Number Of Adherent Leukocytes Research Articles

PI-10: DIETARY IRON RESTRICTION PROTECTS AGAINST VASO-OCCLUSION AND ORGAN DAMAGE IN SICKLE CELL DISEASE

Purpose: Sickle cell disease (SCD) is the most commonly inherited monogenic hemoglobinopathy and results from a mutation in the β-globin gene (HbS), leading to red blood cell (RBC) sickling, vaso-occlusive episodes (VOE), and organ damage. Chronic hemolysis, inflammation, and repeated red blood cell transfusions in SCD can disrupt iron homeostasis. Patients who receive multiple blood transfusions develop iron-overload, and another subpopulation of SCD patients manifest iron deficiency. SCD mice exhibit increased iron absorption via upregulation of the intestinal transcription factor hypoxia inducible factor-2a(HIF-2a) leading to the development of iron overload. Disruption of intestinal HIF-2a or limiting iron intake by dietary iron restriction in SCD mice, significantly reduced systemic iron overload, lowered cellular sickle hemoglobin as reflected by reduced RBC mean corpuscular hemoglobin concentration (MCHC), and consequently improved hemolysis and ameliorated anemia. Understanding iron homeostasis is therefore critical to SCD pathogenesis and holds the promise for identifying novel approaches to treat SCD. Materials and methods: SCD mice were generated by transplanting bone marrow cells from Berkeley SCD (Tg[Hu-miniLCRα1GγAγδβS]Hba−/− Hbb−/−) mice into 8-week old, lethally-irradiated C57BL/6J mice. Immediately after transplantation, SCD mice were fed either a regular-iron diet (Ctrl) containing 185 parts per million (ppm) iron or an otherwise identical iron-restricted diet (IRD) containing 3 ppm iron. VOE and organ damage analysis were done 3-4 months after treatments. Results: IRD resulted in a reduced organ iron concentration (Fig-1A) in SCD compared to Ctrl group, confirming that dietary iron restriction ameliorates organ iron deposition in SCD mice. SCD mice treated with IRD exhibited reduced MCHC (Fig-1B) and serum indirect bilirubin(IDBILC) (Fig-1C) indicating a decrease in cellular sickle hemoglobin and hemolysis. Furthermore, IRD reduced the fraction of aged neutrophils by >50% in SCD mice (Fig-1D), which corresponded with improved VOE parameters. Specifically, intravital microscopy (IVM) demonstrated elevated mean centerline RBC velocity and blood flow rate and decreased leukocyte adhesion in IRD compared to Ctrl mice (Fig-1E-G). Importantly, IRD led to prolonged survival after TNF-a challenge in SCD mice (Fig-1H). IRD resulted in a dramatic reduction in lung leukocyte infiltration, indicating significantly reduced lung inflammation in SCD mice (Fig-2A). Compared to Ctrl liver samples, IRD resulted in significantly less liver necrosis, leukocyte infiltration and fibrosis (Fig-2B,C), decreased serum liver enzymes (ALT, AST; Fig-2D), and reduced levels of total and direct bilirubin (TBILC, DBILC; Fig-2D).We further hypothesized that, in addition to reducing iron concentration in organs, IRD ameliorates organ damage by preventing translocation of microbial compounds.IRD inhibits translocation of FITC-Dextran by 5-fold compared to Ctrl suggesting functionally improved gut permeability (Fig-2E). We detected significantly decreased TLR2 ligands in serum of IRD mice (Fig-2F) suggesting ligands from gram-positive bacteria (TLR2 agonists) may play a role in triggering SCD complications. Conclusion: Our study demonstrates for the first time that dietary iron restriction reduces VOE severity and alleviates chronic organ damage in SCD mice. Novel approaches that target iron availability/absorption in the gut, the gut microbiome and aged neutrophil crosstalk and gut barrier integrity, provide an important area of further investigation with the potential of improving hemolysis, VOE, and organ damage in SCD patients.(A)The bone-marrow/spleen/liver Non-heme iron content. (B)MCHC, (C)IDBILC,(D)aged neutrophils percentage in Ctrl/IRD SCD mice. IVM parameters (E) (Vrbc) (F) blood flow rate (J) the number of adherent leukocytes and (J) survival rate after TNF-a injection(A)Leukocyte infiltration in lung and (B)liver tissue. (C)Liver necrosis, leukocyte infiltration, and fibrosis. (D)Liver ALT and AST, TBILC, and DBILC from sera. (E)serum FITC-Dextran and (F) TLR2 ligands activity in Ctrl/IRD SCD mice The authors do not declare any conflict of interest

Anti-Inflammatory Effects of the Iron Chelator, DIBI, in Experimental Acute Lung Injury.

Iron plays a critical role in the immune response to inflammation and infection due to its role in the catalysis of reactive oxygen species (ROS) through the Haber-Weiss and Fenton reactions. However, ROS overproduction can be harmful and damage healthy cells. Therefore, iron chelation represents an innovative pharmacological approach to limit excess ROS formation and the related pro-inflammatory mediator cascades. The present study was designed to investigate the impact of the iron chelator, DIBI, in an experimental model of LPS-induced acute lung injury (ALI). DIBI was administered intraperitoneally in the early and later stages of lung inflammation as determined by histopathological evaluation. We found that lung tissues showed significant injury, as well as increased NF-B p65 activation and significantly elevated levels of various inflammatory mediators (LIX, CXCL2, CCL5, CXCL10, IL-1𝛽, IL-6) 4 h post ALI induction by LPS. Mice treated with DIBI (80 mg/kg) in the early stages (0 to 2 h) after LPS administration demonstrated a significant reduction of the histopathological damage score, reduced levels of NF-B p65 activation, and reduced levels of inflammatory mediators. Intravital microscopy of the pulmonary microcirculation also showed a reduced number of adhering leukocytes and improved capillary perfusion with DIBI administration. Our findings support the conclusion that the iron chelator, DIBI, has beneficial anti-inflammatory effects in experimental ALI.

Anti-inflammatory iron chelator, DIBI, reduces leukocyte-endothelial adhesion and clinical symptoms of LPS-induced interstitial cystitis in mice.

Interstitial cystitis (IC) is a prevalent and debilitating chronic inflammatory disease of the urinary bladder. Currently there are no fully effective therapeutic agents available, in part due to the still obscure pathogenesis of IC. Lipopolysaccharide (LPS) also known as endotoxin from Gram negative bacteria elicits IC in mice and has formed the basis of model systems for investigation. Excess free iron plays an important role in inflammation through generation of reactive oxygen species (ROS). The novel iron chelator DIBI has been shown to sequester excess free iron and dampen excess inflammatory responses to systemic LPS administration and also to Gram negative bacterial infections. The overall objective of this study was to evaluate the effects of DIBI on LPS induced IC in mice. Leukocyte activation, endothelial adhesion and functional capillary density were assessed by intravital microscopy of the bladder microcirculation following a single intravesical LPS administration with or without intravesical DIBI treatment. Clinical IC symptoms were also assessed through behavioral and pain threshold force measurements. Four groups of female BALB/c mice (n = 5-6/group) were randomized in this study: control group, IC group without therapy, IC group with DIBI therapy and control group with DIBI therapy. The groups were examined using intravital microscopy (IVM) of the bladder for leukocyte-endothelial interactions (adherent leukocytes, temporarily interacting leukocytes) and functional capillary density (FCD). A modified behavioral score by Boucher et al. and Von-Frey-Aesthesiometry were used to evaluate key behavioral indices related to pain and visceral pain perception. LPS introduced intravesically induced an early (≤2h) inflammation of the bladder evidenced by leukocyte activation and adhesion to bladder capillary walls. Intravesical DIBI therapy of mice 30min following LPS administration and assessed after 1.5h treatment showed a significant decrease in the number of adherent leukocytes compared to IC animals without DIBI treatment. DIBI treated mice showed a significantly lowered increase in behavioral distress scores compared to IC mice without therapy. Untreated IC mice exhibited a significantly decreased threshold force value for evoked pain response and DIBI treatment improved the threshold pain response. A significant inverse correlation was found for the two pain and suffering evaluation methods results. DIBI reduced inflammatory endothelial leukocyte adhesion and key indices related to pain and suffering over those observed in untreated IC mice. Our findings suggest a potential therapeutic role for DIBI for IC treatment.

A Microphysiological System to Study Leukocyte-Endothelial Cell Interaction during Inflammation.

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of activated leukocytes across the vascular endothelium into key organs can lead to organ failure. A physiologically relevant biomimetic microfluidic assay (bMFA) has been developed and validated using several experimental and computational techniques, which can reproduce the entire leukocyte rolling/adhesion/migration cascade to study leukocyte-endothelial cell interactions. Microvascular networks obtained from in vivo images in rodents were digitized using a Geographic Information System (GIS) approach and microfabricated with polydimethylsiloxane (PDMS) on a microscope slide. To study the effect of shear rate and vascular topology on leukocyte-endothelial cell interactions, a Computational Fluid Dynamics (CFD) model was developed to generate a corresponding map of shear rates and velocities throughout the network. The bMFA enables the quantification of leukocyte-endothelial cells interactions, including rolling velocity, number of adhered leukocytes in response to different shear rates, number of migrated leukocytes, endothelial cell permeability, adhesion molecule expression and other important variables. Furthermore, by using human-related samples, such as human endothelial cells and leukocytes, bMFA provides a tool for rapid screening of potential therapeutics to increase their clinical translatability.

Inhibition of Factor XII Attenuates Prothrombotic Complications in Sickle Cell Mice

Sickle Cell Disease (SCD) is the most common inherited hemoglobinopathy, affecting millions worldwide. Although characterized by chronic hemolytic anemia and recurrent vaso-occlusive episodes, SCD is increasingly recognized as a hypercoagulable state. Indeed, SCD patients have an 11-25% incidence of venous thromboembolism at a median age of 30 years, associated with a 3-fold increased risk of mortality. Moreover, ischemic stroke and silent cerebral infarctions occur in 7-13% of SCD patients. We have previously shown that tissue factor, an initiator of the extrinsic coagulation pathway, contributes to thrombo-inflammation and microvascular cerebral thrombosis in mouse models of SCD . Recently, the intrinsic coagulation pathway, including Factor XII (FXII), has received significant attention because targeting components of this pathway reduces thrombosis without affecting primary hemostasis. We have shown that FXII deficiency reduces plasma markers of thrombin generation and inflammation in sickle mice. However, the contribution of FXII to thrombosis and prothrombotic complications in SCD is not known. In this study we evaluated the effects of blocking FXII activity on venous thrombosis and ischemia/reperfusion (IR)-induced brain injury in SCD mice.First, Townes HbSS mice (SS) and non-sickle Townes HbAA controls (AA) (male and female, 16 weeks) received anti-FXII antibody or control IgGκ1 (10 mg/kg, IV) 30 minutes prior to subjecting them to venous thrombosis, initiated by applying positive current (3 volts, 90 sec) to the femoral vein. To visualize platelet and fibrin accumulation, mice were injected with rhodamine 6G and anti-fibrin antibody 59D8 labeled with Alexa Fluor 647, respectively. The femoral vein thrombi were imaged by intravital fluorescence microscopy using time-lapse capture every 10 seconds, to acquire images of fibrin and platelets over 60 min. The accumulation of platelets and fibrin was quantified for relative intensity of each fluorophore over the region of the observed thrombus. As previously shown, thrombi of SS/IgG mice showed an increased fibrin and platelet accumulation compared to AA/IgG group. Importantly, 15D10 treatment significantly attenuated both fibrin (p<0.001) and platelet (p<0.05) deposition over time in SS mice compared to SS/IgG group. The same effect of 15D10 treatment was observed in AA mice. At the end of experiment, clots were collected and stained with hematoxylin and eosin, and clot volume was assessed histomorphometrically (Nikon Ti-2, FIJI Software). Surprisingly, despite higher fibrin content, clots from SS/IgG mice had significantly smaller volume than clots from AA/IgG group (0.32 ± 0.04 versus 0.60 ± 0.11 mm 3, p<0.05). Importantly, administration of 15D10 significantly reduced clot volume in both SS (0.086 ± 0.01 mm 3, p<0.05) and AA mice (0.1 ± 0.02 mm 3, p<0.05).Next, AA and SS mice (male and female, 8-10 weeks) were subjected to brain IR injury induced by middle cerebral artery occlusion for 60 minutes followed by 24 hours of reperfusion (mouse model of ischemic stroke). 15D10 or control IgGκ1 (10 mg/kg, IV) were injected 30 minutes before occlusion and again at 6 hours into the reperfusion period to generate 3 experimental groups: AA/IgG, SS/IgG and SS/15D10. All analyzed parameters of brain IR injury were significantly worse in the SS/IgG group compared to the AA/IgG group. Compared to IgG, pre-treatment of SS mice with 15D10 significantly attenuated neuronal damage determined by volume of brain infarction (11.7 ± 3.7 vs 24.9 ± 2.4%, p<0.001) and improved behavioral deficit assessed by mean stroke score (9.0 ± 0.9 vs 14.6 ± 0.9, p<0.01). These changes were accompanied by a significant increase in leukocytes rolling (1978.0 ± 93.5 vs 1517.0 ± 180.3 rolling leukocytes/sec/mm 2, p<0.001), and significant reduction in the number of adherent leukocytes (367.2 ± 49.0 vs 723.4 ± 48.5, adherent leukocytes/mm 2, p<0.001) observed in the brain microvasculature of SS mice treated with 15D10 compared to SS/IgG group.Together, our data indicates that in the mouse model of SCD FXII contributes to the experimental venous thrombosis and ischemic stroke. Given that targeting the intrinsic pathway can reduce thrombosis without affecting hemostasis, our data suggest that targeting FXII might be a beneficial treatment in reducing inflammatory and thrombotic complications in SCD patients without a risk of bleeding. DisclosuresWallisch: Aronora Inc,: Current Employment. Key: Grifols: Research Funding; Takeda: Research Funding; BioMarin: Honoraria, Other: Participation as a clinical trial investigator; Sanofi: Consultancy; Uniqure: Consultancy, Other: Participation as a clinical trial investigator. Gruber: Aronora Inc.: Current Employment, Current equity holder in publicly-traded company; Oregon Health and Science University: Current Employment.

Utility of intestinal intravital microscopy for the study of CNS injury-induced immunodepression syndrome (CIDS).

Stroke, traumatic brain injury, or other forms of central nervous system (CNS) injury initiate a local inflammatory response. Compensatory anti-inflammatory pathways are activated to limit secondary damage due to inflammation. The associated release of immunosuppressing neuromodulators can result in system-wide immune dysregulation (CNS injury-induced immune-depression syndrome -CIDS). To establish an experimental stroke model where CIDS can be studied by intravital microscopy (IVM). We used the photothrombotic stroke (PTS) model in C57BL/6 mice and studied its effects on peripheral immunity following challenge with lipopolysaccharide (LPS). Leukocyte activation, as well as capillary perfusion of the microcirculation, were assessed using intestinal intravital microscopy (IVM). PTS caused a significant reduction in the number of adhering leukocytes in submucosal venules of the terminal ileum of mice challenged with LPS compared to LPS-challenged animals without stroke. Leukocyte rolling was also impacted by PTS in the submucosal venules. Following stroke, we also observed decreased mucosal functional capillary density (FCD). Our results suggest that PTS with subsequent LPS challenge poses as a viable model to further study CIDS using intravital microscopy of the intestinal microcirculation.

Time course of microvalve pathophysiology in high pressure low flow model of venous insufficiency and the role of micronized purified flavonoid fraction.

The aim of this study was to characterize, in an experimental model, the mechanisms involved in the initiation of venous insufficiency at the level of microvenous valve and whether they can be influenced by early treatment with micronized purified flavonoid fraction (MPFF). The external right iliac vein of 78 male golden Syrian hamsters was ligated to induce chronic venous insufficiency. Internal venular diameter as well as leukocyte-endothelium-interaction (leukocytes sticking after staining with rhodamine 6G), were assessed using an intravital microscope. In the second part of the study 30 animals were divided into three groups and underwent: ligation plus MPFF, ligation plus 10% lactose solution (vehicle), or sham operation. Treatment with MPFF 100 mg/kg/day or vehicle started 2 days before ligation and lasted for 7 days. Venular diameter and number of adherent leukocytes were assessed 5 days post-ligature. Venular diameter increased immediately after ligature and reached a maximum at 4 hours (P<0.001 vs. baseline), followed by a plateau before gradually returning to baseline dimensions. The increase in the number of adherent leukocytes was also immediate but attained maximal number at 3 days (P<0.0001), followed by a plateau and then gradual return to baseline numbers. In MPFF-treated animals, leukocyte adhesion to the microvalves was prevented compared with vehicle-treated animals (P<0.0001) and venular diameter was also significantly reduced (P<0.05). Venous hypertension induced immediate venular dilatation followed by an increase in the number of adherent leukocytes at microvalve level. Treatment with MPFF prevented the initiation of microvalve inflammation and may play a protective role in the progression of chronic venous insufficiency.

The Clinical-Stage sGC Stimulator IW-1701 Prevents Increase of Plasma Biomarkers of Intravascular Inflammation and Suppresses Leukocyte-Endothelial Interactions in Tnfα-Treated Mice

Soluble guanylate cyclase (sGC) is a heme containing signaling enzyme that catalyzes the formation of cyclic guanosine 3‘,5‘-monophosphate (cGMP) from GTP in response to nitric oxide (NO) binding to heme. The second messenger cGMP modulates many physiological processes including inflammation. Treatment with NO donors or inhibitors of cGMP-selective phosphodiesterase 9 (PDE9) significantly attenuates TNFα-induced intravascular inflammation in C57BL/6 and sickle cell mice. The anti-inflammatory effect of PDE9 inhibitors was augmented by co-administration of hydroxyurea (HU), which is the current standard of care for sickle cell disease. It has been proposed that HU may exert its anti-inflammatory effect by acting as an NO donor. sGC stimulators are small molecules that bind to sGC and enhance NO-dependent catalytic activity of the enzyme. Here we evaluated the effect of the sGC stimulator IW-1701 in the absence and presence of HU on biomarkers of intravascular inflammation, leukocyte-endothelial cell interactions and neutrophil trafficking in C57BL/6 mice.Treatment of C57BL/6 mice with TNFα (50 ng/mouse, i.p.) increased the plasma levels of biomarkers of endothelial cell activation (sP-selectin, sE-selectin, sICAM-1) and leukocyte activation (MIP-2, sL-selectin). Pretreatment of mice with IW-1701 (10 mg/kg, p.o.) reduced the increase in plasma concentrations of MIP-2 and sL-selectin following the TNFα challenge by 81% and 58%, respectively. The TNFα-induced increase in plasma levels of sP-selectin, sE-selectin and sICAM-1 were also inhibited by 31%, 37% and 34%, respectively in IW-1701-treated mice. Co-treatment with HU augmented the effect of IW-1701 on plasma concentrations of sE-selectin and sICAM-1 in TNFα treated mice.The effects of IW-1701, HU and the combination on leukocyte endothelial cell interactions was assessed using intravital microscopy of post-capillary venules of the mouse cremaster muscle. TNFα treatment of C57BL/6 mice decreased leukocyte velocity from 26.6±3.1 μm/sec to 5.5±0.7 μm/sec and leukocyte rolling flux from 43.3±7.1 cells/min to 16.1±2.7 cells/min as compared to control mice. Pre-treatment of mice with IW-1701 (10 mg/kg) alone increased leukocyte velocity and rolling flux to 10.3±1.1 μm/sec and 24.1±2.3 cells/min, respectively. Similarly, pre-treatment of mice with HU (100 mg/kg, p.o.) alone increased leukocyte velocity to 15.5±1.7 μm/sec and leukocyte rolling flux to 50.2±5.9 cells/min. Co-administration of IW-1701 in combination with HU further increased leukocyte velocity (19.7±1.9 μm/sec) and leukocyte rolling flux (64.5±5.5 cells/min) in post-capillary venules of the mouse cremaster muscle. Treatment with either IW-1701, HU, or the combination did not change the number of adherent leukocytes to the vascular wall following TNFα stimulation. To further evaluate the effect of IW-1701 on neutrophil extravasation and trafficking we used a peritoneal recruitment model. Intraperitoneal injection of TNFα induced accumulation of Gr.1 positive neutrophils in the mouse peritoneal cavity (4±0.4×105 PMNs/peritoneal lavage). Trafficking of PMNs into mouse peritoneum was attenuated by treatment of mice with HU (2.7±0.5×105 PMNs/peritoneal lavage). Co-administration of HU and IW-1701 further decreased the number of neutrophils extravasated into the peritoneum of TNFα-challenged mice (1.4±0.3×105 PMNs/peritoneal lavage).In summary, prophylactic treatment with IW-1701 inhibited the TNFα-induced increase in plasma levels of biomarkers of intravascular inflammation and attenuated the effect of TNFα on transient adhesive interactions between leukocytes and endothelial cells in mice. Co-administration of HU significantly reduced neutrophil trafficking and augmented the effect of IW-1701 on biomarkers of endothelial cell activation and transient adhesive interactions in the mouse cremaster muscle. This study supports further evaluation of the sGC stimulator IW-1701 to reduce complications associated with intravascular inflammation such as vaso-occlusive crisis in patients with sickle cell disease. DisclosuresTchernychev:Ironwood Pharmaceuticals: Employment, Other: own stock options. Feil:Ironwood Pharmaceuticals: Other: This research was sponsored by Ironwood Pharmaceuticals. Germano:Ironwood Pharmaceuticals: Employment, Other: own stock options of Ironwood Pharmaceuticals . Warren:Ironwood Pharmaceuticals: Employment, Other: own stock options of Ironwood Pharmaceuticals . Lonie:Ironwood Pharmaceuticals: Employment, Other: own stock options of Ironwood Pharmaceuticals . Feil:Ironwood Pharmaceuticals: Other: This research was sponsored by Ironwood Pharmaceuticals. Milne:Ironwood Pharmaceuticals: Employment, Equity Ownership. Hadcock:Ironwood Pharmaceuticals: Employment, Other: own stock options of Ironwood Pharmaceuticals . Chien:Ironwood Pharmaceuticals: Employment, Equity Ownership. Currie:Ironwood Pharmaceuticals: Employment, Equity Ownership. Graul:Ironwood Pharmaceuticals: Employment, Other: own stock options of Ironwood Pharmaceuticals .

Effects of Diabetes on Microcirculation and Leukostasis in Retinal and Non-Ocular Tissues: Implications for Diabetic Retinopathy.

Changes in retinal microcirculation are associated with the development of diabetic retinopathy (DR). However, it is unclear whether such changes also develop in capillary beds of other non-retinal tissues. Here, we investigated microcirculatory changes involving velocity of rolling neutrophils, adherence of neutrophils, and leukostasis during development of retinal vascular lesions in diabetes in other non-retinal tissues. Intravital microscopy was performed on post-capillary venules of cremaster muscle and ear lobe of mice with severe or moderate diabetes and compared to those of non-diabetic mice. Additionally, number and velocity of rolling leukocytes, number of adherent leukocytes, and areas of leukostasis were quantified, and retinal capillary networks were examined for acellular capillaries (AC) and pericyte loss (PL), two prominent vascular lesions characteristic of DR. The number of adherent neutrophils and areas of leukostasis in the cremaster and ear lobe post-capillary venules of diabetic mice was increased compared to those of non-diabetic mice. Similarly, a significant increase in the number of rolling neutrophils and decrease in their rolling velocities compared to those of non-diabetic control mice were observed and severity of diabetes exacerbated these changes. Understanding diabetes-induced microcirculatory changes in cremaster and ear lobe may provide insight into retinal vascular lesion development in DR.

Genesis of Antibiotic Resistance (AR) LXI:Turbulence Modelling: Mechanisms of E‐Complex [Ca2+/Mg2+Ecto‐ATPase (EC 3.6.1.15) /E‐NTPDase (NTPDase1) EC 3.6.1.6) regulated E‐kinase/PKA‐induced E‐NTPase∞CD36/E‐NTPDase∞CD39/CD73 Oligomeric Complex (E complex @ Ec)] activated leucocytes‐platelets‐erythrocytes (LPE) Augment “Hairpin Vortices of Tollmien–Schlichting waves(TSW)” for laminar to turbulent transition

Here we present a plausible pathophysiological cascade initiating the transition of laminar to turbulent flow as a cause for, but not limiting other unknown clinical variable for an increased mortality rate in NCT01663701‐SSSP‐2. Based on the correlation between Perfused Boundary Region (PBR) and number of rolling leukocytes in post‐capillary venules in sepsis cohort, the PBR has been suggested as an index of glycocalyx shedding enhancing the leukocyte–endothelium interaction. NCT00793442 observed that average number of rolling leukocytes was 26 in the sepsis group and 9.8 in the non‐infected control group per field. The sepsis cohort, had an increased number of adhered leukocytes in non‐survivors compared to non‐sepsis cohort. Correlation of aforesaid hemodynamic parameters implicating the LPE impacting the damaged endothelial surface, tissue perfusion and organ dysfunction due to septic shock not withstanding “DIC”. After the LPE start forming in the arterial end of the capillary bed, rolling up and formation of vortex. Amplification of LPE involves the formation of a single vortex of greater strength through the pairing of vortices. As the vortices progress deregulating starling forces from the arterial region in the capillary bed towards the venous end, vortices to become heavily distorted and less distinct. Due to presence of damaged endothelial surface with adhered LPE, flow breaks down, generating a large number of small‐scale eddies, and the flow undergoes rapid transition to the fully turbulent regime. Mixing layers and wakes behind bluff bodies exhibit a sequence of events, leading to transition and turbulent flow. It has known that initial linear instability occurs around Rex, crit = 91 000 forming an unstable two‐dimensional disturbances are called TSW waves. Increased LPE adhesion, endothelial damage, possible scar tissue formation and/or necrotic lesions in the capillary bed are likely to be large enough a secondary, non‐linear, instability mechanism causes the TSW to become three‐dimensional and finally evolve into hairpin Λ‐vortices. In the most common mechanism of transition, would be K‐type transition, where the hairpin vortices are aligned. The hairpin vortices with a high shear region is induced which intensifies, elongates and rolls up. Regions of intense and highly localized changes occur at random times and locations near the damaged endothelial wall. These turbulent spots are carried along with the flow and grow by spreading across the capillary bed, which causes increasing amounts of laminar fluid to take part in the turbulent motion. Under progressive hypotensive state induced endothelial damage and abrupt collapse of interstitial space would result CCP ensuing hypoxia. Such scenario may have been the cause for mortality in NCT01663701‐SSSP‐2.Support or Funding InformationSupported by professional development funds and in part CME activities of Subburaj KannanMD PhD for www.aaets.org

Experimental Cannabinoid 2 Receptor Activation by Phyto-Derived and Synthetic Cannabinoid Ligands in LPS-Induced Interstitial Cystitis in Mice.

Interstitial cystitis (IC) is a chronic bladder disorder with unclear etiology. The endocannabinoid system has been identified as a key regulator of immune function, with experimental evidence for the involvement of cannabinoid receptors in bladder inflammation. This study used intravital microscopy (IVM) and behavioral testing in lipopolysaccharide-induced IC, to investigate the anti-inflammatory analgesic effects of a natural dietary sesquiterpenoid, beta-caryophyllene (BCP), which is present in cannabis among other plants, and has reported agonist actions at the cannabinoid 2 receptor (CB2R). BCP’s anti-inflammatory actions were compared to the synthetic CB2R-selective cannabinoid, HU308, and to an FDA-approved clinical treatment (dimethyl sulfoxide: DMSO). IVM data revealed that intravesical instillation of BCP and/or HU308 significantly reduces the number of adhering leukocytes in submucosal bladder venules and improves bladder capillary perfusion. The effects of BCP were found to be comparable to that of the selective CB2R synthetic cannabinoid, HU308, and superior to intravesical DMSO treatment. Oral treatment with BCP was also able to reduce bladder inflammation and significantly reduced mechanical allodynia in experimental IC. Based on our findings, we believe that CB2R activation may represent a viable therapeutic target for IC, and that drugs that activate CB2R, such as the generally regarded as safe (GRAS) dietary sesquiterpenoid, BCP, may serve as an adjunct and/or alternative treatment option for alleviating symptoms of inflammation and pain in the management of IC.

Longitudinal In Vivo Characterization of the Streptozotocin-Induced Diabetic Mouse Model: Focus on Early Inner Retinal Responses.

The goal of this study was to perform an extensive temporal characterization of the early pathologic processes in the streptozotocin (STZ)-induced diabetic retinopathy (DR) mouse model, beyond the vascular phenotype, and to investigate the potential of clinically relevant compounds in attenuating these processes. Visual acuity and contrast sensitivity (CS) were studied in the mouse STZ model until 24 weeks postdiabetes onset. ERG, spectral domain optical coherence tomography (SD-OCT), leukostasis, and immunohistochemistry were applied to investigate neurodegeneration, inflammation, and gliosis during early-, mid- and late-phase diabetes. Aflibercept or triamcinolone acetonide (TAAC) was administered to investigate their efficacy on the aforementioned processes. Visual acuity and CS loss started at 4 and 18 weeks postdiabetes onset, respectively, and progressively declined over time. ERG amplitudes were diminished and OP latencies increased after 6 weeks, whereas SD-OCT revealed retinal thinning from 4 weeks postdiabetes. Immunohistochemical analyses linked these findings to retinal ganglion and cholinergic amacrine cell loss at 4 and 8 weeks postdiabetes onset, respectively, which was further decreased after aflibercept administration. The number of adherent leukocytes was augmented after 2 weeks, whereas increased micro- and macroglia reactivity was present from 4 weeks postdiabetes. Aflibercept or TAAC showed improved efficacy on inflammation and gliosis. STZ-induced diabetic mice developed early pathologic DR hallmarks, from which inflammation seemed the initial trigger, leading to further development of functional and morphologic retinal changes. These findings indicate that the mouse STZ model is suitable to study novel integrative non-vascular therapies to treat early DR.

Progranulin protects the mouse retina under hypoxic conditions via inhibition of the Toll‑like receptor‑4‑NADPH oxidase 4 signaling pathway.

To investigate the function of progranulin on the retina under hypoxic conditions, 8-week-old C57BL/6 mice were divided into normal condition and hypoxic condition groups (n=24 mice/group). The hypoxia model was established through intravitreal injection of 9 mM cobalt chloride. Subsequently, 10 mM progranulin and an equal amount of PBS were injected into the right and left eyes, respectively. Photoreceptor function was examined using electroretinogram (ERG) analysis. Morphological alterations were examined using immunofluorescence co-localization, retinal vascular inflammation was examined using the leukostasis assay, and signaling pathways were screened using immunoblotting. The results revealed that ERG amplitude was significantly lower under hypoxic conditions compared with under normal conditions. Furthermore, the amplitude was significantly reduced in the PBS-injected eyes compared with in the progranulin-injected eyes. Morphological examination demonstrated that the number of rods in the PBS-injected eyes was decreased compared with in the progranulin-injected eyes under hypoxic conditions. In addition, the arrangement of the cones was sparse and the morphology of the outer segments was short and small. Although the number of adherent leukocytes in the progranulin-injected eyes was higher in the hypoxic mice compared with in those under normal conditions, the number was only 52.31% of the number detected in the PBS-injected eyes. Analysis of the signaling pathways demonstrated that the protective effects of progranulin on retinas under hypoxic conditions were regulated by the Toll-like receptor 4 (TLR4)-NADPH oxidase 4 (NOX4) pathway, instead of the caspase and Wnt/β-catenin pathways. In conclusion, progranulin exerted protective effects on the function and morphology of photoreceptors in a hypoxic environment, and could reduce retinal vascular inflammation, through inhibition of the TLR4-NOX4 pathway.

In vivo quantification of rolling and adhered leukocytes in human sepsis

BackgroundThe use of in vivo videomicroscopy at the bedside has demonstrated microcirculatory flow disturbances in sepsis. The ability of in vivo videomicroscopy to detect changes in the prevalence of rolling and adhered leukocytes that occur in sepsis is not well-described in humans. We sought to (1) develop methodology for accessing and quantifying sublingual leukocyte rolling and adherence with sidestream dark field (SDF) imaging; (2) compare the number of rolling and adhered leukocytes between patients with septic shock and non-infected controls; and (3) compare the number of rolling and adhered leukocytes between survivors and non-survivors of septic shock.MethodsWe included adult (age > 18 years) patients in the emergency department presenting with septic shock prospectively enrolled in the ProCESS trial. We recruited comparison non-infected patients as emergency department controls. Using a SDF videomicroscope, we obtained image sequences from the sublingual mucosa, quantifying rolling and adhered leukocytes per 1 mm × 1 mm visual field in a standardized 3-s clip. We report data as median and interquartile range and depicted as box plots. We compared groups using the Mann-Whitney U test, considering a p value < 0.05 significant.ResultsWe included a total of 64 patients with septic shock and 32 non-infected controls. The median number of adhered leukocytes per field in the sepsis group was 1.0 (IQR 0–3.5) compared to 0 (0–0) in the non-infected group (p < 0.001). The median number of rolling leukocytes was 26 (10.3–42) in the sepsis group and 9.8 (4.8–17.3) in the non-infected group (p < 0.001) per field. Among the patients with sepsis (n = 64), there was an increased number of adhered leukocytes in non-survivors compared to survivors (3.0 (1–5.5) vs. 1.0 (0–3.0)) (p < 0.05); however, there was no difference in rolling leukocytes (35 (20–48) vs. 26 (10–41)) (p = 0.31).ConclusionsOur results demonstrated a higher number of rolling and adhered leukocytes in patients with septic shock when compared to non-infected controls, and an increased number of adhered leukocytes in non-survivors.Trial registrationClinicalTrials.gov, NCT00793442; Registered on 19 November 2008PG0GM076659 (US NIH Grant/Contract). First submitted 18 July 2007. First posted 2 August 2007.

Necrotic amplification loop in acute pancreatitis: pancreatic stellate cells and nitric oxide are important players in the development of the disease

Necrotic amplification loop in acute pancreatitis: pancreatic stellate cells and nitric oxide are important players in the development of the disease

Renin-Angiotensin System Inhibitors Can Prevent Intravenous Lipid Infusion-Induced Myocardial Microvascular Dysfunction and Leukocyte Activation.

Levels of triglycerides and free fatty acids (FFAs) are elevated in patients with diabetes and may contribute to endothelial dysfunction through renin-angiotensin system (RAS) activation and oxidative stress. The present study investigated how systemic FFA loading affected myocardial microcirculation during hyperemia via RAS.Methods and Results:Eight healthy men received candesartan, perindopril, or a placebo for 2 days in a double-blind crossover design, and then myocardial microcirculation during hyperemia induced by a 2-h infusion of lipid/heparin was assessed using dipyridamole stress-myocardial contrast echocardiography (MCE). Leukocyte activity and hemorheology were also assessed ex vivo using a microchannel flow analyzer, serum levels of oxidative stress markers, and IκB-α expression in mononuclear cells. Serum FFA elevation by the infusion of lipid/heparin significantly decreased myocardial capillary blood velocity and myocardial blood flow during hyperemia. Both candesartan and perindopril significantly prevented the FFA-induced decrease in capillary blood velocity and myocardial blood flow during hyperemia. Systemic FFA loading also caused an increase in the number of adherent leukocytes and prolonged the whole blood passage time. These effects were blocked completely by candesartan and partially by perindopril. Both agents prevented the FFA-induced enhancement of oxidative stress and IκB-α degradation in mononuclear cells. Both candesartan and perindopril can prevent FFA-induced myocardial microcirculatory dysfunction during hyperemia via modulation of leukocyte activation and microvascular endothelial function.

In vivo observation of cerebral microcirculation after experimental subarachnoid hemorrhage in mice

Acute brain injury caused by subarachnoid hemorrhage is the major cause of poor prognosis. The pathology of subarachnoid hemorrhage likely involves major morphological changes in the microcirculation. However, previous studies primarily used fixed tissue or delayed injury models. Therefore, in the present study, we used in vivo imaging to observe the dynamic changes in cerebral microcirculation after subarachnoid hemorrhage. Subarachnoid hemorrhage was induced by perforation of the bifurcation of the middle cerebral and anterior cerebral arteries in male C57/BL6 mice. The diameter of pial arterioles and venules was measured by in vivo fluorescence microscopy at different time points within 180 minutes after subarachnoid hemorrhage. Cerebral blood flow was examined and leukocyte adhesion/albumin extravasation was determined at different time points before and after subarachnoid hemorrhage. Cerebral pial microcirculation was abnormal and cerebral blood flow was reduced after subarachnoid hemorrhage. Acute vasoconstriction occurred predominantly in the arterioles instead of the venules. A progressive increase in the number of adherent leukocytes in venules and substantial albumin extravasation were observed between 10 and 180 minutes after subarachnoid hemorrhage. These results show that major changes in microcirculation occur in the early stage of subarachnoid hemorrhage. Our findings may promote the development of novel therapeutic strategies for the early treatment of subarachnoid hemorrhage.

QiShenYiQi Pills, a Compound Chinese Medicine, Prevented Cisplatin Induced Acute Kidney Injury via Regulating Mitochondrial Function.

Nephrotoxicity is a serious adverse effect of cisplatin chemotherapy that limits its clinical application, to deal with which no effective management is available so far. The present study was to investigate the potential protective effect of QiShenYiQi Pills (QSYQ), a compound Chinese medicine, against cisplatin induced nephrotoxicity in mice. Pretreatment with QSYQ significantly attenuated the cisplatin induced increase in plasma urea and creatinine, along with the histological damage, such as tubular necrosis, protein cast, and desquamation of epithelial cells, improved the renal microcirculation disturbance as indicated by renal blood flow, microvascular flow velocity, and the number of adherent leukocytes. Additionally, QSYQ prevented mitochondrial dysfunction by preventing the cisplatin induced downregulation of mitochondrial complex activity and the expression of NDUFA10, ATP5D, and Sirt3. Meanwhile, the cisplatin-increased renal thiobarbituric acid-reactive substances, caspase9, cleaved-caspase9, and cleaved-caspase3 were all diminished by QSYQ pretreatment. In summary, the pretreatment with QSYQ remarkably ameliorated the cisplatin induced nephrotoxicity in mice, possibly via the regulation of mitochondrial function, oxidative stress, and apoptosis.

Anti-inflammatory effects of a novel iron chelator, DIBI, in experimental sepsis.

Iron catalyzes the generation of reactive oxygen species (ROS) as part of the innate antimicrobial defense. During sepsis, the dysregulated systemic inflammatory response to infection, iron homeostasis becomes disrupted, generating an excess of ROS causing damage to tissues. This can be potentially suppressed using iron chelators that selectively bind iron to prevent its participation in ROS-related inflammatory reactions. We hypothesize that administration of DIBI, a novel iron-chelator, attenuates the dysregulated systemic immune response and reduces tissue damage in experimental endotoxemia. Five groups of animals (n = 5-10) were included in this study: control, untreated endotoxemia, and endotoxemia animals treated with either DIBI-A, MAHMP, or DIBI-B. Intravital microscopy was performed on the intestine of anesthesized mice to observe leukocyte endothelial interactions and evaluate the intestinal microcirculation. Treatment of endotoxemic mice with DIBI-B reduced the number of adhering leukocytes in submucosal collecting (V1) venules by 68%. DIBI-B, MAHMP, and DIBI-A were able to restore functional capillary density (FCD) in the intestinal muscle layer by 74%, 44%, and 11%, respectively. DIBI-B reduces leukocyte recruitment and improves FCD in experimental endotoxemia, outperforming other chelators tested. These findings suggest a potential role for DIBI-B as a candidate drug for sepsis treatment.

LRP-1 Pathway Targeted Inhibition of Vascular Abnormalities in the Retina of Diabetic Mice

ABSTRACTPurpose: The cell surface LDL (low-density lipoprotein) receptor-related protein-1 (LRP-1) is important for lipid transport and several cell signaling processes. Human apolipoprotein E (apoE) is a ligand of LRP-1. We previously reported that a short peptide (apoEdp) mimicking the LRP-1 binding region of apoE prevents hyperglycemia-induced retinal endothelial cell dysfunction in vitro. The in-vivo outcome of apoE-based peptidomimetic inhibition of LRP-1 in the treatment of diabetic retinopathy is unknown.Methods: Six months after streptozotocin induction of diabetes, male C57Bl/6 mice were intravitreally inoculated with apoEdp in a controlled release formulation. On the 15th day post-apoEdp treatment, mouse retinas were harvested to examine (1) blood–retinal–barrier (BRB) permeability by Evans blue dye, inflammatory leukostasis by concanavalin staining of leukocytes and LRP-1 pathway-related protein expression by Western blot analysis and gelatin zymography.Results: Intravitreal apoEdp treatment of diabetic mice significantly reduced Evans blue extravasation and the number of adherent leukocytes in the diabetic mouse retinas. ApoEdp treatment inhibited the expression of extracellular matrix (ECM) degrading proteases heparanase and MMP-2, and restores the BRB tight junction proteins occludin and ZO-1. ApoEdp treatment also inhibited Wnt/β-catenin-related expression of pro-inflammatory molecules ICAM-1, HIF-1α, and VEGF through negative regulation by LRP-1.Conclusion: Intravitreal apoEdp treatment of diabetic mice resulted a significant decrease in retinal vascular abnormalities through downregulation of LRP-1-related ECM protein degradation and Wnt/β-catenin-related pro-angiogenic molecules.