Yeeran

Following the previous issue on abdominal organ ultrasound, we have systematically established the normal anatomical locations, standardized scanning planes, and ultrasound evaluation system for the liver, kidneys, spleen, pancreas, and major abdominal blood vessels in mice and rats. In the small animal ultrasound research system, accurately mastering normal imaging features is the foundation for identifying pathological changes, while the core value of scientific research experiments lies in dynamically capturing disease pathological phenotypes, precisely quantifying the degree of organ injury, and objectively evaluating the efficacy of drug interventions.

This issue is the second part of the abdominal organ ultrasound series. It focuses on the abdominal organ disease models most widely used in current research and systematically breaks down the pathogenic pathological features, typical ultrasound imaging phenotypes, dedicated quantitative evaluation indicators, and detection protocols of various models. At the same time, in response to pain points such as numerous interferences, data dispersion, poor reproducibility, and ambiguous lesion interpretation in abdominal ultrasound experiments, we provide some suggestions to help you obtain better experimental results.

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I. Core Research Pain Points of Abdominal Organ Ultrasound in Mice and Rats

Compared with cardiac ultrasound and tumor ultrasound imaging systems, abdominal organ ultrasound is more affected by anatomical structures and physiological motion. It is the module with the most errors, the least stable data, and the highest risk of experimental failure in small animal ultrasound experiments. The core research pain points are mainly concentrated in five aspects:

1.  Numerous interference artifacts, early lesions easily masked: The abdominal cavity of mice and rats contains densely packed intestines, and gastrointestinal gas interference is severe. Combined with high-frequency respiratory motion and minimal organ movement, this easily produces large amounts of reverberation artifacts and motion artifacts, completely obscuring superficial and deep tiny lesions in organs and leading to missed detection of early injury.

2.  Blurred anatomical boundaries, high interpretation threshold: Abdominal organs are closely adjacent, and soft tissue echogenicity is similar. Novices find it difficult to distinguish physiological heterogeneous organ echogenicity from early pathological injury, and false-positive and false-negative interpretation errors are highly likely.

3.  Early injury is mostly functional change and cannot be identified by conventional 2D ultrasound: Early abdominal organ diseases mainly involve functional injuries such as microcirculatory perfusion disorders, tissue mechanical changes, and microvascular remodeling. There are no obvious structural abnormalities on 2D grayscale imaging, and relying only on conventional ultrasound can easily miss key early phenotypes.

4.  No unified standards for experiments, poor data comparability: Most experiments do not standardize scanning depth, instrument gain, focus position, and anatomical sampling sites. Data from different time points, different groups, and different operators are highly dispersed and cannot be used for statistical analysis and high-quality publication.

5.  Insufficient use of advanced functions, limited project innovation: Relying only on basic 2D and Doppler indicators, without combining advanced technologies such as contrast-enhanced ultrasound (CEUS), shear wave elastography (SWE), ultrasound super-resolution imaging (URM), and ultrasound cavitation, makes it impossible to mine microscopic, deep, and mechanistic research data. Project depth and innovation are difficult to meet the requirements of high-impact journals.

II. Ultrasound Phenotypes and Standardized Evaluation Protocols for Mainstream Abdominal Organ Disease Models

2.1 Fatty Liver Model (High-Fat Diet/Alcoholic Fatty Liver)

Core pathological features: Extensive hepatocellular steatosis and abnormal lipid accumulation lead to altered echo characteristics of liver parenchyma and changes in liver tissue texture. In the early stage of the disease, there are no obvious fibrotic structural changes, and occult microcirculatory perfusion disorders are present. This is a classic model for metabolic disease and nutritional toxicology research.

Typical ultrasound phenotype: 2D grayscale ultrasound shows diffusely and uniformly increased liver parenchymal echogenicity, presenting characteristic ground-glass-like imaging changes, and the liver-to-kidney echogenicity ratio is significantly increased. As the modeling period prolongs and steatosis worsens, typical manifestations such as posterior field echo attenuation, blurred intrahepatic ductal structures, and decreased visibility of vascular course may appear.

Figure 1 Fatty liver ultrasound: liver-to-kidney echogenicity ratio is significantly increased.

Multimodal evaluation is recommended: Shear wave elastography (SWE) can stably measure liver tissue elastic modulus. Liver stiffness is not significantly increased in simple fatty liver, so SWE can accurately distinguish simple steatosis from steatotic liver fibrosis, addressing the limitation that 2D ultrasound cannot stage the disease. Contrast-enhanced ultrasound (CEUS) can capture occult microcirculatory perfusion disorders in early fatty liver and identify subtle hemodynamic changes in the early modeling stage. Ultrasound super-resolution imaging (URM) can observe early subtle remodeling features of intrahepatic microvessels, providing advanced microvascular data support for research on lipid metabolism disorders and fat injury mechanisms.

Core quantitative indicators: Liver-to-kidney echogenicity ratio, liver parenchymal echo homogeneity, CEUS perfusion timing parameters, SWE elastic modulus value, and intrahepatic microvascular distribution density.

2.2 Liver Fibrosis/Cirrhosis Model

Core pathological features: Persistent abnormal collagen deposition in liver tissue, hepatic lobular architectural remodeling, and fibrotic cord proliferation are accompanied by progressively increased liver tissue stiffness, tortuous and rarefied intrahepatic microvessels, and increased portal vein pressure. In the late stage, it can progress to secondary pathological changes such as cirrhosis, splenic congestion, and ascites.

Typical ultrasound phenotype: 2D ultrasound shows coarsened and unevenly distributed liver parenchymal echoes, with cord-like and reticular hyperechoic fibrous structures appearing in the liver. The liver capsule changes from smooth to rough and uneven, and the overall liver morphology becomes stiff. Doppler blood flow examination indicates slowed portal venous flow velocity and increased vascular resistance index. In the middle and late stages, secondary phenotypes such as splenomegaly and free abdominal fluid may occur.

Figure 2 Gross liver images and ultrasound images after four weeks of dimethylnitrosamine (DMN) treatment.

(Reference: Chen W, Chen J Y, Tung Y T, et al. High-frequency ultrasound imaging to evaluate liver fibrosis progression in rats and yi guan jian herbal therapeutic effects [J]. Evidence-Based Complementary and Alternative Medicine, 2013, 2013.)

Key points of multimodal evaluation: SWE is the core quantitative gold standard for liver fibrosis staging. It can accurately quantify the stepwise increase in liver stiffness and enable discrimination of mild, moderate, and severe fibrosis stages. CEUS can display microcirculatory remodeling features such as uneven intrahepatic perfusion, delayed contrast agent time to peak, and focal perfusion defects. URM can intuitively present subtle structural changes such as tortuous, rarefied, reduced branching, and spatially disorganized hepatic microvessels, providing innovative data for research on the mechanism of vascular remodeling in liver fibrosis

Figure 3 Liver vascular disorganization observed by ultrasound super-resolution (URM).

2.3 Drug-Induced Liver Injury/Acute Liver Inflammation Model

Core pathological features: Drug toxic stimulation induces acute hepatocyte edema, extensive inflammatory cell infiltration, and acute congestion and edema of liver tissue, accompanied by transient microcirculatory disturbance. This belongs to acute, reversible organ injury and is mainly used for efficacy evaluation and toxicology research of hepatoprotective drugs and anti-inflammatory and detoxifying drugs.

Ultrasound phenotype: 2D ultrasound shows diffusely decreased liver parenchymal echogenicity, uneven echo distribution, mild enlargement of liver volume, and full morphology. CDFI blood flow imaging indicates a compensatory increase in intrahepatic blood flow signals and hyperperfusion. CEUS can accurately capture subclinical injury phenotypes such as local intrahepatic microcirculatory perfusion disorder and local perfusion heterogeneity, and can evaluate drug injury and repair effects at an early stage when structural changes are not obvious.

Figure 4 Liver contrast-enhanced ultrasound image.

2.4 Diabetic Nephropathy and Obesity-Related Kidney Injury Models

Core pathological features: In the early stage, the disease is mainly characterized by tubular injury, renal hyperperfusion, and compensatory microcirculatory disturbance. As the disease progresses, glomerulosclerosis, renal interstitial collagen deposition, interstitial fibrosis, and thinning and atrophy of the renal cortex occur. This is a mainstream model for research on metabolic nephropathy and renal fibrosis mechanisms.

Ultrasound phenotype: In the early stage, compensatory enlargement of kidney volume, mildly increased renal cortical echogenicity, and cortical hyperperfusion can be seen. In the late stage, renal cortical thickness decreases, the corticomedullary echogenic boundary becomes blurred, renal blood flow signals decrease, and the renal arterial resistance index significantly increases, presenting typical degenerative changes of chronic kidney disease.

Figure 5 Kidney ultrasound of obese mice.

Value of advanced modalities: SWE can identify increased tissue stiffness caused by renal interstitial fibrosis at an early stage when 2D structure shows no obvious abnormality. CEUS can accurately capture abnormal renal cortical microcirculatory perfusion, with sensitivity far higher than conventional structural indicators. URM super-resolution imaging can visualize microscopic vascular remodeling changes such as rarefaction and reduced branching of intrarenal microvessels, revealing early kidney injury mechanisms at the microvascular level. The combination of the three is a core approach for early kidney injury and early drug intervention evaluation

Figure 6 Shear wave elastography.

Figure 7 Left: ultrasound super-resolution vascular image of a normal kidney; right: ultrasound super-resolution vascular image of a kidney with acute kidney injury.

2.5 Obstructive Nephropathy and Hydronephrosis Model

Urinary tract obstruction can directly cause separation of the renal pelvis, hydronephrosis, and renal pelvic dilation. Long-term compression can lead to compressed thinning of the renal cortex, renal parenchymal atrophy, and irreversible impairment of renal function. Ultrasound can dynamically monitor the degree of obstruction, rate of hydronephrosis progression, and changes in compressed cortical thickness. At the same time, it can dynamically follow up the organ repair process after relief of obstruction. It is a classic observational method for urology, kidney injury repair, and obstructive nephropathy research.

Figure 8 Left: 2D ultrasound image of a normal kidney; right: 2D ultrasound image of a kidney with unilateral ureteral obstruction.

(Reference: Wei S, Xu C, Zhang Y, Shi Z, Wu M, Yang B. Ultrasound Assisted a Peroxisome Proliferator-Activated Receptor (PPAR)γ Agonist-Loaded Nanoparticle-Microbubble Complex to Attenuate Renal Interstitial Fibrosis. Int J Nanomedicine. 2020 Oct 2;15:7315-7327.)

2.6 Acute Pancreatitis Model

Ultrasound phenotype: In acute inflammation, pancreatic tissue is enlarged and thickened, parenchymal echogenicity is decreased, and structures are blurred. Anechoic fluid areas formed by inflammatory exudation can be seen around the pancreas, accompanied by tissue edema and infiltration. Severe acute pancreatitis may show characteristic manifestations such as focal pancreatic necrosis, uneven parenchymal echogenicity, and patchy non-perfusion areas. Small animal ultrasound can dynamically monitor the progression of pancreatic inflammation and absorption of inflammatory exudate, providing a dynamic quantitative basis for efficacy evaluation of anti-inflammatory drugs and pancreatic protective drugs.

2.7 Immune Inflammatory Splenomegaly and Hepatic Congestion Model

Systemic inflammatory stimulation and cirrhotic portal hypertension can cause compensatory splenomegaly, increased splenic thickness, and decreased or uneven splenic parenchymal echogenicity. Combined with portal vein hemodynamic parameters and ascites, it can indirectly evaluate the body's systemic inflammation level, severity of portal hypertension, and organ congestion status. It is an important auxiliary evaluation system for research on immune inflammation and secondary injury in liver disease.

III. Multimodal Combination Research Strategies for Abdominal Organ Ultrasound

Based on the complete multimodal ultrasound technology system updated in the earlier issues of this series, customized detection combination protocols can be arranged for different research directions and project depths, balancing the completeness of basic data and project innovation:

1.  Conventional pharmacodynamic and basic mechanism projects: Use a combination of 2D structural imaging + CDFI blood flow imaging + spectral blood flow parameters, meeting the needs of routine experimental data output and basic paper publication, and covering evaluation of organ morphology, size, structure, and basic hemodynamics.

2.  Early injury and subclinical lesion mechanism projects: Use a combination of CEUS microcirculatory perfusion + URM microvascular fine structure, focusing on early functional injury of disease, mining early microcirculatory disorders and microvascular remodeling phenotypes that cannot be detected by conventional ultrasound, and significantly enhancing project innovation.

3.  Fibrosis, sclerosis, and organ structural remodeling projects: Use SWE elasticity quantification as the core evaluation indicator, combined with 2D structural and Doppler blood flow parameters, to evaluate organ fibrosis progression and drug reversal effects from multiple dimensions of morphology, blood flow, and mechanical microenvironment.

4.  Frontier projects on novel physical interventions and drug enhancement: Ultrasound cavitation effects can be introduced to conduct research on ultrasound-mediated targeted intervention, drug penetration enhancement, and combined treatment of tumors and organ injury, creating an innovative entry point for high-impact projects.

IV. Full-Process Standardized Quality Control and Error Avoidance for Abdominal Ultrasound

The core reason for poor reproducibility of abdominal ultrasound data is not insufficient equipment precision, but nonstandard scanning and lack of quality control. In view of the characteristics of abdominal imaging, a dedicated standardized quality control system should be established to thoroughly solve the problems of data dispersion and poor reproducibility:

1.  Standardized quality control for gas artifacts: Strictly perform fasting pretreatment of animals before experiments, and standardize abdominal hair removal and coupling agent application procedures. Gently push the abdominal wall to displace local intestinal gas, and uniformly acquire images at a stable moment during steady respiration and between breaths, maximizing avoidance of gastrointestinal gas and respiratory artifact interference.

2.  Standardized quality control of scanning planes: For all groups and all time points, strictly standardize scanning depth, instrument gain, focus position, and imaging magnification. Strictly use the standard organ planes defined in Issue 7, with fixed anatomical landmarks as positioning anchors, and eliminate structural misjudgment and measurement errors caused by off-axis, oblique, and inconsistent-depth scanning.

3.  Standardized quality control of probe pressure: The abdominal organs of mice and rats are soft and easily deformed under pressure. Use a light-contact imaging mode throughout, relying only on the coupling agent to conform to the skin, and do not apply pressure with the probe. Before freezing and saving images, confirm that there is no compression deformation of organs and no pseudo-stenosis of blood vessels, avoiding false-positive blood flow and abnormal stiffness caused by manual compression.

4.  Standardized quality control of animal status: Standardize anesthesia protocol, anesthesia depth, and post-anesthesia waiting time. Monitor body temperature, heart rate, and respiration stability throughout. Avoid abdominal organ blood flow fluctuation, perfusion disorder, and imaging deviation caused by excessive anesthesia, animal stress, and abnormal body temperature.

5.  Standardized quality control of measurement data: For all quantitative indicators, continuously select 3–5 stable imaging cycles for measurement and take the average. Uniformly exclude invalid data caused by respiratory artifacts, motion interference, and plane offset. For advanced modality examinations, standardize ROI sampling location, sampling area, and analysis parameters, ensuring full comparability of data between groups.

V. Full-Text Summary and Future Series Preview

Combined with the previous six issues of this series on cardiovascular ultrasound and multimodal tumor ultrasound, this two-part issue has fully completed the mouse and rat abdominal organ ultrasound research system, officially achieving coverage of the three core small animal ultrasound research modules: "heart–tumor–abdominal organs." It establishes a complete research methodology from normal anatomy, standard scanning planes, multimodal indicators, disease pathological phenotypes, multidimensional efficacy evaluation, to full-process experimental quality control.

Under the current trend of refined research, single endpoint pathological and biochemical indicators are no longer sufficient to support high-level research output. Integrating multimodal small animal ultrasound technologies involving 2D structure, hemodynamics, microcirculatory perfusion, microvascular fine structure, tissue mechanical properties, and ultrasound physical intervention can comprehensively mine in vivo dynamic research data, providing strong technical support for various projects such as metabolic diseases, organ injury, fibrotic lesions, anti-inflammatory, hepatoprotective, and renoprotective studies.

In the future, we will continue to delve into the field of small animal ultrasound research and continue to share practical content series such as bone and joint ultrasound, soft tissue ultrasound, hands-on ultrasound intervention, multimodal high-impact data mining, and analysis of difficult experimental problems. We will continuously improve the entire small animal ultrasound research system, helping researchers standardize experimental procedures, avoid experimental errors, and produce high-quality research results. You are welcome to continue following and learning.