Number Of Adherent Leukocytes Research Articles

COMPARISON OF THE PROTECTIVE EFFECTS OF TYPE III PHOSPHODIESTERASE (PDE3) INHIBITOR (CILOSTAZOL) AND ACETYLSALICYLIC ACID ON INTESTINAL MICROCIRCULATION AFTER ISCHEMIA REPERFUSION INJURY IN MICE

Antiplatelet therapy has been proposed as the treatment of choice for ischemia/reperfusion injury. The aim of this study is to elucidate the difference in effect between cilostazol (CZ) and acetylsalicylic acid (ASA) on microcirculatory disturbance in ischemia/reperfusion injury. Either 10 mg/kg of CZ (n = 14) or 100 mg/kg of ASA (n = 14) was administered orally to mice. Thereafter, 20 min of intestinal ischemia, followed by 60-min reperfusion, was applied; then, the status of submucosal microcirculation was observed under intravital microscopy. The blood cell counts and organ damage markers were examined in the portal blood. Next, 5 mm of the ileum was excised and was then histologically examined. Platelet-leukocyte aggregates were often observed in the postcapillary venules, and this formation was significantly reduced by both CZ and ASA. The number of adherent leukocytes was significantly lesser in the CZ-treated mice than in the ASA-treated mice (P < 0.01). The leukocyte number, lactate dehydrogenase, and lactate levels were best maintained in the CZ-treated mice (P < 0.05). The villus height was best preserved in the CZ-treated mice. Cilostazol inhibited not only the platelet aggregation but also the leukocyte adhesion to the endothelium, thereby inducing organ protection.

Dynamic Changes of Post-Ischemic Hepatic Microcirculation Improved by a Pre-Treatment of Phosphodiesterase-3 Inhibitor, Milrinone

Phosphodiesterase-3 inhibition has been shown to attenuate hepatic warm ischemia-reperfusion injury. The aim of this study was to investigate the effect of milrinone, phosphodiesterase-3 inhibitor, on post-ischemic microcirculation of rat livers by intravital microscopy. Male Wistar rats were randomly assigned to three groups; group A, milrinone pre-treatment; group B, ischemic pre-conditioning; and group C, no pre-treatment. All animals underwent a 60-min warm ischemia of the left lateral liver lobe. Microvascular perfusion and leukocyte-endothelial interaction were observed by intravital videomicroscopy. Hepatocellular viability and cellular damage were quantified by adenosine triphosphate tissue concentration as well as alanine aminotransferase and lactate dehydrogenase blood levels, respectively. In groups A and B, cyclic AMP hepatic tissue concentration was elevated significantly. After reperfusion, microvascular perfusion in hepatic sinusoids was significantly better maintained, and the number of adherent leukocytes was reduced in sinusoids and in post-sinusoidal venules in these rats. Serum transaminase blood levels were suppressed significantly in these groups compared with controls. The demonstrated improvement of hepatic microcirculation is certainly derived from milrinone induced cell protection in ischemia reperfusion of the liver. This effect is outlined by improved energy status and reduced liver enzyme liberation and mimics the effect of ischemic pre-conditioning.

Recurrent obstructive apneas trigger early systemic inflammation in a rat model of sleep apnea

Obstructive sleep apnea (OSA) is associated with vascular disorders possibly due to systemic inflammation. To determine whether repeated episodes of OSA in a rat model lead to endothelial cell activation and systemic leukocyte recruitment in the microcirculation. Three experimental groups (apnea, sham and naive) were studied. The apnea group was instrumented and subjected to repeated obstruction for 3h (rate 60/h, length 5s) using a special device. The sham group was only instrumented and the naive group was used as a control. Leukocyte-endothelial cell interactions (intravital microscopy) and expression of P-selectin (immunohistochemistry) were determined in colonic venules. The apnea group induced a significant increase in the flux of leukocytes rolling, number of rolling leukocytes and number of adherent leukocytes when compared with the sham or naive groups. P-selectin was up-regulated only in the apnea group. This experimental model of recurrent obstruction demonstrates rapid endothelial cell activation, suggesting the onset of an inflammatory response.

Effects of fractionated radiation on the brain vasculature in a murine model: Blood–brain barrier permeability, astrocyte proliferation, and ultrastructural changes

Radiation therapy of CNS tumors damages the blood-brain barrier (BBB) and normal brain tissue. Our aims were to characterize the short- and long-term effects of fractionated radiotherapy (FRT) on cerebral microvasculature in mice and to investigate the mechanism of change in BBB permeability in mice. Intravital microscopy and a cranial window technique were used to measure BBB permeability to fluorescein isothiocyanate (FITC)-dextran and leukocyte endothelial interactions before and after cranial irradiation. Daily doses of 2 Gy were delivered 5 days/week (total, 40 Gy). We immunostained the molecules to detect the expression of glial fibrillary acidic protein and to demonstrate astrocyte activity in brain parenchyma. To relate the permeability changes to endothelial ultrastructural changes, we used electron microscopy. Blood-brain barrier permeability did not increase significantly until 90 days after FRT, at which point it increased continuously until 180 days post-FRT. The number of adherent leukocytes did not increase during the study. The number of astrocytes in the cerebral cortex increased significantly; vesicular activity in endothelial cells increased beginning 90 days after irradiation, and most tight junctions stayed intact, although some were shorter and less dense at 120 and 180 days. The cellular and microvasculature response of the brain to FRT is mediated through astrogliosis and ultrastructural changes, accompanied by an increase in BBB permeability. The response to FRT is delayed as compared with single-dose irradiation treatment, and does not involve leukocyte adhesion. However, FRT induces an increase in the BBB permeability, as in the case of single-dose irradiation.

Anti-inflammatory effects of low-dose radiotherapy in an experimental model of systemic inflammation in mice

The aim of this study was to determine the effects of low-dose radiotherapy (LD-RT) on the inflammatory response and to characterize the potential mechanisms underlying these effects. Mice were irradiated with 0.1, 0.3, 0.6 Gy, or sham radiation before lipopolysaccharide (LPS) challenge. Leukocyte-endothelial cell interactions in intestinal venules were assessed using intravital microscopy. Intercellular adhesion molecule-1 (ICAM-1) expression was determined using radiolabeled antibodies 5 h after irradiation. Production of transforming growth factor-beta1 (TGF-beta1) was measured by enzyme-linked immunosorbent assay and its in vivo functional relevance by immunoneutralization. Compared with vehicle treated animals, LPS induced a marked increase in leukocyte adhesion (0.13+/-0.59 vs. 5.89+/-1.03, p<0.0001) in intestinal venules. The number of adherent leukocytes was significantly reduced by the 3 doses of LD-RT tested; the highest inhibition was observed with 0.3 Gy (0.66+/-1.96, p<0.0001). LPS-induced ICAM-1 upregulation was not modified by LD-RT. Circulating levels of TGF-beta1 were significantly increased in response to LD-RT in controls and LPS challenged animals. Neutralization of TGF-beta1 partially restored LPS-induced adhesion (4.83+/-1.41, p<0.05). LD-RT has a significant anti-inflammatory effect, inhibiting leukocyte recruitment, which is maximal at 0.3 Gy. This effect results in part from increased TGF-beta1 production and is not related to modulation of ICAM-1 expression.

Lactobacillus caseiprevents the upregulation of ICAM-1 expression and leukocyte recruitment in experimental colitis

Lactobacillus casei has been shown to attenuate the severity of experimental colitis. The objective of the present study was to determine whether the effects of L. casei on colitis are related to modulation of leukocyte recruitment into the inflamed intestine. Rats with a colonic segment excluded from fecal transit were surgically prepared. The segment was decontaminated with antibiotics and recolonized with normal flora isolated from the inflamed rat colon, associated or not to L. casei. Control and colitic [2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced] animals were studied. Leukocyte-endothelial cell interactions were characterized in the colonic microcirculation by intravital microscopy, and ICAM-1 and VCAM-1 expression was measured by the radiolabeled antibody technique. Compared with the noninflamed colonic segment, induction of colitis by TNBS provoked a marked increase in the number of leukocytes firmly adherent to the venular wall (0.5 +/- 0.1 vs. 2.1 +/- 0.6 leukocytes/100 mum, P < 0.01). Colonization with L. casei significantly reduced the number of adherent leukocytes (1.3 +/- 0.4 leukocytes/100 mum; P < 0.05) but did not affect the increased rolling interactions associated with the induction of colitis. Compared with the noncolitic group, induction of colitis was associated with a marked increase in ICAM-1 expression (117 +/- 4 vs. 180 +/- 3 ng antibody/g tissue) that was abrogated when the colitic segment was colonized by L. casei (117 +/- 3 ng antibody/g tissue, P < 0.05). However, L. casei administration did not modify VCAM-1 upregulation in colitic animals. L. casei attenuates leukocyte recruitment observed in experimental colitis induced by TNBS. This effect is possibly related to abrogation of ICAM-1 upregulation.

COMPARISON OF THE PROTECTIVE EFFECTS OF CILOSTAZOL AND ACETYLSALICYLIC ACID ON INTESTINAL MICROCIRCULATION AFTER ISCHEMIA REPERFUSION INJURY

Objective: The aim of this study is to elucidate the difference of effects between cilostazol (CZ) and acetyl-salicylic acid (ASA) on microcirculatory disturbance in the I/R injury. Methods: Either 10 mg/kg of CZ (n = 14) or 100 mg/kg of ASA (n = 14) was administered orally to mice. 20 min of intestinal ischemia followed by 60 min reperfusion was applied and the status of submucosal microcirculation was observed under the intravital microscopy. Blood cell counts and organ damage markers were examined in the portal blood. 5 mm of ileum was excited and served for the histological examination. Results: Platelet-leukocyte aggregates were often observed in post-capillary venule and this formation was significantly reduced both by CZ and ASA. The number of adherent leukocytes was significantly less in CZ treated mice compared to ASA treated mice (P < 0.01). Leukocyte number, LDH and lactate levels were best maintained in CZ treated mice (P < 0.05, respectively). Villus height was preserved best in CZ treated mice. Conclusion: CZ inhibited not only the platelet aggregation but also leukocyte adhesion to the endothelium and thus, reached to the organ protection.Figure

Influence of polyclonal anti-thymocyte globulins upon ischemia–reperfusion injury in a non-human primate model

Background Polyclonal anti-thymocyte globulins (ATGs) are used to induce immunosuppression and to treat acute rejection after transplantation. ATGs induce apoptosis and in peripheral T-lymphocytes having the potential to inhibit leukocyte adhesion. We analysed the influence of three different ATGs upon the microvasculature and the different cell-subpopulations after ischemia/reperfusion (IRI). Materials and methods Extremities of cynomolgus monkeys were surgically isolated and flushed with Ringer's lactate at 4 °C. After 60 min of ischemia the limbs were reperfused with matching human blood. ATGs were added to the blood 30 min prior to the reperfusion. Four groups were generated: Tecelac-ATG group, Fresenius(S)-ATG group, Thymoglobulin-ATG group and a control group. Blood analyses were performed in blood samples taken after the beginning of the reperfusion. Biopsies from muscular tissue were obtained after the experiments. Results The number of circulating leukocytes was lower in the ATG-groups than in control. Morpho-cytological analyses showed depletion of peripheral lymphocytes. Histological examination showed less tissue damage, reduced presence of fibrin and adherent thrombocytes in the ATG-treated groups. Leukocyte infiltration, both in muscle and vascular structures, was significantly diminished in the ATG-groups in comparison to control. Discussion Our results show that ATGs have a favourable impact on early mechanisms of IRI. ATGs showed a reduction of the number of adherent leukocytes and muscle infiltrates suggesting that preoperative therapy with ATGs may have an advantageous effect on primary non-function and on chronic rejection as well as a positive influence upon IRI.

Role of the coagulation system in experimental colitis

Growing evidence indicates that there is significant “cross-talk” between inflammatory and coagulation pathways. While patients with inflammatory bowel disease exhibit an increased incidence of thrombosis, it remains unclear whether the coagulation cascade contributes to the intestinal inflammation. The objective of this study was to determine whether tissue factor (TF) contributes to leukocyte/platelet-endothelial adhesion, tissue injury, and disease severity in experimental murine colitis induced by feeding C57Bl/6 mice 3% dextran sodium sulfate (DSS) in drinking water for 6 days. Some mice were treated with a monoclonal antibody (mAb) against murine tissue factor (muTF-mAb) two times (20mg/kg, ip) during the 6 days of DSS ingestion. Colitis was assessed by a disease activity index (DAI) and histology. Intravital fluorescence microscopy was used to quantify leukocyte and platelet adhesion in venules. In untreated mice, DSS resulted in an increased DAI, colon shortening, an increased colon weight/length ratio, and a profound increase in the number of adherent leukocytes and platelets in colonic venules. All of these responses to DSS were significantly attenuated in DSS mice treated with muTF-mAb. These findings support a link between the coagulation and inflammatory pathways in experimental colitis and implicate tissue factor as a major pro-inflammatory agent in this model. (Supported by DK65649)

The uptake of a Klebsiella pneumoniae capsule polysaccharide mutant triggers an inflammatory response by human airway epithelial cells

The means by which airway epithelial cells sense a bacterial infection and which intracellular signalling pathways are activated upon infection are poorly understood. A549 cells and human primary airway cells (NHBE) were used to investigate the response to infection with Klebsiella pneumoniae. Infection of A549 and NHBE with K. pneumoniae 52K10, a capsule polysaccharide (CPS) mutant, increased the surface levels of ICAM-1 and caused the release of IL-8. By contrast, the wild-type strain did not elicit these responses. Consistent with a functional role for these responses, there was a correlation between ICAM-1 levels and the number of adherent leukocytes on the epithelial cell surface. In addition, treatment of neutrophils with IL-8 enhanced their ability to kill K. pneumoniae. Strain 52K10 was internalized by A549 cells more efficiently than the wild-type, and when infections with 52K10 were performed in the presence of cytochalasin D the inflammatory response was abrogated. These findings suggest that cellular activation is mediated by bacterial internalization and that CPS prevents the activation through the blockage of bacterial adhesion and uptake. Collectively, the results indicate that bacterial internalization by airway epithelial cells could be the triggering signal for the activation of the innate immune system of the airway. Infection of A549 cells by 52K10 was shown to trigger the nuclear translocation of NF-kappaB. Evidence is presented showing that 52K10 activated IL-8 production through Toll-like receptor (TLR) 2 and TLR4 pathways and that A549 cells could use soluble CD14 as TLR co-receptor.

CD44, but not l‐selectin, is critically involved in leucocyte migration into the skin in a murine model of allergic dermatitis

CD44 and l-selectin (CD62L) are major adhesion receptors that mediate leucocyte recruitment at inflammatory sites and lymph nodes, by supporting cell rolling under blood flow. Both CD44 and CD62L have been implicated in inflammatory skin disorders, but their specific involvement in an immediate-type allergic reaction remains uncertain. We used mice deficient in CD44 or CED62L or both in order to determine whether one or both of these molecules were required for leucocyte extravasation in an atopic dermatitis-like allergic response. Wild-type (WT) mice and mice deficient in CD44, CD62L or both were immunized with ovalbumin (OVA). Inflammatory reaction in the ear was elicited once by means of intradermal injection of OVA. Effective sensitization of CD62L knockout (KO) mice required intraperitoneal antigen injection; however, OVA-specific T helper 2 (Th2)-type immune responses and IgE production in mice lacking CD44, CD62L or both were comparable to those in WT mice following intraperitoneal immunization. We employed intravital videomicroscopy to monitor the recruitment of fluorescence-labelled leucocytes to the ear tissue following challenge with OVA. The number of adherent leucocytes was significantly reduced in CD44 KO and CD44/CD62L double KO mice, indicating that CD44 was involved in firm adhesion, the committed step of leucocyte extravasation. Histology of the OVA-challenged ears showed a diminished leucocyte infiltration in the ears of CD44 KO and double KO mice. The results of our study demonstrate that CD44, but not CD62L, is required for leucocyte extravasation during a Th2-type inflammatory response.

Protective effects of a selective neutrophil elastase inhibitor (sivelestat) on lipopolysaccharide-induced acute dysfunction of the pulmonary microcirculation

The purpose of this study was to evaluate the effect of a neutrophil elastase inhibitor, sivelestat, on lipopolysaccharide-induced acute lung injury through analysis of hemodynamic changes in the pulmonary microcirculation. Randomized animal study. Medical school laboratory. Twenty-seven Wistar rats (15 rats for microspectroscopic observations, 12 rats for measurements of neutrophil elastase activity and wet-to-dry ratio). Thoracosternotomy was performed on male Wistar rats under continuous anesthesia and mechanical ventilation. Rats were divided into three groups (n = 5 each groups) on the basis of the reagent used: lipopolysaccharide group (100 microg/kg lipopolysaccharide intravenously), sivelestat group (10 mg/kg sivelestat; 100 microg/kg lipopolysaccharide intravenously), and control group (saline only, intravenously). We measured morphologic changes and hemodynamic variables, including tissue blood flow, erythrocyte velocity, erythrocyte count, thickness of interalveolar septa, and leukocyte adhesion in the pulmonary microcirculation, with a video-rate (33 msec/frame) dual-spot microspectroscopy system (DSMSS) and a laser-Doppler flowmeter. Blood-free wet-to-dry ratio and neutrophil elastase activity in bronchoalveolar lavage fluid, serum, and supernatant of lung homogenate were measured in another set of experiments (n = 4 for each group). Sixty minutes after lipopolysaccharide administration, severe thickening of the interalveolar septa was observed in the lipopolysaccharide but not the sivelestat group. In the lipopolysaccharide group, DSMSS measurements of erythrocyte velocity and hemoglobin oxygenation in single capillaries were decreased significantly (vs. control p < .05, vs. sivelestat p < .01), whereas tissue blood flow and erythrocyte velocity measurements from laser-Doppler flowmeter were increased significantly (vs. control p < .05, vs. sivelestat p < .01). The number of adherent leukocytes was increased significantly in the lipopolysaccharide group at 30, 45, and 60 mins after lipopolysaccharide administration (vs. control p < .01, vs. sivelestat p < .05). The number of adherent leukocytes did not increase in the sivelestat group. The wet-to-dry ratio was significantly higher in the lipopolysaccharide group than in control (p < .05) and sivelestat (p < .05) groups. Neutrophil elastase activities in the bronchoalveolar lavage fluid, serum, and lung tissue were all significantly lower in the sivelestat group than in the lipopolysaccharide group (p < .05). Lipopolysaccharide induces leukocyte adhesion in the pulmonary microcirculation, resulting in decreased tissue hemoglobin oxygen and alveolar and interstitial edema. The selective neutrophil elastase inhibitor sivelestat reduces neutrophil elastase activity and attenuates acute changes in the pulmonary microcirculation.

In vivo effects of hypothermia on the microcirculation during extracorporeal circulation

Induced hypothermia has been shown to be protective during cardiac surgery, but also in traumatic, ischemic, burn, and neurological injury. In previous in vivo animal experiments, we documented increased leukocyte/endothelial (L/E) cell interaction following normothermic extracorporeal blood circulation (ECC). This study was carried out to investigate whether reduced core temperature during ECC affects the damage to the microcirculation as evidenced by leukocyte adherence and edema formation. Intravital fluorescence microscopy was used on the dorsal skinfold chamber preparation in Syrian golden hamsters. ECC was introduced via a micro-rollerpump (1 ml/min) and a 60 cm silicon tube (1mm inner diameter) shunted between the carotid artery and the jugular vein after application of 300IE Heparin/kg per body weight. Experiments were performed in chronically instrumented, awake animals (age 10-14 weeks, weight 65-75 g). Animals of the experimental group were cooled to 18 degrees C body temperature while ECC, followed by a rewarming period (n=7), controls experienced ECC under normothermia (37 degrees C, n=7). 30 min ECC at 18 degrees C resulted in a decrease of rolling and adherent leucocytes (stickers) in postcapillary venules after 1, 4 and 8h compared with the control group (119+/-46 vs. 274+/-113 n/mm2, P<0.05, mean+/-SD; n=7 in each group). Functional capillary density was significantly reduced during hypothermia (80+/-16 vs. 148+/-16 cm/cm2, P<0.05), but restored after rewarming. In contrast, edema formation was markedly increased during hypothermia. Hypothermia during ECC significantly reduced L/E cell interaction in the early post-ECC period. Hypothermia markedly reduced microvascular perfusion, but was completely restored upon rewarming. Despite a reduced number of adherent leukocytes, no protection of endothelial barrier function was seen as a consequence of induced hypothermia.

Hydroxymethylglutaryl co-enzyme A reductase inhibition attenuates endotoxin-mediated inflammatory responses

The aim of this study was to investigate whether inhibition of hydroxymethylglutaryl co-enzyme A reductase attenuates leucocyte-endothelial cell interactions and alters expression of endothelial constitutive nitric oxide synthase (ecNOS) and inducible nitric oxide synthase (iNOS) following exposure to endotoxin. Male Sprague-Dawley rats were randomized into control, lipopolysaccharide (LPS) and pravastatin + LPS groups (seven per group). Pravastatin sodium was gavaged at 0.4 mg per kg per day for 5 days, after which LPS 15 mg/kg was administered via the jugular vein. Intravital microscopy was used to determine leucocyte-endothelial cell interactions. Following the administration of LPS there was a significant reduction in leucocyte rolling velocity at 10 min (mean(s.e.m.) 69(3) versus 102(6) per cent of baseline value; P = 0.041), an increase in the number of adherent leucocytes at 10 min (4.5(0.5) versus 2.8(0.3) per 100 microm; P = 0.044) and an increase in the number of leucocytes undergoing transendothelial migration at 30 min (4.2(0.4) versus 1.7(0.4) per field; P = 0.008) compared with controls. Pretreatment with pravastatin significantly attenuated LPS-induced leucocyte-endothelial cell interactions (rolling velocity 89(6) per cent at 10 min, P = 0.038; adherent leucocytes 3.0(0.5) per 100 microm at 10 min, P = 0.038; migrating leucocytes 1.9(0.5) per field at 30 min, P = 0.001). This endothelial protection was associated with maintenance of ecNOS and reduced iNOS expression within mesenteric tissues. These data show that pravastatin produces anti-inflammatory effects in response to injurious stimuli by attenuation of leucocyte-endothelial cell interactions.

Radiation-Induced Up-regulation of Adhesion Molecules in Brain Microvasculature and their Modulation by Dexamethasone

Little is known about the time course and magnitude of the up-regulation of endothelial cell adhesion molecules (ECAMs) in irradiated brain vasculature and the mechanisms by which dexamethasone modulates this up-regulation. We used antibody-conjugated microspheres and a rat closed cranial window model to determine the time course of functional up-regulation of radiation (20 Gy)-induced ICAM1, E-selectin and P-selectin in the pial vasculature of the rat brain and to determine the relationship between suppression of inflammation by dexamethasone and the expression of these ECAMs. The results indicate that ICAM1, E-selectin and P-selectin were up-regulated to a functional level in the microvasculature with distinct time-course patterns. The number of adherent anti-E-selectin and anti-P-selectin microspheres was 5- 12 times greater than that of IgG microspheres 3-6 h postirradiation, and their expression returned to normal at 48 h. The number of adherent anti-ICAM1 microspheres was five and nine times greater than that of IgG at 24 and 48 h, respectively, and returned to baseline by 7 days. Dexamethasone significantly reduced the number of adhering leukocytes and the number of adhering anti-ICAM1, anti-E-selectin and anti-P-selectin microspheres to background levels. Our findings partially identify a key sequence in radiation-induced inflammatory response and provide a potential means to limit radiation-induced inflammatory responses and their potential side effects in the brain.

Platelet–endothelial cell interactions in murine antigen-induced arthritis

Growing evidence supports the substantial pathophysiological impact of platelets on the development of rheumatoid arthritis. At present there are no methods for studying these cellular mechanisms in vivo. The aim of this study was to visualize and investigate platelet-endothelial cell interaction in the knee joint of mice with antigen-induced arthritis (AiA) by means of intravital microscopy. In 14 mice (Balbc) intravital microscopic assessment was performed on day 8 after AiA induction in two groups (controls, AiA). The severity of AiA was assessed by measuring knee joint swelling and by histological scoring. Ex vivo fluorescently labelled rolling and adherent platelets and leucocyte-endothelium interactions were investigated by intravital fluorescence microscopy. Swelling of the knee joint as well as histological score was significantly enhanced in arthritic animals compared with controls. In control mice intravital microscopy revealed low baseline rolling and sticking of leucocytes and fluorescently labelled platelets. AiA induced a significant increase in the fraction of rolling leucocytes (3 times) and rolling platelets (6 times) compared to the control group. Furthermore, AiA induction resulted in a significantly enhanced number of adherent leucocytes (3-fold) and adherent platelets (12-fold) in comparison with control animals. Platelet kinetics were directly analysed using intravital microscopy in the arthritic microcirculation in vivo for the first time. We provide the first evidence that platelets accumulate in arthritic vessels, indicating platelet activation due to AiA. Platelet recruitment and subsequent activation might play an important role in the pathogenesis of rheumatoid arthritis.

Leukocytes with Bright Fluorescence in Rats

In Response: We thank Sato et al. for their interest in our article (1) and for inquiring into the method we used to visualize leukocytes in our study. Our experimental preparation used the mesentery of the rat. This established model has been used for years by other researchers (2,3) and is well suited for intravital microscopy because it is readily accessible, easily surgically isolated, thin (<200 μm thick), and transparent, thus allowing for direct transillumination of vessels and cellular morphotic elements. In the present study (1), we used an upright epifluorescence microscope (Olympus BX51) with water immersion lens (magnification 20× or 40×; numerical aperture = 0.5 or 0.8, respectively) operating in bright field mode. Due to the exceptional clarity of the morphotic elements within the mesenteric microcirculation, no fluorescent dye was needed in our study to observe and quantitate leukocyte-endothelial interactions (adhesion and leukocyte rolling velocities). Leukocytes, as mentioned in the first paragraph of our study (1), is a general term that we and others (4) use to describe polymorphoneutrophils in in vivo flow conditions. Most leukocytes that we observed in the microvessels of our rats were probably granulocytes (5,6). However, the distinction between granulocytes and mononuclear cells could not readily be made during real-time flow conditions when employing intravital microscopy; hence we use the general term leukocyte. The off-line analysis system we used to analyze the number of adherent leukocytes, and the velocity of rolling leukocytes as they adhered to or traveled along the endothelium of a postcapillary venule from videotape, generally allowed us to differentiate granulocytes from the smaller lymphocytes (Fig. 1).Figure 1.: Bright-field transillumination of rat mesentery venule showing granulocyte and smaller lymphocyte.However, on occasions when rolling velocities of a specific cell type is required, or when tracking the movement of leukocytes in tissues or organs is not amiable to transillumination, for example, in parenchymatous or opaque organs of the body such as in the liver, brain and lung, then other techniques, such as using fluorescent markers, may be required to visualize leukocyte dynamics. As pointed out by Sato et al., some fluorescent dyes may induce phototoxic effects especially in leukocytes (7). To avoid these potential problems, many researchers, including ourselves (Fig. 2), use transgenic animal models that express green fluorescent protein (GFP) in certain cell lines, such as the murine lysozyme M GFP mouse (8). These mice express GFP in their mature neutrophils, while other cell lineages are fluorescent negative.Figure 2.: Epifluorescence illumination of lys-M GFP mouse showing GFP labeled neutrophils in a cremasteric venule.John K. Hayes, PhD Dmytro M. Havaleshko, MD Roman V. Plachinta, MD George F. Rich, MD, PhD Department of Anesthesiology University of Virginia Health System Charlottesville, VA [email protected]

Selective Suppression of Pathologic, but Not Physiologic, Retinal Neovascularization by Blocking the Angiotensin II Type 1 Receptor

To investigate the anti-inflammatory and anti-angiogenic effects of telmisartan, an angiotensin II type 1 receptor (AT1-R) antagonist, on ischemia-induced retinal neovascularization. C57BL/6 neonatal mice were reared in an 80% concentration of oxygen from postnatal day (P)7 to P12, followed by room-air breathing until P17, to induce ischemia-initiated retinal neovascularization (i.e., a murine model of ischemic retinopathy). Tissue localization of AT1-R was examined by immunohistochemistry for murine retinal wholemounts and human fibrovascular tissues excised at vitrectomy for proliferative diabetic retinopathy. Animals received intraperitoneal injection of telmisartan or vehicle. A concanavalin A lectin perfusion-labeling technique was used to evaluate the areas of physiological and pathologic retinal new vessels and the number of leukocytes adhering to the vasculature. Retinal mRNA and protein levels of intercellular adhesion molecule (ICAM)-1, vascular endothelial growth factor receptor (VEGFR)-1, and VEGFR-2 were examined by RT-PCR and ELISA. Vessels in human fibrovascular tissues and the murine retinas were positive for AT1-R. Pathologic (P < 0.01), but not physiologic (P > 0.05), retinal neovascularization was significantly suppressed in telmisartan-treated mice compared with vehicle-treated animals. The number of adherent leukocytes (P < 0.01) was also significantly reduced, together with retinal ICAM-1 levels (P < 0.01) in the telmisartan-treated group compared with the control group. No significant difference was detected in retinal VEGFR-2 levels between the two groups, whereas retinal VEGFR-1 levels in the telmisartan-treated group were significantly (P < 0.05) lower than in the vehicle-treated group. The present findings suggest that the AT1-R signaling blockade leads to the selective suppression of pathologic, but not physiological, retinal neovascularization through the inhibition of the inflammatory processes related to pathologic neovascularization.

Role of CD44 and Hyaluronan in Neutrophil Recruitment

Lymphocyte CD44 interactions with hyaluronan localized on the endothelium have been demonstrated to mediate rolling and regulate lymphocyte entry into sites of chronic inflammation. Because neutrophils also express CD44, we investigated the role of CD44 and hyaluronan in the multistep process of neutrophil recruitment. CD44(-/-) and wild-type control mice were intrascrotally injected with the neutrophil-activating chemokine, MIP-2, and leukocyte kinetics in the cremasteric microcirculation were investigated 4 h subsequently using intravital microscopy. Neither the rolling flux nor the rolling velocities were decreased in CD44(-/-) mice relative to wild-type mice. In vitro, neutrophils did not roll on the CD44 ligand hyaluronan, consistent with the in vivo data that CD44/hyaluronan did not mediate rolling. However, the number of adherent leukocytes in the venule was decreased by 65% in CD44(-/-) mice compared with wild-type mice. Leukocyte emigration was also greatly decreased in the CD44(-/-) mice. The same decrease in adhesion and emigration was observed in the wild-type mice given hyaluronidase. Histology revealed neutrophils as being the dominant infiltrating population. We generated chimeric mice that express CD44 either on their leukocytes or on their endothelium and found that CD44 on both the endothelium and neutrophils was important for optimal leukocyte recruitment into tissues. Of those neutrophils that emigrated in wild-type and CD44(-/-) mice, there was no impairment in migration through the interstitium. This study suggests that CD44 can mediate some neutrophil adhesion and emigration, but does not appear to affect subsequent migration within tissues.

Pentastarch in a Balanced Solution Reduces Hepatic Leukocyte Recruitment in Early Sepsis

To characterize the hepatic leukocyte endothelial cell interactions occurring in early sepsis and to determine whether this is influenced by fluid treatment. Sepsis was induced by cecal ligation and perforation in C57Bl/6 mice. One of 6 iv fluid regimes was given immediately postsurgery and at 6 h. The hepatic microcirculation was examined by intravital microscopy at 6 h. All 0.9% saline-based solutions were associated with an increase in leukocyte-endothelial cell interactions as demonstrated by an increase in the rolling flux in the sham and naïve mice. In the septic mice treated with normal saline, there was a 20-fold increase in leukocyte adhesion within the postsinusoidal venules, compared to sham mice. Treatment with lactated Ringer's reduced the sepsis-mediated leukocyte recruitment by 50%. When septic mice received 6% pentastarch in a balanced solution or 3% saline, venular leukocyte adhesion was reduced by an additional 50%. When the pentastarch was prepared in normal saline there was no further reduction in venular leukocyte adhesion compared to the lactated Ringer's-treated mice. All hyperosmolar solutions improved the sepsis-induced reduction in sinusoidal perfusion but only the pentastarch in the balance solution significantly reduced the number of adherent leukocytes within the sinusoids. Hepatic leukocyte recruitment occurs early in sepsis. Pentastarch in a balanced solution but not in normal saline significantly reduces hepatic leukocyte recruitment, suggesting solution composition, as well as osmolarity impact the innate immune response.