This animal study was approved by the local animal ethics committee of Renji Hospital and performed according to the guidelines of the Laboratory Animal Care and Use Committee at School of Medicine, Shanghai Jiao Tong University (Shanghai, China). Fifty female Sprague-Dawley rats weighing 200-220 g underwent ovariectomy before the study. Three weeks after the initial surgery, the rats underwent PPVL or sham operation (SO) as previously described by Reiberger et al[1] and were divided into five groups: SO, PPVL + placebo (PLAC), PPVL + E2, PPVL + ICI and PPVL + E2 + ICI. Briefly, under ketamine anesthesia (intramuscular injection of 10 mg/100 g body weight), the portal vein was isolated, and stenosis was induced by a single ligature of 3-0 silk placed around both the portal vein and a 20-gauge blunt-tipped needle. The needle was then removed, leaving a calibrated stenosis of the portal vein. Portal hypertension was considered present at 14 d after surgery. In SO animals, the portal vein was isolated and similarly manipulated but not ligated. Starting on the day of surgery, the PPVL + E2, PPVL + ICI and PPVL + E2 + ICI groups were subcutaneously administered β-estradiol (2 μg/100 g body weight/d, R and D Systems, United States) or/and ICI182, 780 (2 μg/100 g body weight/d, R and D Systems, United States) for 14 d. The SO and PPVL groups were subcutaneously administered the same volume of the placebo (ethanol).

Under anesthesia induced by an intramuscular injection of ketamine (10 mg/100 g body weight), the velocity and inside diameter of the portal vein (PV) proximal to the ligation and stroke volume (SV) were obtained using a Vevo770 High-Resolution Imaging System (Visual Sonics, Canada). A 22 G catheter filled with heparin saline was inserted into the femoral artery to obtain the mean arterial pressure (MAP) and heart rate (HR). Another 22 G catheter was introduced into the portal vein to measure PP, and one was inserted into the inferior vena cava to measure central venous pressure (CVP) after making an incision at the midline of the abdomen. All parameters were recorded using an SP840 pressure transducer and a multichannel recorder (Philips, United States).

Cardiac output (CO), cardiac index (CI), PV blood flow, portal venous inflow (PVI), systemic vascular resistance (SVR) and splanchnic arteriolar resistance (SAR) were calculated as follows: CO = SV × HR; CI = CO × 100/body weight (g); PVI = PV blood flow × 100/body weight (g); SVR = (MAP - CVP)/CI; SAR = (MAP - PP)/PVI. PV blood flow and CO were normalized by body weight and presented as CI and PVI. Resistances in the vascular systems were calculated from the ratio between the perfusion pressure (P) and blood flow (Q) of each vascular territory.

Following the determination of hemodynamic measurements, the mesenteric arteries and the mesentery were removed. Using an SMZ-168 dissecting microscope (Motic, China), the third-order arterioles in the mesentery were carefully dissected and transferred to a vascular perfusion system containing a 3-(N-morpholino) propanesulfonic acid-buffered physiological salt solution (MOPS-PSS, 0-4 °C, pH 7.4; NaCl 145 mmol/L, KCl 5.0 mmol/L, CaCl₂ 2.0 mmol/L, MgSO₄ 1.0 mmol/L, NaH₂PO₄ 1.0 mmol/L, glucose 5.0 mmol/L, pyruvate 2.0 mmol/L, EDTA 0.02 mmol/L and MOPS 3.0 mmol/L). A glass micropipette containing MOPS-PSS (top diameter, 50 μm) was inserted into one end of the arteriole and fixed with 11-0 single strands. Blood was flushed out at a perfusion pressure of 8 mmHg. Another glass micropipette was then inserted into the other end of the arteriole and fixed. The two glass micropipettes were suspended in organ baths containing 60 mL of MOPS-PSS (37 °C, pH 7.4). The arteriole was equilibrated under a pressure of 80 mmHg for 30 min prior to the experiments. After the equilibration period, cumulative NE concentration response curves (10^-8 mol/L-10^-4 mol/L) were obtained by increasing the concentration in quarter-log increments. The inner diameter was measured using a BA310 microscope camera system (Motic, China). The vasoconstriction rate and the logarithm of the NE concentration were used as the vertical axis and the abscissa, respectively.

The oxidative fluorescent dihydroethidium (DHE, Sigma, United States) was used to evaluate in situ production of superoxide anion (O₂•⁻)[12]. Frozen, enzymatically intact, 10-μm-thick sections of the superior mesenteric arteries were incubated with DHE (10 μmol/L) in PBS for 30 min at 37 °C in a humidified chamber protected from light. DHE is freely permeable to cells and is oxidized in a reaction with O₂•⁻ to ethidium bromide, which binds to DNA in the nucleus and fluoresces red. Images were obtained with an IX71 fluorescence microscope (Olympus, Japan).

The level of hydrogen peroxide (H₂O₂) was measured using a hydrogen peroxide assay kit (Abcam, United States). In the presence of horseradish peroxidase (HRP), the OxiRed Probe reacts with H₂O₂ to produce product with color. The superior mesenteric artery was cleaned of connective tissue, precipitated with RIPA (Beyotime, China; 200 μL RIPA/20 mg tissue) for 15 min and then centrifuged for 15 min at 1000 g. A total of 5 μL of the supernatant was diluted with 46 μL of assay buffer, mixed with 50 μL of the Reaction Mix (assay buffer 46 μL, OxiRed Probe 2 μL, HRP 2 μL) and then incubated at room temperature for 10 min. The OD570 nm was read with a Synergy 4 Multi-Mode Microplate Reader (BioTek, United States), and the H₂O₂ concentration was calculated according to a standard concentration curve.

The change in the reactivity of the mesenteric arteriole in response to NE was presented as a dose-response curve, which was fitted by a nonlinear regression analysis (GraphPad Software Inc., San Diego, CA, United States). Maximal responses (Emax) and effective concentrations causing half maximum responses (EC₅₀, calculated by regression analysis) were obtained from concentration response curves. Values are expressed as the means ± SD. Statistical comparisons were performed using one-way ANOVA. P values < 0.05 were considered significant. All statistical analyses were performed using the Statistical Package for the Social Sciences 13.0 (SPSS Inc., United States).

Compared to SO, PPVL resulted in a lowered HR (P < 0.05) and MBP (P < 0.05) and significantly increased PP (P < 0.01) in the four groups of prehepatic PHT. However, measurement of MAP and HR revealed no significant differences between the four PPVL groups (P > 0.05). PP significantly decreased by 18.5% in PPVL + E2 rats with a mean PP of 12.2 ± 1.6 mmHg compared to a mean PP of 14.9 ± 1.4 mmHg in PPVL + PLAC rats (P < 0.01) (Table 1).

PVI was significantly increased in the four groups of rats that underwent PPVL compared to the corresponding SO group (P < 0.01). Treatment with E2 resulted in a reduction in PVI in PPVL + E2 rats compared to PPVL + PLAC rats (3.98 ± 1.4 mL/min per 100 g vs 4.87 ± 1.6 mL/min per 100 g, P < 0.05) (Table 1).

Although CI was slightly increased in the four groups of PPVL rats compared to the SO group, there were no significant differences among the five different groups (P > 0.05). SVR was lower in the four groups of PPVL rats compared to SO rats due to the decreased MBP (P < 0.05), which was increased 16.9% by E2 treatment in PPVL + E2 rats compared to PPVL + PLAC rats (3.95 ± 0.41 mmHg∙min∙100 g/mL vs 3.38 ± 0.35 mmHg∙min∙100 g/mL, P < 0.05) (Table 1).

SAR was significantly lower in the four groups of PPVL rats compared to SO rats (P < 0.01). Rats treated with E2 showed significantly higher SAR, with a 34.1% increase compared to PPVL + PLAC rats (19.29 ± 1.8 mmHg∙min∙100 g/mL vs 14.39 ± 2.2 mmHg∙min∙100 g/mL, P < 0.01) (Table 1).

ICI182, 780 treatment alone did not influence PP, PVI, CI, SVR or SAR compared to PPVL + PLAC rats, except that the body weight was slightly lower (P < 0.05). However, ICI182, 780 reversed the beneficial effects of E2 on PVI, CI, SVR and SAR. Thereby, the systemic circulation and splanchnic hyperdynamic circulation were more deteriorated (Table 1).

Compared with the SO group, the dose-response curve of the mesenteric arteriole in response to NE was lower and shifted right, with decreased Emax (P < 0.01) and increased EC50 (P < 0.01, Figure 1; Table 1) in the four PPVL groups. In the mesenteric arterioles of PPVL rats treated with E2, the dose-response curve was shifted left, and the EC50 was decreased (P < 0.01), compared with the PPVL + PLAC rats. ICI182, 780 alone did not influence the dose-response curve or EC50 compared to PPVL rats treated with placebo (P > 0.05). However, ICI182, 780 decreased the response to NE of arterioles treated with E2, thereby the dose-response curve was shifted right.

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Figure 1 Dose-response curves for isolated rat mesenteric arteriole contractility in response to norepinephrine at different concentrations. The dose-response curves in the four partial portal vein ligation (PPVL) groups were lowered and shifted to the right compared to the sham operation (SO) group. Treatment with estrogen (E2) reversed the curve to left, whereas ICI182, 780 decreased the response to norepinephrine of arterioles treated with E2. PLAC: Placebo.

Low-intensity fluorescence was observed throughout SO vessels, confirming that all layers of the normal vessel produced low amounts of O₂•⁻. Fourteen d after the surgery, O₂•⁻ production was increased in the four groups of PPVL rats (Figure 2). However, the staining was particularly weak in PPVL + E2 rats, and the administration of ICI182, 780 blocked the beneficial effect that E2 provided.

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Figure 2 In situ detection of superoxide anion. Fluorescence micrographs of superior mesenteric arteries stained with the O₂•⁻-sensitive dye DHE (red fluorescence) were obtained from sham operation (SO) and partial portal vein ligation (PPVL) rats at 14 d after surgery. Images were acquired at identical settings. ^bP < 0.01 vs SO; ^cP < 0.05, ^dP < 0.01 vs PPVL + placobo (PLAC); ^fP < 0.01 vs PPVL + estrogen (E2).

In the superior mesenteric arteries, H₂O₂ generation was nearly 2.5-fold higher in PPVL + PLAC rats than in SO rats (8.2 ± 1.5 μmol/L vs 3.3 ± 0.9 μmol/L, P < 0.01; Figure 3). Exogenous E2 reduced H₂O₂ levels in PPVL + E2 rats when compared with those in PPVL + PLAC rats (4.9 ± 1.0 μmol/L vs 8.2 ± 1.5 μmol/L, P < 0.01). The H₂O₂ levels in mesenteric arteries treated with ICI182, 780 alone were similar to those found in PPVL + PLAC rats (7.9 ± 1.8 μmol/L vs 3.3 ± 0.9 μmol/L, P < 0.01). However, the administration of ICI182, 780 to PPVL + E2 rats suppressed the antioxidant effect of E2 (6.6 ± 1.3 μmol/L vs 4.9 ± 1.0 μmol/L, P < 0.05).

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Figure 3 Hydrogen peroxide determination. The hydrogen peroxide concentrations of the superior mesenteric arteries were obtained from sham operation (SO) and partial portal vein ligation (PPVL) rats 14 d after surgery. ^aP < 0.05, ^bP < 0.01 vs SO; ^cP < 0.05, ^dP < 0.01 vs PPVL + placobo (PLAC); ^eP < 0.05, ^fP < 0.01 vs PPVL + estrogen (E2).

Portal hypertension, a major complication of liver cirrhosis, is associated with the development of hyperdynamic circulation and would be triggered by persistent splanchnic vasodilation. Functional changes can be found in the portal hypertensive splanchnic vasculature, including splanchnic vasodilation and decreased responsiveness to vasoconstrictors as a result of endothelial dysfunction and impaired activation of vasoconstrictive mechanisms.

Increased oxidative stress may lead to impaired endothelium dysfunction and be involved in the pathogenesis of portal hypertension. It has therefore been a subject of interest

The authors found that improvements in oxidative stress after estrogen administration manifest as a functional improvement in the contractile response of mesenteric arteries to vasoconstrictors in partial portal vein ligation (PPVL) rats.

The authors proposed that treatment with estrogen could improve the systemic and splanchnic hyperdynamic circulation in PPVL rats.

The manuscript is an interesting manuscript with novel observations. The authors delineated the effect of estrogen treatment on the systemic and splanchnic hyperdynamic circulation in portal hypertensive rats. The findings are straight forward and manuscript is well written and nicely discussed.