS: 0.9±0.4, GMI-1077: 0.6±0.2, GMI-1070: 0.07±0.05 and PES: 0.01±0.01 RBC interactions/WBC/min, p< 0.05) and markedly improved the blood flow in venules (PBS: 312±24, GMI-1077: 398±41, GMI-1070: 710±68 and PES: 683±75 nL/s, p< 0.001), to levels observed in non-sickle mice. The increased leukocyte rolling fluxes by these glycomimetics suggest that they inhibit E-selectin > P-selectin. Since the hallmark of E-selectin-mediated adhesion is the slow leukocyte rolling, we analyzed leukocyte rolling velocities in the various group and indeed found a 2-fold increase in rolling velocities in sickle mice treated with GMI-1070 compared to PBS control (PBS: 21±1 μm/s, GMI-1070: 38±1 μm/s, p<0.001). Consistent with these results, other studies using a parallel plate flow chamber (0.9 dynes/cm2) revealed that GMI-1070 was much more potent (1000-fold difference) in inhibiting the binding of human PMNs to TNF-α-stimulated (to induce E-selectin) endothelial cells (HUVEC) than with IL-4 and histamine stimulated HUVECs (to induce P-selectin). Further, competitive inhibition assays revealed that the IC50 of GMI-1070, relative to the standard glycyrrhizin, was much lower for E-selectin than P-selectin. These studies suggest that E-selectin-mediated adhesion/signaling may play a more important role than previously appreciated in the pathophysiology of SCD, and suggest that GMI-1070 may be beneficial for the treatment of sickle cell vaso-occlusion.

Effects of Pan-Selectin Antagonist GMI-1070 on the Treatment of Vaso-Occlusion in Sickle Cell Mice

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 535-535 ◽  
Author(s):  
Jungshan Chang ◽  
John T Patton ◽  
Paul S. Frenette ◽  
John L. Magnani
Keyword(s):  
Blood Flow ◽  
Red Blood Cells ◽  
Sickle Cell ◽  
Small Molecule ◽  
Vascular Endothelium ◽  
Intravital Microscopy ◽  
Blood Cells ◽  
Tnf Α ◽  
Sickle Red Blood Cells ◽  
Adherent Leukocytes

Abstract Acute vaso-occlusion (VOC) in patients with sickle cell disease (SCD) induces intense pain arising from organ damage and is the major cause of morbidity and mortality. Hypoxia and abnormal sickle red blood cells (RBC) induce inflammatory mediators and activation of the vascular endothelium leading to the recruitment of adherent leukocytes and sickle RBC followed by aggregates that eventually occlude blood flow. Previous studies have implicated the critical roles of cell adhesion molecules E- and P-selectins by using intravital microscopy in SCD mice (Berkeley strain) with altered genetic backgrounds (SCD transplanted in recipients lacking E-and P-selectins), or antibodies against endothelial selectins, or small molecules directed against the selectins. Here, we designed a treatment protocol for this SCD mouse model, in which a small molecule pan-selectin antagonist (GMI-1070) is administered to sickle cell mice late in the process of established vaso-occlusion in order to test the effects of GMI-1070 in a more clinically relevant model. GMI-1070 is a small molecule pan-selectin antagonist designed on the bioactive conformation of the carbohydrate ligand and inhibits leukocyte adhesion to activated endothelium in vitro with particularly strong activity against E-selectin (IC50 = 3.4 μM). Berkeley SCD mice were generated by bone marrow transplantation into lethally irradiated C57BL/6 male mice and the fully engrafted (100% donor RBC chimerism) mice were used for intravital microscopy experiments. VOC events were induced by injection with TNF-α at time 0 and the formation of occlusions were allowed to proceed as long as possible just prior to the death of the control mice. GMI-1070 (20 mg/kg) or vehicle (PBS pH 7.4) were administered at t = 110 min. Post-capillary and collecting venules in the cremaster muscle were analyzed for effects on an established VOC event. Under these conditions, GMI-1070 significantly increased the microcirculatory blood flow to levels observed in non-sickle cell mice (vehicle: 237 ± 15 nL/sec; GMI-1070: 533 ± 58 nL/sec; p<0.0001). The recruitment of adherent leukocytes to the vascular endothelium was also significantly reduced (vehicle: 2235 ± 156; GMI-1070: 1270 ± 203 cells/mm2; p=0.0013), and there were significant and dramatic reductions in the capture of sickle red blood cells to adherent leukocytes (vehicle: 0.68 ± 0.27; GMI-1070: 0.03 ± 0.01 interactions/WBC, min, 100ml; p=0.0003). Mice began to succumb to VOC within 2.5 hours after injection of TNF-α and surgical trauma which continued until all of the control SCD mice died. Administration of GMI-1070 prevented the death of half of the treated mice within the timeframe of the experiment and extended the median survival of mice from 5 hours (control, vehicle-treated) to greater than 9 hours for the GMI-1070- treated SCD mice (p = 0.0067). These studies show that GMI-1070 can significantly and dramatically improve the condition and survival of the animals with a severe VOC even when dosed well after the initiating challenge. Thus these data strongly support the use of GMI-1070 for the treatment of patients in acute vaso-occlusive crisis. GMI-1070 is currently in a Phase I clinical trial.

scholarly journals TNF-α activation of arterioles and venules alters distribution and levels of ICAM-1 and affects leukocyte-endothelial cell interactions

2006 ◽  
Vol 291 (5) ◽  
pp. H2116-H2125 ◽  
Author(s):  
Ronen Sumagin ◽  
Ingrid H. Sarelius
Keyword(s):  
Spatial Distribution ◽  
Endothelial Cell ◽  
Inflammatory Responses ◽  
Leukocyte Adhesion ◽  
Leukocyte Rolling ◽  
Cremaster Muscle ◽  
Rolling Velocity ◽  
Tnf Α ◽  
Arteriolar Wall ◽  
Microvessel Wall

The observation that leukocyte-endothelial cell (EC) interactions are localized to specific regions on the microvessel wall suggests that adhesion molecule distribution is not uniform. We investigated ICAM-1 distribution and leukocyte-EC interactions in blood-perfused microvessels (<80 μm) in cremaster muscle of anesthetized mice, using intravital confocal microscopy and immunofluorescent labeling. Variability of ICAM-1 expression directly determines leukocyte adhesion distribution within the venular microcirculation and contributes to leukocyte rolling in arterioles during inflammation. The number of rolling interactions increased with ICAM-1 intensity ( r2 = 0.69, P < 0.05), and rolling velocity was lower in regions of higher ICAM-1 intensity. In controls, venular ICAM-1 expression was approximately twofold higher than in arterioles. After TNF-α treatment, ICAM-1 expression was significantly increased, 2.8 ± 0.2-fold in arterioles and 1.7 ± 0.2-fold in venules ( P < 0.05). ICAM-1 expression on activated arteriolar ECs only reached the level of control venular ICAM-1. Arteriolar but not venular ECs underwent redistribution of ICAM-1 among cells; some cells increased and some decreased ICAM-1 expression, magnifying the variability of ICAM-1. TNF-α treatment increased the length of bright fluorescent regions per unit vessel length (42%, control; 70%, TNF-α) along the arteriolar wall, whereas no significant change was observed in venules (60%, control; 63%, TNF-α). The spatial distribution and expression levels of adhesion molecules in the microcirculation determine the timing and placement of leukocyte interactions and hence significantly impact the inflammatory response. That arteriolar ECs respond to TNF-α by upregulation of ICAM-1, although in a different way compared with venules, suggests an explicit role for arterioles in inflammatory responses.

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

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.

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