Proinflammatory liver and antiinflammatory intestinal mediators involved in portal hypertensive rats.(RESEARCH COMMUNICATION)

Proinflammatory (TNF-[alpha], IL-1[beta], and NO) and antiinflammatory (IL-10, CO) levels were assayed in serum, liver, and small bowel in order to verify a hypothetic inflammatory etiopathogeny of portal hypertension that could be the cause of its evolutive heterogeneity. Male Wistar rats were divided into one control group (n = 11) and one group with a triple stenosing ligation of the portal vein (n = 23) after 28 days of evolution. In one subgroup of portal hypertensive rats, portal pressure, collateral venous circulation, mesenteric vasculopathy, and liver and spleen weights were determined. In the remaining rats with portal hypertension TNF-[alpha], IL-1[beta], and IL-10 were quantified in liver and ileum by enzyme-linked immunosorbent assay. NO synthase activity was studied in liver and ileum. CO and NO were measured in portal and systemic blood by spectrophotometry and Griess reaction, respectively. Portal hypertensive rats with mayor spleen weight show hepatomegaly and mayor development of collateral circulation. Ileum release of IL-10 (0.30 [+ or -] 0.12 versus 0.14 [+ or -] 0.02 pmol/mg protein; P < .01) is associated with a liver production of both proinflammatory mediators (TNF-[alpha]: 2 [+ or -] 0.21 versus 1.32 [+ or -] 0.60 pmol/mg protein; P < .05, IL-1[beta]: 19.17 [+ or -] 2.87 versus 5.96 [+ or -] 1.84 pmol/mg protein; P = .005, and NO: 132.10 [+ or -] 34.72 versus 61.05 [+ or -] 8.30 nmol/mL; P = .005) and an antiinflammatory mediator (CO: 6.49 [+ or -] 2.99 versus 3.03 [+ or -] 1.59 pmol/mL; P = .005). In short-term prehepatic portal hypertension a gut-liver inflammatory loop, which could be fundamental in the regulation both of the portal pressure and of its complications, could be proposed.

INTRODUCTION

Portal hypertension (PHI) is a clinical syndrome which is usually secondary to intrahepatic or extrahepatic obstruction of portal flow. It is characterized by a pathological increase in portal pressure, associated with splenomegaly, and by the development of portosystemic collateral circulation which diverts portal flow to the systemic circulation bypassing the liver [1, 2, 3]. Moreover, PHT is the main complication of cirrhosis and is responsible for most of its common complications: variceal hemorrhage, ascites, and portosystemic encephalopathy [4]. Making an effort to create new PHT models or to improve previously existing ones is justified by the need to study the physiopathological mechanisms of this frequent and severe clinical syndrome for which several different types of medical [5, 6] and surgical [7] treatments have been proposed.

Partial portal vein ligation (PVL) in the rat is the most frequently used experimental model to study in the short term the pathophysiology of prehepatic PHT [8, 9]. Constriction of the portal vein (PV) is immediately followed by increased portal resistance and portal pressure as well as decreased portal venous inflow [10]. Partial development of portosystemic collaterals is found after 4 days of PVL and, after two weeks of evolution most (95%) of the increased portal blood is diverted from the liver by an extensive portocollateral vascular bed [9, 10]. Then, portal resistance decreases to control values and the splanchnic blood flow, secondary to a decrease in the splanchnic arteriolar resistance, increases, all of which contributes to the maintenance of increased portal pressure [10, 11].

Therefore, until now PHT in the rat has always been considered to be a hemodynamic impairment with much more homogeneous alterations than those described in human PHT because of a narrow range of PHT, grade of portosystemic shunts, and hepatic atrophy [12]. However, this evolutive uniformity could not be verified from our previous studies using a modified technique of PV calibrated stenosis in the rat since the degree of hepatic atrophy, splenomegaly, and portosystemic collateral circulation that developed were variable [13]. A possible inflammatory etiopathogeny of PHT could be one cause of this evolutive heterogeneity. Thus, an increased infiltration of the intestinal mucosa and submucosa by mast cells has been described in PHT rats [14]. These inflammatory cells could be considered to be primed to a noxious stimulus and this mechanism could be responsible for the increased susceptibility of the PHT mucosa. If so, the anaphylactic degranulation of the mast cells may cause inflammatory episodes that vary in number and duration in each individual. This difference could explain the great variability observed in prehepatic PHT in the rat.

In order to verify a hypothetic inflammatory etiopathogeny of PHT we have studied some mediators involved in an inflammatory response. These included tumor necrosis factor-[alpha] (TNF-[alpha]), Interleukin-1[beta] (IL-1[beta]), interleukin-10 (IL-10), nitric oxide synthase (NOS) isoforms in tissue (liver and/or intestine), and nitric oxide (NO) and carbon monoxide (CO) in serum in rats with short-term prehepatic PHT.

MATERIAL AND METHODS

Animals

Male Wistar rats from the Complutense University Vivarium in Madrid with body weights from 230 to 250 g were used. The experimental procedures employed in this study are in accordance with the Guidelines for the care and use of Laboratory Animals (1986) published in Spain (Royal Decree 223/1988).

Experimental design

The animals were divided for their study into three groups: one control group (I), in which the animals did not undergo any intervention, and two-subgroups (IIa and IIb), in which prehepatic PHT by triple stenosing ligation of the portal vein (TSLP) was carried out. All the animals were sacrificed by ether overdose after 28 days of evolution. In Group IIa portal pressure, collateral venous circulation, mesenteric vasculopathy, and hepatic and spleen weights were studied. In Group IIb TNF-[alpha], IL-1[beta], and IL-10 levels were assayed in ileum, endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) activity were measured in liver and in intestine (duodenum, jejunum, and ileum). Finally, NO and CO concentrations were quantified in PV, suprahepatic inferior vena cava (SH-IVC), and infrahepatic inferior vena cava (IH-IVC) blood.

Portal vein stenosis technique

The surgical technique used to produce PHT by TSLP has been described previously [15]. In brief, while rats were under ketamine hydrochloride (80mg/kg) and Xylacin (12 mg/kg) im anesthesia, the PV was isolated and three stenosing ligations were performed in its superior, medial, and inferior portions. The stenoses were calibrated by a simultaneous ligation (4-0 silk) around the PV and a 20-gauge blunt-tipped needle. The midline incision was closed on two layers with catgut and 3-0 silk.

Portal vein pressure measurement

The portal pressure is measured by an indirect technique of intrasplenic punction [16], inserting a 20G needle in the splenic parenchyma which is, in turn, connected to a PE-50 tube. After verifying that free back blood flow was obtained, the catheter was then connected to a pressure recorder (PowerLab 200ML 201) and to a transducer (Sensonor SN-844) with a Chart V 4.0 computer program (ADI Instruments). The system is calibrated before each experiment. The zero reference point was established at 1 cm above the operating table. Previous studies have demonstrated an excellent correlation between the indirect measurement of the portal pressure by intrasplenic punction and the…