Leukocyte Adhesion In Postcapillary Venules Research Articles

E3 Ubiquitin Ligase Midline 1 Regulates Endothelial Cell ICAM-1 Expression and Neutrophil Adhesion in Abdominal Sepsis

Septic lung damage is associated with endothelial cell and neutrophil activation. This study examines the role of the E3 ubiquitin ligase midline 1 (Mid1) in abdominal sepsis. Mid1 expression was increased in endothelial cells derived from post-capillary venules in septic mice and TNF-α challenge increased Mid1 levels in endothelial cells in vitro. The siRNA-mediated knockdown of Mid1 decreased TNF-α-induced upregulation of ICAM-1 and neutrophil adhesion to endothelial cells. Moreover, Mid1 silencing reduced leukocyte adhesion in post-capillary venules in septic lungs in vivo. The silencing of Mid1 not only decreased Mid1 expression but also attenuated expression of ICAM-1 in lungs from septic mice. Lastly, TNF-α stimulation decreased PP2Ac levels in endothelial cells in vitro, which was reversed in endothelial cells pretreated with siRNA directed against Mid1. Thus, our novel data show that Mid1 is an important regulator of ICAM-1 expression and neutrophil adhesion in vitro and septic lung injury in vivo. A possible target of Mid1 is PP2Ac in endothelial cells. Targeting the Mid1-PP2Ac axis may be a useful way to reduce pathological lung inflammation in abdominal sepsis.

TLR4 mutation protects neurovascular function and cognitive decline in high-fat diet-fed mice

BackgroundMetabolic syndrome (MS) is defined as a low-grade proinflammatory state in which abnormal metabolic and cardiovascular factors increase the risk of developing cardiovascular disease and neuroinflammation. Events, such as the accumulation of visceral adipose tissue, increased plasma concentrations of free fatty acids, tissue hypoxia, and sympathetic hyperactivity in MS may contribute to the direct or indirect activation of Toll-like receptors (TLRs), specifically TLR4, which is thought to be a major component of this syndrome. Activation of the innate immune response via TLR4 may contribute to this state of chronic inflammation and may be related to the neuroinflammation and neurodegeneration observed in MS. In this study, we investigated the role of TLR4 in the brain microcirculation and in the cognitive performance of high-fat diet (HFD)-induced MS mice.MethodsWild-type (C3H/He) and TLR4 mutant (C3H/HeJ) mice were maintained under a normal diet (ND) or a HFD for 24 weeks. Intravital video-microscopy was used to investigate the functional capillary density, endothelial function, and endothelial–leukocyte interactions in the brain microcirculation. Plasma concentrations of monocyte chemoattractant protein-1 (MCP-1), adipokines and metabolic hormones were measured with a multiplex immunoassay. Brain postsynaptic density protein-95 and synaptophysin were evaluated by western blotting; astrocytic coverage of the vessels, microglial activation and structural capillary density were evaluated by immunohistochemistry.ResultsThe HFD-induced MS model leads to metabolic, hemodynamic, and microcirculatory alterations, as evidenced by capillary rarefaction, increased rolling and leukocyte adhesion in postcapillary venules, endothelial dysfunction, and less coverage of astrocytes in the vessels, which are directly related to cognitive decline and neuroinflammation. The same model of MS reproduced in mice deficient for TLR4 because of a genetic mutation does not generate such changes. Furthermore, the comparison of wild-type mice fed a HFD and a normolipid diet revealed differences in inflammation in the cerebral microcirculation, possibly related to lower TLR4 activation.ConclusionsOur results demonstrate that TLR4 is involved in the microvascular dysfunction and neuroinflammation associated with HFD-induced MS and possibly has a causal role in the development of cognitive decline.

Abstract A195: Mechanosensory mechanisms and in vivo tissue topology contribute to rheology of circulating leukocytes resulting in efficient post-capillary vessel wall adhesion and recruitment

Abstract Efficient recruitment of circulating immune cells to various tissues plays a critical role in homeostasis and immune surveillance, a process that serves as the basis of any successful cell-based immunotherapeutic strategies in cancer, particularly for solid tumors and cancers residing in body sites outside of blood vessels and sinusoid network. Clinical and experimental observations suggest that in vivo leukocyte adhesion and extravasation are maximal near the transition from capillary to post-capillary venule, through a multistep process that includes the intravascular capture, rolling, arrest, crawling of cells through interactions of adhesion molecules (selectins, integrins, chemokines/receptors, for example), eventually leading to transcellular or paracellular transmigration through intact endothelium. These cellular and molecular processes are strongly influenced by a confluence of scale-dependent physical effects. Mimicking the scale of physiologic vessels using in vitro microfluidic systems allows the capture and investigation of these effects on leukocyte adhesion assays, but imposes practical limits on reproducibility and reliable quantification. We have developed a microfluidic platform that provides multiple (54-512) technical replicates within a 15-minute sample collection time, coupled with an automated computer vision analysis pipeline that captures leukocyte adhesion probabilities as a function of not only shear stress imposed on leukocytes within the vessels as conventional wisdom dictates, but also of the extensional stresses imposed by the topology of post-capillary venules and the rheology of circulating leukocytes in these vessels. We identified that in post-capillary channels of physiologic scale, efficient leukocyte adhesion requires erythrocytes forcing leukocytes against the wall, a phenomenon that is promoted by the transitional flow in post-capillary venule expansions and highly dependent on the adhesion molecule ICAM-1. These studies help identified a mechanosensory mechanism that determines the increased likelihood of leukocyte adhesion in post-capillary venules, and further suggests a significant role of mechanosensory channel(s) in influencing leukocyte integrin affinity for cellular capture to the vessel wall. Through a series of truncation mutants, we have narrowed down a small region of putative interacting domain between integrin and a candidate mechanosensory channel—PIEZO1—on leukocytes. These ongoing investigations offer new insights into immune cellular recruitment and new molecular targets to enhance leukocyte recruitment to peripheral tumor sites. Citation Format: Alex Y. Huang, Bryan L. Benson, Luis Correa, Lucy Li, Jay T. Myers, Umut A. Gurkan, Richard Ransohoff. Mechanosensory mechanisms and in vivo tissue topology contribute to rheology of circulating leukocytes resulting in efficient post-capillary vessel wall adhesion and recruitment [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A195.

Cerebral tissue oxygenation impairment during experimental cerebral malaria

Ischemia and hypoxia have been implicated in cerebral malaria (CM) pathogenesis, although direct measurements of hypoxia have not been conducted. C57BL/6 mice infected with Plasmodium berghei ANKA (PbA) develop a neurological syndrome known as experimental cerebral malaria (ECM), whereas BALB/c mice are resistant to ECM. In this study, intravital microscopy methods were used to quantify hemodynamic changes, vascular/tissue oxygen (O2) tension (PO2), and perivascular pH in vivo in ECM and non-ECM models, employing a closed cranial window model. ECM mice on day 6 of infection showed marked decreases in pial blood flow, vascular (arteriolar, venular), and perivascular PO2, perivascular pH, and systemic hemoglobin levels. Changes were more dramatic in mice with late-stage ECM compared with mice with early-stage ECM. These changes led to drastic decreases in O2 delivery to the brain tissue. In addition, ECM animals required a greater PO2 gradient to extract the same amount of O2 compared with non-infected animals, as the pial tissues extract O2 from the steepest portion of the blood O2 equilibrium curve. ECM animals also showed increased leukocyte adherence in postcapillary venules, and the intensity of adhesion was inversely correlated with blood flow and O2 extraction. PbA-infected BALB/c mice displayed no neurological signs on day 6 and while they did show changes similar to those observed in C57BL/6 mice (decreased pial blood flow, vascular/tissue PO2, perivascular pH, hemoglobin levels), non-ECM animals preserved superior perfusion and oxygenation compared with ECM animals at similar anemia and parasitemia levels, resulting in better O2 delivery and O2 extraction by the brain tissue. In conclusion, direct quantitative assessment of pial hemodynamics and oxygenation in vivo revealed that ECM is associated with severe progressive brain tissue hypoxia and acidosis.

Prolyl Hydroxylase Inhibition Attenuates Mast Cell Degranulation During Systemic Hypoxia Via Upregulation of Heme Oxygenase‐1

Acute systemic hypoxia (Hx) causes mast cell degranulation and increased leukocyte adherence in post‐capillary venules of rats. When animals are chronically hypoxic, microvascular inflammation resolves in part due to upregulation of heme oxygenase‐1 (HO‐1), which prevents mast cell activation. Inhibition of prolyl hydroxylase (PHD) increases hypoxia‐inducible factor‐1 (HIF‐1) levels, which promotes HO‐1 expression.ObjectiveOur goal was to evaluate whether inhibition of PHD attenuated Hx‐induced mast cell degranulation, and if upregulation of HO‐1 was involved.MethodsIntravital microscopy was used to measure mast cell degranulation and leukocyte adherence in mesenteric venules of anesthetized rats. The PHD inhibitor ethyl dihydroxybenzoate (EDHB) was injected s.c. to rats 24 hrs prior to experiments. Hx was produced in rats breathing 10% O2.ResultsHx caused mast cell degranulation and increased leukocyte adherence in the mesenteric microcirculation of vehicle‐ but not EDHB‐treated rats. Superfusion of the mesentery with the HO‐1 inhibitor zinc protoporphyrin caused a significant increase in mast cell degranulation and leukocyte adherence during Hx in EDHB‐ but not in vehicle‐treated rats.ConclusionInhibition of PHD reduces Hx‐induced mast cell degranulation and leukocyte adherence in part through HO‐1 upregulation, which is consistent with a HIF‐1‐dependent mechanism.

Curcumin prevents indomethacin-induced gastropathy in rats

To investigate the effects of curcumin on gastric microcirculation and inflammation in rats with indomethacin-induced gastric damage. Male Sprague-Dawley rats were randomly divided into three groups. Group 1 (control group, n = 5) was fed with olive oil and 5% NaHCO(3) (-) (vehicle). Group 2 [indomethacin (IMN) group, n = 5] was fed with olive oil 30 min prior to indomethacin 150 mg/kg body weight (BW) dissolved in 5% NaHCO(3) (-) at time 0th and 4th h. Group 3 (IMN + Cur group, n = 4) was fed with curcumin 200 mg/kg BW dissolved in olive oil 0.5 mL, 30 min prior to indomethacin at 0th and 4th h. Leukocyte-endothelium interactions at postcapillary venules were recorded after acridine orange injection. Blood samples were determined for intercellular adhesion molecule (ICAM)-1 and tumor necrosis factor (TNF)-α levels using enzyme linked immunosorbent assay method. Finally, the stomach was removed for histopathological examination for gastric lesions and grading for neutrophil infiltration. In group 2, the leukocyte adherence in postcapillary venules was significantly increased compared to the control group (6.40 ± 2.30 cells/frame vs 1.20 ± 0.83 cells/frame, P = 0.001). Pretreatment with curcumin caused leukocyte adherence to postcapillary venule to decline (3.00 ± 0.81 cells/frame vs 6.40 ± 2.30 cells/frame, P = 0.027). The levels of ICAM-1 and TNF-α increased significantly in the indomethacin-treated group compared with the control group (1106.50 ± 504.22 pg/mL vs 336.93 ± 224.82 pg/mL, P = 0.011 and 230.92 ± 114.47 pg/mL vs 47.13 ± 65.59 pg/mL, P = 0.009 respectively). Pretreatment with curcumin significantly decreased the elevation of ICAM-1 and TNF-α levels compared to treatment with indomethacin alone (413.66 ± 147.74 pg/mL vs 1106.50 ± 504.22 pg/mL, P = 0.019 and 58.27 ± 67.74 pg/mL vs 230.92 ± 114.47 pg/mL, P = 0.013 respectively). The histological appearance of the stomach in the control group was normal. In the indomethacin-treated group, the stomachs showed a mild to moderate neutrophil infiltration score. Gastric lesions were erosive and ulcerative. In rats treated with indomethacin and curcumin, stomach histopathology improved and showed only a mild neutrophil infiltration score and fewer erosive lesions in the gastric mucosa. The results indicate that curcumin prevents indomethacin-induced gastropathy through the improvement of gastric microcirculation by attenuating the level of ICAM-1 and TNF-α.

Antimyeloperoxidase antibodies rapidly induce α4-integrin–dependent glomerular neutrophil adhesion

AbstractPatients with antineutrophil cytoplasmic antibodies (ANCAs) frequently develop severe vasculitis and glomerulonephritis. Although ANCAs, particularly antimyeloperoxidase (anti-MPO), have been shown to promote leukocyte adhesion in postcapillary venules, their ability to promote adhesion in the glomerular vasculature is less clear. We used intravital microscopy to examine glomerular leukocyte adhesion induced by anti-MPO. In mice pretreated with LPS, 50 μg anti-MPO induced LFA-1–dependent adhesion in glomeruli. In concert with this finding, in mice pretreated with LPS, more than 80% of circulating neutrophils bound anti-MPO within 5 minutes of intravenous administration. However, even in the absence of LPS, more than 40% of circulating neutrophils bound anti-MPO in vivo, a response not seen in MPO−/− mice. In addition, a higher dose of anti-MPO (200 μg) induced robust glomerular leukocyte adhesion in the absence of LPS. The latter response was β2-integrin independent, instead requiring the α4-integrin, which was up-regulated on neutrophils in response to anti-MPO. These data indicate that anti-MPO antibodies bind to circulating neutrophils, and can induce glomerular leukocyte adhesion via multiple pathways. Lower doses induce adhesion only after an infection-related stimulus, whereas higher doses are capable of inducing responses in the absence of an additional inflammatory stimulus, via alternative adhesion mechanisms.

Attenuation of changes in capillary fine structure and leukocyte adhesion improves muscle performance following chronic ischaemia in rats

Acute ischaemia-reperfusion disrupts capillary fine structure and increases leukocyte adhesion in postcapillary venules. We determined whether chronic muscle ischaemia has similar consequences, and whether it is possible to ameliorate its effect on muscle performance. Following ischaemia (unilateral ligation, common iliac artery) rat hindlimb muscles were examined without other intervention or following treatment with an xanthine oxidase inhibitor (allopurinol), a Na(+)/H(+) exchange blocker (amiloride), or an oxygen free radical scavenger (vitamin E). No significant leukocyte adhesion or rolling, nor changes in capillary fine structure were observed 3 days postsurgery, when limb use was limited. However, leukocyte rolling and adhesion almost trebled by 7 days (P < 0.001), when normal gait was largely restored. Capillary fine structure was disturbed over a similar time course, e.g. relative endothelial volume (control 46%, 7 days 61%; P < 0.05), that resolved by 5 weeks. Where activity was increased by mild electrical stimulation 3 days after ligation muscles showed enhanced capillary swelling (endothelial volume 66% versus 50%, P < 0.005), but improved fatigue index (52% versus 16%, P < 0.001) as a result of greater blood flow. Muscle fatigue after ligation was related to the extent of contraction-induced hyperaemia (R(2) = 0.725), but not capillary swelling. Amiloride, and to a lesser extent allopurinol but not vitamin E, significantly decreased leukocyte rolling and adhesion, as well as capillary endothelial swelling. We conclude that increased activity of ischaemic muscles on recovery is likely to accentuate acidosis accompanying changes in microcirculation and contribute to enhanced muscle fatigue, whereas formation of oxygen free radicals may be attenuated by endogenous protective mechanisms.

Pancreatic Ischemia/Reperfusion Injury

Pancreatic ischemia/reperfusion injury (IRI) can influence the results after transplantation. Temperature during ischemia can affect IRI. A temperature of 4 degrees C is assumed as optimal for graft preservation. There are no data about the impact of different ischemia temperatures in pancreatic IRI. Ischemia/reperfusion injury was induced in pancreatic tail segments (2-hour ischemia, 2-hour reperfusion), with rats (7/group) without ischemia served as control. Animals were randomized to the different experimental groups. To achieve the desired temperature (4, 18, or 37 degrees C and 37 degrees C control), pancreatic tail segments were superfused with temperated saline. After reperfusion, microcirculation was observed by intravital fluorescence microscopy. Functional capillary density (FCD), leukocyte adherence in post-capillary venules, and histological damage were analyzed. In IRI groups, decrease of FCD 1 and 2 hours after reperfusion compared with baseline measurements was significant. Functional capillary density in 4 degrees C was better as compared with 18 and 37 degrees C after reperfusion. Lower adherent leukocytes were seen in 4 and 18 degrees C, compared with 37 degrees C and also to CO. In 4 degrees C, histological damage was lower as compared with 18 and 37 degrees C. We could demonstrate that also in pancreatic IRI, tissue injury is temperature dependent. Compared with 37 degrees C, although a protective effect is established already at 18 degrees C, more protection is achieved with storage at 4 degrees C. Our data suggest that 4 degrees C has the best protective effect on pancreatic IRI.

Prolyl Hydroxylase Inhibition Attenuates Hypoxia‐Induced Microvascular Inflammation Via iNOS Upregulation

Acute systemic hypoxia (Hx) promotes leukocyte adherence in post‐capillary venules of rats. When animals are chronically hypoxic, the initial microvascular inflammation resolves in part due to upregulation of inducible nitric oxide synthase (iNOS). Inhibition of prolyl hydroxylase (PHD) has been shown to increase hypoxia‐inducible factor‐1 (HIF‐1) levels, which can increase iNOS expression. Objective: This study evaluated whether inhibition of PHD attenuated Hx‐induced microvascular inflammation, and if upregulation of iNOS was involved. Methods: Intravital microscopy was used to measure leukocyte adherence in mesenteric venules of anesthetized rats. The PHD inhibitor EDHB was administered to rats 24 hrs prior to experiments. Results: Hx caused a rapid increase in leukocyte adherence in vehicle‐ but not in EDHB‐treated rats. Superfusion of the mesentery with the iNOS inhibitor L‐NIL caused a small but significant increase in adherent leukocytes during Hx in EDHB‐ but not vehicle‐treated rats. Conclusion: These results show that inhibition of PHD reduces Hx‐induced microvascular inflammation in part through iNOS upregulation, and are consistent with a HIF‐1‐dependent mechanism.

Remote Preconditioning Reduces Microcirculatory Disorders in Pancreatic Ischemia/Reperfusion Injury

Remote preconditioning (RPC) can protect from ischemia/reperfusion injury (IRI). We investigated the influence of RPC in pancreatic IRI. Wistar rats were randomized to 2 hours of ischemia and 2 hours of reperfusion of a pancreatic tail segment with or without 15 minutes of infrarenal ischemia 60 minutes before IRI. Microcirculatory measurements before ischemia and 1 and 2 hours after reperfusion included functional capillary density and leukocyte adherence in postcapillary venules, quantified by intravital fluorescence microscopy. Histology and tissue myeloperoxidase activity were further parameters of pancreatic injury. Remote preconditioning caused an improvement of microcirculation (functional capillary density: 1 hour after reperfusion, 460 +/- 13 vs 350 +/- 9 cm/cm2; 2 hours after reperfusion, 437 +/- 13 vs 295 +/- 13 cm/cm2; P < 0.01) and reduced inflammatory tissue response (leukocyte adherence in postcapillary venules: 2 hours after reperfusion, 155 +/- 55 vs 748 +/- 187 cells/mm2; P < 0.01). Histology was significantly better in preconditioned animals (IR, 8.1+/- 1.3 score points; RPC, 6.2 +/- 1.3 score points; P < 0.05). The difference in myeloperoxidase activity was not significant (ischemia/reperfusion [IR], 105 +/- 72; RPC, 245 +/- 209 mU x min(-1) x mg(ti)(-1); P = 0.13). With our dynamic functional microcirculatory measurements, we could demonstrate that RPC is a feasible method to reduce experimental pancreatic IRI. This was seen in an attenuation of nutritive tissue perfusion and a reduction of inflammatory tissue response and a lower histological damage. Because it is easy to perform before organ harvest, RPC could be a step to improve organ procurement in pancreas transplantation. Clinical studies are the next step to evaluate RPC in pancreas transplantation.

P‐selectin mediates both arteriolar dysfunction and venular inflammation associated with cytomegalovirus and hypercholesterolemia

Cytomegalovirus (CMV), a β-herpesvirus, has been implicated in the inflammation associated with several vascular diseases. CMV upregulates adhesion molecules and induces leukocyte adhesion in vitro. This study examines the role of P-selectin in the microvascular inflammation that occurs in hypercholesterolemic mice infected with CMV. C57Bl/6 mice were mock-inoculated or infected with murine CMV, and 5wk later placed on normal diet (ND) or high cholesterol diet (HC) for 4wk. Separate CMV-HC groups received P-selectin blocking Ab 18 h before observation. Acetylcholine-induced arteriolar vasodilation and leukocyte adhesion in postcapillary venules were monitored in the cremaster muscle. CMV alone, but not HC alone, impaired arteriolar function. However, leukocyte adhesion was only elevated when CMV was combined with HC. Pre-treatment of CMV-HC mice with P-selectin Ab reduced leukocyte adhesion to levels observed in mock-ND mice. Blocking P-selectin also conferred protection against arteriolar dysfunction in this group. These findings implicate P-selectin in the leukocyte recruitment elicited by CMV infection combined with hypercholesterolemia, and suggest that P-selectin contributes to arteriolar dysfunction through a pathway that is independent of leukocyte adhesion in these vessels. (Supported by AHA 0565285B).

Influence of nitric oxide on microcirculation in pancreatic ischemia/reperfusion injury: an intravital microscopic study

Abstract Recently, protective effects of nitric oxide donors in pancreatic ischemia/reperfusion (IRI) injury have been described. Their role in post-ischemic microcirculation was previously not investigated. Ischemia reperfusion was induced in an isolated pancreatic tail segment in situ. Animals were randomized to four experimental groups (n= 7 animals/group), the control group (CO) received saline as placebo. Treatment groups received either sodium nitroprusside (SN) 5 min before until 2 h after reperfusion, l-arginine (LA) 30 min before reperfusion until 2 h after reperfusion or sodium nitroprusside and l-arginine (SNLA) together. After induction of ischemia (2 h) post-ischemic microcirculation was observed for 2 h by intravital-fluorescence microscopy. Functional-capillary density (FCD), leukocyte adherence in post-capillary venules (LAV) and histological damage were analysed. After reperfusion FCD decreased in all groups (p<0.05). FCD was significantly restored in all groups with administration of nitric oxide donors after reperfusion (p<0.05) as compared to CO without significant difference between the individual nitric oxide donor groups. Leukocyte adherence was significantly increased 1 h and 2 h after reperfusion (p<0.001) as compared to baseline, which was lower in all nitric oxide donor groups. Histological damage in the pancreatic tail-segment was significantly reduced in nitric oxide donor groups (p<0.01). Administration of nitric oxide donors might be useful in ischemia-reperfusion injury of the pancreas by its protective effect on microcirculation and inflammatory reaction.

Influence of nitric oxide on microcirculation in pancreatic ischemia/reperfusion injury: an intravital microscopic study

Recently, protective effects of nitric oxide donors in pancreatic ischemia/reperfusion (IRI) injury have been described. Their role in post-ischemic microcirculation was previously not investigated. Ischemia reperfusion was induced in an isolated pancreatic tail segment in situ. Animals were randomized to four experimental groups (n=7 animals/group), the control group (CO) received saline as placebo. Treatment groups received either sodium nitroprusside (SN) 5 min before until 2 h after reperfusion, L-arginine (LA) 30 min before reperfusion until 2 h after reperfusion or sodium nitroprusside and L-arginine (SNLA) together. After induction of ischemia (2 h) post-ischemic microcirculation was observed for 2 h by intravital-fluorescence microscopy. Functional-capillary density (FCD), leukocyte adherence in post-capillary venules (LAV) and histological damage were analysed. After reperfusion FCD decreased in all groups (P<0.05). FCD was significantly restored in all groups with administration of nitric oxide donors after reperfusion (P<0.05) as compared to CO without significant difference between the individual nitric oxide donor groups. Leukocyte adherence was significantly increased 1 h and 2 h after reperfusion (P<0.001) as compared to baseline, which was lower in all nitric oxide donor groups. Histological damage in the pancreatic tail-segment was significantly reduced in nitric oxide donor groups (P<0.01). Administration of nitric oxide donors might be useful in ischemia-reperfusion injury of the pancreas by its protective effect on microcirculation and inflammatory reaction.

Ischemic preconditioning attenuates capillary no-reflow and leukocyte adherence in postischemic pancreatitis.

Ischemic preconditioning (IPC) has been shown to protect several organs from ischemia-reperfusion injury. Postischemic microvascular dysfunction is considered to be the key mechanism of early graft pancreatitis after transplantation. The aim of the study was to determine whether brief ischemia and reperfusion before prolonged ischemia followed by reperfusion is protective in respect to microcirculatory derangement in postischemic pancreatitis. In an in-situ model of ischemia-reperfusion was induced in the isolated pancreatic tail segment. Wistar rats were randomized to one group ( n=7/group) with 2-h ischemia and reperfusion (I/R) and another group with 10-min ischemia and 10-min reperfusion (IPC) before the prolonged ischemia time. Microcirculation was observed for 2 h by intravital-fluorescence microscopy that analyzed functional capillary density and leukocyte adherence in postcapillary venules. Histological damage was quantified by a semiquantitative score (edema, vacuolization, PMN infiltration, necrosis). IPC resulted in a significant improvement of functional capillary density (248+/-20 vs 372+/-8 cm(-1), P<0.001), a significant reduction in leukocyte adherence in postcapillary venules (476+/-79 vs 179+/-15 cells/mm(2), P<0.001) and in significantly lower histological damage (score 9+/-0.8 vs 5+/-1.4, P<0.001), when compared with the ischemia-reperfusion group. IPC reduces pancreatic inflammatory reaction by preservation of postischemic microcirculation. Therefore, it might become a useful procedure before organ procurement in pancreas transplantation.

Characterization of microcirculatory disturbance in a novel model of pancreatic ischemia-reperfusion using intravital fluorescence-microscopy.

Microcirculatory disturbances caused by ischemia-reperfusion injury (IRI) are the crucial hallmarks of pancreatitis following pancreas transplantation. To develop a novel rodent model of normothermic in situ ischemia of a pancreatic tail-segment that simulates the clinical situation of pancreas transplantation by flushing the organ via an inserted microcatheter and thus enables selective treatment of the organ via this access. Four experimental groups were investigated (n = 7 Wistar rats/group): sham animals without ischemia and dissection of the pancreas; control animals with dissection of a pancreatic tail segment pedunculated on the splenic vessels and flushing od this segment with saline via a microcatheter; and two groups of animals treated like controls with a pancreatic ischemia time of 1 hour or 2 hours. With use of intravital epifluorescence microscopy, the microcirculatory damage was characterized by investigation of functional capillary density (FCD) and leukocyte adherence in postcapillary venules (LAV) before ischemia and during a reperfusion time of 2 hours. Dry:wet ratio determinations, light microscopy, and electron microscopic investigations were performed to characterize the histologic organ damage. FCD decreased significantly (p < 0.05) 2 hours after reperfusion in the groups of 1-hour (-29.21%) and 2-hour ischemia (-42.73%), in comparison with baseline values. LAV increased significantly (p < 0.05), 4.3- and 5.8-fold, after 1-hour and 2-hour ischemia during the observation time. The histologic damage was similar to posttransplantation pancreatitis in humans 1 hour after reperfusion. In sham and control animals these alterations were not significant. The rodent in situ model of pancreatic IRI showed standardized microcirculatory damage dependent on the ischemia time. Offering the possibility of selective treatment by the direct artery access to the ischemic pancreatic area, the model enables investigations of questions related to human pancreas transplantation.

Protective effect of ketoprofen lysine salt on interleukin-1beta and bradykinin induced inflammatory changes in hamster cheek pouch microcirculation.

The purpose of this study was to assess the efficacy of topically applied ketoprofen lysine salt (KLS), a cyclooxygenase inhibitor, against the inflammatory changes induced by interleukin-1beta (IL-1beta) and bradykinin (BK) in hamster cheek pouch microcirculation. In addition, we characterised the pharmacological regulation of IL-1beta activity in this model. Male Syrian hamsters were used. Microcirculation was visualised by fluorescent microscopy. Leukocyte adhesion, permeability, perfused capillary length (PCL) and capillary red blood cell (RBC) velocity were evaluated. KLS (25 microg/ml/min to 1.6 mg/ml/min) was topically applied for 3 min before topically administered IL-1beta (1microg/ml) and BK (10(-4) M). Monoclonal anti-mouse IL-1beta receptor antagonist (200 ng/ml), BK-B2 receptor antagonist (10(-6) M), PAF inhibitor (10(-5) M) and cycloheximide (10 microg/ml) were added topically 15, 10, 15 and 60 min, respectively, before IL-1beta (1 microg/ml). IL-1beta caused a significant increase in microvascular permeability, a decrease in capillary RBC velocity followed by increased leukocyte adhesion in postcapillary venules. BK caused a marked increase in leukocyte adhesion and no decrease in PCL and RBC velocity. Treatment with KLS significantly inhibited both the leukocyte adhesion and microvascular leakage induced by the two mediators. The inflammatory effects induced by IL-1beta were reduced by blockade of IL-1beta receptors and by a BK-B2 receptor antagonist but were not affected by a PAF antagonist and protein synthesis inhibition. These results demonstrate that KLS is effective in preventing early inflammatory changes induced by both IL-1beta and BK in the capillary network. Prostaglandin release and BK are essential components for IL-beta mediated responses, whereas neither PAF nor new protein synthesis appear to be linked to the early inflammatory changes induced by IL-1beta.

Effects of [formula omitted]-NA and Sodium Nitroprusside on Ischemia/Reperfusion-Induced Leukocyte Adhesion and Macromolecular Leakage in Hamster Cheek Pouch Venules

Our objective was to study how the topical application of a nitric oxide synthase inhibitor (l-NA, Nω-nitro-l-arginine) and a nitric oxide donor, sodium nitroprusside (SNP), could modulate leukocyte adhesion (sticking) and microvascular permeability as altered by ischemia/reperfusion (I/R) and topically applied histamine after I/R. Golden hamsters were prepared for intravital microscopy. Ischemia was induced by an inflatable silicon rubber cuff mounted around the neck of the cheek pouch prepared for intravital microscopy. Saline, l-NA, sodium nitroprusside, and histamine were applied in the superfusion solution. FITC–dextran was injected iv 30 min before initiation of ischemia as a marker of microvascular permeability. l-NA 10−5 M inhibited both the increase in number of sticking leukocytes and the increase in vascular permeability after I/R compared with the untreated control group of hamsters. SNP neutralized this effect of l-NA on leukocytes and vascular permeability and caused arteriolar dilation at the concentration used, 10−6 M. Both SNP and l-NA + SNP enhanced the I/R-induced macromolecular leakage. The topical application of SNP and SNP + l-NA did not modify the response to histamine after I/R compared with the untreated control group. In hamsters not subjected to I/R, histamine-induced macromolecular leakage was inhibited by l-NA and l-NA + SNP but was unchanged by SNP. It is concluded that inhibition of nitric oxide formation by l-NA reduced both leukocyte adhesion in postcapillary venules and the increase in macromolecular leakage and that a NO donor such as SNP could enhance the macromolecular leakage response to I/R.

Reduction of inflammatory response in composite flap transfer by local stress conditioning-induced heat-shock protein 32

Background. The failure of composite flaps despite anastomotic patency is thought to be mediated by the inflammatory response within the microvasculature, which results from unavoidable surgical trauma and transfer-related ischemia-reperfusion. Evidence suggests that stress conditioning may improve flap survival; however, the molecular mechanisms of protection are far from being clear. Therefore, we analyzed whether stress conditioning-induced heat-shock protein 32 is effective to prevent the inflammatory response in transferred osteomyocutaneous flaps. Methods. In a rat model, leukocyte-endothelial cell interaction and endothelial integrity disruption as early indicators of the inflammatory response were quantitatively analyzed in muscle, subcuticular tissue, and periosteum of microvascularly transferred osteomyocutaneous flaps by using intravital fluorescence microscopy. Twenty-four hours before flap transfer, stress conditioning was induced by local heating of the left hindlimb up to 42.5°C for 30 minutes. In additional animals, stress conditioning-induced activity of heat-shock protein 32 was inhibited by tin protoporphyrin-IX. Unconditioned flaps served as controls. Results. In all tissues analyzed, control flaps showed significant leukocyte adherence in postcapillary venules, increased intercellular adhesion molecule-1 (ICAM-1) expression, and endothelial integrity disruption, but a lack of heat-shock protein 32. In contrast, stress conditioning induced marked heat-shock protein 32 expression, which was associated with a significant reduction (P <.05) of leukocyte adherence, ICAM-1 expression, and endothelial hyperpermeability. The inhibition of heat-shock protein 32 by tin protoporphyrin-IX completely abolished the stress conditioning-induced amelioration of the inflammatory response in all tissues analyzed. Conclusions. Stress conditioning by local heat-shock priming reduces the inflammatory response in osteomyocutaneous flaps. The protective effect is predominantly mediated by the induction of heat-shock protein 32. (Surgery 2001;129:292-301.)

Regulation of Adhesion Molecules: A New Target for the Treatment of Chronic Venous Insufficiency

As more insight into the mechanisms leading to chronic venous insufficiency (CVI) is gained, novel targets for drug treatment of the disease, or of its complications, become available. Studies using chemical entities capable of inhibiting leukocyte adhesion in postcapillary venules have led to the discovery of selective inhibitors of cell adhesion mechanisms. The aim of the current review is to describe the pharmacology, biochemistry, and molecular biology studies performed with some new inhibitors of adhesion molecule expression. Compounds such as hydroxy triallyl farnisine (S 17834) may offer new and efficient treatment of the microcirculatory complications that accompany chronic venous disease.