[Establishment and applicability comparison of four models of acute liver ischemia/reperfusion injury in rat].

Abstract

To clarify the preparation methods of four rat models of liver ischemia/reperfusion injury (IRI) and to determine a liver IRI animal model that is consistent with clinical conditions, has stable pathological and physiological injury, and is easy to operate. A total of 160 male Sprague-Dawley (SD) rats were randomly divided into four groups using an interval grouping method: 70% IRI (group A), 100% IRI (group B), 70% IRI with 30% hepatectomy (group C), and 100% IRI with 30% hepatectomy (group D), with 40 rats in each group. Each model was further divided into sham operation group (S group) and ischemia groups of 30, 60, and 90 minutes, with 10 rats in each group. After surgery, the survival status and awakening time of the rats were observed, and the liver lobectomy weight, bleeding volume, and hemostasis time of groups C and D were recorded. Blood samples were collected by cardiac puncture after 6 hours of reperfusion for determination the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), blood urea nitrogen (BUN), serum creatinine (SCr), and γ-glutamyl transpeptidase (γ-GT) in the serum to assess liver and kidney function. Hematoxylin-eosin (HE) staining and immunohistochemical staining of macrophages were performed to analyze the liver tissue structure damage from a pathological perspective. Rats in group A exhibited earlier awakening and acceptable mental status, while rats in the other groups showed delayed awakening and poor mental status. The hemostasis time in group D was approximately 1 second longer than that in group C. The mortality of rats subjected to 60 minutes of 70% hepatic ischemia was 0. Compared to the sham operation group, rats in each experimental group showed significant increases in serum levels of AST, ALT, ALP, BUN, SCr, and γ-GT, indicating impaired liver and kidney function in the rat models of liver IRI. In groups A, B, and C, the 90-minute ischemia subgroup exhibited more pronounced elevation in AST, ALT, ALP, BUN, SCr, and γ-GT levels compared to the 30-minute ischemia subgroup [AST (U/L): group A, 834.94±56.73 vs. 258.74±18.33; group B, 547.63±217.40 vs. 277.67±57.92; group C, 930.38±75.48 vs. 640.51±194.20; ALT (U/L): group A, 346.78±25.47 vs. 156.58±13.25; group B, 408.40±138.25 vs. 196.80±58.60; group C, 596.41±193.32 vs. 173.76±72.43; ALP (U/L): group A, 431.21±34.30 vs. 315.95±15.64; group B, 525.88±62.13 vs. 215.63±17.31; group C, 487.53±112.37 vs. 272.46±92.33; BUN (U/L): group A, 18.35±5.63 vs. 14.32±2.30; group B, 30.21±4.55 vs. 17.41±8.14; group C, 20.50±3.64 vs. 15.93±3.22; SCr (U/L): group A, 27.47±8.91 vs. 22.37±5.66; group B, 43.60±15.57 vs. 36.80±7.95; group C, 63.81±20.24 vs. 42.47±7.03; γ-GT (U/L): group A, 15.64±3.57 vs. 6.82±1.48; group B, 9.28±1.91 vs. 5.62±1.21; group C, 10.98±3.18 vs. 5.67±1.10; all P < 0.05]. The 100% IRI 90-minute group and 100% IRI 90-minute group with 30% hepatectomy exhibited more pronounced increases in the above-mentioned indicators compared to the corresponding 70% IRI control group, indicating increased liver and kidney damage in rats subjected to combined blood flow occlusion and hepatectomy. HE staining showed clear liver tissue structure with intact and orderly arranged cells in the sham operation group, while the experimental groups exhibited cell structure damage, including cell rupture or collapse, cell swelling, nuclear pyknosis, deep cytoplasm staining, cell shedding, and necrosis. The interstitium showed infiltration of inflammatory cells. Immunohistochemical staining revealed a higher number of macrophages in the experimental groups compared to the sham operation group. Four models of liver IRI in rat were successfully established. As the duration and severity of hepatic ischemia increased, liver cell ischemia worsened, leading to increased hepatocellular necrosis and exhibiting characteristic features of liver IRI. These models can effectively simulate liver IRI following liver trauma, with the group subjected to 100% ischemia and 30% hepatectomy showing the most severe liver injury. The designed models are reasonable, easy to perform, and exhibit good reproducibility. They can be used for investigating the mechanisms, therapeutic efficacy, and diagnostic methods related to clinical liver IRI.