Myeloid Mineralocorticoid Receptor Transcriptionally Regulates P-Selectin Glycoprotein Ligand-1 and Promotes Monocyte Trafficking and Atherosclerosis.

Objective: MR (mineralocorticoid receptor) activation associates with increased risk of cardiovascular ischemia while MR inhibition reduces cardiovascular-related mortality and plaque inflammation in mouse atherosclerosis. MR in myeloid cells (My-MR) promotes inflammatory cell infiltration into injured tissues and atherosclerotic plaque inflammation by unclear mechanisms. Here, we examined the role of My-MR in leukocyte trafficking and the impact of sex. Approach and Results: We confirm in vivo that My-MR deletion (My-MR-KO) in ApoE-KO mice decreased plaque size. Flow cytometry revealed fewer plaque macrophages with My-MR-KO. By intravital microscopy, My-MR-KO significantly attenuated monocyte slow-rolling and adhesion to mesenteric vessels and decreased peritoneal infiltration of myeloid cells in response to inflammatory stimuli in male but not female mice. My-MR-KO mice had significantly less PSGL1 (P-selectin glycoprotein ligand 1) mRNA in peritoneal macrophages and surface PSGL1 protein on circulating monocytes in males. In vitro, MR activation with aldosterone significantly increased PSGL1 mRNA only in monocytes from MR-intact males. Similarly, aldosterone induced, and MR antagonist spironolactone inhibited, PSGL1 expression in human U937 monocytes. Mechanistically, aldosterone stimulated MR binding to a predicted MR response element in intron-1 of the PSGL1 gene by ChIP-qPCR. Reporter assays demonstrated that this PSGL1 MR response element is necessary and sufficient for aldosterone-activated, MR-dependent transcriptional activity. Conclusions: These data identify PSGL1 as a My-MR target gene that drives leukocyte trafficking to enhance atherosclerotic plaque inflammation. These novel and sexually dimorphic findings provide insight into increased ischemia risk with MR activation, cardiovascular protection in women, and the role of MR in atherosclerosis and tissue inflammation.

Vascular Endothelial Galectins in Leukocyte Trafficking.

Leukocyte recruitment to the site of injury is a crucial event in the regulation of an inflammatory response. Tight regulation of interactions between the endothelium and circulating leukocytes is necessary to ensure a protective response to injury does not result in inflammatory disease. Rising interest in the broad immunoregulatory roles displayed by members of the glycan-binding galectin family suggests that these proteins could be an attractive target for therapeutic intervention, since their expression is significantly altered in disease. The focus of this review is to summarize current knowledge on the role of galectins in leukocyte trafficking during inflammation and the clinical approaches being taken to target these interactions for treatment of inflammatory disease.

Immunometabolic Endothelial Phenotypes: Integrating Inflammation and Glucose Metabolism.

[Figure: see text].

Reelin Depletion Protects Against Atherosclerosis by Decreasing Vascular Adhesion of Leukocytes.

[Figure: see text].

Analgesic and anti-inflammatory articular effects of essential oil and camphor isolated from Ocimum kilimandscharicum Gürke leaves.

ETHNOPHARMACOLOGICAL RELEVANCE: Leaves from Ocimum kilimandscharicum Gürke (Lamiaceae) are popularly used against articular pain. AIM OF STUDY: The aim of this study was to test the anti-inflammatory and anti-hyperalgesic (analgesic) properties of the essential oil and camphor isolated from O. Kilimandscharicum leaves (EOOK) in 4 models including zymosan induced-articular inflammation model in mice. MATERIAL AND METHODS: For in vivo models, EOOK was tested in carrageenan-induced paw edema model with oral doses of 30, 100, and 300 mg/kg (oral administration = p.o.) and in zymosan-induced articular inflammation (including knee edema, leukocyte infiltration, mechanical hyperalgesia and nitric oxide), EOOK (100 mg/kg, p. o.) and camphor (30 mg/kg, p. o.) were tested. EOOK (100 mg/kg, p. o.) was tested in the rolling and also in the adhesion of leukocytes to the mesenteric microcirculation in situ model of carrageenan induced inflammation and EOOK (1, 3, 10, 30, and 60 µg/mL) was tested in vitro against neutrophils chemotaxis induced by N-formyl methionyl leucyl phenylalanine (fMLP). RESULTS: The treatment with EOOK significantly inhibited the carrageenan-induced edema, mechanical and cold hyperalgesia. Both, EOOK and camphor inhibited all articular parameters induced by zymosan. In situ intravitral microscopy analysis, EOOK significantly inhibited carrageenan-induced leukocyte rolling and adhesion. In vitro neutrophils chemotaxis, EOOK inhibited the leukocyte chemotaxis induced by fMLP. CONCLUSIONS: The present study showed that EOOK inhibited pain and inflammatory parameters contributing, at least in part, to explain the popular use of this plant as analgesic natural agent. This study also demonstrates that camphor and some known anti-inflammatory compounds present in EOOK could contribute for analgesic and anti-inflammatory articular properties.

Antithrombin III ameliorates post-traumatic brain injury cerebral leukocyte mobilization enhancing recovery of blood brain barrier integrity.

BACKGROUND: Acute traumatic coagulopathy often accompanies traumatic brain injury (TBI) and may impair cognitive recovery. Antithrombin III (AT-III) reduces the hypercoagulability of TBI. Antithrombin III and heparinoids such as enoxaparin (ENX) demonstrate potent anti-inflammatory activity, reducing organ injury and modulating leukocyte (LEU) activation, independent of their anticoagulant effect. It is unknown what impact AT-III exerts on cerebral LEU activation and blood-brain barrier (BBB) permeability after TBI. We hypothesized that AT-III reduces live microcirculatory LEU-endothelial cell (EC) interactions and leakage at the BBB following TBI. METHODS: CD1 mice (n = 71) underwent either severe TBI (controlled cortical impact (CCI), 6-m/s velocity, 1-mm depth, and 4-mm diameter) or sham craniotomy and then received either AT-III (250 IU/kg), ENX (1.5 mg/kg), or vehicle (saline) every 24 hours. Forty-eight hours post-TBI, cerebral intravital microscopy visualized in vivo penumbral microvascular LEU-EC interactions and microvascular leakage to assess BBB inflammation/permeability. Body weight loss and the Garcia neurological test (motor, sensory, reflex, balance) served as surrogates of clinical recovery. RESULTS: Both AT-III and ENX similarly reduced in vivo penumbral LEU rolling and adhesion (p < 0.05). Antithrombin III also reduced live BBB leakage (p < 0.05). Antithrombin III animals demonstrated the least 48-hour body weight loss (8.4 ± 1%) versus controlled cortical impact and vehicle (11.4 ± 0.5%, p < 0.01). Garcia neurological test scores were similar among groups. CONCLUSION: Antithrombin III reduces post-TBI penumbral LEU-EC interactions in the BBB leading to reduced neuromicrovascular permeability. Antithrombin III further reduced body weight loss compared with no therapy. Further study is needed to determine if these AT-III effects on neuroinflammation affect longer-term neurocognitive recovery after TBI.

GEF-H1 Is Required for Colchicine Inhibition of Neutrophil Rolling and Recruitment in Mouse Models of Gout.

Gout is a painful arthritic inflammatory disease caused by buildup of monosodium urate (MSU) crystals in the joints. Colchicine, a microtubule-depolymerizing agent that is used in prophylaxis and treatment of acute gout flare, alleviates the painful inflammatory response to MSU crystals. Using i.p. and intra-articular mouse models of gout-like inflammation, we found that GEF-H1/GEF-H1/AHRGEF2, a microtubule-associated Rho-GEF, was necessary for the inhibitory effect of colchicine on neutrophil recruitment. GEF-H1 was required for neutrophil polarization in response to colchicine, characterized by uropod formation, accumulation of F-actin and myosin L chain at the leading edge, and accumulation of phosphorylated myosin L chain, flotillin-2, and P-selectin glycoprotein ligand-1 (PSGL-1) in the uropod. Wild-type neutrophils that were pre-exposed to colchicine failed to roll or accumulate on activated endothelial monolayers, whereas GEF-H1 knockout (GEF-H1-/-) neutrophils were unaffected by treatment with colchicine. In vivo, colchicine blocked MSU-induced recruitment of neutrophils to the peritoneum and the synovium in wild-type mice, but not in GEF-H1-/- mice. Inhibition of macrophage IL-1ß production by colchicine was independent of GEF-H1, supporting a neutrophil-intrinsic mode of action. Our results suggest that the anti-inflammatory effects of colchicine in acute gout-like inflammation can be accounted for by inhibition of neutrophil-rolling interactions with the inflamed vasculature and occurs through GEF-H1-dependent neutrophil stimulation by colchicine. These results contribute to our understanding of the therapeutic action of colchicine, and could inform the application of this drug in other conditions.

A Novel Experimental Approach for In Vivo Analyses of the Salivary Gland Microvasculature.

Microvascular dysfunction plays a fundamental role in the pathogenesis of salivary gland disorders. Restoring and preserving microvascular integrity might therefore represent a promising strategy for the treatment of these pathologies. The mechanisms underlying microvascular dysfunction in salivary glands, however, are still obscure, partly due to the unavailability of adequate in vivo models. Here, we present a novel experimental approach that allows comprehensive in vivo analyses of the salivary gland microvasculature in mice. For this purpose, we employed different microscopy techniques including multi-photon in vivo microscopy to quantitatively analyze interactions of distinct immune cell subsets in the submandibular gland microvasculature required for their infiltration into the surrounding parenchyma and their effects on microvascular function. Confocal microscopy and multi-channel flow cytometry in tissue sections/homogenates complemented these real-time analyses by determining the molecular phenotype of the participating cells. To this end, we identified key adhesion and signaling molecules that regulate the subset- and tissue-specific trafficking of leukocytes into inflamed glands and control the associated microvascular leakage. Hence, we established an experimental approach that allows in vivo analyses of microvascular processes in healthy and diseased salivary glands. This enables us to delineate distinct pathogenetic factors as novel therapeutic targets in salivary gland diseases.

Frontline Science: A flexible kink in the transmembrane domain impairs ß2 integrin extension and cell arrest from rolling.

ß2 integrins are the main adhesion molecules in neutrophils and other leukocytes and are rapidly activated by inside-out signaling, which results in conformational changes that are transmitted through the transmembrane domain (TMD). Here, we investigated the biologic effect of introducing a proline mutation in the ß2 integrin TMD to create a flexible kink that uncouples the topology of the inner half of the TMD from the outer half and impairs integrin activation. The ß2 integrin alpha chains, αL, αM, αX, and αD, all contain an inserted (I) domain with homology to von Willebrand factor A domain. ß2 activation was monitored in a homogenous binding assay of 2 reporter monoclonal antibodies: KIM127 reporting extension (E+ ) and mAb24 reporting the high-affinity (H+ ) conformation of the ß2 I-like domain. The proline mutation partially diminished chemokine-induced extension, but not the high-affinity conformation. The proline mutation in the TMD of ß2 completely inhibited arrest of rolling HL-60 cells in response to the chemokine IL-8. TMD mutant HL-60 cells rolling on P-selectin and ICAM-1 were unable to reduce their rolling velocity in response to IL-8. Quantitative dynamic footprinting live-cell imaging showed that blocking TMD topology transmission impaired the chemokine-induced activation of ß2, limiting the appearance of extended high-affinity (E+ H+ ) ß2. This also resulted in a defect in early spreading (3 min after arrest), which could be overcome by forced integrin activation using Mn2+ . We conclude that the TMD proline mutation severely impairs ß2 integrin extension, cell arrest, and early spreading.

High-mannose intercellular adhesion molecule-1 enhances CD16+ monocyte adhesion to the endothelium.

Human monocytes have been classified into three distinct groups, classical (anti-inflammatory; CD14+/CD16-), nonclassical (patrolling; CD14+/CD16++), and intermediate (proinflammatory; CD14++/CD16+). Adhesion of nonclassical/intermediate monocytes with the endothelium is important for innate immunity, and also vascular inflammatory disease. However, there is an incomplete understanding of the mechanisms that regulate CD16+ versus CD16- monocyte adhesion to the inflamed endothelium. Here, we tested the hypothesis that a high-mannose (HM) N-glycoform of intercellular adhesion molecule-1 (ICAM-1) on the endothelium mediates the selective recruitment of CD16+ monocytes. Using TNF-α treatment of human umbilical vein endothelial cells (HUVECs), and using proximity ligation assay for detecting proximity of specific N-glycans and ICAM-1, we show that TNF-α induces HM-ICAM-1 formation on the endothelial surface in a time-dependent manner. We next measured CD16- or CD16+ monocyte rolling and adhesion to TNF-α-treated HUVECs in which HM- or hybrid ICAM-1 N-glycoforms were generated using the α-mannosidase class I and II inhibitors, kifunensine and swainsonine, respectively. Expression of HM-ICAM-1 selectively enhanced CD16+ monocyte adhesion under flow with no effect on CD16- monocytes noted. CD16+ monocyte adhesion was abrogated by blocking either HM epitopes or ICAM-1. A critical role for HM-ICAM-1 in mediating CD16+ monocyte rolling and adhesion was confirmed using COS-1 cells engineered to express HM or complex ICAM-1 N-glycoforms. These data suggest that HM-ICAM-1 selectively recruits nonclassical/intermediate CD16+ monocytes to the activated endothelium.NEW & NOTEWORTHY Monocyte subsets have been associated with cardiovascular disease, yet it is unknown how different subsets are recruited to the endothelium. This study demonstrates the formation of distinct ICAM-1 N-glycoforms in the activated endothelium and reveals a key role for high mannose ICAM-1 in mediating proinflammatory CD16+ monocyte adhesion. Presented data identify roles for endothelial N-glycans in recruiting specific monocyte subsets during inflammation.

Identification of an activated neutrophil phenotype in polymyalgia rheumatica during steroid treatment: a potential involvement of immune cell cross-talk.

We have reported the existence of a distinct neutrophil phenotype in giant cell arteritis (GCA) patients arising at week 24 of steroid treatment. In the present study, we investigated whether longitudinal analysis of neutrophil phenotype in patients with polymyalgia rheumatica (PMR) could reveal a novel association with disease status and immune cell cross-talk. Thus, we monitored PMR patient neutrophil phenotype and plasma microvesicle (MV) profiles in blood aliquots collected pre-steroid, and then at weeks 1, 4, 12 and 24 post-steroid treatment.Using flow cytometric and flow chamber analyses, we identified 12-week post-steroid as a pivotal time-point for a marked degree of neutrophil activation, correlating with disease activity. Analyses of plasma MVs indicated elevated AnxA1+ neutrophil-derived vesicles which, in vitro, modulated T-cell reactivity, suggesting distinct neutrophil phenotypic and cross-talk changes at 24 weeks, but not at 12-week post-steroid.Together, these data indicate a clear distinction from GCA patient neutrophil and MV signatures, and provide an opportunity for further investigations on how to 'stratify' PMR patients and monitor their clinical responses through novel use of blood biomarkers.

Omega-9 Oleic Acid, the Main Compound of Olive Oil, Mitigates Inflammation during Experimental Sepsis.

The Mediterranean diet, rich in olive oil, is beneficial, reducing the risk of cardiovascular diseases and cancer. Olive oil is mostly composed of the monounsaturated fatty acid omega-9. We showed omega-9 protects septic mice modulating lipid metabolism. Sepsis is initiated by the host response to infection with organ damage, increased plasma free fatty acids, high levels of cortisol, massive cytokine production, leukocyte activation, and endothelial dysfunction. We aimed to analyze the effect of omega-9 supplementation on corticosteroid unbalance, inflammation, bacterial elimination, and peroxisome proliferator-activated receptor (PPAR) gamma expression, an omega-9 receptor and inflammatory modulator. We treated mice for 14 days with omega-9 and induced sepsis by cecal ligation and puncture (CLP). We measured systemic corticosterone levels, cytokine production, leukocyte and bacterial counts in the peritoneum, and the expression of PPAR gamma in both liver and adipose tissues during experimental sepsis. We further studied omega-9 effects on leukocyte rolling in mouse cremaster muscle-inflamed postcapillary venules and in the cerebral microcirculation of septic mice. Here, we demonstrate that omega-9 treatment is associated with increased levels of the anti-inflammatory cytokine IL-10 and decreased levels of the proinflammatory cytokines TNF-α and IL-1ß in peritoneal lavage fluid of mice with sepsis. Omega-9 treatment also decreased systemic corticosterone levels. Neutrophil migration from circulation to the peritoneal cavity and leukocyte rolling on the endothelium were decreased by omega-9 treatment. Omega-9 also decreased bacterial load in the peritoneal lavage and restored liver and adipose tissue PPAR gamma expression in septic animals. Our data suggest a beneficial anti-inflammatory role of omega-9 in sepsis, mitigating leukocyte rolling and leukocyte influx, balancing cytokine production, and controlling bacterial growth possibly through a PPAR gamma expression-dependent mechanism. The significant reduction of inflammation detected after omega-9 enteral injection can further contribute to the already known beneficial properties facilitated by unsaturated fatty acid-enriched diets.

A systems biology network analysis of nutri(epi)genomic changes in endothelial cells exposed to epicatechin metabolites.

Although vasculo-protective effects of flavan-3-ols are widely accepted today, their impact on endothelial cell functions and molecular mechanisms of action involved is not completely understood. The aim of this study was to characterize the potential endothelium-protective effects of circulating epicatechin metabolites and to define underlying mechanisms of action by an integrated systems biology approach. Reduced leukocyte rolling over vascular endothelium was observed following epicatechin supplementation in a mouse model of inflammation. Integrative pathway analysis of transcriptome, miRNome and epigenome profiles of endothelial cells exposed to epicatechin metabolites revealed that by acting at these different levels of regulation, metabolites affect cellular pathways involved in endothelial permeability and interaction with immune cells. In-vitro experiments on endothelial cells confirmed that epicatechin metabolites reduce monocyte adhesion and their transendothelial migration. Altogether, our in-vivo and in-vitro results support the outcome of a systems biology based network analysis which suggests that epicatechin metabolites mediate their vasculoprotective effects through dynamic regulation of endothelial cell monocyte adhesion and permeability. This study illustrates complex and multimodal mechanisms of action by which epicatechin modulate endothelial cell integrity.

The polysaccharide-rich tea of Ximenia americana barks prevents indomethacin-induced gastrointestinal damage via neutrophil inhibition.

ETHNOPHARMACOLOGICAL RELEVANCE: Barks of Ximenia americana are used by the population to treat gastrointestinal inflammatory disorders. Indomethacin is a non-selective non-steroidal anti-inflammatory drug that induces marked gastrointestinal damage. AIMS OF THE STUDIES: To evaluate the gastroprotective activity of total polysaccharides contained in the extract (TPL-Xa) or tea (Tea-Xa) of Ximenia americana barks in the mice gastric damage induced by indomethacin. MATERIALS AND METHODS: TPL-Xa was obtained by a combination of NaOH extraction and ethanol precipitation. Tea-Xa was prepared in distilled water boiled during 5 min. Animals received p.o. 0.9% NaCl (saline - control group), TPL-Xa (1-90 mg/kg) or Tea-Xa 1 h before gastritis induction by indomethacin (20 mg/kg). Mice were sacrificed 7 h after gastritis induction and analyzed for the following parameters: stomach lesions measurement; histological evaluation; myeloperoxidase (MPO) activity; nitrate/nitrite and cytokine levels; leukocyte adhesion and rolling by intravital microscopy. RESULTS: TPL-Xa reduced macroscopic and microscopic damage, MPO activity (59%), leukocyte rolling (86%) and adhesion (84%), nitrite/nitrate ratio (100%) and IL-8 (69%), but increased IL-4 (50%). Tea-Xa (12.8 yield; 39.3% carbohydrate, including 25.8% uronic acid; 4% protein) reduced macroscopic damage (62%) and MPO activity (50%). CONCLUSION: TPL and Tea of Ximenia americana barks ameliorate the gastric injury induced by indomethacin in mice, an effect that was dependent on the reduction of neutrophil infiltration.

Impact of lipid modulation on the intestinal microcirculation in experimental sepsis.

It has been observed, that patients who were treated medically for dyslipoproteinemia had a potentially lower risk of complications during infection and sepsis, regarding both morbidity and mortality. Aim of this study in experimental sepsis was to elucidate the impact of lipid metabolism modulation by simvastatin, HDL, or bezafibrate, respectively, on the intestinal microcirculation which plays a crucial role in the development of multiple organ failure in sepsis. Experimental sepsis was induced in Lewis rats by intravenous lipopolysaccharide (LPS) administration. Animals were treated with simvastatin, HDL or bezafibrate. By means of intestinal intravital microscopy (IVM), the inflammatory response in the microcirculation was studied by leukocyte adherence assessment (LA) and functional capillary density (FCD) measurements. In addition, plasma levels of pro-inflammatory cytokines were determined. Bezafibrate treatment led to a reduction in leukocyte adherence, improved functional capillary density (FCD), and a reduction in interleukin-1α (IL-1α), tumour necrosis factor α (TNF-α) and granulocyte macrophage colony stimulating factors (GM-CSF) plasma levels in experimental sepsis. Contrary to this, the administration of HDL increased leukocyte adherence as well as the number of rolling leukocytes. Only IL-1α plasma levels were decreased by HDL. No significant changes were observed following simvastatin treatment. In summary, only bezafibrate showed anti-inflammatory effects in endotoxemia. This effect cannot be explained by the HDL-enhancing effect of the bezafibrate, since the direct administration of HDL showed opposite effects. Bezafibrate induced reduction of inflammation in sepsis should be investigated in further studies.

Microvascular Dysfunction Following Multiwalled Carbon Nanotube Exposure Is Mediated by Thrombospondin-1 Receptor CD47.

Pulmonary exposure to multiwalled carbon nanotubes (MWCNTs) disrupts peripheral microvascular function. Thrombospondin-1 (TSP-1) is highly expressed during lung injury and has been shown to alter microvascular reactivity. It is unclear exactly how TSP-1 exerts effects on vascular function, but we hypothesized that the TSP-1 receptor CD47 may mediate changes in vasodilation. Wildtype (WT) or CD47 knockout (CD47 KO) C57B6/J-background animals were exposed to 50 µg of MWCNT or saline control via pharyngeal aspiration. Twenty-four hours postexposure, intravital microscopy was performed to assess arteriolar dilation and venular leukocyte adhesion and rolling. To assess tissue redox status, electron paramagnetic resonance and NOx measurements were performed, while inflammatory biomarkers were measured via multiplex assay.Vasodilation was impaired in the WT + MWCNT group compared with control (57 ± 9 vs 90 ± 2% relaxation), while CD47 KO animals showed no impairment (108 ± 8% relaxation). Venular leukocyte adhesion and rolling increased by >2-fold, while the CD47 KO group showed no change. Application of the antioxidant apocynin rescued normal leukocyte activity in the WT + MWCNT group. Lung and plasma NOx were reduced in the WT + MWCNT group by 47% and 32%, respectively, while the CD47 KO groups were unchanged from control. Some inflammatory cytokines were increased in the CD47 + MWCNT group only. In conclusion, TSP-1 is an important ligand mediating MWCNT-induced microvascular dysfunction, and CD47 is a component of this dysregulation. CD47 activation likely disrupts nitric oxide (•NO) signaling and promotes leukocyte-endothelial interactions. Impaired •NO production, signaling, and bioavailability is linked to a variety of cardiovascular diseases in which TSP-1/CD47 may play an important role.

Microcirculatory effects of sildenafil in experimental testicular torsion in rats.

PURPOSE: Investigate the short-term effect of sildenafil on microcirculation, especially the velocity, the pattern of the flow and the recruitment of the leukocyte in postcapillaries. METHODS: In male Sprague-Dawley rats, the microcirculatory consequences of 60 min experimental testicular torsion, followed by 240 min of reperfusion, were examined. Using fluorescence intravital microscopy, changes in red blood cell velocity in post-capillary venules and rolling as well as adhesion of leukocytes in the postcapillary venules were examined before the torsion and every hour during the reperfusion period. Sildenafil was given 10 min prior to reperfusion (iv 0.7 mg/kg, n = 6), while control animals received saline vehicle (n = 5). RESULTS: The characteristic flow motion disappeared in the affected testicular during the torsion. Red blood cell velocity values were dramatically decreased (by > 50%) and both rolling and adhesion of leukocytes increased during the reperfusion phase. Sildenafil treatment resulted in significantly higher red blood cell velocity values during the entire reperfusion period, but exerted only a temporary positive effect on the plost-ischaemic leukocyte-endothelial interactions. CONCLUSIONS: Intraoperative administration of sildenafil during surgical detorsion may provide marked testicular microperfusion benefits, but failed to influence the overall leukocyte-driven microcirculatory inflammatory reactions.

Early low-anticoagulant desulfated heparin after traumatic brain injury: Reduced brain edema and leukocyte mobilization is associated with improved watermaze learning ability weeks after injury.

BACKGROUND: Unfractionated heparin administered immediately after traumatic brain injury (TBI) reduces brain leukocyte (LEU) accumulation, and enhances early cognitive recovery, but may increase bleeding after injury. It is unknown how non-anticoagulant heparins, such as 2,3-O desulfated heparin (ODSH), impact post-TBI cerebral inflammation and long-term recovery. We hypothesized that ODSH after TBI reduces LEU-mediated brain inflammation and improves long-term neurologic recovery. METHODS: CD1 male mice (n = 66) underwent either TBI (controlled cortical impact [CCI]) or sham craniotomy. 2,3-O desulfated heparin (25 mg/kg [25ODSH] or 50 mg/kg [50ODSH]) or saline was administered for 48 hours after TBI in 46 animals. At 48 hours, intravital microscopy visualized rolling LEUs and fluorescent albumin leakage in the pial circulation, and the Garcia Neurologic Test assessed neurologic function. Brain edema (wet/dry ratio) was evaluated post mortem. In a separate group of animals (n = 20), learning/memory ability (% time swimming in the Probe platform quadrant) was assessed by the Morris Water Maze 17 days after TBI. Analysis of variance with Bonferroni correction determined significance (p < 0.05). RESULTS: Compared with CCI (LEU rolling: 32.3 ± 13.7 LEUs/100 µm per minute, cerebrovascular albumin leakage: 57.4 ± 5.6%), both ODSH doses reduced post-TBI pial LEU rolling (25ODSH: 18.5 ± 9.2 LEUs/100 µm per minute, p = 0.036; 50ODSH: 7.8 ± 3.9 LEUs/100 µm per minute, p < 0.001) and cerebrovascular albumin leakage (25ODSH: 37.9 ± 11.7%, p = 0.001, 50ODSH: 32.3 ± 8.7%, p < 0.001). 50ODSH also reduced injured cerebral hemisphere edema (77.7 ± 0.4%) vs. CCI (78.7 ± 0.4 %, p = 0.003). Compared with CCI, both ODSH doses improved Garcia Neurologic Test at 48 hours. Learning/memory ability (% time swimming in target quadrant) was lowest in CCI (5.9 ± 6.4%) and significantly improved in the 25ODSH group (27.5 ± 8.2%, p = 0.025). CONCLUSION: 2,3-O desulfated heparin after TBI reduces cerebral LEU recruitment, microvascular permeability and edema. 2,3-O desulfated heparin may also improve acute neurologic recovery leading to improved learning/memory ability weeks after injury.

Visualizing Leukocyte Rolling and Adhesion in Angiotensin II-Infused Mice: Techniques and Pitfalls.

Epifluorescence intravital video microscopy (IVM) of blood vessels is an established method to evaluate the activation of immune cells and their ability to role and adhere to the endothelial layer. Visualization of circulating cells by injection of fluorescent dyes or fluorophore-coupled antibodies is commonly used. Alternatively, fluorescent reporter mice can be used. Interactions of leukocytes, in particular lysozyme M+ (LysM+) monocytes, with the vessel wall play pivotal roles in promoting vascular dysfunction and arterial hypertension. We here present the technique to visualize and quantify leukocyte rolling and adhesion in carotid arteries in angiotensin II (AngII)-induced hypertension in mice by IVM. The implantation of a catheter damages the vascular wall and leads to altered blood cell responses. We compared different injection techniques and administration routes to visualize leukocytes in a LysMCre+IRG+ mouse with widespread expression of red fluorescent protein and conditional expression of green fluorescent protein in LysM+ cells. To study LysM+ cell activation, we used AngII infused mice in which rolling and adhesion of leukocytes to the endothelium is increased. We either injected acridine orange using a jugular catheter or directly though the tail vein and compared the amount of rolling and adhering cells. We found that jugular catheter implantation per se increased the number of rolling and adhering LysM+ cells in sham-infused LysMCre+IRG+ mice compared to controls. This activation was augmented in AngII-infused mice. Interestingly, injecting acridine orange directly through the tail vein did not increase LysM+ cell adhesion or rolling in sham-infused mice. We thereby demonstrated the importance of transgenic reporter mice expressing fluorescent proteins to not interfere with in vivo processes during experimentation. Furthermore, tail vein injection of fluorescent tracers might be a possible alternative to jugular catheter injections.