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Study of Morphological Changes in Rat Liver Caused by Occlusion of Inferior Vena Cava

      Background/objectives

      Up to date, there are no reports on animal survival rate and morphological changes in the liver caused by the impairment of blood outflow from the liver and its time dependence. Moreover, the impact of duration and degree of occlusion of inferior vena cava on pathological changes was not investigated yet. This study aimed at the assessment of the survival rate and morphological changes in the liver with varying degrees of occlusion of inferior vena cava. The exact timing of the reversibility of pathological processes was determined.

      Methods

      Rats (n = 160) were randomly divided into five groups: I – control group (CG) (n = 20); II – sham group (SG) (n = 20); III – intervention group (IG-1) (narrowing of the lumen of the inferior vena cava by 25%) (n = 40); IV intervention group (IG-2) (narrowing by 50%) (n = 40); and intervention group V (IG-3) (narrowing by 75%) (n = 40). The level of postoperative pain, the body and liver weight of the animals, histological examination, morphometry, and macroscopic evaluation of abdominal organs were carried out on the 1, 3, 7, 14, and 30 days following the surgical intervention. The survival rate of animals was assessed using the Kaplan–Meier method.

      Results

      On the 30th day, the rat grimace scale indices in the IG-1 (P ≤ 0.05), IG-2, and IG-3 (P ≤ 0.001) groups were higher. By body weight, this indicator on the 30th day was lower in the IG-1 (P ≤ 0.05), IG-2, and IG-3 (P ≤ 0.001) groups compared to the CG and SG groups. In the IG1 and IG2 groups, the survival rates were 72.5% and 65.0%, respectively. The lowest survival rate was observed in the IG3 group (22.5%).

      Conclusions

      Compression of the inferior vena cava by 75% led to an increase in animal mortality and the development of persistent morphological changes in the liver. At the same time, the survival rate of animals and the extent of changes in the liver with narrowing of the inferior vena cava by 25% and 50% had similar results. The results acquired possess scientific and practical importance.

      Keywords

      Abbreviations:

      CG (Control group), IG (Interventional group), SG (Sham group)
      The impaired venous outflow can cause portal hypertension leading to an increase in vascular resistance to hepatic blood flow.
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      • Erden I.
      • Yurdaydin C.
      • Karayalçin S.
      Hepatic outflow obstruction: enhancement patterns of the liver on MR angiography.
      The obstruction of the blood outflow from the liver can trigger a whole spectrum of clinical disorders, ranging from acute liver failure to passive liver congestion, depending on the severity and level of impediment.
      • Bayraktar U.D.
      • Seren S.
      • Bayraktar Y.
      Hepatic venous outflow obstruction: three similar syndromes.
      The most common cause of occlusion of inferior vena cava is a Budd-Chiari syndrome.
      • Li Y.
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      • Li H.
      • et al.
      Epidemiology of Budd-Chiari syndrome: a systematic review and meta-analysis.
      Such syndrome is associated with the obstruction of hepatic venous outflow at the level of the hepatic veins, inferior vena cava, and/or right atrium.
      • Chen S.
      • Gao Y.
      • Yu C.
      • Zhang M.
      • Nie Z.
      A canine model for IVC occlusive form of Budd-Chiari syndrome using endovascular technique.
      There is a range of attempts to reproduce the impaired blood outflow from the liver, including Budd-Chiari syndrome, on animal model.
      • Chen S.
      • Gao Y.
      • Yu C.
      • Zhang M.
      • Nie Z.
      A canine model for IVC occlusive form of Budd-Chiari syndrome using endovascular technique.
      ,
      • Murad S.D.
      • Dom V.A.L.
      • Ritman E.L.
      • et al.
      Early changes of the portal tract on microcomputed tomography images in a newly-developed rat model for Budd–Chiari syndrome.
      The development of Budd-Chiari syndrome model was mainly achieved via surgical reduction of the diameter of the inferior vena cava or the occlusion of the hepatic branches by the endovascular method.
      • Chen S.
      • Gao Y.
      • Yu C.
      • Zhang M.
      • Nie Z.
      A canine model for IVC occlusive form of Budd-Chiari syndrome using endovascular technique.
      • Murad S.D.
      • Dom V.A.L.
      • Ritman E.L.
      • et al.
      Early changes of the portal tract on microcomputed tomography images in a newly-developed rat model for Budd–Chiari syndrome.
      • Shen B.
      • Zhang Q.
      • Wang X.
      • et al.
      Development of a canine model with diffuse hepatic vein obstruction (Budd-Chiari syndrome) via endovascular occlusion.
      There are two options for imitating the Budd-Chiari syndrome: hepatic vein thrombosis (the so-called classic Budd-Chiari syndrome) and hepatic vena cava–Budd-Chiari syndrome.
      • Shin N.
      • Kim Y.H.
      • Xu H.
      • et al.
      Redefining Budd-Chiari syndrome: a systematic review.
      The effect of hypertension on hepatic vessels has been well described for classical models,
      • Vlad M.
      • Ionescu N.
      • Ispas A.T.
      • Stoica C.
      • Ungureanu E.
      The histological changes of digestive organs in experimental decreases of hepatic venous outflow at the rat.
      including models based on the endovascular approach.
      • Shen B.
      • Zhang Q.
      • Wang X.
      • et al.
      Development of a canine model with diffuse hepatic vein obstruction (Budd-Chiari syndrome) via endovascular occlusion.
      There is a range of reports on the link between the development of persistent morphological changes in the vessels and the liver with the degree of narrowing of the caudal inferior vena cava as well as the duration of violations of blood outflow.
      • Murad S.D.
      • Dom V.A.L.
      • Ritman E.L.
      • et al.
      Early changes of the portal tract on microcomputed tomography images in a newly-developed rat model for Budd–Chiari syndrome.
      ,
      • Orloff M.J.
      • Daily P.O.
      • Girard B.
      Treatment of Budd-Chiari syndrome due to inferior vena cava occlusion by combined portal and vena caval decompression.
      However, there are no data on the effect of increased sinusoidal pressure on the portal tract, morphological changes over time, and survival rate depending on the degree of vasoconstriction. Such information might useful for the effective translation of the results of basic scientific research into clinical practice.
      The objective of this study was to investigate the morphological changes in the liver over time and the survival rate depending on the degree of the narrowing of the caudal inferior vena cava.

      Methods

      Ethical Issues

      The study was carried out in the laboratory of experimental medicine of the NJSC “S. D. Asfendiyarov Kazakh National Medical University”, Almaty city, Kazakhstan. The study was approved by the Local Ethics Committee of S. D. Asfendiarov Kazakh National Medical University (protocol of the Local Ethical Commission No. 2 (93) dated by 23.02.2020).
      The keeping of animals was carried out in accordance with the international rules “Guide for the Care and Use of Laboratory Animals” (National Research Council, 2011), as well as with the ethical principles of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (Strasbourg, 2006). The experimental study was performed on the rats (n = 160) weighing 190–220 g. Animals before and after the operation were kept in the Vivarium of the B. Atchabarov Research Institute of Fundamental and Applied Medicine (Almaty city, Kazakhstan) with a standard diet and care.

      Study Design

      • Laboratory animals (n = 160) were sub-divided (randomization) into five groups (Figure 1):
        Figure 1
        Figure 1Flowchart illustrating the animal experiment and grouping.
      • Group I: Control group (CG): without performing any intervention (n = 20);
      • Group II: Sham group (SG): opening of the chest cavity by right-sided lateral approach, without narrowing of the inferior vena cava (n = 20);
      • Group III: Interventional group (IG-1): narrowing of the lumen of the inferior vena cava above the diaphragm by 25% (n = 40);
      • Group IV: Interventional group (IG-2): narrowing of the lumen of the inferior vena cava above the diaphragm by 50% (n = 40);
      • Group V: Interventional group (IG-3): narrowing of the lumen of the inferior vena cava above the diaphragm by 75% (n = 40).
      There were four runs for each experimental group: 3 days (n = 10); 7 days (n = 10); 14 days (n = 10); and 30 days (n = 10). For each run, five animals were withdrawn from the experiment after 3, 7, 14, and 30 days from the beginning of the experiment. The remaining five animals from series 3, 7, and 14 days were withdrawn from the experiment (who survived for 30 days).
      In the SG group, the lateral opening of the chest cavity was performed in order to exclude the negative effect of surgery and anesthesia. However, the narrowing of the inferior vena cava was not simulated.

      Surgical Intervention

      Experiments on laboratory animals were carried out in the operating unit of the Laboratory of Experimental Medicine B. Atchabarov Research Institute of Fundamental and Applied Medicine (Almaty city, Kazakhstan).
      All rats were kept under standard pathogen-free conditions at room temperature (15–25 °C), humidity 50–60% with a 12-h light/dark cycle, and had free access to food and water.
      • Cheng D.L.
      • Zhu N.
      • Li C.L.
      • et al.
      Significance of malondialdehyde, superoxide dismutase and endotoxin levels in Budd-Chiari syndrome in patients and a rat model.
      The animals were fasting for 12 h (no water restriction). A surgical site was carefully shaved, under general anesthesia (ketamine 80 mg/kg + xylazine 10 mg/kg).
      • Jungebluth P.
      • Ostertag H.
      • Macchiarini P.
      An experimental animal model of postobstructive pulmonary hypertension.
      The dose and time of administration of the anesthetics were recorded in the experiment log. The laboratory animals were fixed on the bench in the position on the left side. All operations were performed using a binocular loupe (type 1.2–3.5×, China).
      Modeling of hepatic hypertension was performed by one surgeon to ensure technical uniformity. Modeling of occlusion of inferior vena cava in the experiment was carried out according to the previously reported technique (“Method for modeling of occlusion of inferior vena cava in experiment”/No. of patent of Republic of Kazakhstan for model: 5796). Briefly: after treatment of the surgical field, a right-sided lateral thoracotomy was performed, with preliminary infiltration with a 0.25% solution of Novocain of the inferior vena cava. After this, the inferior vena cava part above the diaphragm was separated from the adjacent tissues in a blunt way. Thus, the trunk of the inferior vena cava was isolated and placed close to the site where the branches of the hepatic veins enter it.
      According to the results of previous studies, the average diameter of the inferior vena cava in rats was taken into account, which was 3.2 ± 0.1 mm according to the calculations of Doppler analysis,
      • Alkan M.
      • Çelik A.
      • Bilge M.
      • et al.
      The effect of levosimendan on lung damage after myocardial ischemia reperfusion in rats in which experimental diabetes was induced.
      and 3.1 ± 0.4 mm in an in vitro study.
      • Langford D.J.
      • Bailey A.L.
      • Chanda M.L.
      • et al.
      Coding of facial expressions of pain in the laboratory mouse.
      Depending on the planned model of 25%, 50%, and 75% of the degree of narrowing of the inferior vena cava, plastic semicircular rings of the required size were prepared.
      • In group I (CG): there was no intervention (n = 20);
      • In group II (SG): the thoracic cavity was opened with a right-sided lateral approach, and narrowing of the inferior vena cava was not performed (n = 40);
      • In group III (IG-1): the lumen of the inferior vena cava was narrowed with a plastic ring by 25% in the region located above the diaphragm (n = 40);
      • In group IV (IG-2): the lumen of the inferior vena cava was narrowed with a plastic ring by 50% in the region located above the diaphragm (n = 40);
      • In group V (IG-3): the lumen of the inferior vena cava was narrowed by 75% in the region located above the diaphragm (n = 40).
      • Anesthesia was maintained via repeated injections of 20 mg/kg ketamine in case of a positive response to surgical stress or intermittent tail clamping observed.
        • Alkan M.
        • Çelik A.
        • Bilge M.
        • et al.
        The effect of levosimendan on lung damage after myocardial ischemia reperfusion in rats in which experimental diabetes was induced.
        After the completion of the procedure, the muscle and skin layers were closed with 4–0 polypropylene.
      All animals of the experimental groups were injected intramuscularly with penicillin (20 U/day) for five days. Animals from the CG were kept under normal conditions and were not subjected to any procedures.

      PostOperative Management

      After the completion of the operations, the animals were placed in individual cages until anesthesia was restored. Then, the animals were grouped into collective cages and examined at regular intervals for approximately 4 h until the end of the experiment. Animals that died immediately after hepatic vein ligation due to exsanguination, iatrogenic liver injury, pneumothorax, and cardiac and respiratory arrest, or that did not show good recovery from surgical anesthesia in the first eight postoperative hours were excluded from the study (n-5).

      A Visual Assessment of the Animals

      The level of postoperative pain was assessed using the rat grimace scale. Such indicators as periorbital contraction, flattening of the nose and cheeks, changes in the position of the ears and whiskers, decreased activity, retraction of the sides, arching of the back, piloerection, and abdominal edema were evaluated.
      • Langford D.J.
      • Bailey A.L.
      • Chanda M.L.
      • et al.
      Coding of facial expressions of pain in the laboratory mouse.
      ,
      • Sotocinal S.G.
      • Sorge R.E.
      • Zaloum A.
      • et al.
      The Rat Grimace Scale: a partially automated method for quantifying pain in the laboratory rat via facial expressions.
      These criteria were assessed by points: 0 (invisible), 1 (noticeable), and 2 (marked).
      • Tian M.
      • Alimujiang M.
      • Chen J.D.
      Ameliorating effects and mechanisms of intra-operative vagal nerve stimulation on postoperative recovery after sleeve gastrectomy in rats.

      Estimation of Body Weight of Animals

      The body weights of the animals were measured on days 1, 3, 7, 14, and 30 using a scale recording with an accuracy of 1 g (MonoBloc Mettler, Toledo, USA). Individual weight loss greater than 20% of baseline weight at any point in the experiment was identified as a humane endpoint.
      • Fontoura-Andrade J.L.
      • Pinto L.M.
      • Carneiro F.P.
      • Sousa J.B.
      Effect of preconditioning and postoperative hyperbaric oxygen therapy on colonic anastomosis healing with and without ischemia in rats.

      Macroscopic Assessment of the Abdominal Organs

      After opening the abdominal cavity, the presence of varicose veins of the anterior abdominal wall, ascetic fluid in the abdominal cavity, and the presence of changes in the size of the liver and spleen were visually assessed. Indicators of abdominal circumference, the development of abdominal varicose veins, pathological changes in the liver and spleen, changes in liver function, and hypertension in the inferior vena cava were regarded as signs of a simulated Budd-Chiari syndrome,
      • Shen B.
      • Zhang Q.
      • Wang X.
      • et al.
      Development of a canine model with diffuse hepatic vein obstruction (Budd-Chiari syndrome) via endovascular occlusion.
      as a control for this assessment, previously described information was also taken into account.
      • Vdoviaková K.
      • Petrovová E.
      • Maloveská M.
      • et al.
      Surgical anatomy of the gastrointestinal tract and its vasculature in the laboratory rat.

      Liver Weight

      The wet weight of the liver lobes was measured separately using an analytical laboratory balance (Mettler-Toledo AG 245; Mettler-Toledo; LLC; Columbus; OH).
      • Lauber D.T.
      • Tihanyi D.K.
      • Czigány Z.
      • et al.
      Liver regeneration after different degrees of portal vein ligation.

      Histological Evaluation

      After removing the animals from the experiment (in accordance with the established practice), pieces were taken from different parts of the liver (in three planes) for histological examination, with their subsequent fixation in 10% neutral formalin. After fixation in formalin, the liver tissue was kept in alcohol of ascending strength, followed by embedding in paraffin. According to Van Gieson, microtome sections of the liver with a thickness of 5–7 μm were stained with hematoxylin-eosin.
      • Ypsilantis P.
      • Lambropoulou M.
      • Tentes I.
      • et al.
      Impaired liver regeneration following partial hepatectomy using the Pringle maneuver: protective effect of mesna.
      Pieces of the small intestine were resected in its middle third, after which they were fixed in 10% neutral formalin. After fixation in formalin, the tissue of the small intestine was held in alcohols of increasing strength, followed by embedding in paraffin. Microtome sections of the small intestine 4 μm thick were cut and then stained with hematoxylin and eosin.
      • Akdogan R.A.
      • Kalkan Y.
      • Tumkaya L.
      • Alacam H.
      • Erdivanli B.
      • Aydin İ.
      Influence of infliximab pretreatment on ischemia/reperfusion injury in rat intestine.
      Two independent pathologists have performed the histological evaluation. Such an evaluation procedure was carried out immediately after confirmation of death in order to avoid degenerative changes in organs.

      Morphometry

      A Leica DM 2000 binocular light microscope (Leica Microsystems, Wetzlar, Germany) and digital software (Image-Pro plus 6.0; Media Cybernetics) were used to analyze the slides. The quantitative parameters of the histo- and angio-architectonics of the hepatic vessels were studied using an object and an eyepiece micrometer. The morphometry of all vessels caught in the cut was measured with an MOV-1-15 × U4.2. Eyepiece ruler, determining the diameter of the lumen of micro-vessels and the thickness of their middle shell, and the division value of the ocular micrometer was determined using an object micrometer.
      Using a calibrated eyepiece grid, the following parameters were determined: the number of mitoses (BM), the number of bi-nucleated cells (BNC), the number of intact nucleated cells (CK), and the number of grid intersection points (GCP) that did not fall on the sections of hepatocytes and their nuclei. The following indexes were calculated: the parenchymal density index (PP = 1-PST/Nnod) of functional cell mass (FCM = (NK/Sset) × PP × 100,000), which characterizes the parenchymal-stromal relationship per unit volume of tissue; nuclear mass indicator (NM = (CHK + CHDK)/Sset) × PP × 100,000), reflecting the nuclear-cytoplasmic ratio per unit of tissue volume; the mass index of bi-nucleated cells (IMDK = ((NPK/CHK)/Sset) × PP × 100,000), indicating the degree of implementation of the restorative reserves of a unit volume of hepatic tissue; mass mitotic index (MMI = ((FM/CHK)/Sset) × PP × 100,000), showing the proliferative activity of a unit volume of hepatic tissue; an indicator of the functional karyo-cellular index (FCKI = NM/FCM), which characterizes the amount of nuclear material in a cell per unit volume of hepatic tissue; indicator of the average section area of hepatocytes (SPSG = (Sset/PC) × PP), proportional to its functional activity.

      Statistical Analysis

      To calculate the sample size of animals, we used the G × Power v. 9.4 (Germany). The rationale for the sample choice and analysis was based on the previously published method.
      • Festing M.F.
      On determining sample size in experiments involving laboratory animals.
      Statistical analysis was performed using SPSS 25.0 software for Windows. Numerical data were presented as mean ± standard deviation (SD). The significance of the differences was determined by ANOVA. The results were considered reliable if the error of the mean did not exceed 5% (р < 0.05).

      Results

      Grimace Scale Assessment

      The indicators of the level of postoperative pain on the rat grimace scale are presented in Figure 2. On day 1 on the grimace scale, the CG group with a score of 0.05 ± 0.07 was statistically significantly different from the other groups (P ≤ 0.001). However, the indicators on the grimace scale in the groups SG (1.67 ± 0.12), IG1 (1.66 ± 0.13), IG2 (1.54 ± 0.23), and IG3 (1.77 ± 0.15) did not have statistically significant differences between themselves (P ≥ 0.05).
      Figure 2
      Figure 2Level of postoperative pain in animals according to the rat grimace scale. CG: Control group; IG: Interventional group; SG: Sham group.
      On day 3, the CG group (0.04 ± 0.06) had statistically significant differences from the other four groups (P ≤ 0.001). Indicators on this scale in the SG group (0.98 ± 0.38) are statistically lower in contrast to the three intervention groups (IGs): IG1 (1.54 ± 0.25), IG2 (1.62 ± 0.18), and IG3 (1.84 ± 0.12) (P ≤ 0.05). On day 3, there were no statistically significant differences in scores according to the grimace scale in the IG1, IG2, and IG3 groups among themselves (P ≥ 0.05).
      On day 7, the points on the grimace scale in the CG group (0.03 ± 0.06) were statistically significantly lower in comparison with the SG group (0.49 ± 0.17) (P ≤ 0.05) and in contrast to the IG1 (1.12 ± 0.42), IG2 (1.69 ± 0.21), and IG3 (1.77 ± 0.13) (P ≤ 0.001). In addition, differences in the grimace scale of the SG group in comparison with the IG1 (P ≤ 0.05), IG2, and IG3 (P ≤ 0.001) groups were considered statistically significant. However, the indices of the IG2 and IG3 groups did not have statistically significant differences between themselves (P ≥ 0.05).
      On the 14th day, the indices of the CG group (0.02 ± 0.04) on the grimace scale in comparison with the SG group (0.27 ± 0.2, P ≤ 0.05) and in contrast to the other three IGs: IG1 (0.74 ± 0.33), IG2 (1.74 ± 0.18), and IG3 (1.83 ± 0.11) were statistically significantly lower (P ≤ 0.001). In addition, the indicators of the SG group with a statistically significant difference equal to P ≤ 0.05 were lower in contrast to the points on the grimace scale in the IG1 group from group 3 and with a reliability equal to P ≤ 0.001 had lower indicators in comparison with the IG2 and IG3 groups. The IG1 group also had lower scores on the grimace scale in contrast to IG2 and IG3 (P ≤ 0.001) groups; however, no statistically significant difference was found among themselves in the parameters of the IG2 and IG3 groups (P ≥ 0.05).
      On day 30, no statistically significant difference was found between the CG (0.02 ± 0.04) and SG (0.23 ± 0.1) groups on the grimace scale (P ≥ 0.05). The indices of the CG and SG groups were statistically significantly lower in comparison with the IG1 group (0.55 ± 0.23) (P ≤ 0.05) and in contrast to the IG2 (1.67 ± 0.23) and IG3 (1.86 ± 0.09) groups with the reliability equal to P ≤ 0.001. There was no statistically significant difference between the indicators of the IG2 and IG3 groups (P ≥ 0.05).

      Body Weight Assessment

      The body weight indices of animals depending on the day of the postoperative period are shown in Figure 3. At the first day after the start of the experiment, the body weight of animals in the CG group was 205.9 ± 8.5 (n-20) and did not significantly differ from the weight of animals in the SG group equal to 202.6 ± 8.4 (n-20) (P ≥ 0.05). The body weight of animals of the CG and SG groups was significantly higher in comparison with the groups IG1 (194.2 ± 3.45) (n-40), IG2 (195.0 ± 3.02) (n-40), and IG3 (195.6 ± 4.36) (n-40), which was regarded as a statistically significant difference. At the indicated time, statistically significant differences were not determined between groups IG1, IG2, and IG3 (P ≥ 0.05).
      Figure 3
      Figure 3Indicators of body weight of animals depending on the day of the postoperative period. CG: Control group; IG: Interventional group; SG: Sham group.
      On day 3, the body weight of the animals of the CG group (204.9 ± 7.86) (n-20) was statistically significantly higher in comparison with the weight of animals from the SG group (195.1 ± 3.3) (n-20) (P ≤ 0.05) and in contrast to the groups IG1 (176.8 ± 1.28) (n-39), IG2 (172.8 ± 1.63) (n-38), and IG3 (172.3 ± 1.43) (n-36) (P ≤ 0.001). Moreover, the body weights of the animals of the SG group with a confidence index equal to P ≤ 0.01 were statistically significantly higher in comparison with the IG1, IG2, and IG3 groups. The body weight of animals from the IG1 group was also statistically significantly higher in comparison with the IG2 and IG3 groups (P ≤ 0.05).
      On day 7, the body weight of animals in the CG group was 202.5 ± 9.29 (n-20), which was statistically significantly higher in comparison with the SG group (194.1 ± 2.74) (n-19) (P ≤ 0.01) and in contrast to the IG1 (177.8 ± 1.45) (n-37), IG2 (172.1 ± 1.48) (n-37), and IG3 (172.5 ± 1.3) (n-26) (P ≤ 0.001) groups. The body weight of animals of the SG group with a statistically significant difference was greater in comparison with the IG1 group (P ≤ 0.05), as well as in contrast to the IG2 and IG3 groups (P ≤ 0.05). The body weight of the animals of the IG2 and IG3 groups was statistically significantly lower in comparison with the body weight of the animals of the IG1 group (P ≤ 0.05); however, no significant differences were found between themselves in the IG2 and IG3 groups in terms of body weight of animals (P ≥ 0.05).
      It should be noted that on the 14th day, the body weight of the animals in the group was higher than SG (207.4 ± 8.4) (n-19) in contrast to the CG group (202.05 ± 8.65) (n-20) (P ≤ 0.05). The body weights of the animals of the CG and SG groups were statistically significantly higher in contrast to the IG1 (184.7 ± 3.16) (n-37), IG2 (172.2 ± 1.37) (n-37), and IG3 (172.6 ± 1.31) (n-16) groups (P ≤ 0.001). The body weight of the animals of the IG1 group was statistically significantly higher than the body weight of the animals from the IG2 and IG3 groups (P ≤ 0.01), but there were no statistically significant differences between the body weight of the animals of the IG2 and IG3 groups (P ≥ 0.05).
      On the 30th day, there were no statistically significant differences in body weight of animals of the CG (204.2 ± 8.81) (n-20) and SG (205.3 ± 8.12) (n-17) groups (P ≥ 0.05). The weights of animals in the CG and SG groups were statistically significantly more in contrast to the IG1 groups (186.3 ± 2.3) (n-30) (P ≤ 0.05), as well as in comparison with the IG2 (172.1 ± 1.62) (n-25) and IG3 (171.1 ± 1.55) (n-8) groups (P ≤ 0.001). In addition, the body weights of the animals of the IG2 and IG3 groups were statistically significantly lower in comparison with the animals of the IG1 group (P ≤ 0.001). Nevertheless, no significant differences were found out between the body weights of the animals of the IG2 and IG3 groups (P ≥ 0.05).

      Analysis of Survival Rate

      The analysis of survival rate was performed using the Kaplan–Meier method (Figure 4). The results indicate that in the CG group the survival rate was 100%, and in the SG group, this indicator was 90% (n = 18). In groups IG1 and IG2, survival rates were 72.5% and 65.0%, respectively. The lowest survival rates were observed in the IG3 group, where this rate was 22.5%.
      Figure 4
      Figure 4Analysis of animal survival rate (Kaplan-Meier scale). CG: Control group; IG: Interventional group; SG: Sham group.
      In the CG group, the median time and mean time to death were 30 days. In the SG group, the mean time to death was 28.6 ± 1.3 days, and in the IG1 group, this indicator was 26.6 ± 1.2 days, respectively. The mean survival time in the IG2 group was 26.6 ± 1.2 days. In the IG3 group, where the median time to death was 8.0 ± 2.37 days, and the mean time to death was 13.7 ± 1.7 days.
      The data on liver masses of animals are presented in Table 1. Animals of groups CG, IG1, IG2, and IG3, depending on the time of death, were divided into three quarters: from 1 to 10, from 11 to 20, and from 21 to 30 days. In the CG group, depending on the duration of the experiment, no significant changes in liver mass were observed (0–1%), since by 21–30 days (57.4 ± 1.6) in comparison with 1–10 days from the beginning of the experiment (58.2 ± 2.4), a slight decrease in liver mass, without a statistically significant difference (P = 0.863). The simulating of occlusion of inferior vena cava in the IG1 group (1–10 days) led to a decrease in liver mass by −21% (0.001) compared to animals that died on days 21–30, which was regarded as a statistically significant difference (P ≤ 0.001). The highest rates of reduction in liver tissue mass by 21–30 days (42.0 ± 3.2) compared with 1–10 days (59.0 ± 1.3) were found in deceased animals of the IG3 group, where the liver mass was reduced in the range from −33% to – 49%, with a statistically significant difference (P ≤ 0.001).
      Table 1Indicators of the Mass of the Liver of Animals.
      GroupsFrom 1 to 10From 11 to 20From 21 to 30P-value
      M±SDM±SDM±SD
      CG58.2 ± 2.458.7 ± 3.057.4 ± 1.60.863
      IG157.6 ± 1.749.5 ± 1.047.7 ± 1.40.001
      IG258.2 ± 2.649.1 ± 3.147.2 ± 3.10.001
      IG359.0 ± 1.345.4 ± 1.242.0 ± 3.20.001
      P-value0.97500.0410.001
      Abbreviation: CG, Control group; IG-1, Intervention group III; IG-2, Intervention group IV; IG-3, Intervention group V.

      Morphometry Analysis

      The morphometric parameters of the liver of animals are presented in Table 2. According to the results obtained on the functional cell mass examination, in the CG group, this parameter was equal to 75.1 ± 1.02. In the IG-1 group, in comparison with the first time period (1–10 days) (70.2 ± 1.1), at the third period (21–30 days) (60.1 ± 1.7), there was a statistically significant decrease in the functional cell mass (P = 0.014). In IG-2 at third period compared with period 1 (70.4 ± 1.3), the functional cell mass decreased to 51.1 ± 5.9 (P = 0.012). At the same time, in the IG-3 group, this indicator for the first period (67.4 ± 2.4) was higher in comparison with the third period (41.4 ± 4.7) (P = 0.01).
      Table 2Morphometric Parameters of Animal Liver.
      IndexControlIG-1PIG-2PIG-3P
      1 period2 period3 period1 period2 period3 period1 period2 period3 period
      Functional cell mass75.1 ± 1.0270.2 ± 1.165.5 ± 1.960.1 ± 1.70.0170.4 ± 1.359.7 ± 2.651.1 ± 5.90.0167.4 ± 2.449.8 ± 5.441.4 ± 4.70.001
      Nuclear mass80.5 ± 2.1977.4 ± 0.974.2 ± 1.570.0 ± 3.40.0477.3 ± 1.772.3 ± 2.365.1 ± 1.10.0366.6 ± 3.851.5 ± 6.045.8 ± 5.10.001
      Mass mitotic index0.020 ± 0.0010.031 ± 0.0040.037 ± 0.0120.049 ± 0.0340.0010.038 ± 0.0020.021 ± 0.0010.016 ± 0.050.0010.047 ± 0.0110.011 ± 0.0010.007 ± 0.0070.001
      Parenchymal density index0.6 ± 0.020.50 ± 0.010.54 ± 0.120.58 ± 0.780.140.50 ± 0.010.43 ± 0.90.38 ± 1.10.0010.47 ± 3.10.34 ± 2.50.29 ± 4.80.001
      Functional cariocellular index1.01 ± 0.031.45 ± 0.081.58 ± 0.871.27 ± 0.320.041.29 ± 0.750.87 ± 2.40.61 ± 0.540.0011.24 ± 0.90.55 ± 1.40.37 ± 4.50.001
      Abbreviation: IG-1, Intervention group III; IG-2, Intervention group IV; IG-3, Intervention group V. Bold values denote statistical significance at the p ≤ 0.05 level.
      The nuclear mass in the CG group was 80.5 ± 2.19, and in the IG-1 group at the third period (70.0 ± 3.4), this indicator was statistically significantly lower in comparison with the first period (77.4 ± 0.9) (P = 0.042). In addition, in comparison with the first period at the third period in the IG-2 (65.1 ± 1.1) and IG-3 (45.8 ± 5.1) groups, there was a decrease in nuclear masses with statistically significant differences equal to P = 0.038 and P = 0.001, respectively.
      The mass mitotic index in the CG group was 0.02 ± 0.001. In the IG-1 group at the third period (0.049 ± 0.034) in comparison with the first period (0.031 ± 0.004), there was a statistically significant decrease in this indicator (P = 0.01). Also, in the third period in the IG-2 (0.016 ± 0.05) and IG-3 (0.007 ± 0.007) groups, in contrast to the first period, there was a statistically significant increase in the mass mitotic index with equal reliability P = 0.001 in both groups, respectively.
      The parenchymal density index in the CG group was 0.6 ± 0.02. In the IG-1 group, in contrast to period 1 (0.5 ± 0.01), in period 3 (0.58 ± 0.78), an increase in this parameter was noted, but without a statistically significant difference (P = 0.14). However, in groups IG-2 and IG-3 at period 3, a decrease in parenchymal density to 0.38 ± 1.1 and 0.29 ± 4.8 was determined, with a significance equal to P = 0.001 in both groups, respectively.
      In the CG group, the functional karyo-cellular index was 1.01 ± 0.03. In the IG-1 group, in the second period in comparison with the first period, there is an increase in this indicator from 1.45 ± 0.08 to 1.58 ± 0.87, followed by its decrease to 1.27 ± 0.32 in the third period (P = 0.045). In the IG-2 and IG-3 groups, in comparison with the first period in the third period, the indicators of the functional karyo-cellular index were reduced to 0.61 ± 0.54 and 0.37 ± 4.5, respectively (P = 0.001).

      Macroscopic Evaluation

      In the SG group of animals, the macroscopic picture of the abdominal organs was determined as normal. The liver was light, bright, and wine-colored, without signs of necrosis or any pathological changes. Enlargement of the spleen, as well as the presence of ascetic effusion, was not observed.
      In the group of animals IG-1 (first day after the experiment), edema and hyperemia of the tissues of the abdominal wall organs were detected. We observed the fullness of the liver and spleen tissue persisted on the third day of the postoperative period. On the seventh day, the surface of the liver and spleen was dark, and congestion was more pronounced in the peripheral areas. Moderate hepato- and splenomegaly were determined. On the 14th day, there was a small amount of serous fluid in the abdominal cavity, and a decrease in liver mass was visually noted. After 30 days, the liver was full-blooded, with a decrease in the volume of serous fluid.
      The macroscopic picture of the abdominal cavity organs of the IG-2 group of animals on the first day after modeling the narrowing of the lumen of the inferior vena cava by 50% also differed in plethora. The liver was dark wine color. These changes persisted for subsequent periods of observation (at 3.7 days). Both the liver and spleen were enlarged. On the 14th and 30th days from the beginning of the experiment, the size of the liver and spleen was visually reduced (both were dark).
      In the group of animals IG-1, one day after the experiment, the tissues of the abdominal cavity organs were significantly full-blooded. On the third day, an increase in the liver and size of the spleen was visually noted, and the liver was maroon in color. On the seventh day, the impression of liver tuberosity was noted, and the liver looked overloaded and heterogeneous. On days 14 and 30 from the start of the experiment, the liver was also full-blooded. On the 30th day, macroscopically on the anterior abdominal wall, there was an expansion and tortuosity of the saphenous veins.

      Histological Evaluation

      Histological picture of the liver in experimental rats in studied groups was presented in Figure 5. In the CG (Figure 5, A, B, C, D) and in the SG group (Figure 5, E, F, G, H), the histological characteristics of the liver corresponded to the normal state (for all period of observation). In general histological structure, the beam structure of the liver was preserved, and binuclear hepatocytes were determined. In space of Disse, terminal portal veins were not dilated.
      Figure 5
      Figure 5Histological picture of liver in experimental rats in CG and SG groups, after occlusion of inferior vena cava by 25% (IG-1), 50% (IG-2), and 75% (IG-3), depending on the observation time (A, E, I, M, Q - after 3 days; B, F, J, N, R - after 7 days; C, G, K, O, S - after 14 days; D, H, L, P, T -after 30 days). Hematoxylin-eosin. Magnification ×100. CG: Control group; IG: Interventional group; SG: Sham group.
      In group IG-1 (third day from the start), with a narrowing of the lumen of the inferior vena cava by 25%, there is an expansion of the lumen of the central veins, with blood filling. However, the periphery of the lobules retains a normal character. The portal vein showed the signs of a plethora (Figure 5, I). After seven days, the blood repletion of the hepatic vessels was preserved, and the expansion of the space of Disse was observed. The phenomena of parenchymal dystrophy were also determined in the center of the lobules (Figure 5, J). After 14 days, the central zones and adjacent intralobar capillaries became more dilated and congested with blood, which led to compression of hepatocytes (Figure 5, K). After 30 days, due to the increase in stagnant processes, the phenomena of parenchymal dystrophy and hepatocytes continued to increase (Figure 5, L).
      In the IG-2 group (day 3), with 50% occlusion of the lumen of the inferior vena cava in the experiment, congestion was observed only in the central parts of the hepatic lobules (Figure 5, M). On the seventh day after the experiment, the expansion of the sinusoids and the phenomenon of parenchymal dystrophy in the center of the lobules also persisted and progressed (Figure 5, N). On the 14th day, the terminal venules of the portal system were dilated along with the vessels of the portal tract (Figure 5, O). After 30 days, the proliferation and thickening of the walls were noted around the central and collecting veins due to the proliferation of adventitious fibroblasts (Figure 5, P).
      At the same time, in the IG-3 group (day 3), with a narrowing of the lumen of the inferior vena cava by 75%, we detected a pronounced plethora of hepatic vessels not only in the central zones but also in the peripheral parts of the lobules and sinusoids (Figure 5, Q). On the seventh day, there was a continuing trend toward the growth of foci of hemorrhage with signs of atrophy and death of hepatocytes (Figure 5, R). On the 14th day, over-distended and fibrously altered central veins were observed, and the signs of focal lymphostasis were noted (Figure 5, S). After 30 days, there was also a thickening of the walls of the central veins, a pronounced expansion of the terminal venules, with a violation of the histological structure due to the discomplexation of hepatocytes (Figure 5, T).

      Discussion

      To the best of our knowledge, this is the first study to determine morphological changes in tissue, hepatic vessels of the liver, as well as the survival rate of animals with the occlusion of inferior vena cava. In our experiment, we surgically narrowed the lumen of the inferior vena cava by 25%, 50%, and 75% of its normal diameter, respectively. Using microsurgical techniques, all three experimental models proved to be feasible and reproducible.
      The weight of the animals of the three IGs (compared to the CG and SG groups) on the first day after the operation was lower.
      Such a difference can be explained by the time spent for surgical manipulation to establish the constricting ring (IG-1, IG-2, and IG-3 groups). The prolonged time led to an increase in blood loss (compared to the SG group). This circumstance conforms with the results of previous studies, where, after one day after the operation, a comparatively maximum loss of body weight of animals was observed.
      • Dahmen U.
      • Madrahimov N.
      • Madrahimova F.
      • Ji Y.
      • Schenk A.
      • Dirsch O.
      Small-for-size syndrome in the rat: does size or technique matter?.
      According to the available data, weight recovery to preoperative levels often occurs between 25 and 28 days.
      • Higgins G.M.
      Experimental pathology of the liver. Restoration of the liver of the white rat following partial surgical removal.
      It is worth noting that on the 14th day, the body weight of the animals of the SG group was significantly higher than the body weight of the animals of the CG group. This finding could be attributed to the thoracotomy in animals of the SG group in the postoperative period. It led to an increase in the processes of postoperative regeneration, and consequently, to the growth of the volume of food consumption.
      The development of portal hypertension is a common endpoint in chronic liver disease and is a significant contributor to morbidity and mortality.
      • Hilscher M.B.
      • Sehrawat T.
      • Arab J.P.
      • et al.
      Mechanical stretch increases expression of CXCL1 in liver sinusoidal endothelial cells to recruit neutrophils, generate sinusoidal microthombi, and promote portal hypertension.
      ,
      • Micev M.
      • Basaric D.
      • Micev M.
      • Galun D.
      Histopathology of hepatic sinusoidal obstruction syndrome after neoadjuvant oxaliplatin-based chemotherapy.
      The results obtained indicate that, with the narrowing of the inferior vena cava lumen by 25% and 50%, the survival rates varied from 72.5% to 65.0%. At the same time, when the lumen of the inferior vena cava narrowed by 75%, the survival rate of animals was 22.5%, where the mean time to death was 13.7 ± 1.7 days. It indicates an increase in the chances of death as the result of the functioning of the lumen of the inferior vena cava by only 1/3.
      de Aguiar et al. conducted a study on the assessment of the effect of portal hypertension using the modeling of the occlusion of hepatic venous drainage by 25%, 50%, 75%, and 100% on the regeneration of rat liver remnants after partial hepatectomy.
      • de Aguiar L.R.F.
      • Pan Nassif
      • Ribas C.A.P.M.
      • et al.
      Liver regeneration after partial hepatectomy in rats submitted to post – hepatic portal hypertension.
      It was found out that at the occlusion of the inferior vena cava and right hepatic veins (up to 25% of diameter), two animals died between 96 and 144 h, and three animals died with 50% occlusion between 96 and 168 h. The death of two animals was recorded after 75% occlusion between 96 and 120 h, and eight animals died at 100% occlusion in the first 48 h.
      • de Aguiar L.R.F.
      • Pan Nassif
      • Ribas C.A.P.M.
      • et al.
      Liver regeneration after partial hepatectomy in rats submitted to post – hepatic portal hypertension.
      In addition, simulated stenosis of inferior vena cava (in the right hepatic vein and inferior vena cava) provoked the development of portal hypertension, including the congestion and vascular ectasia ten days later.
      • de Aguiar L.R.F.
      • Pan Nassif
      • Ribas C.A.P.M.
      • et al.
      Liver regeneration after partial hepatectomy in rats submitted to post – hepatic portal hypertension.
      The authors noted that the partial obstruction of hepatic venous drainage caused intrahepatic venous congestion, even after the restoration of liver volume.
      It has been shown that the blockage of the hepatic veins disrupts portal circulation, increasing sinusoidal resistance.
      • Erden A.
      • Erden I.
      • Yurdaydin C.
      • Karayalçin S.
      Hepatic outflow obstruction: enhancement patterns of the liver on MR angiography.
      For the chronic impairment of venous outflow, passive venous congestion leads to expansion and blockage of sinusoidal vessels, damage to the endothelium, atrophy of hepatocytes, and deposition of fibrosis.
      • Brancatelli G.
      • Furlan A.
      • Calandra A.
      • Dioguardi Burgio M.
      Hepatic sinusoidal dilatation.
      Akiyoshi and Terada reported that when the inferior vena cava narrowed by ¼ its size in rats one week after ligation, slight fibrosis was observed in the corresponding central lobular necrotic areas. After six weeks, those changes spread to the mid-spherical zone of the central vein, where incomplete fibrous septa appeared.
      • Akiyoshi H.
      • Terada T.
      Centrilobular and perisinusoidal fibrosis in experimental congestive liver in the rat.
      Blockage of the hepatic vein disrupts portal circulation, increasing sinusoidal resistance, and thereby, contributes to the development of lobar or segmental hypertrophy, as a result of the compensation for atrophy or stagnation of sinusoidal flow due to sluggish venous outflow through collateral vessels.
      • Van Beers B.
      • Pringot J.
      • Trigaux J.P.
      • Dautrebande J.
      • Mathurin P.
      Hepatic heterogeneity on CT in Budd-Chiari syndrome: correlation with regional disturbances in portal flow.
      Apart from that, it is known that after modeling hepatic venous outflow disturbances in the remnant of the resected liver leads to significant liver dysfunction, and, ultimately, to a significant decrease in survival rate.
      • Bockhorn M.
      • Benkö T.
      • Opitz B.
      • et al.
      Impact of hepatic vein deprivation on liver regeneration and function after major hepatectomy.
      As a matter of fact, hepatic encephalopathy is a common complication of portal hypertension and liver cirrhosis affecting the mental status and central nervous system.
      • Jawaro T.
      • Yang A.
      • Dixit D.
      • Bridgeman M.B.
      Management of hepatic encephalopathy: a primer.
      It has been shown that the oxidative stress and cellular hypoxia play an important role in the tissue damage as a result of the violation of liver hemodynamic (outflow and inflow).
      • Polat E.
      • Topaloglu S.
      • Sokmensuer C.
      • et al.
      Heterogeneity of damage between segments of rat liver after inflow-outflow obstruction.
      The oxidative stress has been considered as the cause of morphological changes in the liver tissue.
      • Pinaeva O.G.
      • Lebed'ko O.A.
      • Pinaev S.K.
      • Sazonova E.N.
      Hepatoprotective effect of neonatal administration of non-opioid Leu-Enkephalin analogue in adult albino rats subjected to antenatal hypoxia.
      In our study, we observed a significant decrease in functional cell mass in the IGs in the period of 21–30 days (P ≤ 0.05). This fact may indicate the development of plethora and blood stagnation in the liver tissue.
      A reduced number of hepatocytes with progressive fibrosis is associated with liver cirrhosis.
      • Ryoo J.W.
      • Buschmann R.J.
      A morphometric analysis of the hypertrophy of experimental liver cirrhosis.
      However, it should be noted that liver atrophy or hypertrophy (as a result of cirrhosis) affects the hepatocytes and the metabolic capacity of the liver.
      • Matsui Y.
      • Okuda Y.
      • Nakagawa M.
      • et al.
      Effect of hepatocyte volume on energy status in the cirrhotic rat liver.
      We observed an increase in nuclear mass was observed, which indicates the development of degenerative and destructive processes in the liver tissue. It has been shown that hepatic outflow obstruction or Budd-Chiari syndrome is a vascular problem that mimics liver cirrhosis.
      • Erden A.
      • Erden I.
      • Yurdaydin C.
      • Karayalçin S.
      Hepatic outflow obstruction: enhancement patterns of the liver on MR angiography.
      Lebedeva et al. conducted the study using a model of cirrhosis in rats and data of patients with alcoholic cirrhosis.
      • Lebedeva E.I.
      Morphometric indicators of white rat and human hepatocytes in toxic cirrhosis of the liver.
      Twelve weeks after the beginning of the experiment, there was an increase in the average area of hepatocytes and nuclear hypertrophy. In patients with alcoholic cirrhosis, the ratio between the area of the nucleus and the cytoplasm shifted toward a significant decrease in the part of the cytoplasm. These findings were explained by fact that the regeneration of liver hepatocytes occurred due to hyperplasia and hypertrophy of organelles.
      • Lebedeva E.I.
      Morphometric indicators of white rat and human hepatocytes in toxic cirrhosis of the liver.
      According to the results obtained, in the IGs, there was an increase in the mass mitotic index up for period 2 (P = 0.001), and a sharp decline in period 3 in all experimental groups (P = 0.001). An increase in the mass mitotic index reflects a rise in the current (relatively short-term) intensity of functioning and a decrease in the depth of reparative potential. Interestingly, in the first group, the liver was able to adjust, and the increase in the index masses continues, while in the IG-2 and IG-3 groups, there are high sharp indicators and a sharp drop (P = 0.001). This fact may indicate the zeroing of the reparative reserves of the IG-3 group.
      In terms of parenchymal density indicators, in all studied groups, there was a decrease in this indicator. Nonetheless, only in the IG-1 group for the period 3 was observed an increase in parenchymal density up to 0.58 ± 0.78 (P = 0.14) without statistical significance. At the same time, in other groups IG-2 and IG-3, a decrease in this indicator was recorded (P = 0.001). A fall in the parenchymal density index indicates a decrease in the functionally active parenchyma. This process can be explained by increased blood and lymph filling of the liver. On the other hand, it can be caused by growing degenerative and necrotic processes, leading to a real and reliable decrease in the number of fully functioning hepatocytes per unit volume of hepatic tissue.
      In the IG-1 group, during the second period (compared to the first one), there was an increase in karyo-cellular index up to 1.58 ± 0.87. However, in the third period, it was changed by the decrease down to 1.27 ± 0.32 (P = 0.0452) indicating some preservation of the compensatory mechanisms of the liver. At the same time, in groups IG-2 and IG-3, in comparison with the first period, a sharp decrease in the functional karyo-cellular index (P = 0.001) was detected for period 3. This finding indicates a depletion of the reparative potential of the liver tissue. In fact, the indicator of the functional karyo-cellular index reflects the average amount of nuclear material per cell per unit volume of liver tissue. So that this indicator shows the activity of reparative processes in liver tissue.
      To summarize, our data indicate that the compression of the inferior vena cava by 75% led to an increase in animal mortality and the development of persistent morphological changes in the liver. At the same time, the survival rate of animals and the extent of changes in the liver with narrowing of the inferior vena cava by 25% and 50% had similar results. The results acquired possess scientific and practical importance in the context of the management of portal hypertension after the occlusion of inferior vena cava. However, the clinical relevance and efficacy of the proposed model require further intensive research.

      Credit authorship contribution statement

      Shynar Tanabayeva: conceptualization, methodology, investigation, analysis and interpretation, critical revision, writing - original draft. Ydyrys Almabayev: conceptualization, methodology, investigation, analysis and interpretation, critical revision. Marat Kamyspaev: methodology, analysis, and interpretation, writing - original draft. Ruslan Kulmanbetov: validation, analysis, writing - original draft. Maira Kopbayeva: writing - original draft, analysis, and interpretation. Nurgulim Akhmad: visualization, data curation, writing - review & editing. Gulnara Altynbekova: translation, text editing, methodology, and interpretation. Ildar Fakhradiyev: writing - review & editing, analysis, interpretation, critical revision.

      Conflicts of interest

      The authors have none to declare.

      Acknowledgements

      The authors express their gratitude for the administrative and technical support provided by the S.D. Asfendiyarov Kazakh National Medical University.

      Funding

      This research received no external funding. The work was carried out within the framework of the Ph.D. dissertation.

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