A Novel Approach for Non-Invasive Liver Research
App Note / Case Study
Last Updated: July 1, 2024
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Published: May 17, 2024
Credit: iStock
Almost all chronic liver diseases lead to fibrosis, resulting in downstream complications such as cirrhosis and liver failure. Consequently, therapeutic strategies often prioritize mitigating or reversing fibrotic processes.
However, the absence of effective non-invasive in vivo tools for monitoring liver disease progression has significantly hindered therapeutic development.
This case study utilizes non-invasive ultrasound imaging to visualize and quantify liver echogenicity and stiffness, providing critical insights into both disease progression and the efficacy of therapeutic interventions in mouse models.
Download this app note to explore:
- How ultrasound imaging can advance the development of targeted therapies for liver disease
- The phenotypic response of liver health to CRISPR-based therapy
- An automated and high-throughput approach for preclinical research
CASE STUDY
Temporal tracking of an effective
intervention in rodent liver fibrosis.
The liver, as the largest solid internal organ of the body,
plays a central role in multiple physiological processes,
including metabolism, detoxification, nutrient storage, and
protein synthesis. Damage to the liver, whether arising from
metabolic disorders, viral infections, or exposure to toxic
substances, can manifest in various forms and severity,
ranging from abnormal fat accumulation (steatosis) and mild
inflammation to the development of scar tissue (fibrosis),
progressing to more severe conditions such as cirrhosis or
liver failure. The repercussions of liver injury span across
various research domains, including metabolic disease,
toxicology, cancer, infectious disease, alcoholic disease, and
rare genetic conditions like cystic diseases.
Since nearly all chronic liver diseases result in fibrosis,
which later causes even worse downstream prognosis
(e.g., cirrhosis and liver failure), many different therapeutic
approaches to treating liver disease focus either wholly
or in part on addressing and reversing fibrosis. Despite
the urgency and absence of a cure for liver fibrosis,
preclinical research tools for liver studies have been lacking,
hindering progress in therapeutic development. The lack
of widespread research tools to non-invasively observe
critical phenotypes in liver tissue echoes the challenges
researchers in oncology faced in the early 2000s; at that
time, tracking tumor growth and therapeutic response relied
on very crude assessments (e.g., caliper measurements or
ex vivo tumor weights). The oncology research field struggled
with these crude measurements until the introduction of
in vivo bioluminescence imaging systems, which offered a
non-invasive, real-time observation platform, revolutionizing
preclinical cancer research.
In a similar vein, a new and fundamentally enabling tool
has now become available to preclinical liver disease
researchers. The Vega® system has emerged as a significant
advancement in non-invasive in vivo technologies, enabling
researchers to gain a deep understanding of critical liver
disease phenotypes. Functioning as a hands-free, automated,
high-throughput preclinical ultrasound imaging system, the
Vega has demonstrated effectiveness in noninvasively staging
liver disease phenotypes across an array of diverse liver
disease models. The list of rodent models tested with the
Vega includes chemical injury models (e.g., CCl41
), genetic
models such as cholestasis,2
and metabolic disease models
(e.g., western diet,3
high fat diet and choline-deficient high fat
diet4
) providing critical insights into the natural progression
of liver disorders. While the Vega has been used to track
these different liver disease models evolving longitudinally,
until recently it had not been thoroughly tested tracking
interventions to these diseases. This ability to not only
observe the onset of liver disease phenotypes but also their
resolution after therapeutic intervention is a critical piece of
the puzzle for labs looking to implement this technology on
their own novel therapies for liver disease.
A case study using a new ultrasonic research tool for rapid and non-invasive
3D tissue assessment
Temporal tracking of an effective intervention in rodent liver fibrosis.
www.revvity.com 2
In this study, researchers set out to evaluate the efficacy of the Vega system’s automated ultrasound for non-invasively
monitoring therapy response in rodent models of non-alcoholic steatohepatitis (NASH). The system, equipped with 3D B-mode
and Shear Wave Elastography (SWE) modes, facilitates the tracking of changes in liver echogenicity (brightness) and stiffness,
respectively, providing valuable insights into fat accumulation and fibrosis severity.
This study not only confirms the Vega’s ability to provide non-invasive insights into disease progression, but also underscores its
capacity to monitor disease regression, which is important for evaluating drug efficacy and therapeutic interventions.
Rationale for using in vivo imaging
• Provides non-disruptive insights into longitudinal disease
progression within individual mice.
• Essential for studies with extended diet-induced models
where mistimed endpoints could mean thousands of
dollars wasted in lab resources and months of time.
• Helps in eliminating outliers and reducing variability within
experimental groups.
• Ensures good baseline normalization between animals
or cages, ensuring they are at the same level of disease
severity at the commencement of the study thereby
reducing downstream biological variability.
Motivation for longitudinal in vivo imaging
Methodology and study design
To assess the Vega system’s efficacy for monitoring therapy
response, the researchers tested two different therapeutic
interventions for NASH: diet reversal and CRISPR therapy.
In the first study, mice were fed choline deficient high-fat
diets (CDAHFD) for 8 weeks. At this timepoint, half of the
mice were transitioned to a less damaging standard high fat
diet, simulating effective therapy, while the remaining mice
continued on the CDAHFD. A third control group received a
standard chow diet for the whole study period. Imaging was
performed every 2 weeks for 18 weeks. In the second study,
mice were fed a Gubra Amylin NASH diet (GAN) and treated
with liver-directed AAV-CRISPR against three candidate
genes. Two control groups were included, representing a
GAN-only and a vehicle injection group, and imaging was
performed every 8 weeks for 32 weeks.
The diets in both treatment approaches were selected
for their known ability to induce liver damage, providing a
platform for assessing therapeutic responses. Longitudinal
imaging using 3D ultrasound was utilized to measure liver
echogenicity and stiffness, enabling the researchers to
evaluate the impact of therapeutic interventions on liver
health over time.
Results
Diet reversal
Researchers presented compelling evidence that liver
disease instantiation and resolution can be tracked within
individual mice noninvasively by the Vega system. Both
qualitative and quantitative liver assessments are shown
in Figures 1 and 2, respectively. Figure 1 demonstrates how
mice on the CDAHFD exhibited increased liver stiffness over
time, which was clearly reversed following the dietary switch
at 8 weeks. This reversal is further illustrated in Figure 2,
where both liver echogenicity and stiffness rapidly decline
following the timepoint where the diet was switched.
Temporal tracking of an effective intervention in rodent liver fibrosis.
www.revvity.com 3
Figure 1: CDAHFD Image Data. Representative liver images in the three groups across the course of the CDAHFD diet-reversal study.
CDAHFD elicited a strong response in noninvasive markers of liver echogenicity and stiffness, with clear reversal at the diet switch timepoint.
Figure 2: CDAHFD Noninvasive Quantification. Longitudinal progression of liver echogenicity and stiffness over 18 weeks in mice fed CDAHFD,
standard chow, or switch from CDAHFD -> chow. Error bars represent mean ± std.
Temporal tracking of an effective intervention in rodent liver fibrosis.
www.revvity.com 4
CRISPR therapy
Figure 3 shows how both brightness and stiffness of the livers of mice with no CRISPR treatment increased over time in response
to GAN diet feeding, confirming the Vega’s ability to track changes in liver disease phenotypes over time.
Figure 3: GAN + CRISPR Image Data. Representative images showing both brightness and stiffness images of the livers in mice on a GAN diet
with no CRISPR treatment (“Control” group). Body weight, liver volume, echogenicity, and stiffness all increased substantially in response to
GAN diet feeding.
Figure 4: GAN Noninvasive Quantification. Longitudinal progression of liver echogenicity and liver stiffness readings over 32 weeks in mice
fed a GAN diet with CRISPR treatment. Echogenicity increased over time across all groups in a similar fashion, plateauing after 24-32 weeks.
Stiffness also increased over time, however CRISPR-2 treatment appeared to delay the onset of stiffness rise, suggesting less liver damage.
Error bars represent median ±IQR.
While liver echogenicity readings (a marker of fatty liver) increased over time in all groups, Figure 4 demonstrates how
differences in liver stiffness readings were identified between the CRISPR treatment groups. Specifically, liver stiffness was
delayed and lower in the CRISPR-2 treatment group, suggesting that this treatment knocked out or edited a gene that protected
against liver stiffness associated with the GAN diet.
Temporal tracking of an effective intervention in rodent liver fibrosis.
www.revvity.com 5
Importantly, Figure 5 illustrates the Vega’s ability to identify mice that developed spontaneous tumors following CRISPR
treatment (an unintended consequence of the genetic alterations in the treated groups). At week 8, there is no visible tumor
but the echotexture of the liver appears heterogenous and irregular, hinting at underlying changes. By week 16 a small mass
is visible and by week 24 the mass undergoes significant expansion. This information is essential in the field of cell and gene
therapy, so that unintended outcomes can be identified early and to ensure that that genetic interventions are refined.
Figure 5: Spontaneous Liver Tumors. Representative images of a spontaneous liver tumor developing over time. At week 8, the tumor is not
visible however the echotexture of the liver appears heterogenous and irregular. By week 16, a small echolucent mass is visible (yellow arrow,
<1 mm3
), and by week 24 the mass had expanded significantly (716 mm3
).
Study relevance
Visualizing and quantifying the progression and regression of
liver echogenicity and stiffness through non-invasive imaging
offers a valuable means to monitor liver disease over time in
response to therapeutic interventions. The diet intervention
study, which illustrated disease progression and regression,
highlighted the Vega system’s potential for showing the
dynamic changes in liver phenotypes in response to
effective therapy. The delayed rise in liver stiffness following
CRISPR-2 treatment in the second study not only supports
the Vega’s ability to identify effective treatments, but also
emphasizes how this finding might not have been evident
from echogenicity changes alone. The Vega also proved
invaluable in detecting unintended side effects, particularly
the development of spontaneous liver tumors following
CRISPR treatment. This capability is crucial in the context of
genetic interventions, where unexpected consequences need
to be promptly identified and addressed.
Conclusion
Overall, this study marks a significant step forward in
validating a cutting edge tool for liver disease researchers,
namely its ability to noninvasively assess liver fibrosis and
steatosis in response to effective therapeutic interventions.
The study demonstrates the ability of the Vega system to
provide valuable, noninvasive insights into liver disease
progression and the efficacy of therapeutic interventions in
mouse models.
SWE is an ultrasound-based stiffness quantification
technology that is used routinely for noninvasive liver
fibrosis assessment in the clinic. However, it is not widely
utilized in rodent models. As demonstrated in this study, the
combination of echogenicity and stiffness measurements
enables more informed decisions regarding treatment
efficacy, disease progression, and potential interventions.
This dual approach proves instrumental in identifying
treatments that positively influence both echogenicity and
stiffness, offering a more comprehensive understanding for
managing liver diseases effectively.
Looking ahead, future work will focus on further automating
the workflow, leveraging machine learning approaches
to streamline data acquisition and analysis. This move
reflects the commitment of the Revvity team to enhance
the efficiency and precision of non-invasive assessments
in liver research, ensuring the continued advancement of
therapeutic development.
https://www.revvity.com/category/ultrasound-imaging
Temporal tracking of an effective intervention in rodent liver fibrosis.
For a complete listing of our global offices, visit www.revvity.com
Copyright ©2024, Revvity, Inc. All rights reserved. 1347293
Revvity, Inc.
940 Winter Street
Waltham, MA 02451 USA
www.revvity.com
References
1. Shear Wave Elastography of Liver Fibrosis Mouse Model
Using an Automated Preclinical Ultrasound System.
Gandham V, et al. in Imaging in 2020, Jackson Hole,
Wyoming, USA 2022
2. Monitoring treatment response of nor-ursodeoxycholic
acid in Mdr2 (Abcb4)-/- mice with automated shear wave
elastography, Czernuszewicz TJ et al. in Proceedings of
the AASLD: The Liver Meeting, Washington DC, USA, 2022
3. Automated ultrasound for noninvasive evaluation of
NAFLD progression in a murine Western Diet model,
Czernuszewicz TJ et al. in Proceedings of the AASLD:
The Liver Meeting, Washington DC, USA, 2022
4. Development of a Robotic Shear Wave Elastography
System for Noninvasive Staging of Liver Disease in Murine
Models. Czernuszewicz, T.J., et al., 2022. Hepatology
Communications, 6(7), pp.1827-1839
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