Capillary oxygen regulates demand-supply coupling by triggering connexin40-mediated conduction: Rethinking the metabolic hypothesis.

Coupling red blood cell (RBC) supply to O2 demand is an intricate process requiring O2 sensing, generation of a stimulus, and signal transduction that alters upstream arteriolar tone. Although actively debated, this process has been theorized to be induced by hypoxia and to involve activation of endothelial inwardly rectifying K+ channels (KIR) 2.1 by elevated extracellular K+ to trigger conducted hyperpolarization via connexin40 (Cx40) gap junctions to upstream resistors. This concept was tested in resting healthy skeletal muscle of Cx40-/- and endothelial KIR2.1-/- mice using state-of-the-art live animal imaging where the local tissue O2 environment was manipulated using a custom gas chamber. Second-by-second capillary RBC flow responses were recorded as O2 was altered. A stepwise drop in PO2 at the muscle surface increased RBC supply in capillaries of control animals while elevated O2 elicited the opposite response; capillaries were confirmed to express Cx40. The RBC flow responses were rapid and tightly coupled to O2; computer simulations did not support hypoxia as a driving factor. In contrast, RBC flow responses were significantly diminished in Cx40-/- mice. Endothelial KIR2.1-/- mice, on the other hand, reacted normally to O2 changes, even when the O2 challenge was targeted to a smaller area of tissue with fewer capillaries. Conclusively, microvascular O2 responses depend on coordinated electrical signaling via Cx40 gap junctions, and endothelial KIR2.1 channels do not initiate the event. These findings reconceptualize the paradigm of blood flow regulation in skeletal muscle and how O2 triggers this process in capillaries independent of extracellular K+.

Genetic ablation of smooth muscle KIR2.1 is inconsequential to the function of mouse cerebral arteries.

Cerebral blood flow is a finely tuned process dependent on coordinated changes in arterial tone. These changes are strongly tied to smooth muscle membrane potential and inwardly rectifying K+ (KIR) channels are thought to be a key determinant. To elucidate the role of KIR2.1 in cerebral arterial tone development, this study examined the electrical and functional properties of cells, vessels and living tissue from tamoxifen-induced smooth muscle cell (SMC)-specific KIR2.1 knockout mice. Patch-clamp electrophysiology revealed a robust Ba2+-sensitive inwardly rectifying K+ current in cerebral arterial myocytes irrespective of KIR2.1 knockout. Immunolabeling clarified that KIR2.1 expression was low in SMCs while KIR2.2 labeling was remarkably abundant at the membrane. In alignment with these observations, pressure myography revealed that the myogenic response and K+-induced dilation were intact in cerebral arteries post knockout. At the whole organ level, this translated to a maintenance of brain perfusion in SMC KIR2.1-/- mice, as assessed with arterial spin-labeling MRI. We confirmed these findings in superior epigastric arteries and implicated KIR2.2 as more functionally relevant in SMCs. Together, these results suggest that subunits other than KIR2.1 play a significant role in setting native current in SMCs and driving arterial tone.

Spectroscopy detects skeletal muscle microvascular dysfunction during onset of sepsis in a rat fecal peritonitis model.

Sepsis is a dysregulated host inflammatory response to infection potentially leading to life-threatening organ dysfunction. The objectives of this study were to determine whether early microvascular dysfunction (MVD) in skeletal muscle can be detected as dynamic changes in microvascular hemoglobin (MVHb) levels using spectroscopy and whether MVD precedes organ histopathology in septic peritonitis. Skeletal muscle of male Sprague-Dawley rats was prepared for intravital microscopy. After intraperitoneal injection of fecal slurry or saline, microscopy and spectroscopy recordings were taken for 6 h. Capillary red blood cell (RBC) dynamics and SO2 were quantified from digitized microscopy frames and MVHb levels were derived from spectroscopy data. Capillary RBC dynamics were significantly decreased by 4 h after peritoneal infection and preceded macrohemodynamic changes. At the same time, low-frequency oscillations in MVHb levels exhibited a significant increase in Power in parts of the muscle and resembled oscillations in RBC dynamics and SO2. After completion of microscopy, tissues were collected. Histopathological alterations were not observed in livers, kidneys, brains, or muscles 6 h after induction of peritonitis. The findings of this study show that, in our rat model of sepsis, MVD occurs before detectable organ histopathology and includes ~ 30-s oscillations in MVHb. Our work highlights MVHb oscillations as one of the indicators of MVD onset and provides a foundation for the use of non-invasive spectroscopy to continuously monitor MVD in septic patients.

National Preclinical Sepsis Platform: developing a framework for accelerating innovation in Canadian sepsis research.

Despite decades of preclinical research, no experimentally derived therapies for sepsis have been successfully adopted into routine clinical practice. Factors that contribute to this crisis of translation include poor representation by preclinical models of the complex human condition of sepsis, bias in preclinical studies, as well as limitations of single-laboratory methodology. To overcome some of these shortcomings, multicentre preclinical studies-defined as a research experiment conducted in two or more research laboratories with a common protocol and analysis-are expected to maximize transparency, improve reproducibility, and enhance generalizability. The ultimate objective is to increase the efficiency and efficacy of bench-to-bedside translation for preclinical sepsis research and improve outcomes for patients with life-threatening infection. To this end, we organized the first meeting of the National Preclinical Sepsis Platform (NPSP). This multicentre preclinical  research collaboration of Canadian sepsis researchers and stakeholders was established to study the pathophysiology of sepsis and accelerate movement of promising therapeutics into early phase clinical trials. Integrated knowledge translation and shared decision-making were emphasized to ensure the goals of the platform align with clinical researchers and patient partners. 29 participants from 10 independent labs attended and discussed four main topics: (1) objectives of the platform; (2) animal models of sepsis; (3) multicentre methodology and (4) outcomes for evaluation. A PIRO model (predisposition, insult, response, organ dysfunction) for experimental design was proposed to strengthen linkages with interdisciplinary researchers and key stakeholders. This platform represents an important resource for maximizing translational impact of preclinical sepsis research.

Role and Molecular Mechanisms of Pericytes in Regulation of Leukocyte Diapedesis in Inflamed Tissues.

Leukocyte recruitment is a hallmark of the inflammatory response. Migrating leukocytes breach the endothelium along with the vascular basement membrane and associated pericytes. While much is known about leukocyte-endothelial cell interactions, the mechanisms and role of pericytes in extravasation are poorly understood and the classical paradigm of leukocyte recruitment in the microvasculature seldom adequately discusses the involvement of pericytes. Emerging evidence shows that pericytes are essential players in the regulation of leukocyte extravasation in addition to their functions in blood vessel formation and blood-brain barrier maintenance. Junctions between venular endothelial cells are closely aligned with extracellular matrix protein low expression regions (LERs) in the basement membrane, which in turn are aligned with gaps between pericytes. This forms preferential paths for leukocyte extravasation. Breaching of the layer formed by pericytes and the basement membrane entails remodelling of LERs, leukocyte-pericyte adhesion, crawling of leukocytes on pericyte processes, and enlargement of gaps between pericytes to form channels for migrating leukocytes. Furthermore, inflamed arteriolar and capillary pericytes induce chemotactic migration of leukocytes that exit postcapillary venules, and through direct pericyte-leukocyte contact, they induce efficient interstitial migration to enhance the immunosurveillance capacity of leukocytes. Given their role as regulators of leukocyte extravasation, proper pericyte function is imperative in inflammatory disease contexts such as diabetic retinopathy and sepsis. This review summarizes research on the molecular mechanisms by which pericytes mediate leukocyte diapedesis in inflamed tissues.

Antimicrobial Efficacy of a New Chlorhexidine-based Device Against Staphylococcus aureus Colonization of Venous Catheters.

Vascular catheters are a major cause of nosocomial bloodstream infections. ChloraLock (ATTWILL Medical Solutions, Inc, West Jordan, UT, and ICU Medical, Inc, San Clemente, CA) is a novel antimicrobial device containing chlorhexidine digluconate (CHG) that is fitted onto a syringe and infuses CHG into the catheter lumen during locking. The objective of this study was to evaluate the antimicrobial efficacy of ChloraLock with in vitro tests and its ability to reduce Staphylococcus aureus contamination of catheters in the external jugular veins of Yorkshire swine. ChloraLock significantly reduced the bacterial load in the in vitro tests by up to 6 log10 colony-forming units (CFU) and by 3 to 4 log10 CFU/lumen in vivo in a swine model with 0.9% NaCl catheter locks.

Syndecan-1 (CD138) deficiency increases Staphylococcus aureus infection but has no effect on pathology in a mouse model of peritoneal dialysis.

BACKGROUND: Technique failure in peritoneal dialysis (PD) due to fibrosis and angiogenesis is complicated by peritonitis. Staphylococcus aureus infection is one of the most common causes of peritonitis in PD. The heparan sulfate proteoglycan, syndecan-1 (CD138), was reported to regulate fibrosis, angiogenesis, inflammation and S. aureus infection. The objectives of this study were to examine the effects of syndecan-1 on S. aureus infection and histopathology in a PD model. RESULTS: Syndecan-1(-/-) and wild type mice were dialyzed for 4 weeks and infected intraperitoneally with S. aureus. Tissues were collected after 4 h for histomorphometric analysis. Intravital microscopy was used to observe leukocyte recruitment and to quantify syndecan-1 in the parietal peritoneum microcirculation. The dialyzed syndecan-1(-/-) mice were more susceptible to S. aureus infection than undialyzed syndecan-1(-/-) controls and wild type animals. However, peritoneal fibrosis and neovascularization due to PD did not differ between syndecan-1(-/-) and wild type mice. Intravital microscopy showed that in S. aureus infection, syndecan-1 was removed from the subendothelial layer of peritoneal venules but syndecan-1 deficiency did not affect leukocyte recruitment. CONCLUSIONS: This study indicates that, while syndecan-1 is important for providing a barrier to acute S. aureus infection in PD, it does not affect peritoneal fibrosis and angiogenesis.

Peritoneal Dialysis Catheter Increases Leukocyte Recruitment in the Mouse Parietal Peritoneum Microcirculation and Causes Fibrosis.

UNLABELLED: ♦ BACKGROUND: The objective of this study was to examine the effects of a conventional dialysis solution and peritoneal catheter on leukocyte-endothelial cell interactions in the microcirculation of the parietal peritoneum in a subacute peritoneal dialysis (PD) mouse model. ♦ METHODS: An intraperitoneal (IP) catheter with a subcutaneous injection port was implanted into mice and, after a 2-week healing period, the animals were injected daily for 6 weeks with a 2.5% dextrose solution. Intravital microscopy (IVM) of the parietal peritoneum microcirculation was performed 4 hours after the last injection of the dialysis solution. Leukocyte-endothelial cell interactions were quantified and compared with catheterized controls without dialysis treatment and naïve mice. ♦ RESULTS: The number of rolling and extravascular leukocytes along with peritoneal fibrosis and neovascularization were significantly increased in the catheterized animals compared with naïve mice but did not significantly differ between the 2 groups of catheterized animals with sham injections or dialysis solution treatment. ♦ CONCLUSION: The peritoneal catheter implant increased leukocyte rolling and extravasation, peritoneal fibrosis and vascularization in the parietal peritoneum independently from the dialysis solution treatment.

Syndecan-1 in the mouse parietal peritoneum microcirculation in inflammation.

BACKGROUND: The heparan sulfate proteoglycan syndecan-1 (CD138) was shown to regulate inflammatory responses by binding chemokines and cytokines and interacting with adhesion molecules, thereby modulating leukocyte trafficking to tissues. The objectives of this study were to examine the expression of syndecan-1 and its role in leukocyte recruitment and chemokine presentation in the microcirculation underlying the parietal peritoneum. METHODS: Wild-type BALB/c and syndecan-1 null mice were stimulated with an intraperitoneal injection of Staphylococcus aureus LTA, Escherichia coli LPS or TNFα and the microcirculation of the parietal peritoneum was examined by intravital microscopy after 4 hours. Fluorescence confocal microscopy was used to examine syndecan-1 expression in the peritoneal microcirculation using fluorescent antibodies. Blocking antibodies to adhesion molecules were used to examine the role of these molecules in leukocyte-endothelial cell interactions in response to LTA. To determine whether syndecan-1 co-localizes with chemokines in vivo, fluorescent antibodies to syndecan-1 were co-injected intravenously with anti-MIP-2 (CXCL2), anti-KC (CXCL1) or anti-MCP-1 (CCL2). RESULTS AND CONCLUSION: Syndecan-1 was localized to the subendothelial region of peritoneal venules and the mesothelial layer. Leukocyte rolling was significantly decreased with LPS treatment while LTA and TNFα significantly increased leukocyte adhesion compared with saline control. Leukocyte-endothelial cell interactions were not different in syndecan-1 null mice. Antibody blockade of ß2 integrin (CD18), ICAM-1 (CD54) and VCAM-1 (CD106) did not decrease leukocyte adhesion in response to LTA challenge while blockade of P-selectin (CD62P) abrogated leukocyte rolling. Lastly, MIP-2 expression in the peritoneal venules was not dependent on syndecan-1 in vivo. Our data suggest that syndecan-1 is expressed in the parietal peritoneum microvasculature but does not regulate leukocyte recruitment and is not necessary for the presentation of the chemokine MIP-2 in this tissue.

Lactobacillus for preventing recurrent urinary tract infections in women: meta-analysis.

INTRODUCTION: Urinary tract infections (UTIs) are the most common infections affecting women, and often recur. Lactobacillus probiotics could potentially replace low dose, long term antibiotics as a safer prophylactic for recurrent UTI (rUTI). This systematic review and meta-analysis was performed to compile the results of existing randomized clinical trials (RCTs) to determine the efficacy of probiotic Lactobacillus species in preventing rUTI. MATERIALS AND METHODS: MEDLINE and EMBASE were searched from inception to July 2012 for RCTs using a Lactobacillus prophylactic against rUTI in premenopausal adult women. A random-effects model meta-analysis was performed using a pooled risk ratio, comparing incidence of rUTI in patients receiving Lactobacillus to control. RESULTS: Data from 294 patients across five studies were included. There was no statistically significant difference in the risk for rUTI in patients receiving Lactobacillus versus controls, as indicated by the pooled risk ratio of 0.85 (95% confidence interval of 0.58-1.25, p = 0.41). A sensitivity analysis was performed, excluding studies using ineffective strains and studies testing for safety. Data from 127 patients in two studies were included. A statistically significant decrease in rUTI was found in patients given Lactobacillus, denoted by the pooled risk ratio of 0.51 (95% confidence interval 0.26-0.99, p = 0.05) with no statistical heterogeneity (I2 = 0%). CONCLUSION: Probiotic strains of Lactobacillus are safe and effective in preventing rUTI in adult women. However, more RCTs are required before a definitive recommendation can be made since the patient population contributing data to this meta-analysis was small.

Intravital microscopy of the murine urinary bladder microcirculation.

OBJECTIVE: To establish an in vivo mouse model of the urinary bladder microcirculation, and characterize the molecular mechanisms of endotoxin-induced leukocyte recruitment. METHODS: The murine model was adapted from a technique previously reported for the rat. Mouse bladder microcirculation was observed using intravital microscopy, four hours after intravesical challenge with lipopolysaccharide (LPS) and leukocyte-endothelial interactions were examined. Molecular mechanisms of leukocyte recruitment were identified using antibodies to adhesion molecules and chemokines. RESULTS: LPS from Escherichia coli administered intravesically resulted in a significant increase in leukocyte adhesion and rolling at four hours post stimulation. LPS from Pseudomonas aeruginosa administered at similar doses resulted in a significant, but lower increase in leukocyte adhesion after four hours compared with E. coli LPS. Leukocyte adhesion within the bladder microcirculation was dependent on α(4) -integrins and ICAM-1, whereas leukocyte rolling was P-selectin dependent, but α(4) -integrin independent. Blockade of MIP-2 and KC did not alter leukocyte-endothelial interactions. The bladder endothelium expressed P-selectin, ICAM-1, VCAM-1, MIP-2, and MCP-1. Only VCAM-1 endothelial expression was significantly increased after LPS stimulation. CONCLUSION: The mouse model of the urinary bladder microcirculation is suitable for the study of inflammatory responses during urinary tract infection (UTI) in vivo.

Innate immunity of the liver microcirculation.

The liver is a complex organ with a unique microcirculation and both synthetic and immune functions. Innate immune responses have been studied in response to single inflammatory mediators and several clinically relevant models of infection and injury. While standard histological techniques have been used in many models, the liver microcirculation is also amenable to in vivo examination using epifluorescent, confocal and transillumination intravital microscopy. These techniques have begun to clarify not only the molecular mechanisms but also the specific cell populations involved in the liver inflammation. In this review, we discuss the cells and mediators involved in hepatic innate immunity in simple and complex models of injury and infection, and present the view that the liver microcirculation utilizes non-classical pathways for leukocyte recruitment.