Mechanism of Ascorbate Protection Against Sepsis-Induced Capillary Blood Flow Impairment

Sepsis is a systemic inflammatory response to an infection. The overwhelming inflammation has many deleterious effects, including cessation of capillary blood flow. This cessation may lead to organ failure and subsequent death, but the cause of cessation during sepsis is not fully understood. Ascorbate (reduced vitamin C) has been shown to restore capillary blood flow by an unknown mechanism. I hypothesized that activation of both platelets and the coagulation pathway in sepsis contributes to the cessation of capillary blood flow and that ascorbate protects against cessation by reducing platelet activation. Using intravital microscopy in the mouse hindlimb skeletal muscle in vivo, I observed that sepsis impairs capillary blood flow and increases both adhesion of platelets/platelet aggregates to the capillary wall and deposition of fibrin plaques in the same capillaries. Platelet depletion, blocking of P-selectin (a key adhesion molecule), antithrombin, and eptifibatide (anti-aggregatory agent) all reduced the capillary blood flow impairment and platelet adhesion. Intravenous bolus injection of ascorbate reduced platelet adhesion in capillaries, via the endothelial nitric oxide synthase (eNOS) system. To study any direct effects of ascorbate on platelet function, I used an ex vivo model (isolated mouse platelets) examining platelet aggregation under septic conditions. Here, thrombin, ADP, and thromboxane (agents released into the blood during sepsis), but not lipopolysaccharide (LPS), tumor necrosis factor (TNFa) or septic plasma, increased platelet aggregation and surface P-selectin protein expression. Ascorbate inhibited the increased aggregation and P-selectin expression. Next, an in vitro mouse microvascular endothelial cells model was used to study the effect of ascorbate on platelet-endothelial cell adhesion. LPS and TNFa increased platelet adhesion and P-selectin mRNA expression in endothelial cells. LPS also increased P-selectin-containing endothelial granule secretion. Ascorbate prevented the increased adhesion and granule secretion but did not affect mRNA expression. Thus, I conclude that impairment of blood flow in the septic microvasculature requires platelets and is reduced by anti-coagulant/anti-aggregatory agents. Ascorbate prevents platelet-endothelial adhesion and platelet aggregation, partly through reducing P-selectin protein expression at the platelet/endothelial cell surface. Thus ascorbate reduces adhering platelets in septic capillaries leading to restoration of blood flow

Effect of ascorbate on plasminogen activator inhibitor-1 expression and release from platelets and endothelial cells in an in-vitro model of sepsis.

The microcirculation during sepsis fails due to capillary plugging involving microthrombosis. We demonstrated that intravenous injection of ascorbate reduces this plugging, but the mechanism of this beneficial effect remains unclear. We hypothesize that ascorbate inhibits the release of the antifibrinolytic plasminogen activator inhibitor-1 (PAI-1) from endothelial cells and platelets during sepsis. Microvascular endothelial cells and platelets were isolated from mice. Cells were cultured and stimulated with lipopolysaccharide (LPS), tumor necrosis factor alpha (TNFα), or thrombin (agents of sepsis), with/without ascorbate for 1-24 h. PAI-1 mRNA was determined by quantitative PCR. PAI-1 protein release into the culture medium was measured by ELISA. In platelets, PAI-1 release was measured after LPS, TNFα, or thrombin stimulation, with/without ascorbate. In endothelial cells, LPS and TNFα increased PAI-1 mRNA after 6-24 h, but no increase in PAI-1 release was observed; ascorbate did not affect these responses. In platelets, thrombin, but not LPS or TNFα, increased PAI-1 release; ascorbate inhibited this increase at low extracellular pH. In unstimulated endothelial cells and platelets, PAI-1 is released into the extracellular space. Thrombin increases this release from platelets; ascorbate inhibits it pH-dependently. The data suggest that ascorbate promotes fibrinolysis in the microvasculature under acidotic conditions in sepsis

Voluntary running exercise protects against sepsis-induced early inflammatory and pro-coagulant responses in aged mice

Background: Despite many animal studies and clinical trials, mortality in sepsis remains high. This may be due to the fact that most experimental studies of sepsis employ young animals, whereas the majority of septic patients are elderly (60 - 70 years). The objective of the present study was to examine the sepsis-induced inflammatory and pro-coagulant responses in aged mice. Since running exercise protects against a variety of diseases, we also examined the effect of voluntary running on septic responses in aged mice. Methods: Male C57BL/6 mice were housed in our institute from 2-3 to 22 months (an age mimicking that of the elderly). Mice were prevented from becoming obese by food restriction (given 70-90% of ad libitum consumption amount). Between 20 and 22 months, a subgroup of mice ran voluntarily on wheels, alternating 1-3 days of running with 1-2 days of rest. At 22 months, mice were intraperitoneally injected with sterile saline (control) or 3.75 g/kg fecal slurry (septic). At 7 h post injection, we examined (1) neutrophil influx in the lung and liver by measuring myeloperoxidase and/or neutrophil elastase in the tissue homogenates by spectrophotometry, (2) interleukin 6 (IL6) and KC in the lung lavage by ELISA, (3) pulmonary surfactant function by measuring percentage of large aggregates, (4) capillary plugging (pro-coagulant response) in skeletal muscle by intravital microscopy, (5) endothelial nitric oxide synthase (eNOS) protein in skeletal muscle (eNOS-derived NO is putative inhibitor of capillary plugging) by immunoblotting, and (6) systemic blood platelet counts by hemocytometry. Results: Sepsis caused high levels of pulmonary myeloperoxidase, elastase, IL6, KC, liver myeloperoxidase, and capillary plugging. Sepsis also caused low levels of surfactant function and platelet counts. Running exercise increased eNOS protein and attenuated the septic responses. Conclusions: Voluntary running protects against exacerbated sepsis-induced inflammatory and pro-coagulant responses in aged mice. Protection against pro-coagulant responses may involve eNOS upregulation. The present discovery in aged mice calls for clinical investigation into potential beneficial effects of exercise on septic outcomes in the elderly

The importance of migratory connectivity for global ocean policy.

The distributions of migratory species in the ocean span local, national and international jurisdictions. Across these ecologically interconnected regions, migratory marine species interact with anthropogenic stressors throughout their lives. Migratory connectivity, the geographical linking of individuals and populations throughout their migratory cycles, influences how spatial and temporal dynamics of stressors affect migratory animals and scale up to influence population abundance, distribution and species persistence. Population declines of many migratory marine species have led to calls for connectivity knowledge, especially insights from animal tracking studies, to be more systematically and synthetically incorporated into decision-making. Inclusion of migratory connectivity in the design of conservation and management measures is critical to ensure they are appropriate for the level of risk associated with various degrees of connectivity. Three mechanisms exist to incorporate migratory connectivity into international marine policy which guides conservation implementation: site-selection criteria, network design criteria and policy recommendations. Here, we review the concept of migratory connectivity and its use in international policy, and describe the Migratory Connectivity in the Ocean system, a migratory connectivity evidence-base for the ocean. We propose that without such collaboration focused on migratory connectivity, efforts to effectively conserve these critical species across jurisdictions will have limited effect