Microvascular Stasis Research Articles

Fucosylation Inhibitor, 2-Fluorofucose, Inhibits NF-κB Activation and Vaso-Occlusion In Transgenic Sickle Mice

Polymorphonuclear cells (PMNs) roll along vessel walls by binding to E- and P-selectins on the endothelial surface. These vascular adhesion molecules are involved in the initial tethering of PMNs on endothelium rather than their firm adhesion. Endothelial cell–expressed P- and E-selectins interact with leukocyte counterreceptors P-selectin glycoprotein ligand-1 (PSGL-1) and E-selectin ligand-1 (ESL-1). These glycoprotein counterreceptors are active only when modified posttranslationally by fucosylated oligosaccharides represented by the sialyl Lewis x tetrasaccharide (sLex) or its structural variants. Because of the important role that fucose plays in protein glycosylation and cellular functions, specific inhibitors of its incorporation could have significant applications in basic research and therapy. One such inhibitor, 2-fluorofucose peracetate, was recently reported to block fucosylation of cellular proteins and to inhibit binding of PMNs to P- and E-selectin. One potential application is the inhibition of leukocyte recruitment in pro-inflammatory disease states such as sickle cell disease (SCD). P-selectin expression on endothelium has been demonstrated to play an important role in vaso-occlusion, a hallmark of SCD. Inhibition of P-selectin binding inhibits vaso-occlusion in transgenic sickle mice. Therefore, we hypothesized that 2-fluorofucose would inhibit tissue inflammation and vaso-occlusion in SCD mice. MethodsNY1DD sickle mice were given plain water or 20 or 100 mM 2-fluorofucose in their drinking water ad libitum. The first cohort of mice (n=8/group) was treated for 7 days. On day 7 the mice were sacrificed with CO2, an EDTA blood sample was collected from the heart and the liver was removed and frozen in liquid nitrogen. Total white blood cell counts and differentials were performed by manual counting using a hemocytometer and Wright-stained blood smears, respectively. Nuclear extracts were prepared from nuclei isolated from liver homogenates. Western blots of the nuclear extracts were immunostained with antibodies to NF-ĸB phospho-p65 a marker of NF-ĸB activation. The second cohort of mice (n=4/group) was also treated for 7 days. On day 4, dorsal skin fold chambers were implanted onto the mice. On day 7, flowing venules in the dorsal skin-fold chamber window were selected and mapped using intravital microscopy. Thereafter, the mice were infused via the tail vein with human stroma-free hemoglobin (0.32 µmols heme/kg), a known inducer of vascular stasis in SCD mice. At 1 and 4 hours after infusion the same venules were re-examined and the percentage of vessels that had become static (no flow) was recorded. A positive control group of NY1DD mice (n=3) with implanted dorsal skin-fold chambers was given water to drink and pretreated with hemin (40 µmols/kg X 3 days, i.p.), a known inhibitor of vascular stasis. ResultsWhite blood cells counts were 16.3±3.2 (K/µL, mean±SD) in SCD mice treated with water. The white counts increased to 22.1±5.2 in SCD mice treated with 20 mM 2-fluorofucose and 34.2±7.2 in SCD mice treated with 100 mM 2-fluorofucose, respectively (p<0.05 for all pairwise comparisons). NF-ĸB in liver and other organs is activated in SCD mice compared to normal C57BL/6 mice. Nuclear NF-ĸB phospho-p65 was partially diminished in mice treated with 20 mM 2-fluorofucose and markedly decreased in mice treated with 100 mM 2-fluorofucose or pretreated with heme (Fig. 1).Infusion of hemoglobin induced ∼30% microvascular stasis at 1 and 4 hours in SCD mice treated with water (Fig. 2). Treatment with 20 mM 2-fluorofucose partially inhibited stasis at 1 and 4 hours (p<0.05 compared to water). Treatment with 100 mM 2-fluorofucose or heme inhibited stasis to 6.7% or less at 1 and 4 hours (p<0.025 compared to water). [Display omitted] [Display omitted] ConclusionsOral administration of 2-fluorofucose has significant anti-inflammatory and anti-vaso-occlusive properties in SCD mice. Disclosures:Belcher:Seattle Genetics, Inc.: Research Funding. Chen:Seattle Genetics, Inc.: Research Funding. Nguyen:Seattle Genetics, Inc.: Research Funding. Okeley:Seattle Genetics, Inc.: Employment. Senter:Seattle Genetics, Inc.: Employment. Benjamin:Seattle Genetics, Inc.: Employment. Vercellotti:Seattle Genetics, Inc.: Research Funding.

Interference With TNFα Using Long-Term Etanercept In S+SAntilles Sickle Transgenic Mice Ameliorates Abnormal Endothelial Activation, Vasoocclusion, and Pulmonary Hypertension Including Its Pulmonary Arterial Wall Remodeling

A systemic inflammatory state is a prominent feature of sickle cell anemia. Using sickle transgenic mice, we previously identified a blood monocyte-TNF-endothelium activation axis using multiple complementary approaches. For example, endothelial activation (identified by abnormal expression of VCAM-1 and tissue factor (TF) by pulmonary vessel endothelium) was evident in unstressed BERK and S+SAntilles sickle mice. In NY1DD sickle mice, this was triggered by transient enhanced sickling and vasoocclusion resulting from transient exposure to hypoxia/reoxygenation (H/R). Endothelial activation depended upon Egr-1 and NFkB(p50) in peripheral blood mononuclear cells, and transfusion experiments clarified that the peripheral blood monocytes (PBM) were the responsible effecter cells. Finally, interference with TNFa using etanercept (Etn) prevented this PBM-induced endothelial activation. Based on these background data, we have now tested the “clinical” efficacy of long-term, anti-inflammatory TNFa interference via treatment of S+SAntilles sickle mice with Etn (started at weaning, 3 mg/kg sc, once/wk x 6 wk). In parallel, littermate controls received equimolar scrambled peptide version of Etn's TNF-receptor moiety. In response we observed significant improvement in multiple parameters relevant to inflammation-induced vessel wall disease in this model, as follows (data shown in inset). Etn decreased blood markers of inflammation: plasma levels of sVCAM-1 and SAP (by ELISA), and MCP-1 and TNFa (by FACS cytobead assay). Etn also decreased the % of PBM positive for TNFa (by FACS). Etn diminished the abnormal pulmonary vessel endothelial expression of VCAM-1 and TF (by histochemical and immunofluorescent staining). Etn eliminated the enhanced vasoocclusion seen as dermal microvascular stasis in the live mouse (using the dorsal skin fold chamber) measured at 1 and 4 hours post-H/R (7%O2x1hr, followed by 21%O2x 1-4hr). Instructively, Etn did not significantly improve the similar stasis induced by heme infusion, a TNF-independent direct endothelial stressor. Finally, Etn diminished evidence for pulmonary hypertension, assessed via three of its hallmark features: elevated right ventricular mean systolic pressure (by RV catheterization), abnormal peri-vascular inflammatory cell aggregates (by immuno-histochemical staining for CD11b), and abnormal pulmonary arterial wall muscularization (by immuno-histochemical staining for SM actin). Remarkably, Etn actually normalized RVSP, and Etn's partial improvement in pulmonary arterial muscularization resulted not only from prevention of its progression over duration of this study but also from some reversal of the abnormal remodeling that had already developed by the time Etn was started. In parallel, Etn reduced the number of abnormal peri-vascular aggregates of CDllb+ cells in the pulmonary arterial system.These results enable several conclusions. In the S+SAntilles sickle model, endothelial activation and aspects of vascular wall pathobiology derive from TNFa produced by activated PBM, a monocyte-TNF-endothelial activation axis. Interference with TNFa using etanercept exerts multiple salubrious vascular effects that predict possible clinical benefit. And results suggest that inflammation involving TNFa plays a proximate, generative role in development of pulmonary hypertension in this model. Assuming these benefits are independently corroborated, a controlled test of etanercept's efficacy for aspects of human sickle vascular pathobiology perhaps should be considered. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

MP4CO, a pegylated hemoglobin saturated with carbon monoxide, is a modulator of HO-1, inflammation, and vaso-occlusion in transgenic sickle mice

AbstractTransgenic sickle mice expressing βS hemoglobin have activated vascular endothelium in multiple organs that exhibits enhanced expression of NF-ĸB and adhesion molecules and promotes microvascular stasis in sickle, but not normal, mice in response to hypoxia/reoxygenation (H/R), or heme. Induction of heme oxygenase-1 (HO-1) or administration of its products, carbon monoxide (CO) or biliverdin, inhibits microvascular stasis in sickle mice. Infusion of human hemoglobin conjugated with polyethylene glycol and saturated with CO (MP4CO) markedly induced hepatic HO-1 activity and inhibited NF-ĸB activation and H/R-induced microvascular stasis in sickle mice. These effects were mediated by CO; saline or MP4 saturated with O2 (MP4OX) had little to no effect on H/R-induced stasis, though unmodified oxyhemoglobin exacerbated stasis. The HO-1 inhibitor, tin protoporphyrin, blocked MP4CO protection, consistent with HO-1 involvement in the protection afforded by MP4CO. MP4CO also induced nuclear factor-erythroid 2 p45-related factor 2 (Nrf2), an important transcriptional regulator of HO-1 and other antioxidant genes. In a heterozygous (hemoglobin-AS) sickle mouse model, intravenous hemin induced cardiovascular collapse and mortality within 120 minutes, which was significantly reduced by MP4CO, but not MP4OX. These data demonstrate that MP4CO induces cytoprotective Nrf2 and HO-1 and decreases NF-ĸB activation, microvascular stasis, and mortality in transgenic sickle mouse models.

H-Ferritin Ferroxidase Activity Induces Cytoprotective Pathways and Inhibits Microvascular Stasis in Transgenic Sickle Mice

AbstractAbstract 378Hemolysis, oxidative stress, inflammation, vaso-occlusion and organ infarction are hallmarks of sickle cell disease (SCD). We hypothesize that intravascular heme, liberated from hemoglobin S derived from hemolyzed sickle red blood cells, is fundamental to inflammation and vaso-occlusion in SCD. Heme can be degraded by heme-oxygenase-1 (HO-1), releasing Fe2+, carbon monoxide (CO), and biliverdin/bilirubin. We have previously shown that increases in HO-1 activity inhibit vascular inflammation and vaso-occlusion in transgenic mouse models of SCD. HO-1 products, CO and biliverdin/bilirubin, also are protective in SCD, but Fe2+ released from the heme ring requires further processing. The released Fe2+ from heme is oxidized by ferritin heavy chain (FHC) ferroxidase activity and safely stored as catalytically-inactive Fe3+ inside ferritin clusters. FHC overexpression has been shown to be cytoprotective in response to inflammation and oxidative stress in vivo and in vitro. In this study, we hypothesize that overexpression of FHC with its ferroxidase activity will inhibit inflammation and microvascular stasis in transgenic sickle mice in response to stroma-free hemoglobin. We utilized a Sleeping Beauty transposase plasmid to deliver a human wt-ferritin heavy chain (wt-FHC) transposable element by hydrodynamic tail vein injections to NY1DD SCD mice. Control mice were infused with the same volume of lactated Ringer’s solution LRS) or a triple missense (ms-) human FHC plasmid encoding no ferroxidase enzyme activity. Eight weeks after injection, the mice were implanted with dorsal skin-fold chambers to access microvascular blood flow. LRS-treated mice had 40% microvascular stasis (% non-flowing venules) when infused with stroma-free hemoglobin, while wt-FHC overexpressing mice had only 5% stasis (p<0.05), suggesting vascular protection by wt-FHC. Ferroxidase activity was critical in this protection as mice overexpressing ms-FHC were not protected from microvascular stasis (33%). The wt-FHC SCD mice had marked increases in FHC mRNA and protein, light chain ferritin, 5-aminolevulinic acid synthase (5-ALA-synthase), cellular heme content, ferroportin, nuclear factor erythroid 2-related factor 2 (Nrf2), nuclear FHC and microsomal HO-1 activity and protein, and a decrease in activated phosho- and total nuclear factor-kappa B (NF-κB) p65 in the nuclei of the livers. We conclude that wt-FHC ferroxidase activity enhances 5-ALA-synthase activity and cytoprotective Nrf2-regulated proteins including HO-1 resulting in decreased NF-κB-activation, inflammation and microvascular stasis in transgenic SCD mice. Therapies directed at increasing ferritin heavy chain production and decreasing reactive iron availability may be beneficial in limiting sickle cell crises and end organ damage. Disclosures:Vercellotti:Sangart Inc: Consultancy, Research Funding. Chen:Sangart Inc: Research Funding. Hebbel:Sangart Inc: Consultancy, Research Funding. Belcher:Sangart Inc: Research Funding.

MP4CO, a Pegylated Hemoglobin Saturated with Carbon Monoxide, Inhibits Microvascular Stasis in Transgenic Sickle Mice Through Heme Oxygenase-1.

AbstractAbstract 2112Our laboratory has shown in murine models of sickle cell disease (SCD) that intravascular heme promotes oxidative stress, inflammation and microvascular stasis through toll-like receptor-4 (TLR4) signaling. Furthermore, the heme degrading enzyme, heme-oxygenase-1 (HO-1) and its by-products biliverdin and carbon monoxide (CO), inhibit these effects. CO may induce salutary effects in SCD to decrease vaso-occlusion by inhibiting hemoglobin S polymerization, vasodilation and anti-inflammatory actions, including induction of HO-1. MP4CO is a 4.3 g/dL solution of human hemoglobin conjugated with polyethylene glycol and saturated with CO. In the current studies, we tested the hypothesis that MP4CO would induce HO-1 in transgenic sickle mice and inhibit microvascular stasis in response to hypoxia/reoxygenation (H/R). Microvascular stasis (% non-flowing venules) was examined by intravital microscopy following 1hr of hypoxia (7% O2) and 1hr of reoxygenation (room air) in NY1DD transgenic sickle mice implanted 3 days earlier with a dorsal skin fold chamber window (DSFC). Five treatment groups of 3–6 mice were studied initially: 1) lactated Ringer’s solution (LRS); 2) MP4OX (oxygen saturated MP4); 3) MP4CO; 4) oxygen-saturated stroma-free hemoglobin (SFH); 5) hemin chloride, 40 nmols/g i.p. × 3 days was administered as a positive control based on the previously-demonstrated induction of HO-1. Other than hemin chloride, all solutions (LRS, MP4CO, MP4OX, SFH) were administered i.v., 0.008 mL/g. In the first study, LRS, MP4OX, MP4CO or SFH were infused 24hr prior to H/R and in the second study the same solutions were infused 30min after hypoxia, during the reoxygenation phase of the experiment.In sickle mice treated with LRS or MP4OX 24hr prior to H/R, 25% and 22% of the venules, respectively, became static in response to H/R. However, in sickle mice treated with MP4CO 24hr prior to H/R, only 9% of the venules became static (p<0.05 MP4CO vs. LRS and MP4OX). In contrast, sickle mice treated with SFH 24hr prior to H/R developed significantly more stasis (37% stasis) than sickle mice in the other treatment groups (p<0.05). As we have previously shown, pretreatment with hemin abrogated vascular stasis in sickle mice (3% stasis, p<0.05 vs. all other groups). In additional groups of sickle mice, LRS, MP4OX, MP4CO and SFH were administered 30min after hypoxia during the reoxygenation phase. After H/R, LRS-treated animals had 26% stasis, MP4OX-treated mice had 18% stasis (p<0.05 vs. LRS) and MP4CO-treated mice had11% stasis (p<0.05 vs. LRS). Infusion of SFH 30min post-hypoxia markedly worsened stasis compared to the other treatments (44% stasis, p<0.05 vs. MP4CO, LRS and MP4OX).Infusion of MP4CO, but not LRS, MP4OX or SFH, markedly induced expression of microsomal HO-1 activity and protein, suggesting HO-1 was responsible for inhibition of stasis by MP4CO. Indeed, the HO-1 inhibitor SnPP reversed the effect of MP4CO on H/R-induced stasis in sickle mice (27% stasis with SnPP + MP4CO vs. 10% with LRS + MP4CO, p<0.05). The mechanism of HO-1 induction by MP4CO was likely due to an increased expression of nuclear factor erythroid 2-related factor 2 (Nrf2), an important transcriptional regulator of HO-1. MP4CO induced strikingly more Nrf2 in liver nuclei than LRS, MP4OX or SFH. Induction of HO-1 by MP4CO decreased the inflammatory response in sickle mice as evidenced by a decrease in activated nuclear factor-kappa B (NF-kB) phospho-p65 in liver nuclei following H/R.We conclude that MP4CO enhances cytoprotective Nrf2-regulated proteins including HO-1 resulting in decreased NF-kB activation, inflammation and microvascular stasis in transgenic SCD mice. CO delivery via MP4CO may be beneficial in patients with sickle cell anemia. Disclosures:Belcher:Sangart Inc: Research Funding. Chen:Sangart Inc: Research Funding. Young:Sangart Inc: Employment. Burhop:Sangart Inc: Employment. Vercellotti:Sangart Inc: Consultancy, Research Funding.

Control of neutrophil pseudopods by fluid shear: role of Rho family GTPases

Blood vessels and blood cells are under continuous fluid shear. Studies on vascular endothelium and smooth muscle cells have shown the importance of this mechanical stress in cell signal transduction, gene expression, vascular remodeling, and cell survival. However, in circulating leukocytes, shear-induced signal transduction has not been investigated. Here we examine in vivo and in vitro the control of pseudopods in leukocytes under the influence of fluid shear stress and the role of the Rho family small GTPases. We used a combination of HL-60 cells differentiated into neutrophils (1.4% dimethyl sulfoxide for 5 days) and fresh leukocytes from Rac knockout mice. The cells responded to shear stress (5 dyn/cm2) with retraction of pseudopods and reduction of their projected cell area. The Rac1 and Rac2 activities were decreased by fluid shear in a time- and magnitude-dependent manner, whereas the Cdc42 activity remained unchanged (up to 5 dyn/cm2). The Rho activity was transiently increased and recovered to static levels after 10 min of shear exposure (5 dyn/cm2). Inhibition of either Rac1 or Rac2 slightly but significantly diminished the fluid shear response. Transfection with Rac1-positive mutant enhanced the pseudopod formation during shear. Leukocytes from Rac1-null and Rac2-null mice had an ability to form pseudopods in response to platelet-activating factor but did not respond to fluid shear in vitro. Leukocytes in wild-type mice retracted pseudopods after physiological shear exposure, whereas cells in Rac1-null mice showed no retraction during equal shear. On leukocytes from Rac2-null mice, however, fluid shear exerted a biphasic effect. Leukocytes with extended pseudopods slightly decreased in length, whereas initially round cells increased in length after shear application. The disruption of Rac activity made leukocytes nonresponsive to fluid shear, induced cell adhesion and microvascular stasis, and decreased microvascular density. These results suggest that deactivation of Rac activity by fluid shear plays an important role in stable circulation of leukocytes.

Microvascular blood flow and stasis in transgenic sickle mice: utility of a dorsal skin fold chamber for intravital microscopy.

Vascular inflammation, secondary to ischemia-reperfusion injury, may play an essential role in vaso-occlusion in sickle cell disease (SCD). To investigate this hypothesis, dorsal skin fold chambers (DSFCs) were implanted on normal and transgenic sickle mice expressing human alpha and beta(s)/beta(s-Antilles) globin chains. Microvessels in the DSFC were visualized by intravital microscopy at baseline in ambient air and after exposure to hypoxia-reoxygenation. The mean venule diameter decreased 9% (P < 0.01) in sickle mice after hypoxia-reoxygenation but remained constant in normal mice. The mean RBC velocity and wall shear rate decreased 55% (P < 0.001) in sickle but not normal mice after hypoxia-reoxygenation. None of the venules in normal mice became static at any time during hypoxia-reoxygenation; however, after 1 hr of hypoxia and 1 hr of reoxygenation, 11.9% of the venules in sickle mice became static (P < 0.001). After 1 hr of hypoxia and 4 hr of reoxygenation, most of the stasis had resolved; only 3.6% of the subcutaneous venules in sickle mice remained static (P = 0.01). All of the venules were flowing again after 24 hr of reoxygenation. Vascular stasis could not be induced in the subcutaneous venules of sickle mice by tumor necrosis factor alpha (TNF-alpha). Leukocyte rolling flux and firm adhesion, manifestations of vascular inflammation, were significantly higher at baseline in sickle mice compared to normal (P < 0.01) and increased 3-fold in sickle (P < 0.01), but not in normal mice, after hypoxia-reoxygenation. Plugs of adherent leukocytes were seen at bifurcations at the beginning of static venules. Misshapen RBCs were also seen in subcutaneous venules.

Investigation of the thermal and tissue injury behaviour in microwave thermal therapy using a porcine kidney model

Minimally invasive microwave thermal therapies are being developed for the treatment of small renal cell carcinomas (RCC, d<3 cm). This study assessed the thermal history and corresponding tissue injury patterns resulting from microwave treatment of the porcine renal cortex. Three groups of kidneys were evaluated: (1) in vitro treated, (2) in vivo with 2-h post-treatment perfusion (acute) and (3) in vivo with 7-day post-treatment perfusion (chronic). The kidneys were treated with an interstitial water-cooled microwave probe (Urologix, Plymouth, MN) that created a lesion centered in the renal cortex (50 W for 10 min). The thermal histories were recorded at 0.5 cm radial intervals from the probe axis for correlation with the histologic cellular and vascular injury. The kidneys showed a reproducible 2 cm chronic lesion with distinct histologic injury zones identified. The thermal histories at the edge of these zones were found using Lagrangian interpolation. The threshold thermal histories for microvascular injury and stasis appeared to be lower than that for renal epithelial cell injury. The Arrhenius kinetic injury models were fit to the thermal histories and injury data to determine the kinetic parameters (i.e. activation energy and frequency factor) for the thermal injury processes. The resultant activation energies are consistent in magnitude with those for thermally induced protein denaturation. A 3-D finite element thermal model based on the Pennes bioheat equation was developed and solved using ANSYS (V7.0). The real geometry of the kidneys studied and temperature dependent thermal properties were used in this model. The specific absorption rate (SAR) of the microwave probe required for the thermal modelling was experimentally determined. The results from the thermal modelling suggest that the complicated change of local renal blood perfusion with temperature and time during microwave thermal therapy can be predicted, although a first order kinetic model may be insufficient to capture blood flow changes. The local blood perfusion was found to be a complicated function of temperature and time. A non-linear model based on the degree of vascular stasis was introduced to predict the blood perfusion. In conclusion, interstitial microwave thermal therapy in the normal porcine kidney results in predictable thermal and tissue injury behaviour. Future work in human kidney tissue will be necessary to confirm the clinical significance of these results.

１．脳梗塞超急性期の病態

Through many experimental brain ischemia studies, it has been suggested that all of the cellular elements in the central nervous system show dynamic stress responses depending on the degree of environmental changes induced by ischemia and reperfusion. In this symposium, first I reviewed the pathogenic role of microvascular stasis (i. e., secondary ischemia) caused by the primary ischemic event and demonstrated the important role of cell adhesion molecules through the experiments using ICAM-1 knock-out mouse as a model of brain ischemia/reperfusion. Next, I discussed the ischemia-induced neuronal cell responses in relation to the apoptosis-like selective neuronal death and the induction of adopted stress responses including stress protein synthesis and 'ischemic tolerance' phenomenon. A variety of stress proteins induced by ischemic stress have been reviewed in relation to their pathophysiological roles in the ischemic brain. Finally, I reviewed the important pathogenic roles of endoplasmic reticulum (ER) stress as well as adaptive responses of ubiquitin-proteasome system in ischemia-induced neuronal cell death. For the development of a novel therapeutic agent against ischemic stroke, it is quite important to clarify both the negative and positive cellular responses induced by brain ischemia/reperfusion.

Apoptosis and leucocyte-endothelium interactions contribute to the delayed effects of cryotherapy on tumours in vivo.

In dermatology, cryotherapy is commonly used to treat benign and malignant skin lesions. However, studies investigating the time-course of the direct and delayed effects on the microvasculature and subsequent tissue destruction are lacking. Amelanotic melanomas (A-Mel-3) were implanted into the dorsal skinfold chamber of Syrian Golden hamsters (n=51). Tumour and normal tissue were frozen to -20 masculine C (cooling rate 150 masculine C/min). Intravital fluorescence microscopy was performed to assess microvascular changes and leucocyte-endothelium interactions up to 24 h. The relative degrees of necrosis and apoptosis and the accumulation of leucocytes were investigated by histology and immunohistochemistry. Apoptosis was quantified using the TUNEL assay in combination with computer-assisted image analysis. After cryotherapy, red blood cell velocity (RBCV) decreased in postcapillary venules and tumour vessels, whereas functional vessel density (FVD) was significantly reduced in tumour vessels as compared with postcapillary venules. Leucocyte-endothelium interaction increased first in normal and tumour tissue, and then after 24 h leucocytes accumulated in normal tissue close to the tumour margin. Necrosis was induced in the cryolesions directly after freezing and remained constant over the entire observation period. In contrast, apoptosis increased in the periphery of the tumour tissue up to 24 h following cryotherapy. In conclusion, tissue destruction by cryotherapy is not only induced by direct necrosis and microvascular stasis, but also by the inflammatory infiltrate and subsequent apoptosis. This could be an important finding regarding the generation of an immune response.

Functional Role for Selectins in the Pathogenesis of Cerebral Ischemia.

Neutrophil accumulation and neutrophil-mediated tissue damage in the postischemic brain have been well documented in numerous animal studies and in humans during the past three decades. Activation of vascular endothelial cells and leukocytes by local and humoral factors increases the cell surface expression of a family of adhesion molecules termed selectins, which promote low-affinity leukocyte rolling over the surface of endothelial cells. This process is an obligatory preliminary step leading to subsequent firm attachment, at which point leukocytes can exert numerous cytotoxic effects leading to microvascular plugging, stasis and thrombosis. Advances in our understanding of the role of inflammation and neutrophil­endothelial cell interactions in the pathophysiology of tissue ischemia and reperfusion have provided the current impetus for pharmacologic approaches that treat acute ischemic stroke by interrupting selectin­ligand interaction. (c) 2002 Prous Science. All rights reserved.

Dynamic Intrahepatic Flow and Cellular Alterations during Radiofrequency Ablation of Liver Tissue in Mice

The purpose of this study was to identify microvascular and other associated changes that occur in the liver during focal heating with monopolar radiofrequency (RF). Intravital video microscopy was performed on exteriorized transilluminated livers of 15 live mice during RF-induced heating of liver parenchyma. Microvascular flow parameters, flow reversibility, microbubble formation, phagocytic activity, and endothelial permeability were recorded throughout a range of tip temperatures (40 degrees C-95 degrees C). During RF application, five discrete zones extended outward from the electrode surface: (i) tissue coagulation, (ii) cellular edema/necrosis, (iii) sinusoidal stasis, (iv) parenchymal shunting, and (v) normal liver tissue. Reversal of stasis in sinusoids and small (<25 microm) vessels occurred at tip temperatures below 50 degrees C. This zone of stasis corresponded to the hyperemic zone on histologic analysis. Although alterations in permeability and phagocytic activity were first identified at 43 degrees C, tip temperatures higher than 55 degrees C always produced local endothelial leakiness to carbon microparticles at the periphery and always inhibited phagocytic activity. At tip temperatures higher than 95 degrees C, microbubble formation occurred with bubbles ultimately tracking through necrotic tissue into patent sinusoids. Larger peripheral vessels (>30 microm) limited extension of coagulation. Although coagulation occurs at tip temperatures higher than 50 degrees C, RF heating induced reversible microvascular stasis at temperatures lower than 50 degrees C. Increased sinusoidal endothelial permeability occurs at near-coagulative temperatures. Therefore, targeted endovascular microparticle delivery through this leaky endothelium may provide an additional and complimentary adjunct for RF ablation therapy.

Comparison of thermal damage calculated using magnetic resonancethermometry, with magnetic resonance imaging post-treatment and histology,after interstitial microwave thermal therapy of rabbit brain

Clinical application of high-temperature thermal therapy as a treatment forsolid tumours requires an accurate and close to real-time method forassessing tissue damage. Imaging methods that detect structural changesduring heating may underestimate the extent of thermal damage. This is dueto the occurrence of delayed damage manifested at tissue locations exposedto temperatures lower than those required to cause immediate structuralchanges. An alternative approach is to measure temperature and thencalculate the expected damage based on the temperature history at eachtissue location. Magnetic resonance (MR) imaging methods now allowtemperature maps of the target and surrounding tissues to be generated inalmost real-time. The aim of this work was to evaluate whether thermaldamage zones calculated on the basis of MR thermometry maps measured duringheating correspond to actual tissue damage as measured after treatment byhistological methods and MR imaging.Four male rabbits were treated with high-temperature thermal therapydelivered in the brain by a single microwave antenna operating at 915 MHz.MR scanning was performed before, during and after treatment in a 1.5 Twhole-body scanner. Temperature maps were produced using the protonresonance frequency (PRF) shift method of MR thermometry. In addition,conventional T1-weighted and T2-weighted spin-echo images wereacquired after treatment. Thermal damage zones corresponding to cell death,microvascular blood flow stasis and protein coagulation were calculatedusing an Arrhenius analysis of the MR temperature/time course data. Thecalculated zones were compared with the lesions seen on histopathologicalexamination of the brains which were removed within 6-8 h of treatment.The results showed that calculated damage zones based on MR thermometryagreed well with areas of damage as assessed using histology after heatingwas completed. The data suggest that real-time calculations of finalexpected thermal damage based on an Arrhenius analysis of MR temperaturedata may provide a useful method of real-time monitoring of thermal therapywhen combined with conventional T2-weighted images taken aftertreatment.

Inflammatory mediators of cerebral endothelium: a role in ischemic brain inflammation.

Brain inflammation has been implicated in the development of brain edema and secondary brain damage in ischemia and trauma. Adhesion molecules, cytokines and leukocyte chemoattractants released/presented at the site of blood-brain barrier (BBB) play an important role in mobilizing peripheral inflammatory cells into the brain. Cerebral endothelial cells (CEC) are actively engaged in processes of microvascular stasis and leukocyte infiltration by producing a plethora of pro-inflammatory mediators. When challenged by external stimuli including cytokines and hypoxia, CEC have been shown to release/express various products of arachidonic acid cascade with both vasoactive and pro-inflammatory properties, including prostaglandins, leukotrienes, and platelet-activating factor (PAF). These metabolites induce platelet and neutrophil activation and adhesion, changes in local cerebral blood flow and blood rheology, and increases in BBB permeability. Ischemic CEC have also been shown to express and release bioactive inflammatory cytokines and chemokines, including IL-1beta, IL-8 and MCP-1. Many of these mediators and ischemia in vitro and in vivo have been shown to up-regulate the expression of both selectin and Ig-families of adhesion molecules in CEC and to facilitate leukocyte adhesion and transmigration into the brain. Collectively, these studies demonstrate a pivotal role of CEC in initiating and regulating inflammatory responses in cerebral ischemia.

Dynamic cellular response following brain ischemia and reperfusion

Since the first documentation of the induction of heat shock protein following transient cerebral ischemia, much experimental evidence suggested that all of the cellular elements in the central nervous system show dynamic stress responses depending on the degree of environmental changes induced by ischemia and reperfusion. In this review, first I focused on the importance of the usage of an appropriate experimental model for brain ischemia and reperfusion, and I presented our work on mouse models of transient global and focal ischemia. Next, I reviewed the pathogenic role of microvascular stasis (i.e., secondary ischemia) caused by the primary ischemic event and demonstrated the important role of cell adhesion molecules through the experiments using ICAM-1 knock-out mouse as a model of brain ischemia/reperfusion. Thirdly, I discussed the ischemia-induced neuronal cell responses in relation to the apoptosis-like selective neuronal death and the induction of adopted stress responses including stress protein synthesis and 'ischemic tolerance' phenomenon. A variety of stress proteins induced by ischemic stress have been reviewed and a pivotal role of tyrosine kinase system in selective neuronal death has been suggested in the gerbil model of transient forebrain ischemia. Finally, I showed the important pathophysiological roles of glial cells such as astrocytes and oligodendrocytes in the cellular cross-talk triggered by an ischemic event. For the development of a novel therapeutic agent against ischemic stroke, it is quite important to clarify both the negative and positive cellular responses induced by brain ischemia/reperfusion.

IL-1 is an important mediator for microcirculatory changes in endotoxin-induced intestinal mucosal damage.

Although small intestine is frequently injured in endotoxin shock, the exact pathological sequence has not been fully understood. The major objective of this study is to elucidate the role of interleukin (IL)-1 in endotoxin-induced microcirculatory disturbance of rat small intestine. Mucosal and submucosal microvessels of the rat ileum were observed by intravital microscope with a high speed video camera system and the attenuating effect of E5090, an inhibitor of IL-1 generation, on endotoxin-induced intestinal microcirculatory disturbances was investigated. Endotoxin infusion produced significant mucosal damage, but before these morphological changes became significant, microvascular stasis in villi, decreased red blood cell velocity, and increased leukocyte adherence to venular walls were observed in intestinal microcirculatory beds 30 min after endotoxin administration. Intestinal IL-1alpha levels were also significantly increased at that time. Endotoxin treatment enhanced chemiluminescence activity from neurophils and rapidly mobilized CD18 on leukocytes. E5090, which suppressed the IL-1 production in intestinal mucosa, attenuated the microcirculatory disturbances induced by endotoxin, and significantly reduced the subsequent mucosal damage. E5090 also attenuated the increased chemiluminescence activity and CD18 expression on leukocytes. In conclusion, the production of IL-1alpha is enhanced in the intestinal mucosa during endotoxin infusion. IL-1 may be an important mediator of microcirculatory changes, including decreased red blood cell velocity and increased leukocyte sticking and its activation, leading to the mucosal damage.

Vascular effects of photodynamic therapy.

Vascular damage and blood flow stasis are consequences of photodynamic therapy (PDT) of solid tumors using many photosensitizers. Microvascular stasis and resulting hypoxia are effective means to produce cytotoxicity and tumor regression. The observation of blood flow stasis after photodynamic therapy results from a combination of damage to sensitive sites within the microvasculature and the resulting physiological responses to this damage. A generalized hypothesis for the mechanisms leading to vessel stasis begins with perturbation and damage to endothelial cells during light treatment of photosensitized tissues. Endothelial cell damage leads to the establishment of thrombogenic sites within the vessel lumen and this initiates a physiological cascade of responses including platelet aggregation, the release of vasoactive molecules, leukocyte adhesion, increases in vascular permeability, and vessel constriction. These effects from damage combine to produce blood flow stasis.

Intestinal Microcirculation and Leukocyte Behavior in Ischemia-Reperfusion Injury

Polymorphonuclear leukocytes are thought to play a pivotal role in the generation of ischemia-reperfusion-induced mucosal injury; however, their behavior in the intestinal microvasculature following this injury has not been directly examined. In this study intravital microscopy was used to investigate the dynamics of blood flow and leukocyte behavior in the villus, serosal, and mesenteric microcirculation during ischemia-reperfusion in anesthetized hamsters. Thirty minutes of ischemia and 90 min of reperfusion resulted in almost complete microvascular stasis in the villi of control animals, whereas only a few serosal and mesenteric microvessels exhibited stasis. Following reperfusion, there was a significant increase in leukocyte accumulation in all three tissues; however, significantly fewer leukocytes adhered in the villus microvasculature than in the rest of either the mucosa, serosa, or mesentery. Treatment with gamma-hydroxybutyrate (GHB), a compound that we have previously demonstrated to be highly effective in preventing ischemia-reperfusion injury, significantly reduced both the microvascular stasis and leukocyte accumulation in all three vascular beds. This study demonstrates that there are significant differences in the response to ischemia-reperfusion in various intestinal layers and that areas most susceptible to damage are not necessarily those exhibiting the greatest leukocyte accumulation.

Involvement of Nitric Oxide and Cyclooxygenase Products in Photoactivation-Induced Microvascular Occlusion

Photoactivation of intravascular dyes with high doses of light is a technique used clinically to treat tumors. This procedure results in arteriolar constriction, mast cell degranulation, platelet thrombus formation, and, ultimately, microvascular stasis. In vivo microscopy was utilized in the current study to examine if the endothelial release of prostaglandins and nitric oxide could participate in the microvascular effects of photoactivation. Diameter changes and thrombus formation of arterioles and venules of the cremaster muscle of male Sprague-Dawley rats were quantitated during continuous light activation of intravascular fluorescein isothiocyanate conjugated to bovine serum albumin. Vasoconstriction and thrombus development were assessed separately, using the relationships between the width of the red blood cell column, the inner wall diameter, and the thickness of the plasma layer. Venular photoactivation resulted in thrombus growth which reached 30% of the maximum size by 16.8 ± 3.71 min and a subsequent growth rate of 6.2 ± 1.64 μm/min. In arterioles, 30% thrombus growth occurred at 14.0 ± 2.02 min with a growth rate of 3.0 ± 0.57 μm/min. Continuous arteriolar photoactivation led to a vasoconstriction of 34.4 ± 6.87% of the initial vessel diameter. Thirty percent of the maximal constriction occurred after 10.6 ± 1.26 min of photoactivation. Constriction proceeded at a rate of 3.8 ± 1.32 μm/min. Topically applied mefenamic acid (a cyclooxygenase inhibitor) and N W -nitro-L-arginine methyl ester (L-NAME, a nitric oxide synthase inhibitor) each enhanced both the arteriolar and the venular thrombus growth due to photoactivation. Photoactivation-induced arteriolar constriction was augmented by L-NAME and inhibited by mefenamic acid. These data suggest that the photoactivation of intravascular dyes is accompanied both by the release of nitric oxide, which inhibits thrombus development and arteriolar constriction, and by the release of cyclooxygenase products, which inhibit thrombus growth and induce vasoconstriction. Rats treated with busulfan to induce thrombocytopenia exhibited a 90% decrease in circulating platelets. In these animals, photoactivation caused significantly delayed thrombus growth in arterioles and venules, while arteriolar constriction remained unaltered, suggesting that the vasoconstrictor prostanoid is not of platelet origin.

Isoproterenol prevents ethanol-induced microvascular stasis and deep histologic injury in rat gastric mucosa.

The effect of topical isoproterenol (5.8 x 10(-5) M) on the microvascular stasis and histologic injury produced in the rat gastric mucosa by absolute ethanol was investigated using in vivo microscopic and histologic techniques. Our findings show that in control animals, absolute ethanol applied to the gastric mucosa caused a rapid decrease in microvascular blood flow, leading to complete stasis by 3 min. In contrast, prior treatment of the gastric mucosa with topical isoproterenol increased microvascular blood flow initially and then maintained microvascular flow in the majority of microvessels studied after the application of absolute ethanol. Treatment with topical isoproterenol also reduced the histologic injury score and percent mucosal necrosis caused by absolute ethanol. Pretreatment of animals with a beta-adrenergic antagonist propranolol (2 mg/kg subcutaneously), completely blocked these protective effects. We observed no systemic effects attributable to the topical use of isoproterenol. These results indicate that prior treatment of the gastric mucosa with topical isoproterenol maintains microvascular blood flow and reduces the deep histologic injury in the stomach caused by 100% ethanol.