Cutaneous absorption of xenobiotics is considered a significant route of environmental exposure.5,6 Blood flow is an important factor in cutaneous absorption kinetics of xenobiotics.3 A proposed in vitro model for cutaneous absorption in anurans is the perfused amputated anuran pelvic limb. This model requires the amputation of the pelvic limb, cannulation of the common iliac artery, and perfusion at a physiologic flow rate. The objective of this study is to establish baseline values for the normal perfusion of the pelvic limb of anurans. We hypothesize that flow rate does not differ between bullfrogs (Rana catesbeiana) and marine toads (Bufo marinus).
Five wild-caught bullfrogs and five marine toads were obtained from commercial sources and acclimated for 3 weeks. Animals were manually restrained in dorsal recumbency on a water-moistened cloth towel, and water soluble contrast gel was applied bilaterally to the inguinal region. A 7.5 MHz linear transducer was gently applied over this region until the pulsing common iliac artery was identified just proximal to its bifurcation using color Doppler ultrasound. Measurements were taken at times when animals were not breath-holding to minimize the impact of increased intra-coelomic pressure on arterial diameter. Three successive diameter measurements were obtained in a transverse plane at times when the common iliac artery appeared most circular and while animals were respiring normally. This was followed by three successive time-averaged mean velocity measurements in a longitudinal plane.
Maximum volumetric flow rate (ml/min) was calculated from the product of the mean time-average mean velocity (TAMx) and the mean cross-sectional area of the artery.1,2 Flow rate per kg body weight was then calculated. A Wilcoxon signed rank test was used for nonparametric statistical comparison between left and right pelvic limb data and between frog and toad data (p<0.05).
No significant differences were noted between left and right volumetric flow rates for frogs or toads. Volumetric flow rates did not differ between frogs and toads at 24.7±11.0 ml/min and 19.1±9.6 ml/min, respectively. However, when flow rates were adjusted for body mass, toads had a significantly greater flow rate of 294.4±112.9 ml/min.kg compared to 86.4±37.5 ml/min.kg for frogs. Higher volumetric flow rates in toads can be explained by differences in metabolism or breath-holding in toads.
This study demonstrates the value of performing color Doppler ultrasound on an artery of interest prior to constructing an amputated perfused limb preparation. A flow rate of 1 ml/min (standard flow rate for isolated perfused porcine skin flaps) is sufficient to maintain patency of the common iliac artery of anurans, however, it does not represent a physiologic perfusion rate.4 Small permutations in arterial radius (r) and errors in measurement have a large effect on cross-sectional area (πr2) and resultant volumetric flow calculations. Therefore, higher flow rates can increase oncotic pressure and result in extravasation of the perfusate. The square root of the measured flow rates would account for increases in TAMx due to stress and increases in intracoelomic pressure, as well as variation in angle of the transducer in relation to blood flow and measurements of radii.
1. Kantrowitz BM, TG Nyland, P Fisher. 1989. Estimation of portal blood flow using duplex real-time and pulsed Doppler ultrasound imaging in the dog. Vet. Radiol. 30(5):222-226.
2. Raisis AL, LE Young, HB Meire, PM Taylor, KJ Blissitt, D Marlin, P Lekeux. 2000. Measurements of hindlimb blood flow recorded using Doppler ultrasound during administration of vasoactive agents in halothane-anesthetized horses. Vet. Radiol. & Ultrasound. 41(1):42-72.
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4. Riviere JE, KF Bowman, NA Monteiro-Riviere, LP Dix, MP Carver. 1986. The Isolated Perfused Porcine Skin Flap (IPPSF) I. A Novel in vitro Model for Percutaneous Absorption and Cutaneous Toxicology Studies. Fund. Appl. Toxicol., 7: 444-453.
5. Taylor SK., ES Williams, KW Mills. 1999. Effects of malathion on disease susceptibility in Woodhouse's toads. J. Wildl. Dis., 35(3): 536-541.
6. Wallace KB. 1992. Species-selective toxicity of organophosphorus insecticides: A pharmacodynamic phenomenon. In: Chambers, J., and P. Levi (eds.). Organophosphates: Chemistry, Fate, and Effects. Academic Press, San Diego. Pp. 79-105.