Norman R. Harris

Age-related responses of the microcirculation to ischemia-reperfusion and inflammation.

Aging is a major risk factor for a variety of ischemic disorders including ischemic heart disease and stroke. Intense research over the past decade into ischemia-reperfusion (I/R) injury has implicated a general mechanism whereby reactive oxygen species produced at the onset of reperfusion overwhelm endogenous antioxidants, resulting in a cascade of events including mast cell degranulation, recruitment of neutrophils to the endothelial wall, arteriolar constriction that limits tissue perfusion, and increased vascular permeability that leads to inflammation and edema. Much of our knowledge regarding I/R injury comes from animal models; however, despite the fact that I/R disproportionately affects older individuals, young animals are usually chosen in models of I/R injury due to their greater availability, lower cost, and fewer health problems. Results obtained from young animals demonstrate a central role for both neutrophils and mast cells in I/R-induced increases in microvascular permeability and arteriolar constriction; however, it is not clear that a role for neutrophils is extended to older animals. A growing body of evidence indicates that neutrophils isolated from elderly individuals exhibit attenuated chemotaxis, oxidant release, and phagocytosis, and it has been suggested that these deficiencies are related to an age-associated increase in glucocorticoid production and oxidative stress. Therefore, neutrophils may have a limited capacity to influence microcirculatory tissue in the elderly compared to in the young. In support of this hypothesis, I/R-induced increases in microvascular permeability and decreases in vascular perfusion have been found to occur in older rats despite the absence of a significant increase in leukocyte-endothelial cell adhesion. Furthermore, elimination of circulating neutrophils attenuates I/R-induced mesenteric permeability only in young rats. Therefore, it appears that neutrophil-independent mechanisms of inflammation may be responsible for much of the microvascular dysfunction initiated by I/R in older animals.

L‐Arginine and Antineutrophil Serum Enable Venular Control of Capillary Perfusion in Hypercholesterolemic Rats

Objective: The purpose of this study was to investigate and counteract dysfunctional control of capillary flow in hypercholesterolemia. Capillary flow is controlled by arteriolar tone, which in turn is influenced by mediators released from closely paired venules in a mechanism that involves nitric oxide (NO). However, venular control of capillary flow is altered with hypercholesterolemia.

Nitric oxide measurements in rat mesentery reveal disrupted venulo-arteriolar communication in diabetes.

Objective: Arteriolar tone is partially controlled by diffusing mediators released by closely paired venules and is reported to depend on venular shear and venular leukocyte adherence. In healthy rat mesentery, venule‐initiated arteriolar dilation and consequent enhanced capillary flow appear to be tightly regulated by nitric oxide (NO). In contrast, diabetes inhibits NO‐dependent vasodilation and is associated with dysfunctional microcirculation. The objective of this study was to investigate venule‐dependent NO in diabetes.

Reperfusion-induced changes in capillary perfusion and filtration: effects of hypercholesterolemia.

Fluid filtration rate (J(v)/S) and red blood cell velocity (V(RBC)) in individual mesenteric capillaries of normocholesterolemic (NC) and hypercholesterolemic (HC) rats were measured before and after ischemia and reperfusion (I/R). In NC rats, a correlation was found between baseline J(v)/S and the percent of the feeding arteriole length that was paired (<15 micrometer) with a postcapillary venule (A-V pairing), but not in the HC group. Additionally, in NC rats only, a correlation was found between baseline V(RBC) and A-V pairing. In capillaries in which A-V pairing was substantial (>20%), V(RBC) dropped after reperfusion in the HC group (54% of baseline; P < 0.05), but not in the NC group (79%). The decrease in V(RBC) in HC rats could be attenuated by a P-selectin antibody (PB1.3). PB1.3 was also able to attenuate the increase in I/R-induced capillary J(v)/S in HC rats (median increase = 1.26-fold vs. 1.53-fold without PB1.3). These data suggest a role for A-V pairing in capillary perfusion in NC rats and a potential role for P-selectin in I/R-induced microvascular dysfunction in HC rats.Fluid filtration rate ( J v/ S) and red blood cell velocity ( V RBC) in individual mesenteric capillaries of normocholesterolemic (NC) and hypercholesterolemic (HC) rats were measured before and after ischemia and reperfusion (I/R). In NC rats, a correlation was found between baseline J v/ Sand the percent of the feeding arteriole length that was paired (<15 μm) with a postcapillary venule (A-V pairing), but not in the HC group. Additionally, in NC rats only, a correlation was found between baseline V RBC and A-V pairing. In capillaries in which A-V pairing was substantial (>20%), V RBCdropped after reperfusion in the HC group (54% of baseline; P < 0.05), but not in the NC group (79%). The decrease in V RBC in HC rats could be attenuated by a P-selectin antibody (PB1.3). PB1.3 was also able to attenuate the increase in I/R-induced capillary J v/ Sin HC rats (median increase = 1.26-fold vs. 1.53-fold without PB1.3). These data suggest a role for A-V pairing in capillary perfusion in NC rats and a potential role for P-selectin in I/R-induced microvascular dysfunction in HC rats.

Extravascular transport of fluorescently labeled albumins in the rat mesentery.

Objective: Fluorescently labeled albumin is used frequently as a tracer when monitoring microvascular permeability. Several fluorescent dyes are available for labeling protein, including fluorescein isothiocyanate (FITC) and Texas Red (TR). Because differences in leakage of dye‐labeled proteins have been reported, the objective of the present study was to compare the accumulation of these two tracers in interstitium and lymph after the inflammatory event of ischemiareperfusion.

Inhibition of Leukocyte Adherence Enables Venular Control of Capillary Perfusion in Streptozotocin-Induced Diabetic Rats

Objective: Vasoactive molecules can diffuse from venules to dilate closely paired arterioles and enhance capillary perfusion. Venular control of capillary flow has been found to be dependent on nitric oxide (NO), which might be scavenged rapidly in diabetic microvasculature due to the presence of activated leukocytes. This study attempts to improve venular control of capillary flow using fucoidan, which inhibits venular leukocyte adhesion.

Requirement of Arteriovenular Pairing for Increased Capillary Filtration During Acute Inflammation

Objective: The purpose of this study was to determine whether the leukocyte chemoattractant N‐formyl methionyl‐leucyl‐phenylalanine (FMLP) increases capillary fluid filtration rate (Jv/S), and if so, to determine whether the mechanism involves arteriovenular communication.

Control of the arteriolar myogenic response by transmural fluid filtration

Mechanisms of the myogenic response have not been completely established. We hypothesize that transmural fluid filtration from plasma across smooth muscle cells and into the surrounding interstitium helps regulate arteriolar myogenic tone. Arteriolar diameters in the rat mesentery were monitored prior to and following vascular occlusion with a glass micropipette. Arteriolar occlusion not only gave an increase in hydrostatic pressure that initiated a myogenic response upstream of the pipette, but also allowed measurement of fluid filtration rate by monitoring the movement of vascular red blood cells. A statistically significant correlation existed between basal myogenic tone and fluid filtration. Additionally, the myogenic response was attenuated when an osmotic solution of albumin and ficoll was infused into the bloodstream to decrease fluid filtration. These results are consistent with the hypothesis that shear stress on arteriolar smooth muscle, induced by transmural fluid filtration, is a contributing factor that helps control myogenic tone.

Effect of nitric oxide synthase inhibition on arteriovenular control of capillary perfusion

Postcapillary venules contribute to the control of arteriolar tone and therefore capillary perfusion. In a previous study of the rat mesentery, we found a significant correlation between capillary red blood cell velocity (V/sub RBC/) and the percent of the feeding arteriole length that was paired (proximity < 15 microns) with a postcapillary venule (% pairing). In the present study using the same model, we test whether nitric oxide (NO) is involved in the arteriovenular control of capillary perfusion. The baseline relationship between V/sub RBC/ and % pairing (p<0.001; positive slope of 0.079 mm/sec/%) not only is eliminated following NO synthase inhibition with L-NAME, but also becomes negative (p=0.004; -0.035 mm/sec/%). The negative correlation may indicate that in the absence of NO, venules (or venular leukocytes) promote constriction of nearby arterioles, therefore limiting capillary perfusion. For individual capillaries, the decrease in VRBC (L-NAME minus baseline) was highly correlated with % pairing (p<0.001; r/sup 2/=0.81), with the intercept near zero. This suggests that L-NAME has no effect on capillary perfusion when the arteriolar pathway lacks nearby venules, and that L-NAME has a maximal effect when arteriovenular pairing is high. These results confirm a role for NO in the control of capillary perfusion by arteriovenular communication.

Modeling FMLP-induced capillary filtration: potential role of oxidatively activated leukocytes

Microvascular arterioles and venules are typically found in a closely paired countercurrent arrangement that allows diffusional communication. Increases in capillary fluid filtration rate due to acute inflammation appear to utilize this structural arrangement, and are dependent on leukocytes accumulating at arteriovenular pairing sites (Microcirculation 7:259-268, 2000). We have preliminary evidence that following exposure of the rat mesentery to the leukcoyte chemoattractant formyl-met-leu-phe (FMLP), leukocytes that extravasate from postcapillary venules release a signal that is delivered by nearby arterioles to downstream branching capillaries. We hypothesize that such a mechanism can be described in a mathematical model that relates the diffusion distance from a tissue leukocyte to the nearest arteriole, and also the transit time in the bloodstream from the arteriole to capillary. We also hypothesize that leukocytes that are oxidatively activated are the most likely to participate in this mechanism. Following localized FMLP exposure at arteriovenular pairing sites, /spl sim/30-50% of tissue leukocytes were oxidativley activated, as defined by cellular fluorescence of the oxidant-sensitive dye dihydrorhodamine 123. However, when we measured diffusion distances from those cells to the nearby arteriole, we found no correlation between the estimated effect on capillary filtration (using our mathematical model) and the actual measured effect.