Evaluating criteria for intact capillaries in Rana pipiens using hydraulic conductivity (Lp), rolling and sticking white blood cells (WBC), and an index of systemic inflammation

Abstract

WBC rolling/sticking along capillary walls have been used for decades to identify inflammation and exclude microvessels from studies of basal capillary function in vivo. For microcirculatory preparations a broader view of animal health state also is required, but often remains undefined. We measured activation of WBC obtained from a peripheral blood sample as an index of systemic inflammation in Rana pipiens and then measured Lp on individual capillaries always devoid of rolling/sticking WBC. We hypothesized that capillary Lp following our standard protocol (abrupt change in shear stress (Δτ)) would be higher in frogs with activated WBC (oxidative burst) compared to control. Frogs (Rana pipiens, N=38) were pithed and mesentery was exteriorized and superfused with frog Ringer's (14–16°C). True capillaries were cannulated at 10 cm H2O, perfused with 10 mg·ml−1BSA/Ringer's, stimulated with Δτ, and Lp assessed at 30 cm H2O (modified Landis technique). Mean±SE Lp for control capillaries (no activated WBC; within 95% confidence intervals (CI) of Δτ/Lp response curve) was 2.7±0.3 × 10−7 cm·s−1·cm H2O−1 (n=15). In frogs with activated WBC, Lp was higher (11.4±1.6 × 10−7, n=9; P<0.0001) compared to control. A subset of frogs with no activated WBC displayed Lp values outside CI that averaged 8.9±1.1 × 10−7 (n=12), were higher than control, and did not differ from the activated WBC group. These data suggest that capillary rolling/sticking WBC may not provide comprehensive information concerning inflammation of intact capillaries in Rana pipiens. Systemic activated WBC accounted for half of the identified outliers. Supported by RO1 HL63125.