Intravital Videomicroscopy Research Articles

Vasopressin Attenuates TNF-Mediated Inflammation in the Rat Cremaster Microcirculation

Our previous study in a swine polytrauma model suggested that equieffective systemic pressor doses of arginine vasopressin (AVP) versus phenylephrine (PE) have differential effects on the systemic and cerebral microcirculation. The purpose of this study was to directly observe the effects of AVP versus PE on inflammatory changes evoked by tumor necrosis factor alpha (TNF) in the skeletal muscle microcirculation. Seventy-five male rats (180-250 g) were anesthetized with isoforane, intubated and mechanically ventilated with 100% oxygen. The cremaster muscle microcirculation was prepared for intravital video microscopy while being suffused with a heated hetastarch-electrolyte solution. Fluorescein isothiocyanate-labeled albumin (100 mg/kg) was administered intravenously (i.v.) before one of five protocols. In series 1 (n = 20), either AVP (0.2 U/mL) or its vehicle was added to the suffusate for 10 minutes, washed out for 30 minutes, then TNF was suffused (5 ng/mL) for 30 minutes. In series 2 (n = 16), the protocol was similar, except AVP (0.2 U/mL) or an equieffective dose of PE (0.04 mg/mL) was administered i.v. (4.5 mL/h) for 15 minutes before, during, and 45 minutes after TNF suffusion. In series 3 (n = 12), the protocol was similar to series 2, except venous hemorrhage preceded i.v. AVP or PE. In series 4 (n = 15), the protocol was similar to series 3, except an AVP antagonist (vaprisol, 1 mg/kg i.v.) or its vehicle was administered after hemorrhage. In the control series (n = 13), inflammation was evaluated either with a different suffusate (lactated Ringers instead of hetastarch solution), different antigen (histamine instead of TNF), or hemorrhage with no antigen. In series 1, the TNF-evoked increase in leukocyte infiltration (i.e., rolling), leukocyte activation (i.e., sticking), and macromolecular permeability (i.e., albumin extravasation) were attenuated with topical AVP versus vehicle (both p < 0.05), with no effect on venular blood flow (which determines sheer stress). In series 2, the TNF-evoked increase in infiltration, activation, and permeability were all attenuated, and arteriolar blood flow (which determines perfused capillary surface area and hydrostatic pressure) was reduced with i.v. AVP versus i.v. PE (all p < 0.05). In series 3, after hemorrhage to mean arterial pressure <50 mm Hg for 30 minutes, the TNF-evoked increase in infiltration and activation was attenuated, and arteriolar and venular blood flow were both reduced with i.v. AVP versus PE (all p < 0.05). In series 4, after hemorrhage, the TNF-evoked increase in leukocyte activation was potentiated with the vaprisol versus vehicle (p < 0.05) with no effect on arteriolar or venular blood flow. In series 5 (controls), suffusion with lactated Ringers' versus hetastarch solution more than doubled the TNF-evoked increase in activation (p < 0.05). (1) AVP can attenuate TNF-evoked leukocyte infiltration, activation or permeability changes in the skeletal muscle microcirculation. (2) The mechanism is probably receptor mediated and does not entirely depend on sheer stress in venules or Starling forces in capillaries. (3) The magnitude of this anti-inflammatory effect is influenced by several conditions, including volume status, the colloid or crystalloid suffusion fluid, and is possibly specific to the antigenic stimulus (TNF vs. histamine).

Functional blocking of specific integrins inhibit colonic cancer migration

For more effective oncological management of disseminated colorectal cancer, therapies must be devised that target the different individual stages of metastasis development. Recent work showed that integrin subunits alpha2, alpha6 and beta4 are involved in the colorectal cancer cell extravasation process. By means of Immunocytochemistry and Western blotting, it was shown that all three integrins are expressed not only in human colorectal cancer cells (HT29) but also in rat colonic cancer cells (DHDK12). Using in vivo models and intravital video microscopy techniques, it was shown that functional blocking of these integrin subunits by specific antibodies produced a significant reduction in cancer cell extravasation and migration. In conclusion, integrin subunits alpha2, alpha6 and beta4 are expressed in unrelated colorectal cancer cell strains and appear to play a key role in cancer cell migration.

Budlein A from Viguiera robusta inhibits leukocyte-endothelial cell interactions, adhesion molecule expression and inflammatory mediators release

Budlein A has been reported to exert some analgesic and anti-inflammatory properties. In this study, we have evaluated its effect on LPS-induced leukocyte recruitment in vivo and the mechanisms involved in its anti-inflammatory activity. In vivo, intravital videomicroscopy was used to determine the effects of budlein A on LPS-induced leukocyte-endothelial cell interactions in the murine cremasteric microcirculation. In vitro, the effects of budlein A on LPS-induced cytokine, chemokine and nitrites release, T-cell proliferative response as well as cell adhesion molecule expression (CAM) were evaluated. In vivo, intraperitoneal administration of budlein A (2.6 mM/kg) caused a significant reduction of LPS-induced leukocyte rolling flux, adhesion and emigration by 84, 92 and 96% respectively. In vitro, T-cell proliferative response was also affected by budlein A. When murine J774 macrophages were incubated with the sesquiterpene lactone, LPS-induced IL-1beta, tumor necrosis factor-alpha (TNF-alpha) and keratinocyte-derived chemokine (KC) release were concentration-dependently inhibited. In human umbilical vein endothelial cells (HUVECs), budlein A also reduced the production of TNF-alpha, monocyte chemoattractant protein-1 (MCP-1), IL-8, nitrites and CAM expression elicited by LPS. Budlein A is a potent inhibitor of LPS-induced leukocyte accumulation in vivo. This effect appears to be mediated through inhibition of cytokine and chemokine release and down-regulation of CAM expression. Thus, it has potential therapeutic interest for the control of leukocyte recruitment that occurs in different inflammatory disorders.

Modulation of circulating cell–endothelial cell interaction by erythropoietin in lean and obese mice with cecal ligation and puncture

Objective: Septic shock related morbidity is increased further in obese patients. Obese mice have exaggerated inflammatory response compared to lean mice when exposed to cecal ligation and puncture (CLP). The current study employs use of erythropoietin (EPO), an anti-inflammatory agent in obese and lean mice with CLP to study inflammation. Method: Intravital videomicroscopy was used to quantify CLP-induced platelet (PA) and leukocyte (LA) adhesion in cerebral microcirculation of WT (C57Bl/6) and ob/ ob (B6.V- Lepob/J) mice with or without EPO (500 units/kg) treatment. P-selectin expression (P-sel) in various tissues, blood–brain barrier (BBB) and behavioral function and serum tumor necrosis factor (TNF) levels were quantified in these animals. LA, PA, BBB function and P-sel were measured in TNF monoclonal antibody (mAb) pre-treated ob/ ob mice. Results: EPO attenuated the LA, PA, BBB and behavioral dysfunctions and serum TNF levels 4 h after CLP in both WT and ob/ ob mice, and P-sel in ob/ ob CLP mice. Furthermore, TNF mAb and EPO decreased the LA, PA, BBB dysfunction and P-sel in the ob/ ob CLP animals to the same degree. Conclusion: EPO exerts significant anti-inflammatory actions in obese and lean septic mice. Modulation of P-selectin expression via attenuation of TNF production is a suggested underlying mechanism.

Myeloid-Specific Deletion of Tumor Suppressor PTEN Augments Neutrophil Transendothelial Migration during Inflammation

Phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) is a second messenger that is involved in a number of cell activities including cell growth, proliferation, and motility. PIP(3) is produced by PI3K and regulated by PTEN (phosphatase and tensin homolog deleted on chromosome 10) and SHIP lipid phosphatases. Evidence from our experiments shows that enhanced PIP(3) production results in elevated neutrophil recruitment under inflammatory conditions. However, the mechanism of this elevation is not well understood. We used intravital video microscopy to investigate neutrophil recruitment in the cremaster venules of wild-type and PTEN knockout (KO) mice. Neutrophil transmigration was augmented in PTEN KO mice 4 h after TNF-alpha intrascrotal injection. PTEN KO neutrophils also showed significantly enhanced transmigration 2 h after MIP-2 intrascrotal injection, an effect that dramatically decreased when PI3K or Src kinase inhibitor treatments preceded MIP-2 stimulation. Similarly, fMLP superfusion of the cremaster muscle lead to enhanced emigration in PTEN KO mice. The observed elevation in neutrophil emigration was likely caused by increased speed of crawling, crossing the venular wall, and migrating through the muscular tissue in PTEN KO mice because the effect of PTEN depletion on neutrophil rolling or adhesion was minimal. Interestingly, chemoattractant-induced release of gelatinase and elastase was also elevated in PTEN null neutrophils, providing a potential mechanism for the enhanced neutrophil migration in the PTEN KO mice. Collectively, these results demonstrate that PTEN deletion in neutrophils enhances their invasivity and recruitment to inflamed sites more likely by raising the cell physical capability to cross the vascular and tissue barriers.

Renal microcirculatory dysfunction in sepsis‐induced acute kidney injury (AKI) in mice

Understanding the sequence of events leading to sepsis‐induced renal injury, especially at the early stages of sepsis, is critical for developing new therapy to reduce mortality that is currently greater than 50%. The cecal ligation and puncture (CLP) model of sepsis was used to study the development of AKI in male 40‐week C57BL/6 mice. Intravital video microscopy (IVVM) was used to monitor and quantitate changes in the peritubular microenvironment in sham surgery mice and at 2, 4, or 6 h following CLP. IVVM revealed a dramatic fall in peritubular capillary perfusion as early as 2 h post CLP. The percentages of cortical peritubular capillaries with continuous flow dropped from 85 ± 4% to 66 ± 6% (P &lt; .05, n = 6) and continued to worsen through 6 h. We also observed that cellular levels of NAD(P)H, an indicator of cellular redox stress measured by IVVM, were elevated following CLP but lagged behind the fall in capillary perfusion. These data show that a compromised renal cortical microcirculation is a very early event in the development of sepsis. The data also indicate a critical relationship between peritubular capillary dysfunction and the development of tubular stress and renal injury during sepsis. This study suggests that therapeutic approaches targeting the peritubular capillary that improve the renal microcirculation could reduce sepsis‐induced AKI. Supported by AHA Grant In Aid #850227Z.

Characterizing the Response of Skeletal Muscle Microvasculature to Imposed Oxygen Variations

Computational modeling of the oxygen regulatory system has shown how signals conducted upstream from capillaries change the flow pattern in the arteriole tree. The simulation showed that the hemodynamic response to higher frequency oscillations (period 30 sec) is attenuated relative to lower frequency oscillations (period 90 sec). To test this prediction in vivo, a custom O2 gas chamber is used to supply O2 at the surface of exposed skeletal muscle and a sequence of 3 oscillations (1st and 3rd 30 sec period, 2nd 90 sec period, see figure) were generated. The hemodynamic response in capillaries to this stimulus was quantified using intravital video microscopy and a functional image analysis system (RBC velocity and hematocrit to yield RBC supply rate, SR). The RBC supply rate response supported the results from the computational model for the 1st and 2nd sine oscillation. The SR response to the higher frequency oscillation was attenuated to ~60‐70% of the lower frequency stimulus. However, the 3rd sinusoidal oscillation showed no response to the O2 signal suggesting that other factors need to be included in the model. Overall, the model and experimental results support the hypothesis that regulation of O2 supply involves a conducted signal along the arteriolar tree controlling both flow resistance and distribution of RBCs at bifurcations. (NIH R33 HL089094).

Angiotensin II‐induced microvascular responses in mouse brain

Although angiotensin II (AngII)‐induced hypertension increases the risk for ischemic stroke, the mechanisms linking AngII to this cardiovascular disease remain poorly understood. We determined whether AngII (delivered over a 2 wk period with an Alzet pump) promotes leukocyte (LECA)‐ and platelet (PECA)‐endothelial cell adhesion in cerebral venules and/or alters blood brain barrier (BBB) permeability in C57Bl/6 (WT), AT1‐R−/− mice, and AT1‐R−/−‐&gt;WT bone marrow chimeras (BMC). Intravital video microscopy was used to monitor blood cell adhesion from a cranial window. AngII infusion induced dose‐dependent (400‐2000 ng/kg/min) increases in mean arterial pressure (MAP), LECA, and PECA. AT1‐R−/−, AT1‐R−/−‐&gt;WT BMC, and WT mice treated with Losartan (25 mg/kg/day) all exhibited no changes in MAP, LECA and PECA in response to AngII. BBB permeability, measured by Evans blue extravasation, was increased in response to AngII in WT and AT1‐R−/−‐&gt;WT BMC mice, but not in AT1‐R−/− mice and WT mice treated with Losartan. These findings implicate AT1‐receptors (AT1‐R) on blood cells in the inflammatory and pro‐thrombogenic phenotype induced during AngII‐induced hypertension. The accompanying BBB dysfunction appears to involve AT1‐R on endothelial cells, rather than blood cells. (Supported by HL26441)

Reduction in endothelial glycocalyx thickness after hemorrhage

Although some evidence suggests that ischemia induces changes in the endothelial glycocalyx (EG), little information is available about the EG following hemorrhagic hypotension (HH). We have employed video analysis to obtain preliminary data on EG changes following hemorrhage. Using intravital videomicroscopy, we studied the spinotrapezius muscle of anesthetized rats subjected to HH (40% blood vol.) for over 1 h. The analog video camera signal was digitized using standard software to DV‐AVI format. About 100 frames of several video clips (3‐5 s each) were processed using specially designed software to derive dimensions of the endothelial glycocalyx from the gap between erythrocytes and the luminal endothelial surface. Frequency distributions of the EG thickness during baseline and during HH showed a shift to lower values following hemorrhage, corresponding to a reduction in EG thickness of nearly 50 % (1.1 µm [range: 0.4 ‐ 4.3 µm] vs 0.6 µm [range: 0.3 ‐ 1.3 µm]). These changes occurred despite similar distributions of anatomic diameters (baseline range: 9 ‐ 24 µm; HH: 10 ‐ 21 µm). The findings suggest that the analysis of EG changes using in vivo video recordings can be used to investigate changes following HH and provide preliminary support for our hypothesis that these low flow states cause reductions in the endothelial surface layer thickness.SupportNIH, NWO, NHS, (HV: NHF grant 2005T037).

Role of AT1‐receptors in hypercholesterolemia‐induced thrombosis.

Hypercholesterolemia is associated with increased platelet aggregation and increased risk for thrombosis. Previous reports have described the ability of diet‐induced hypercholesterolemia (HC) to enhance thromboembolism in arterioles, but not venules, in rabbit mesentery (Broeders; ATBV 22:680, 2002). The objectives of this study were to determine whether: 1) HC promotes thrombus formation in microvessels of mouse cremaster muscle and 2) angiotensin II type‐1 receptors (AT1‐R) mediate the HC‐induced responses. Intravital videomicroscopy was used to monitor thrombus formation induced with a light/dye endothelial injury model in mice placed on either a normal diet (ND), 2 wks high‐cholesterol diet (HCD) or HCD mice treated with the AT1‐R antagonist, losartan (25mg/kg/day) in drinking water for 7 days. Arterioles in HCD mice exhibited an enhanced rate of thrombus formation compared to ND mice, decreasing the time of thrombus onset and the time to flow cessation. As previously reported, light/dye‐induced thrombosis was not altered in venules of HCD mice. The HCD‐enhanced thrombosis in arterioles was not altered by losartan treatment. These findings confirm the vulnerability of arterioles to HC‐induced thrombosis, and indicate that mechanisms other than AT1‐R activation underlie the increased risk for thrombosis during hypercholesterolemia. (Supported by HL26441).

Actinonin, a meprin A inhibitor, protects the renal microcirculation during sepsis

Meprin A, a matrix metalloproteinase highly expressed in the kidney and reported to be an early biomarker of acute kidney injury (AKI), is thought to play an important role in several causes of AKI. To address the role of meprin A in the early microcirculatory dysfunction associated with sepsis‐induced AKI, we studied the effects of the meprin A inhibitor actinonin in the cecal ligation and puncture (CLP) model of sepsis in the mouse. Male C57BL/6 mice (40 weeks) underwent CLP with vehicle (CLPv) or actinonin at 20 mg/kg, ip (CLPact) 30 min prior to CLP. At 7h post CLP, intravital videomicroscopy was utilized to image renal cortical peritubular capillary perfusion. Actinonin prevented the fall in renal capillary perfusion. The percentage of capillaries with continuous perfusion in CLPv and CLPact were 50 ± 9% and 83 ± 10% respectively (P &lt; .05) and the percentage with no perfusion in CLPv and CLPact were 33 ± 10% and 7 ± 2% respectively (P &lt; .05). Capillary perfusion in CLPact was no different from sham mice. Since meprin A is known to activate the proform of IL‐1β, serum IL‐1β was used to assess the inhibitory activity of actinonin. IL‐1β levels decreased in CLPact by 36% compared to CLPv at 7h (P &lt; .05). These data suggest that meprin A may play an important role in the microcirculatory failure associated with sepsis‐induced AKI and that actinonin should be evaluated further as a possible therapeutic agent. Supported by NIH DK075991.

In vivo Imaging of Cutaneous T-Cell Lymphoma Migration to the Skin

Cutaneous T-cell lymphoma (CTCL) is characterized by the accumulation of malignant CD4(+) T cells in the skin. Although the expression of adhesion molecules and chemokine receptors on CTCL cells has been studied extensively on ex vivo isolated cells, very little is known about the dynamics and mechanisms of CTCL trafficking in vivo. However, detailed knowledge of the molecular cues mediating CTCL migration may be used to interfere with their homing to the skin. We made use of real-time intravital epifluorescence video and two-photon microscopy to visualize malignant T cells from Sezary syndrome (SS), a leukemic variant of CTCL, in dermal microvessels in mouse ear skin. We found that SS cells rolled along dermal venules in a P-selectin- and E-selectin-dependent manner at ratios similar to CD4(+) memory T cells from normal donors. We furthermore show that the chemokine CCL17/TARC, but not CCL27/CTACK, was sufficient to induce the arrest of SS cells in the microvasculature. However, a combination of both chemokines was required to induce extravasation of SS cells. Together, our experiments delineate the molecular adhesion cascade operant in SS cell homing to the skin in vivo.

Vessel Diameter Measurement from Intravital Microscopy

The blood vessel diameter is often measured in microcirculation studies to quantify the effects of various stimuli. Intravital video microscopy is used to measure the change in vessel diameter by first recording the video and analyzing it using electronic calipers or by using image shearing technique. Manual measurement using electronic calipers or image shearing is time-consuming and prone to measurement error, and automated measurement can serve as an alternative that is faster and more reliable. In this paper, a new feature-based tracking algorithm is presented for automatically measuring diameter of vessels in intravital video microscopy image sequences. Our method tracks the vessel diameter throughout the entire image sequence once the diameter is marked in the first image. The parameters were calibrated using the intravital videos with manual ground truth measurements. The experiment with 10 synthetic videos and 20 intravital microscopy videos, including 10 fluorescence confocal and 10 non-confocal transmission, shows that the measurement can be performed accurately.

Ischemic limb preconditioning downregulates systemic inflammatory activation

We examined local and systemic antiinflammatory consequences of ischemic preconditioning (IPC) in a rat model of limb ischemia-reperfusion (I-R) by characterizing the leukocyte-endothelial interactions in the periosteum and the expression of adhesion molecules playing a role in leukocyte-mediated inflammatory processes. IPC induction (2 cycles of 10 min of complete limb ischemia and 10 min of reperfusion) was followed by 60 min of ischemia/180 min of reperfusion or sham-operation. Data were compared with those on animals subjected to I-R and sham-operation. Neutrophil leukocyte-endothelial cell interactions (intravital videomicroscopy), intravascular neutrophil activation (CD11b expression changes by flow cytometry), and soluble and tissue intercellular adhesion molecule-1 (ICAM-1; ELISA and immunohistochemistry, respectively) expressions were assessed. I-R induced enhanced leukocyte rolling and adherence in the periosteal postcapillary venules after 120 and 180 min of reperfusion. This was associated with a significantly enhanced CD11b expression (by approximately 80% and 72%, respectively) and moderately increased soluble and periosteal ICAM-1 expressions. IPC prevented the I-R-induced increases in leukocyte adherence and CD11b expression without influencing the soluble and tissue ICAM-1 levels. The results show that limb IPC exerts not only local, but distant antiinflammatory effects through significant modulation of neutrophil recruitment.

CD44-specific antibody treatment and CD44 deficiency exert distinct effects on leukocyte recruitment in experimental arthritis

AbstractCD44, the leukocyte adhesion receptor for hyaluronan, has been considered a therapeutic target on the basis of the robust anti-inflammatory effect of CD44-specific antibodies in animal models of immune-mediated diseases. However, CD44 deficiency does not provide substantial protection against inflammation. Using intravital video microscopy in a murine model of rheumatoid arthritis, we show that CD44 deficiency and anti-CD44 antibody treatment exert disparate effects on leukocyte recruitment in inflamed joints. Leukocyte rolling, which is increased in CD44-deficient mice, is promptly abrogated in anti–CD44-treated wild-type mice. CD44-specific antibodies also trigger platelet deposition on granulocytes and subsequent depletion of this leukocyte subset in the circulation. These in vivo effects require CD44 cross-linking and are reproducible with an antibody against Gr-1, a molecule that, like CD44, is highly expressed on granulocytes. Anticoagulant pretreatment, which prevents platelet deposition, mitigates both granulocyte depletion and the suppressive effect of CD44-specific antibody on joint swelling. Our observations suggest that cross-linking of prominent cell surface molecules, such as CD44 or Gr-1, can initiate a rapid self-elimination program in granulocytes through engagement of the coagulation system. We conclude that the robust anti-inflammatory effect of CD44-specific antibodies in arthritis is primarily the result of their ability to trigger granulocyte depletion.

NOD1 Expression in the Eye and Functional Contribution to IL-1β–Dependent Ocular Inflammation in Mice

NOD1 plays an important role in host defense and recognizes the minimal component of bacterial cell walls, meso-diaminopimelic acid (iE-DAP). Polymorphisms in NOD1 are associated with autoinflammatory diseases characterized by uveitis such as Crohn's disease and sarcoidosis. NOD1 is homologous to NOD2, which is responsible for an autosomal dominant form of uveitis. Nonetheless, the role of NOD1 in intraocular inflammation has not been explored. The induction of uveitis by iE-DAP in mice and the potential contribution of interleukin (IL)-1beta were investigated. BALB/c mice or mice deficient in caspase-1 or IL-1R1 and their congenic controls were injected intravitreally with iE-DAP or saline. The time course, dose response, and contribution of IL-1beta to ocular inflammation were quantified by intravital video microscopy, histology, and immunohistochemistry. NOD1 and IL-1beta were measured in eye tissue by immunoblotting and ELISA. NOD1 protein is expressed in the eye and promotes ocular inflammation in a dose- and time-dependent fashion. The authors previously defined the role of IL-1beta in NOD2 uveitis and tested whether NOD1 and NOD2 used similar mechanisms. Treatment with iE-DAP significantly increased IL-1beta, which was caspase-1 dependent. However, in contrast to NOD2, caspase-1 and IL-1R1 were essential mediators of iE-DAP-induced uveitis, suggesting that NOD1 and NOD2 induce ocular inflammation by distinct mechanisms involving IL-1beta. These findings demonstrate that NOD1 is expressed within the eye and that its activation results in uveitis in an IL-1beta-dependent mechanism. Characterizing the differences between NOD1 and NOD2 responses may provide insight into the pathogenesis of uveitis.

Involvement of Nitric Oxide in Microcirculatory Reactions after Ischemia-Reperfusion of the Rat Urinary Bladder

Background: Nitric oxide (NO) plays a role in inflammation. Our aim was to investigate the role of NO in the microcirculatory changes after ischemia-reperfusion (I/R) of the bladder using intravital videomicroscopy (IVM). Methods: In rats, 60 min of bladder ischemia followed by 30 min of reperfusion was performed in the presence of N^G-nitro-L-arginine methyl ester (L-NAME), the NO precursor L-arginine, or saline pre-treatments. Venular red blood cell velocity (RBCV), functional capillary density (FCD), vessel diameters, and leukocyte-endothelial cell interactions in postcapillary venules were determined. Concentrations of nitrite/nitrate in the plasma and myeloperoxidase (MPO) levels in the lungs and the bladder were measured. Results: Elevations of the numbers of rolling and adherent leukocytes, and of plasma nitrite/nitrate levels were found, while FCD and RBCV decreased. L-NAME pretreatment ameliorated the enhanced leukocyte-endothelial cell interactions without influencing the microcirculatory perfusion. In contrast, the L-arginine pretreatment further increased plasma nitrite/nitrate levels and preserved the FCD and RBCV, but did not affect leukocyte-endothelial interactions. None of these treatments influenced MPO activities. Conclusion: Our results suggest that NO plays an enhancing role in the I/R-induced neutrophil-endothelial interactions of the bladder. Supplementation of NO ameliorates the microcirculatory perfusion deficit without influencing the postischemic microcirculatory inflammatory reactions.

Bimodal Oscillation Frequencies of Blood Flow in the Inflammatory Colon Microcirculation

Rhythmic changes in blood flow direction have been described in the mucosal plexus of mice with acute colitis. In this report, we studied mice with acute colitis induced either by dextran sodium sulfate or by trinitrobenzenesulfonic acid. Both forms of colitis were associated with blood flow oscillations as documented by fluorescence intravital videomicroscopy. The complex oscillation patterns suggested more than one mechanism for these changes in blood flow. By tracking fluorescent nanoparticles in the inflamed mucosal plexus, we identified two forms of blood flow oscillations within the inflammatory mouse colon. Stable oscillations were associated with a base frequency of approximately 2 cycles/sec. Velocity measurements in the upstream and downstream vessel segments indicated that stable oscillations were the result of regional flow occlusion within the mucosal plexus. In contrast, metastable oscillations demonstrated a lower frequency (0.2-0.4 cycles/sec) and appeared to be the result of flow dynamics in vessels linked by the bridging mucosal vessels. These blood flow oscillations were not directly associated with cardiopulmonary movement. We conclude that both the stable and metasable oscillating patterns reflect flow adaptations to inflammatory changes in the mucosal plexus. Anat Rec, 2009. (c) 2008 Wiley-Liss, Inc.

Effects of colloid solutions on ischemia-reperfusion-induced periosteal microcirculatory and inflammatory reactions: Comparison of dextran, gelatin, and hydroxyethyl starch*

To compare the microhemodynamics and possible anti-inflammatory reactions of colloid resuscitation with 4% gelatin, 6% dextran, or 6% hydroxyethyl starch 130/0.4 solutions. A randomized control in vivo animal study in a university research laboratory. Adult male Wistar rats (280 +/- 20 g). Sodium pentobarbital-anesthetized animals were subjected to a 60-min complete hindlimb ischemia and a 180-min reperfusion. Volume resuscitation, either with a colloid (dextran, gelatin, or hydroxyethyl starch, 25 mL kg(-1) during 3 hr intravenously) or with lactated Ringer's solution, was initiated 10 min before reperfusion. Fluorescence intravital videomicroscopy was performed before ischemia and 30, 60, 120, and 180 min postresuscitation to quantify the tibial periosteal functional capillary density, the capillary red blood cell velocity changes, and leukocyte rolling and firm adherence in postcapillary venules. In a separate series blood samples were drawn to determine the release of soluble intercellular adhesion molecule-1 (enzyme-linked immunosorbent assay technique), and the surface expression of CD11b (flow cytometry) on peripheral blood granulocytes. Reperfusion significantly increased the leukocyte rolling and firm adhesion (by 2.6 and 7.1-fold, respectively), evoked marked decreases in periosteal functional capillary density and red blood cell velocity (56% and 39%, respectively), and increased the CD11b expression on the circulating leukocytes (by 85%). Hydroxyethyl starch, but not gelatin or dextran pretreatment, significantly inhibited the firm adherence of the leukocytes and reduced the elevated CD11b expression. Hydroxyethyl starch pretreatment also effectively attenuated the decreases in functional capillary density and red blood cell velocity, whereas gelatin and dextran did not improve the microhemodynamics. Finally, ischemia had no direct effect on the soluble intercellular adhesion molecule-1 levels, whereas gelatin treatment increased significantly this parameter. When compared with gelatin or dextran solutions, hydroxyethyl starch provided a therapeutic advantage in this setting by exerting an inhibitory effect on the ischemia-reperfusion-induced local and systemic leukocyte reactions and the postischemic periosteal microvascular dysfunction.

Kappa-Opioid Receptor Agonist Protects the Microcirculation of Skeletal Muscle From Ischemia Reperfusion Injury

Previous research has demonstrated that pretreatment with kappa opioid receptor (KOR) agonist protects against ischemia reperfusion (I/R) injury in cardiomyocytes and neuron cells through activation of protein kinase C. The purpose of this study is to investigate the KOR agonist's effect on I/R-injured cremaster muscle and its underlying mechanism. Male Sprague Dawley rats were randomized into 3 groups (n = 6 each group). Group I was the ischemia reperfusion (I/R) injury group (4 hours of ischemia followed by 90 minutes of reperfusion). Group II was the U-50488H (selective KOR agonist)-pretreated group (KOR agonist + I/R injury). Group III was pretreated with U-50488H + nor-binaltorphimine (NBI, a selective KOR antagonist) (KOR agonist + antagonist + I/R injury). The numbers of leukocyte rolling, adhering, and transmigrating, functional capillary, and swelling index of the vessel wall of the postcapillary venule were observed under intravital videomicroscopy. Biochemically, the lactate dehydrogenase, creatine phosphokinase isoenzyme, expression of E-selectin, and intercellular adhesion molecule-1 (ICAM-1) were analyzed. The U-50488H-pretreated group had significantly decreased the number of leukocyte sticking (P < 0.001) and transmigrating (P < 0.001) as compared with the I/R-injury group and the U-50488H + NBI-pretreated group. The numbers of functional capillary in the U-50488H-pretreated group were significantly less attenuated compared with the I/R-injury group and U-50488H + NBI-pretreated group (P < 0.001). The expression of the ICAM-1 in the cremaster muscle was evidently reduced in the U-50488H-pretreated group than in the I/R-injury group or the U-50488H + NBI-pretreated group. Administration of KOR agonist protects the muscle flap microcirculation from I/R injury, which can be abolished by concomitant KOR antagonist administration. The KOR agonist-induced protection from ischemia reperfusion injury may be related to decreased expression of adhesion molecule ICAM-1.