1. Introduction

Acute liver failure is a severe consequence of abrupt hepatocyte injury from diverse causes. It has a rapid onset that can evolve rapidly within days or weeks to a lethal outcome. The most prevalent causes of acute liver failure are ischemia, drug-induced liver injury, hepatitis B virus, and autoimmunity [1–3]. The pathology of acute liver failure often shows widespread hepatic necrosis and apoptosis. The clinical manifestations include progressive jaundice, hepatic encephalopathy, and disturbed coagulation. Acute liver failure has a mortality rate as high as 80% [1]. The most common causes of death in patients with acute liver failure are cerebral edema, sepsis, and multisystem organ failure [4–6]. The prognosis of acute liver failure is poor. Therefore, it is of paramount importance to evaluate liver function and commence management promptly. In clinical practice, liver function is usually evaluated using methods such as liver biochemical tests, Child–Pugh classification, and indocyanine green clearance tests. Although these methods have value in a certain aspect of liver function assessment, their clinical development and effect are limited due to the narrow scope of assessment, cumbersome determination methods, the need to purchase special instruments or equipment, and the existence of liver toxicity of drugs. Given the limited assessment scope regarding acute liver failure and tedious measurement protocols, there is a pressing need to develop a more direct, comprehensive, and precise method, such as an imaging method, to assess hepatic function. In recent years, hepatobiliary scintigraphy has been increasingly recognized as an effective method for assessing hepatic function [7–9]. In recent years, various researchers have utilized imaging agents such as ¹¹¹In-octreotide, ^99mTc-sestamibi, and ^99mTc-mebrofenin to evaluate liver function through SPECT. Although some progress has been made, the scope of evaluation remains limited, mostly focusing on surgical cases, healthy populations, or animals. Further research is needed to explore its broader clinical applications [10–12].

Given that previous studies have often focused on a single liver injury model [7,8], we intended to explore more causes of liver injury, so we established separate experimental models for the D-GalN + LPS, partial hepatectomy, and common bile duct ligation. As research on dynamic hepatobiliary scintigraphy grows [7,8], its application in assessing liver function gains increasing attention. However, its precise value in evaluating liver function and its correlation with changes in liver function remain unclear. Furthermore, systematic animal studies are required to validate the efficacy of dynamic hepatobiliary scintigraphy in measuring liver function with visual interpretation of the images and several quantitative functional parameters, such as 5-minute heart/liver index (HLI5) and 15-minute receptor index (LHL15). These nuclear medicine quantitative evaluation indicators have a good correlation with Child–Turcotte criteria score to evaluate the degree of liver function injury [13,14].

Previous studies have shown that proinflammatory cytokines such as interlukin-6 (IL-6), TNF-α, and CXCL10 are responsible for certain pathological manifestations of hepatocyte damage [15–17]. In the present study, we induced acute liver failure using methods corresponding to the common different clinical causes of liver damage, conducted dynamic hepatobiliary single-photon emission computed tomography (SPECT) imaging, and measured the levels of inflammatory cytokines, aiming to elucidate the liver damage mechanisms and provide clinical guidance for diagnosis and treatment. Ultimately, we aimed to establish a comprehensive method including quantitative imaging tools and related cytokines to assess liver function by studying different rat models of liver injury, and thereby reduce the incidence of liver failure and improve the prognosis in patients with acute liver damage.

2. Methods

3. Results

3.1 Dynamic hepatobiliary SPECT

3.1.1 Visual inspection of SPECT images.

Compared with the control group, all of the experimental groups exhibited a decrease in both the accumulation of 99mTc-EHIDA within the liver and the clearance of 99mTc-EHIDA from the liver. These findings indicated impaired liver function in the experimental groups (Fig.1).

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Fig. 1. SPECT/CT images of the control group

(A), D-GalN + LPS group (B), partial hepatectomy group (C), and common bile duct ligation group (D).

https://doi.org/10.1371/journal.pone.0323531.g001

3.1.2 Time–activity curve.

In contrast to the control group, the liver time–activity curves in the experimental groups displayed a lower peak activity of 99mTc-EHIDA, a prolonged time to peak, and a slower clearance. These findings demonstrated gradually impaired liver uptake and excretory functions (Fig.2).

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Fig. 2. Time–activity curves from dynamic SPECT/CT.

A. Control group; B. D-GalN + LPS group; C. Partial hepatectomy group; D. Common bile duct ligation group. The red lines represent the count changes of liver ROI, and green lines represent the count changes of heart ROI.

https://doi.org/10.1371/journal.pone.0323531.g002

3.1.3 SPECT image–derived indexes.

The values of HLI5 for all animals determined by XL and LQH were 0.16 ± 0.09 and 0.17 ± 0.09, respectively (ICC = 0.911, P < 0.05). The values of LHL15 determined by XL and LQH were 0.83 ± 0.08 and 0.83 ± 0.08, respectively (ICC = 0.957, P < 0.05). The agreement of measurements of HLI5 and LHL15 was excellent.

Compared with the control group, with the mean value of 0.06 ± 0.01, all of the experimental groups showed significantly increased HLI5, with the mean values of 0.29 ± 0.05, 0.15 ± 0.07, and 0.18 ± 0.05 for the D-GalN + LPS group (P < 0.05), partial hepatectomy group (P < 0.05), and common bile duct ligation group (P < 0.05), respectively. Compared with the control group, with the mean value of 0.93 ± 0.03, all of the experimental groups showed significantly decreased LHL15, with the mean values of 0.76 ± 0.03, 0.82 ± 0.05, and 0.79 ± 0.01 for the D-GalN + LPS group (P < 0.05), partial hepatectomy group (P < 0.05), and common bile duct ligation group (P < 0.05), respectively (Fig.3A,S1 Table). Additionally, the reference values for the HLI5 and LHL15 parameters in this study were 0.06 ± 0.01 and 0.93 ± 0.03, respectively. We used post-hoc multiple-comparison tests for the HLI5 and LHL15 parameters across the three experimental groups after ANOVA. There was no significant difference observed in HLI5 between the partial hepatectomy group and the common bile duct ligation group (P = 0.7889), while significant differences were found among the other experimental groups (P < 0.05). Furthermore, there was no significant difference in the LHL15 level among the various experimental groups. The HLI5 in the D-GalN + LPS group was the highest, whereas the LHL15 in the partial hepatectomy group was the highest.

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Fig. 3. SPECT image–derived indexes and liver histopathology.

A. The changes in HLI5 and LHL15; B. Hematoxylin–eosin staining showing the liver pathology in the control group and the experimental groups (200×); C. The liver histopathological scores; D. The correlation analysis between the histopathological scores and the SPECT image–derived indexes.

https://doi.org/10.1371/journal.pone.0323531.g003

3.4 Measurement of AST and ALT levels

We analyzed the levels of ALT and AST in serum of each group of rats. Compared with the control group, the experimental groups exhibited decreased levels of ALT, with the mean values of 58.58 ± 6.78, 39.57 ± 4.59, 50.13 ± 4.17, and 28.47 ± 2.09 (U/gprot) for the control group, the D-GalN + LPS group, the partial hepatectomy group, and the common bile duct ligation group, respectively (P < 0.05, for all experimental groups). Compared with the control group, the experimental groups also exhibited decreased levels of AST, with the mean values of 178.77 ± 21.61, 118.59 ± 14.61, 148.69 ± 12.92, and 85.76 ± 13.75 (U/gprot) for the control group, the D-GalN + LPS group, the partial hepatectomy group, and the common bile duct ligation group, respectively (P < 0.05, for all experimental groups) (Fig.4A,S3 Table). Except for the partial hepatectomy group and the common bile duct ligation group, there were no differences in the AST level among the experimental groups (P = 0.3423). However, ALT and AST levels differed among the experimental groups (P < 0.05). The ALT and AST levels in the D-GalN + LPS group were the highest.

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Fig. 4. Measurements of liver biochemical parameters.

A. AST and ALT levels in liver tissue; B. serum IL-6 level; C. TNF-α and CXCL-10 expression levels in liver tissue.

https://doi.org/10.1371/journal.pone.0323531.g004

4. Discussion

Acute liver failure is a rare condition with diverse etiologies. It is characterized by extensive hepatocyte necrosis, severe liver dysfunction, and rapid disease deterioration, and often results in high morbidity and mortality [1,25]. Typical clinical workup relies on serum biochemical tests and Child–Pugh classification for early diagnosis and liver function assessment. However, these commonly used methods assess total liver function indirectly and may not always provide pertinent insights, particularly when assessment of regional liver function is crucial [26].

Therefore, there is a need to explore radiological approaches, with nuclear medicine imaging emerging as a promising avenue for directly assessing liver function. SPECT is a commonly used nuclear medicine imaging modality for liver function assessment [27,28]. SPECT has been used widely in clinical practice because of its various imaging agent options, such as 99mTc-GSA, 99mTc-PMT, 99mTc-disofenin, 99mTc-HBS, and 99mTc-EHID [29–31]. 99mTc-diethyl iminodiacetic acid (99mTc-EHIDA) is a widely used SPECT imaging agent in clinical setting. After intravenous injection, 99mTc-EHIDA binds to serum albumin and is then transported to the liver. In the liver, 99mTc-EHIDA dissociates from albumin and is taken up by hepatocytes in the perisinusoidal space. Subsequently, it is secreted into bile through a metabolism process similar to that of bilirubin and finally excreted into the intestine through the biliary system. Using a gamma camera, the dynamic process of 99mTc-EHIDA being taken up by liver cells, secreted into bile ducts, and entering the intestine can be monitored. Due to its rapid blood clearance, high liver uptake, rapid biliary excretion, and minimal interference from bilirubin, it is most widely used clinically for the diagnosis of various liver and biliary system diseases such as persistent jaundice in infants, acute and chronic cholecystitis, postoperative evaluation of the biliary system, post–liver transplantation monitoring, and bile reflux [32–34]. Gamma rays used to form SPECT images may be absorbed and scattered by the patients’ tissue, potentially affecting image quality.

However, SPECT imaging using a more advanced imaging system owned by the unit enables attenuation and scatter correction, which significantly enhances SPECT image quality and spatial resolution. Moreover, a new-style SPECT allows direct and more reliable quantitative assessment of radioactivity uptake in the liver. Even in obese patients or patients with ascites, the quantification of the radioactivity in the liver is not affected by inflammation and congestion [35]. To explore the utility of 99mTc-EHIDA hepatobiliary dynamic SPECT imaging in assessing liver function, we established animal models of acute liver failure corresponding to common clinical causes of liver failure, including drug-induced liver injury, post-hepatectomy, and obstructive biliary injury. Compared with the control group, significantly reduced liver radioactivity uptake was prominently visible in all of the experimental groups on corresponding SPECT images, directly indicating impaired liver function. Our findings are consistent with literature reports [36]. HLI5 in each experimental group showed varying degrees of increase, while LHL15 showed varying degrees of decrease. Histopathological examination of liver sections revealed varying degrees of hepatocyte swelling, necrosis, congestion, and vacuolar degeneration. The histopathological scores of the experimental groups were significantly higher than that of the control group. Furthermore, both SPECT image–derived indexes, HLI5 and LHL15, demonstrated strong correlations with histopathological findings.

ALT and AST are important indicators of liver damage, and the severity of liver damage can be assessed by measuring ALT and AST levels in liver tissue. Our results confirmed that liver damage induced by varying causes led to significantly decreased ALT and AST levels in the liver tissue. During acute liver injury, many cells in the liver die or become very damaged in a short amount of time. Kupffer cells, hepatic sinusoidal endothelial cells, and other cells release various cytokines and biologically active mediators, such as TNF-α, IL-6, and TGF. These mediators can cause further damage to the liver cells through various pathways. Among these cytokines, IL-6 and TNF-α are considered key factors that can increase liver toxicity [37,38]. IL-6 is an inflammatory cytokine that participates in many inflammatory processes in the body. Elevated serum IL-6 often indicates the presence of inflammatory responses [39,40]. Our results showed that the serum IL-6 levels were significantly higher in all experimental groups compared with the control group. IL-6 may play several roles in the pathological process. First, IL-6 activates hepatic endothelial cells and Kupffer cells to release other cytokines, resulting in indirect tissue and cell damage. Second, IL-6 activates neutrophils to produce reactive oxygen species, enhancing the adhesion of neutrophils to endothelium and promoting inflammatory reactions [41], eventually leading to hepatitis, fibrosis, and cirrhosis. Moreover, IL-6 mediates inflammatory immune responses and chronic inflammation, activates JAK tyrosine kinase, and plays a critical role in liver damage [42,43]. In our acute liver failure animal models, TNF-α level in liver tissue increased, suggesting that TNF-α may partake in liver damage. TNF-α is a proinflammatory cytokine secreted by activated monocytes/macrophages. TNF-α enhances the phagocytic function of neutrophils and promotes the differentiation of the inflammatory cells. The rapid increase in blood TNF-α levels causes acute liver injury. TNF-α directly induces cytotoxic effects on liver sinusoidal endothelial cells, resulting in endothelial cell swelling, vessel constriction, and microcirculation dysfunction [44,45]. Additionally, TNF-α impairs mitochondrial function, triggers hepatocyte apoptosis and necrosis, and further exacerbates liver tissue damage [44]. TNF-α and IL-6 synergistically induce the expression of nitric oxide synthase and promote peroxynitrite production, subsequently activating hepatocytes apoptosis pathways and perpetuating a vicious cycle [46]. CXCL10 interacts with its corresponding chemokine receptor, coordinating and mediating the entry of immune cells into damaged or diseased organs [47]. It triggers conformational changes in leukocyte integrins through cascading reactions, facilitating their interaction with adhesion molecules expressed on endothelial cells, thereby promoting leukocyte adhesion and subsequent extravasation. It plays a crucial regulatory role in liver inflammation and hepatocyte necrosis processes [47 ,48]. In our study, the levels of CXCL-10 in the experimental groups were significantly higher than that in the control group, confirming its critical roles in acute liver damage. In our study, the markers including AST, ALT, TNF-α, IL-6, and CXCL-10 in the experimental groups all showed significant differences from those in the control group. This was consistent with the role of SPECT indicators (HLI5, LHL15) in assessing the liver function in this study. Among them, indicators such as HLI5, AST, ALT, TNF-α, IL-6, and CXCL-10 showed significant differences among the experimental groups, while LHL15 showed relatively small differences. These findings have an important reference value for future research.

This study had some limitations. It was conducted in SD rats, and the sample size of each group was small, which may have led to insufficient statistical power and limited the universality of the study results. In addition, the blank control group in this study did not receive any experimental manipulations, and there was no comparison with known effective treatments, so the effectiveness of experimental manipulations or treatments could not be proven. Therefore, in the future experimental design, larger sample size should be adopted, and positive or negative controls should be added, so as to improve the statistical power and reduce the omission of the evaluation of the real effect of experimental interventions. Additionally, considering the differences between different species and humans in drug response and disease pathogenesis, future research should be conducted on animal models that are more similar to humans in biological characteristics to improve the accuracy and relevance of research. C-reactive protein (CRP), which has been seldom used in similar studies in recent years, is an acute-phase protein synthesized by the liver in response to various inflammatory cytokines secreted by various inflammatory cells. As a highly sensitive indicator of systemic inflammation and tissue damage, CRP levels can rapidly reflect changes in a patient’s condition following trauma, inflammation, or infection. Therefore, the CRP measurement can serve as a valuable tool to monitor liver injury, and it should be considered in future experimental designs [49,50]. Finally, we hope that as research advances, the use of SPECT for evaluating liver injury across various models can be applied to clinical practice. This would enable clinicians to assess liver function early, intuitively, and dynamically, thereby improving patient management and outcomes.

Conclusion

Dynamic hepatobiliary SPECT imaging is a valuable imaging tool for diagnosis and assessing the severity of different types of liver failure. It offers noninvasive and direct monitoring of liver function. SPECT imaging, combined with the calculated indexes and the evaluation of cytokine levels, can become an effective method for assessing liver function of patients with acute liver failure.

Supporting information