Biomedical Research

Hepatic venoconstriction occurs in rat anaphylactic hypotension. However, the exact venoconstrictive site remains unknown, and we therefore attempted to determine its location by using hepatic venography and histology. Anaphylaxis was induced in anesthetized Sprague-Dawley rats by i.v. administration of ovalbumin antigen. Venography of the portal vein (n = 8) was obtained at baseline and maximal hepatic venoconstriction. We separately examined photomicrographs of the liver sections. Along with systemic hypotension, portal venous pressure increased to a peak of 28 ± 3 cmH2O at 2 min after antigen injection. Post-antigen portal venography revealed that 40% of portal venules (76 vessels/total 188 vessels) with diameters from 160 to 300 μm were not visualized, suggesting that stenosis or obliteration occurred distally. The corresponding upstream portal vessels exhibited markedly bulging. Stenosis was also observed in some portal branches with diameters from 180 to 420 μm (9%; 17 vessels/total 188 vessels). Light microscopically, most portal venules with an estimated baseline diameter less than 110 μm showed stenosis, but statistically significant stenosis was found in those with baseline diameters of 20–70 μm. In conclusion, anaphylactic hepatic venoconstriction occurs over a wide range of portal veins with diameters less than 420 μm, and occurs markedly in portal venules with diameters less than 70 μm in anesthetized rats. Anaphylactic hypotension is in part caused by a decrease in effective circulating blood volume, which could be caused by vasodilation in conjunction with the peripheral blood pooling, as well as by a shift of intravascular fluid to the extravascular space due to increased vascular permeability (2). In canine anaphylactic shock, hepatic venoconstriction, especially selective constriction of the postsinusoidal hepatic veins, induces pooling of blood in the upstream splanchnic organs, as well as in the liver itself (5, 6, 20). Likewise, in rat anaphylactic hypotension, hepatic venoconstriction occurs, although the liver blood volume decreases rather than increasing (8, 16, 19). This anaphylactic hepatic venoconstriction plays a significant role in the genesis of blood pressure fall, based on the finding that the elimination of acute portal hypertension by means of surgical perturbations or portacaval shunting attenuates the antigen-induced systemic hypotension in anesthetized rats (10, 16). However, detailed information on the anaphylactic hepatic venoconstriction which causes acute portal hypertension is limited. The anatomical site for anaphylactic venoconstriction of the liver remains unknown. In a previous work using the hepatic vascular occlusion method, we reported that the portal veins are the primary site of anaphylactic hepatic venoconstriction in isolated perfused rat livers (16). Recently this notion was reinforced by measuring the hepatic venular pressure with a servo-null micropipette pressure measurement technique: anaphylactic venoconstriction in the liver venous circulation occurred prior to the hepatic veAddress correspondence to: Dr. Toshishige Shibamoto, Professor and Chairman of Physiology II Department of Physiology II, Kanazawa Medical University, Uchinada 920-0293, Japan Tel: +81-76-286-2211, Fax: +81-76-286-8010 E-mail: shibamo@kanazawa-med.ac. jp These authors contributed equally to this work. M. Kitadate et al. 62 Y-type miniature plastic tube (24); one twig end was connected via a water-filled polyethylene tube to the pressure transducer for measurement of the portal venous pressure (Ppv), and the other was used for the intravenous injection of contrast medium. After closure of the abdomen, the baseline measurements were started. The vascular pressures were measured using transducers (TP-400T; Nihon-Kohden, Tokyo, Japan) with the reference level at the right atrium, and recorded with a thermal physiograph (RJC-4124; Nihon-Kohden). Hepatic venography procedure. For portal venography, warmed nonionic contrast medium (Iomeron 350; Eisai, Tokyo, Japan) was injected into the portal vein with an automated power injector (Medrad Mark V Pro Vis., Warrendale, PA) at 3 mL/s with a total injection volume of 1.7 mL, and images were obtained with pulsed fluoroscopy at 14 frames per second. This injection maneuver does not affect the basal hemodynamic levels, although some rats show a transient fall in Psa, followed by a recovery within 2 min. Baseline venography was performed when hemodynamic parameters were stable for at least 20 min after surgery. After the baseline measurements, ovalbumin antigen (0.6 mg) was intravenously injected. When the Ppv reached the peak levels, the post-antigen venography was performed. Measurements of vessel lumen diameter. The images of portal venography were analyzed in the left lateral lobe and the right portion of the middle lobe because the portal branches of these two lobes were depicted without overlap by those of the other lobes, and therefore were suitable for measuring the vessel diameter (13). A sequence of frames was chosen in which the branches of the portal veins were most intensely filled with contrast agent. A measure with a diameter of 100 μm was present in the side of each frame and was used for software calibration. The images were magnified and printed on standard X-ray films. The vessel diameter was measured by using a digital micrometer caliper, and, finally, corrected for geometric magnification. The measurement was repeated three times by one examiner on separate days, and the mean values were adopted. The intraclass correlation coefficient for the measured values, as assessed with statistical software (SPSs; IBM Corporation, Endicott, NY), was 0.98. Histological investigation. In separate experiments, the sensitized (n = 5) and non-sensitized control (n = nules, that is, the portal veins or the sinusoids (18). However, the exact constrictive site in the portal veins is not known. Thus the purpose of the present study was to determine the primary site of anaphylactic venoconstriction in the liver by using portal venography. We also morphologically examined tissue sections of the liver to compensate for the shortcomings of venography: namely, if constriction of the small portal veins, i.e., portal venules, is strong, the contrast medium in venography will not reach the venoconstrictive site, resulting in a failure to visualize the venoconstrictive veins (11). In addition, the limit of the spatial resolution in the angiography system was 150 μm. MATERIALS AND METHODS Animals. The experiments in the present study were approved by the Animal Research Committee of Kanazawa Medical University, and conducted in keeping with the Principles of Laboratory Animal Care (NIH publication No. 85–23, Revised 1996). Eighteen male Sprague-Dawley rats (Japan SLC, Shizuoka, Japan) weighing 542 ± 41 g were used in this study. Rats were maintained at 23°C under pathogen-free conditions on a 12 : 12-hour dark/light cycle, and allowed food and water ad libitum. Sensitization. Rats were sensitized by the subcutaneous injection of an emulsion made by mixing equal volumes of complete Freud’s adjuvant (0.5 mL) with 1 mg ovalbumin (grade V; Sigma) dissolved in physiologic saline (0.5 mL) (16). Non-sensitized rats were injected with complete Freund’s adjuvant and ovalbumin-free saline. Surgical preparation of animals. After two weeks, the rats (n = 8) were anesthetized with pentobarbital sodium (50 mg/kg, i.p.), and placed on an angiographic flat-panel detector system (Infinix Celeve-I; Toshiba, Tokyo, Japan). The adequacy of anesthesia was monitored by the stability of blood pressure and respiration under control conditions and during a tail-pinch stimulus. Supplemental doses of anesthetic (10% of initial dose) were given intraperitoneally if necessary. Systemic arterial pressure (Psa) was recorded from the right carotid artery. The right femoral vein was catheterized for an intravenous injection of antigen. After midline incision, the catheter (ID 0.47 mm, OD 0.67 mm) was inserted directly into the main portal vein and the catheter tip was positioned at approximately 1 cm away from the hepatic hilus. This catheter was connected to a custom-made Anaphylactic portal hypertension 63