We thank Kari Huppert and Stacey Huppert for their expertise and help regarding bile duct cannulation and their laboratory hospitality. We also thank Nadja Schultz and Charlotte L. Mattsson for their help with common bile duct cannulation.
We thank the following Granting Agencies for their support:
For work in ERA Lab: Karolinska Institutet (2-560/2015-280), Stockholms L&#228;ns Landsting (CIMED (2-538/2014-29)), Ragnar S&#246;derbergs stiftelse (Swedish Foundations&#39; Starting Grant), European Association for the Study of the Liver (Daniel Alagille Award), Swedish Heart-Lung Foundation (20170723), and Vetenskapsr&#229;det (2019-01350).
For work in JK Lab: We acknowledge CzechNanoLab Research Infrastructure supported by MEYS CR (LM2018110). J.K. thanks to the support of grant FSI-S-20-6353.

<ol>
	<li>Narat, J. K., Loef, J. A., Narat, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+preparation+of+multicolored+corrosion+specimens.">On the preparation of multicolored corrosion specimens.</a> The Anatomical Record. 64, 155-160 (1936).</li><li>Ludwig, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+of+the+human+biliary+system+studied+by+quantitative+computer-aided+three-dimensional+imaging+techniques.">Anatomy of the human biliary system studied by quantitative computer-aided three-dimensional imaging techniques.</a> Hepatology. 27, 893-899 (1998).</li><li>Masyuk, T. V., Ritman, E. L., LaRusso, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+the+rat+intrahepatic+biliary+system+by+three-dimensional+reconstruction.">Quantitative assessment of the rat intrahepatic biliary system by three-dimensional reconstruction.</a> American Journal of Pathology. 158, 2079-2088 (2001).</li><li>Masyuk, T. V., Ritman, E. L., LaRusso, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+artery+and+portal+vein+remodeling+in+rat+liver:+Vascular+response+to+selective+cholangiocyte+proliferation.">Hepatic artery and portal vein remodeling in rat liver: Vascular response to selective cholangiocyte proliferation.</a> American Journal of Pathology. 162, 1175-1182 (2003).</li><li>Sparks, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signaling+regulates+formation+of+the+three-dimensional+architecture+of+intrahepatic+bile+ducts+in+mice.">Notch signaling regulates formation of the three-dimensional architecture of intrahepatic bile ducts in mice.</a> Hepatology. 51, 1391-1400 (2010).</li><li>Cuervo, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+notch+signaling+is+essential+to+prevent+hepatic+vascular+malformations+in+mice.">Endothelial notch signaling is essential to prevent hepatic vascular malformations in mice.</a> Hepatology. 64, 1302-1316 (2016).</li><li>Thakurdas, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Jagged1+heterozygosity+in+mice+results+in+a+congenital+cholangiopathy+which+is+reversed+by+concomitant+deletion+of+one+copy+of+Poglut1+(Rumi).">Jagged1 heterozygosity in mice results in a congenital cholangiopathy which is reversed by concomitant deletion of one copy of Poglut1 (Rumi).</a> Hepatology. 63, 550-565 (2016).</li><li>Walter, T. J., Sparks, E. E., Huppert, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3-Dimensional+resin+casting+and+imaging+of+mouse+portal+vein+or+intrahepatic+bile+duct+system.">3-Dimensional resin casting and imaging of mouse portal vein or intrahepatic bile duct system.</a> Journal of Visualized Experiments: JoVE. (68), e4272 (2012).</li><li>Hankeova, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DUCT+reveals+architectural+mechanisms+contributing+to+bile+duct+recovery+in+a+mouse+model+for+Alagille+syndrome.">DUCT reveals architectural mechanisms contributing to bile duct recovery in a mouse model for Alagille syndrome.</a> Elife. 1, 1-29 (2021).</li><li>Andersson, E. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+model+of+Alagille+syndrome+and+mechanisms+of+Jagged1+missense+mutations.">Mouse model of Alagille syndrome and mechanisms of Jagged1 missense mutations.</a> Gastroenterology. 154, 1080-1095 (2018).</li><li>Tanimizu, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrahepatic+bile+ducts+are+developed+through+formation+of+homogeneous+continuous+luminal+network+and+its+dynamic+rearrangement+in+mice.">Intrahepatic bile ducts are developed through formation of homogeneous continuous luminal network and its dynamic rearrangement in mice.</a> Hepatology. 64, 175-188 (2016).</li><li>Ober, E. A., Lemaigre, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+liver:+Insights+into+organ+and+tissue+morphogenesis.">Development of the liver: Insights into organ and tissue morphogenesis.</a> Journal of Hepatology. 68, 1049-1062 (2018).</li></ol>

A separate project in the ERA lab is funded by ModeRNA. ModeRNA had no role in the project/protocol described here.

Several critical steps determine DUCT success, from sample preparation to the parameters of the CT device. To achieve the best results, well contrasted, well-injected, and bubble-free resin should be used to allow straightforward digital processing with automated thresholding to obtain 3D data, images, and movies. With training and following this protocol, 90% of injections are successful and result in reproducible data. It is important to use fresh yellow resin to achieve the best contrast between the two injected systems. The yellow resin has a very strong radiopacity, while the blue resin has undetectable radiopacity. Top results are achieved within the first three months after opening a new yellow resin bottle. With time, resin precipitates, and after longer storage (&#62;6 months), the yellow and the green resins will no longer be distinguishable in CT scans. Images with poor contrast necessitate extensive and time-consuming manual tracing and segmentation of the two systems. Next, well-stretched tubing is indispensable to fit into the common bile duct of adult mice and common bile duct and portal vein of postnatal mice. The entry point for the injection must be created with care. If the common bile duct is cut open transversally, it is likely to detach from the surrounding tissue, preventing successful entry of the tubing. This step is especially delicate for postnatal mice in which the common bile duct retracts and &#34;curls up&#34; if it has detached from its surrounding tissue, making insertion of the tubing extremely challenging. The common bile duct entry and injection may require some practice. While preparing the tubing with resin and throughout injection, avoid bubble formation as bubbles will create negative space in the CT images and require time-consuming manual correction. It is important to gently massage the liver by rolling over its surface with a wetted cotton swab during and after the injection procedure as this facilitates even resin spreading. After the completion of injection and removal of the tubing, the silk suture knot must be tightened quickly and carefully, so the resin does not flow out of the liver before it polymerizes completely. For successful microCT imaging, the sample must be properly fixed in place with agarose and thermally adapted to eliminate movement artifacts in the CT data. The acquisition settings are also of key importance, which should be optimized to reach an adequate spatial resolution to resolve fine structures.
Technical modifications to the injection procedure can be made to achieve injection in younger mice. Currently, resin casting of younger mouse livers is limited by the availability of sufficiently thin tubing, with PE10 being the smallest commercially available tubing. Tanimizu et al. successfully injected carbon ink into embryonic day 17 (E17) common bile duct using glass capillaries11. Future testing of whether resin can be delivered via glass capillary would therefore be of interest. DUCT was further adapted to inject other tubular systems such as the airways and pulmonary artery vasculature of the lungs9. The double resin injection could also be modified to be used with other commercially available resins, or this protocol could be used for injections with carbon ink.
One of the main limiting factors of the DUCT pipeline is the resin viscosity. DUCT can only be used for resin casting of tubular structures above a diameter of 5 &#181;m. In this data set, the resin could penetrate tubes with the smallest diameter of 5 &#181;m9. This size limitation precludes the analysis of fine ductules and small capillaries. To further advance the DUCT pipeline to smaller caliber vessels, other commercially available resins should be tested, or the development of new low-viscosity radiopaque agents may improve the lumen penetration.
In Hankeova&#160;et al.9, DUCT was compared to two other commonly used techniques, double carbon ink injections followed by tissue clearing and standard photography, and iDISCO+ with staining of the blood vessels with alpha-smooth muscle cell actin and bile ducts with cytokeratin 7, followed by 3D imaging9. DUCT outperformed the other two methods in terms of dual analysis (which was challenging for iDISCO+ due to high liver autofluorescence), 3D imaging, and quantification (not possible with carbon ink injection), and lumenization (DUCT provides data for the internal lumen architecture and system perfusion). As mentioned above, the main limitation of DUCT is the minimum lumen size that can be injected and analyzed (5 &#181;m limit), a parameter in which both carbon ink injection and iDISCO+ performed better. DUCT is superior to single system resin casting3,5,6 because it allows analysis of each injected system separately and also facilitates dual 3D investigation to study the architectural relationship between the two systems.
DUCT can be applied to study any two tubular networks in 3D. As proof of principle, DUCT was used to visualize the liver biliary and portal vein systems and the pulmonary artery vasculature and airways in lung9. The intrahepatic bile ducts develop adjacent to the portal vein, and the portal vein provides a structural template and signaling center that regulates the growth and differentiation of the biliary tree12. In Hankeova&#160;et al.9, DUCT explored biliary regeneration in a mouse model for the human pediatric disease Alagille syndrome. DUCT revealed previously unreported architectural mechanisms that the biliary system used to achieve a wild-type-like volume9. The Alagille syndrome mice utilized two different strategies: (1) in the hilar and central regions of the liver, the biliary system increased its branching, and (2) in the liver periphery, the de novo-generated bile ducts were highly tortuous. These two factors combined to yield a near-normal biliary system volume, despite the abnormal architecture. Furthermore, DUCT detected abnormal bile duct branching that occurred independent of portal vein branching and bile ducts forming connecting bridges between two portal veins9. These phenotypes would be impossible to detect in single resin casting and could be misinterpreted in 2D histological sections as bile duct proliferation. DUCT thus provides data describing the 3D architecture of two tubular networks at the whole organ or lobe level with the possibility of qualitative and in-depth quantitative analysis. DUCT could be a new standard for postnatal liver development and liver regeneration analyses in different animal models.

Organ resin casting is a technique that dates back to the 17th century1. One of the first examples of modern resin casting was performed on the human liver from an autopsy. Intrahepatic bile ducts were filled with a contrast agent mixed with gelatin, followed by imaging with an x-ray CT scan2. The aim of the DUCT technique is to visualize, digitalize and analyze two tubular resin-casted networks, in tandem, in 3D.
DUCT is based on the extensive existing knowledge of single-system liver resin casting3,4,5,6,7,8 and extends to simultaneous 3D visualization and analysis of two systems9. DUCT advanced single resin casting to double resin casting by mixing two radiopaque resins of different contrast and injecting these resins into two different networks, specifically the common bile duct and portal vein. DUCT can be applied to young postnatal mice with reproducible results as early as postnatal day 15 (P15). Compared to microscopy-based imaging techniques, the main advantage is that DUCT is faster, antibody-free, and liver tissue autofluorescence does not interfere with imaging. Further, DUCT provides quantitative data describing lumenization status, internal diameter, network connectivity, and perfusion. Differentiating between the presence of lumen-forming cells and their de facto morphogenesis into tubes is essential for analyzing organs in which ductular cells are present but do not form tubes, as can be the case in Alagille syndrome10. The main disadvantage of DUCT is the limited penetration of the resin, which is viscous and does not enter tubes with a small caliber (&#60;5 &#181;m). DUCT can be applied for any tubular structure after determining the injection entry point, such as the arterial and venous circulatory systems, airways, the extrahepatic bile duct, or lymphatic vessels. It could thus facilitate whole organ architecture analysis of other tissues such as lungs and pancreas.
MicroCT segmented images can be processed using commercially available imaging software, such as ImageJ, or custom-written pipelines (e.g., MATLAB). The resin-injected liver can be analyzed qualitatively for network expansion and connectivity or quantitatively for volume, length, branching, tortuosity of a single system, and the interaction between two systems such as the distance between two systems, or branchpoint dependence (does system 1 branch in proximity to system 2 branching?). The DUCT pipeline encompassing resin injection, microCT scanning, and CT data segmentation, combined with detailed quantitative analysis of architectural mechanisms of two tubular systems, could provide a standard for whole liver analysis in animal models.

<table><tbody><tr><td>1.5 mL SafeSeal micro tubes</td><td>Sarstedt</td><td>72.706</td><td></td></tr><tr><td>23 G butterfly needle with tubing</td><td>BD bioscience</td><td>367283</td><td></td></tr><tr><td>23 G needle</td><td>BD bioscience</td><td>305892</td><td></td></tr><tr><td>30 G needle</td><td>BD bioscience</td><td>305106</td><td></td></tr><tr><td>Agarose</td><td>Top-Bio</td><td>P045</td><td></td></tr><tr><td>Benzyl alcohol</td><td>Sigma Aldrich</td><td>108006</td><td></td></tr><tr><td>Benzyl benzoate</td><td>Sigma Aldrich</td><td>B6630</td><td></td></tr><tr><td>Corning 50 mL tubes</td><td>Sigma Aldrich</td><td>CLS430829-500EA</td><td>polypropylene</td></tr><tr><td>Cotton swabs</td><td>Medicarier</td><td>60406</td><td></td></tr><tr><td>Dissection Microscope</td><td>Leica Camera AG</td><td></td><td>Leica M60</td></tr><tr><td>Dulbecco&#039;s phosphate-buffered saline</td><td>ThermoFisher Scientific</td><td>14190144</td><td></td></tr><tr><td>Ethanol 70%</td><td>VWR</td><td>83801.41</td><td></td></tr><tr><td>Falcon tube 15 mL</td><td>Verkon</td><td>331.850.084.006</td><td></td></tr><tr><td>Forceps curved</td><td>Fine Science Tools</td><td>11051-10</td><td>Fine Graefe 10 cm curved</td></tr><tr><td>Forceps straight</td><td>Fine Science Tools</td><td>11050-10</td><td>Fine Graefe 10 cm straight</td></tr><tr><td>Formaldehyde solution</td><td>Sigma Aldrich</td><td>F8775</td><td></td></tr><tr><td>GE Phoenix v|tome|x L 240</td><td>Waygate Technologoies</td><td></td><td>micro computed tomography scanner</td></tr><tr><td>Hanks&#039; Balanced Salt Solution</td><td>ThermoFisher Scientific</td><td>14025092</td><td></td></tr><tr><td>Heparin</td><td>Leo Pharma</td><td>B01AB01</td><td>5000 IE/mL</td></tr><tr><td>Isolfurane</td><td>Baxter</td><td>FDG9623</td><td></td></tr><tr><td>Methanol</td><td>ThermoFisher Scientific</td><td>11413413</td><td></td></tr><tr><td>MICROFIL</td><td>Flowtech</td><td>MV-122</td><td>synthetic resin yellow</td></tr><tr><td>MICROFIL</td><td>Flowtech</td><td>MV-120</td><td>synthetic resin blue</td></tr><tr><td>MICROFIL</td><td>Flowtech</td><td>MV-diluent</td><td>clear resin diluent</td></tr><tr><td>Pasteur pipette</td><td>Verkon</td><td>130.690.424.503</td><td></td></tr><tr><td>Peristaltic pump</td><td>AgnThos</td><td>010.6131.M20</td><td></td></tr><tr><td>phoenix datos|x 2.0 software</td><td>Baker Hughes</td><td></td><td>CT data reconstruction software</td></tr><tr><td>Rocker</td><td>VWR</td><td>444-0142</td><td></td></tr><tr><td>Silk suture</td><td>AgnThos</td><td>14757</td><td>Black silk, 4-0, sterile, 100 m</td></tr><tr><td>Skin scissor</td><td>Fine Science Tools</td><td>14058-09</td><td>Iris straight tip 9 cm</td></tr><tr><td>Spring scissor</td><td>Fine Science Tools</td><td>15000-03</td><td>Vannas micro, straight tip 2 mm</td></tr><tr><td>Syringe 1 mL Luer</td><td>BD bioscience</td><td>303172</td><td></td></tr><tr><td>Tubing PE10</td><td>BD bioscience</td><td>427401</td><td></td></tr><tr><td>Tubing PE50</td><td>BD bioscience</td><td>427411</td><td></td></tr><tr><td>VG Studio MAX 3.3 software</td><td>Volume Graphics GmbH</td><td></td><td>CT data processing and analysis software</td></tr></tbody></table>

duct: double resin casting followed by micro-computed tomography for 3d liver analysis

The liver is the biggest internal organ in humans and mice, and high auto-fluorescence presents a significant challenge for assessing the three-dimensional (3D) architecture of the organ at the whole-organ level. Liver architecture is characterized by multiple branching lumenized structures, which can be filled with resin, including vascular and biliary trees, establishing a highly stereotyped pattern in the otherwise hepatocyte-rich parenchyma. This protocol describes the pipeline for performing double resin casting micro-computed tomography, or &#34;DUCT&#34;. DUCT entails injecting the portal vein and common bile duct with two different radiopaque synthetic resins, followed by tissue fixation. Quality control by clearing one lobe, or the entire liver, with an optical clearing agent, allows for pre-screening of suitably injected samples. In the second part of the DUCT pipeline, a lobe or the whole liver can be used for micro-computed tomography (microCT) scanning, (semi-)automated segmentation, and 3D rendering of the portal venous and biliary networks. MicroCT results in 3D coordinate data for the two resins allowing for qualitative as well as quantitative analysis of the two systems and their spatial relationship. DUCT can be applied to postnatal and adult mouse liver and can be further extended to other tubular networks, for example, vascular networks and airways in the lungs.

What to do 
Successful double resin injection is achieved when both the intrahepatic bile ducts and portal vein vasculature are well filled. As a quality control step, clearing one lobe (for example, the left lateral lobe) allows for verification of a successful injection, followed by imaging of lobes of interest. The optically cleared lobe can be scanned later using microCT; hence it is possible to optically clear the whole liver. In well-injected mouse liver, the portal vein vasculature should be filled with resin until the liver periphery and resin should be visible in side-branches (Figure 4), and this architecture is faithfully recapitulated in microCT scanned and segmented data. Further, well-injected intrahepatic bile ducts should be visible next to the main portal vein branches extending almost to the periphery, and resin should be visible in the major side branches. If the control lobe passes the quality control step, the lobes of interest (included the optically cleared one) can be scanned with microCT. The result of the segmented data from a well-injected liver is shown for a P15 mouse (Figure 4A,B) and an adult mouse (Figure 4C,D).
What not to do 
Intact liver tissue is a prerequisite for successful injection. Take extra care when cutting the abdominal cavity and diaphragm not to accidentally nick the liver tissue. If there is physical damage to the liver during this procedure, the resin is very likely to leak out during portal vein injection (Figure 5A). It is not possible to achieve a good injection of the vascular system if the liver is physically damaged.
One of the common mistakes is underfilling the liver with resin that can lead to challenges for visualization or analysis. One of the causes for system underfilling is resin hardening prematurely in the needle or the tip of the tubing before the injection is completed (Figure 5B, blue arrowheads, brackets depict large bubbles). A good practice is to use one injection set per animal and work fast after the curing agent is added to the resin. If the resin hardens during the injection (which can be observed by a half-filled system, here exemplified with a half-filled portal vein vasculature) remove the tubing, cut the tip of the tubing (always diagonally to create a beveled tip), and push the plunger. If resin begins to drip again, carefully re-insert the tubing and secure it with the suture. If the resin has hardened in the needle, replace the tubing completely, fill it with resin (avoiding bubbles), carefully re-insert the tubing, and secure it with the suture. It can be challenging to replace the tubing, especially in young postnatal mice &#60;P30, as the tissue is more fragile. Another cause of poor resin filling of portal vein vasculature can be insufficient transcardial perfusion (Figure 5C, blue arrowheads denote blood visible in terminal branches). This can be observed when the tips of the vessels are filled with blood instead of resin. To avoid this, ensure that the portal vein (outside the liver) does not contain any blood before the injection. The third cause of underfilled liver is when the tubing is inserted too deep into the liver and enters a branch towards one of the lobes. To prevent this, insert the tubing at a minimum of 0.5 cm from the entry to the liver.
Conversely, one, or both, of the systems become overfilled with resin (Figure 5D). It is necessary to visually monitor the liver throughout the injection. Biliary system casting with resin is more challenging than portal vein resin casting since resin-filled ducts are only faintly visible on the liver surface, and it is difficult to assess when the system is nearly full and when to stop. When small yellow resin dots appear on the liver surface (Figure 2Ci, blue arrowhead), this is a sign that the biliary system is completely filled, and the resin is starting to leak out of the ducts. Minor resin leakage can be manually corrected during microCT data segmentation (Figure 5D, right panels).
If the injection pressure is too high, this can cause vessels or ducts to rupture (Figure 5E), irreversibly damaging vessel or duct architecture. The liver will not be suitable for microCT scanning or analysis. To avoid resin overfilling, optimize the right volume and pressure used for injection in each mouse model. When working with mice that have been challenged with a toxic diet, genetic modification, or liver injury that affects the biliary or venous systems, or liver stiffness, the injection pressure and volume may need to be adjusted as volume and pressure tolerated may be different from the wild type mice. This protocol describes the manual injection of the two systems, but it is possible to connect the syringe to a pump to standardize the injection pressure. Bubbles are another very common injection artifact that leads to sparse filling of the tubular networks (Figure 5F-H, blue arrowheads). To avoid bubble formation, make sure that the syringe and tubing do not contain any bubbles, are completely filled with resin, and the resin is dripping from the tip of the tubing before injection. Small bubbles that appear as negative areas on the microCT data can be manually corrected during post-processing steps, although this is laborious.
Fresh is the best 
Using fresh yellow resin is a crucial factor, significantly affecting the contrast of the two resins and the microCT data segmentation. When the freshly opened resin is used (Figure 6A) there is a clear difference in contrast between the yellow resin-injected bile ducts (bright white) and the green resin-injected portal veins (bright grey). Liver that is injected with fresh resin is easily processed using automated global thresholding. With prolonged storage, the resin precipitates, and the contrast diminishes. After 3 months of storage, the contrast can still be sufficient to distinguish the portal vein from the bile duct (Figure 6B), but precipitation affects the mixing of the two resins, which is visible as a heterogeneous opacity in the filled portal vein (Fig 6B, blue arrowheads). Heterogeneous contrast negatively affects the automated thresholding and necessitates manual corrections, which increases the processing time. If the resin is older than six months, the contrast has degraded to a point at which it is not possible to distinguish the yellow-injected bile duct from the green-injected portal vein based on their contrast alone (Figure 6C). In this case, the bile duct and portal vein must be segmented manually based on their diameter and position in the hilar region and followed manually throughout the entire microCT data. This procedure is extremely time-consuming and best avoided.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62941/62941fig01.jpg" /> 
Figure 1: Injection set for resin casting. (A) Injection set #1 comprises a 30 G needle and PE10 tubing that is ~30 cm long. Injection set #2 is composed of a 23 G needle and PE50 tubing ~30 cm long. (B) The tip of the tubing is stretched and cut at an angle to create a beveled tip. The ruler in A and B is a centimeter ruler, with major increments of 1 cm, intermediate increments of 5 mm, and minor increments of 1 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62941/62941fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62941/62941fig02.jpg" /> 
Figure 2: Double resin casting flow chart. (A) Schematic showing the murine venous circulatory system and the heart with highlighted right atrium, which should be cut away before perfusion, and the left ventricle into which the needle should be inserted for perfusion to wash away the blood from the circulatory system. The inferior vena cava should be severed under the kidneys to relieve vascular pressure. (B) Bile duct resin injection flow chart. (i) Zoom image of (A) depicting where to sever the IVC. (ii) Image depicting the common bile duct (yellow dotted line) from the liver hilar region to the sphincter of Oddi (black arrowheads), with suture thread under the cleared common bile duct. (iii) Suitable position for loose overhand knot around common bile duct. (iv) The yellow dotted line and black arrowheads label the sphincter of Oddi, demonstrating the oblique angle for incision and how the opening should appear after the oblique angle incision. (v) Schematic demonstrating the orientation of the PE10 tubing bevel opening (upwards) upon insertion. (vi) Appearance of yellow resin being injected; resin should easily pass the loosely tied knot. IVC, inferior vena cava; CBD, common bile duct. (C) Portal vein resin injection flow chart. (i) The green dotted line marks the portal vein from the hilar region. The blue arrowheads label the overfilled biliary system. (ii) Suitable location for loose overhand knot around portal vein. (iii) Schematic demonstrating the bevel opening (upward) upon insertion. (iv) Appearance of liver upon injection of green resin and yellow resin; note the resin-filled blood vessel in the liver periphery. PV, portal vein. Figure 2A was created with Biorender.com. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62941/62941fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62941/62941fig03.jpg" /> 
Figure 3: Micro CT data processing in volume graphics software. (A) Surface determination, (i) overestimated isovalue (current preview of selection is shown in yellow color), (ii) underestimated isovalue, (iii) optimal selection of isovalue for proper surface determination of portal vein and bile ducts. (B) Splitting region of interest (ROI) created by surface determination,&#160;(i) set the value in dialog window high enough that only one segment (the largest one) will remain, (ii) in yellow frames the smaller (excluded) particles are shown. (C) Surface smoothing of the data, (i) smoothing function is on the left panel, (ii) set the smoothing strength to 1 (max. 2) and create new smoothed ROI, (iii) smoothed data. (D) Separation of individual tubular systems, (i) in surface determination function set the isovalue so that only the biliary system is included in selection (current preview of selection is shown in yellow color), (ii) mark the ROI of both systems and ROI of only the biliary system and subtract biliary system ROI from ROI of both systems, (iii) Portal vein shown in grey, the biliary system shown in green. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62941/62941fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62941/62941fig04.jpg" /> 
Figure 4: Well-injected bile duct (BD) and portal vein (PV) systems. (A) Optically cleared right medial lobe (RML) of postnatal day 15 (P15) liver injected with two resins into the two systems. Scale bar 1 mm. (B) 3D rendering of P15 RML shown in (A) depicting portal vein vasculature in white and biliary system in green. Scale bar 1 mm. (C) Optically cleared RML of adult liver injected with two resins into the two systems. Scale bar 1 mm. (D) 3D rendering of adult RML shown in (C) depicting portal vein vasculature in white and biliary system in green. H = hilar, P = peripheral. Scale bar 1 mm. Panels A, B, D are adapted with permission from Hankeova&#160;et al.9. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62941/62941fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62941/62941fig05.jpg" /> 
Figure 5: Common challenges of double resin liver injections. (A) The image depicts a liver that was accidentally nicked during the initial opening of the abdominal cavity, and the resin is leaking through the cut (blue arrowhead). (B) Poorly injected portal vein system due to resin hardening. Blue arrowheads label empty terminal branches, and red brackets label large bubbles. Scale bar 1 mm. (C) Poorly injected portal vein system due to poor transcardial perfusion. Blue arrowheads label blood visible in the terminal branches. Scale bar 1 mm. (D) Overfilled biliary system manifested by isolated balls of resin. The left panels show the optically cleared liver, and the right panels show the 3D microCT rendered image. The blue dotted outlines depict zoom-in regions. The black arrowheads label a part of the liver that was damaged during the optical clearing after microCT scanning. Scale bar 1 mm. (E) High pressure during resin injection can cause rupture of the portal vein (the animal in this panel carries a Jag1H268Q mutation), marked by blue arrowheads. Scale bar 1 mm. (F) Bubbles in the resin during portal vein injection (blue arrowheads) and (G) biliary system injection (blue arrowhead), scale bar 1 mm. (H) MicroCT scan of bubbles (blue arrowhead), poorly filled terminal branches (red brackets) and resin leakage (yellow arrowhead), scale bar 1 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62941/62941fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62941/62941fig06.jpg" /> 
Figure 6: Differential resin contrast. (A) Freshly opened yellow resin generates sufficient contrast to distinguish resin injected portal vein (grey) and bile ducts (white). (B) Three months storage of yellow resin lead to precipitation of the resin resulting in heterogeneous opacity (grey-white portal vein, blue arrowhead). (C) Prolonged storage (&#62;6 months) of yellow resin diminishes the contrast between the portal vein (grey) and bile ducts (grey). Scale bar 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62941/62941fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about DUCT: Double Resin Casting followed by Micro-Computed Tomography for 3D Liver Analysis at JoVE.com

DUCT: Double Resin Casting followed by Micro-Computed Tomography for 3D Liver Analysis

Double resin casting micro-computed tomography, or DUCT, enables visualization, digitalization, and segmentation of two tubular systems simultaneously to facilitate 3D analysis of organ architecture. DUCT combines ex vivo injection of two radiopaque resins followed by micro-computed tomography scanning and segmentation of the tomographic data.

The liver is the biggest internal organ in humans and mice, and high auto-fluorescence presents a significant challenge for assessing the ...

duct-double-resin-casting-followed-micro-computed-tomography-for-3d

Nanyang Technological University

Research

JoVE Journal

Medicine

3.3K Views.  Karolinska Institutet. Deze methode stelt ons in staat om de architectuur van levervaat- en galsystemen in 3D te analyseren, visualiseren en kwantificeren. We kunnen nu de ruimtelijke aspecten van leverziekte en regeneratie beter begrijpen. Het belangrijkste voordeel van deze techniek is dat het niet afhankelijk is van het bestaan van antilichamen of fluorescentiebeeldvorming.Het maakt ook directe visualisatie mogelijk van welke netwerken goed lumen zijn. Deze methode is geschikt voor de analyse van buisvorm...

Video: DUCT: Double Resin Casting followed by Micro-Computed Tomography for 3D Liver Analysis

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography (&mu;CT).
High resolution &mu;CT is a very versatile yet accurate (1-2 microns of resolution) technique for 3D examination of ex-vivo biological samples1, 2. As opposed to electron tomography, the &mu;CT allows the examination of up to 4 cm thick samples. This technique requires only few hours of measurement as compared to weeks in histology. In addition, &mu;CT does not rely on 2D stereologic models, thus it may complement and in some cases can even replace histological methods3, 4, which are both time consuming and destructive. Sample conditioning and positioning in &mu;CT is straightforward and does not require high vacuum or low temperatures, which may adversely affect the structure. The sample is positioned and rotated 180&deg; or 360&deg;between a microfocused x-ray source and a detector, which includes a scintillator and an accurate CCD camera, For each angle a 2D image is taken, and then the entire volume is reconstructed using one of the different available algorithms5-7. The 3D resolution increases with the decrease of the rotation step. The present video protocol shows the main steps in preparation, immobilization and positioning of the sample followed by imaging at high resolution.

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography ( CT).

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized ...

Bioengineering

3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System

In organs, the correct architecture of vascular and ductal structures is indispensable for proper physiological function, and the formation and maintenance of these structures is a highly regulated process. The analysis of these complex, 3-dimensional structures has greatly depended on either 2-dimensional examination in section or on dye injection studies. These techniques, however, are not able to provide a complete and quantifiable representation of the ductal or vascular structures they are intended to elucidate. Alternatively, the nature of 3-dimensional plastic resin casts generates a permanent snapshot of the system and is a novel and widely useful technique for visualizing and quantifying 3-dimensional structures and networks.
A crucial advantage of the resin casting system is the ability to determine the intact and connected, or communicating, structure of a blood vessel or duct. The structure of vascular and ductal networks are crucial for organ function, and this technique has the potential to aid study of vascular and ductal networks in several ways. Resin casting may be used to analyze normal morphology and functional architecture of a luminal structure, identify developmental morphogenetic changes, and uncover morphological differences in tissue architecture between normal and disease states. Previous work has utilized resin casting to study, for example, architectural and functional defects within the mouse intrahepatic bile duct system that were not reflected in 2-dimensional analysis of the structure1,2, alterations in brain vasculature of a Alzheimer's disease mouse model3, portal vein abnormalities in portal hypertensive and cirrhotic mice4, developmental steps in rat lymphatic maturation between immature and adult lungs5, immediate microvascular changes in the rat liver, pancreas, and kidney in response in to chemical injury6.
Here we present a method of generating a 3-dimensional resin cast of a mouse vascular or ductal network, focusing specifically on the portal vein and intrahepatic bile duct. These casts can be visualized by clearing or macerating the tissue and can then be analyzed. This technique can be applied to virtually any vascular or ductal system and would be directly applicable to any study inquiring into the development, function, maintenance, or injury of a 3-dimensional ductal or vascular structure.

A method of visualizing and quantifying the 3-dimensional structure of mouse hepatic portal vein or intrahepatic bile duct is described. This resin cast technique can also be applied to other ductal or vascular systems and allows for in situ visualization or quantification of a system's intact communicating architecture.

In  organs, the correct architecture of vascular and ductal structures is  indispensable for proper physiological function, and the formation and  ...

Vascular Casting of Adult and Early Postnatal Mouse Lungs for Micro-CT Imaging

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and behave by observing the surface of a tissue. This method provides a means to visualize the complex 3-dimensional vascular architecture of the lung.
To accomplish this, a catheter is inserted into the pulmonary artery and the vasculature is simultaneously flushed of blood and chemically dilated to limit resistance. Lungs are then inflated through the trachea at a standard pressure and the polymer compound is infused into the vascular bed at a standard flow rate. Once the entire arterial network is filled and allowed to cure, the lung vasculature may be visualized directly or imaged on a micro-CT (&#181;CT) scanner.
When performed successfully, one can appreciate the pulmonary arterial network in mice ranging from early postnatal ages to adults. Additionally, while demonstrated in the pulmonary arterial bed, this method can be applied to any vascular bed with optimized catheter placement and endpoints.

The aim of this technique is ex vivo visualization of pulmonary arterial networks of early postnatal and adult mice through lung inflation and injection of a radio-opaque polymer-based compound via the pulmonary artery. Potential applications for casted tissues are also discussed.

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and ...

Developmental Biology

Visualization of Vascular and Parenchymal Regeneration after 70% Partial Hepatectomy in Normal Mice

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the silicone compound, via a 26 G catheter, into the portal or hepatic vein. After silicone injection, organs were explanted and prepared for ex-vivo micro-CT (&#181;CT) scanning. The silicone injection procedure is technically challenging. Achieving a successful outcome requires extensive microsurgical experience from the surgeon. One of the challenges of this procedure involves determining the adequate perfusion rate for the silicone compound. The perfusion rate for the silicone compound needs to be defined based on the hemodynamic of the vascular system of interest. Inappropriate perfusion rate can lead to an incomplete perfusion, artificial dilation and rupturing of vascular trees.
The 3D reconstruction of the vascular system was based on CT scans and was achieved using preclinical software such as HepaVision. The quality of the reconstructed vascular tree was directly related to the quality of silicone perfusion. Subsequently computed vascular parameters indicative of vascular growth, such as total vascular volume, were calculated based on the vascular reconstructions. Contrasting the vascular tree with silicone allowed for subsequent histological work-up of the specimen after &#181;CT scanning. The specimen can be subjected to serial sectioning, histological analysis and whole slide scanning, and thereafter to 3D reconstruction of the vascular trees based on histological images. This is the prerequisite for the detection of molecular events and their distribution with respect to the vascular tree. This modified silicone injection procedure can also be used to visualize and reconstruct the vascular systems of other organs. This technique has the potential to be extensively applied to studies concerning vascular anatomy and growth in various animal and disease models.

Tools used for visualizing vascular regeneration require methods for contrasting the vascular trees. This film demonstrated a delicate injection technique used to achieve optimal contrasting of the vascular trees and illustrate the potential benefits resulting from a detailed analysis of the resulting specimen using &#181;CT and histological serial sections.

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the ...

3D Printing of Preclinical X-ray Computed Tomographic Data Sets

Three-dimensional printing allows for the production of highly detailed objects through a process known as additive manufacturing. Traditional, mold-injection methods to create models or parts have several limitations, the most important of which is a difficulty in making highly complex products in a timely, cost-effective manner.1 However, gradual improvements in three-dimensional printing technology have resulted in both high-end and economy instruments that are now available for the facile production of customized models.2 These printers have the ability to extrude high-resolution objects with enough detail to accurately represent in vivo images generated from a preclinical X-ray CT scanner. With proper data collection, surface rendering, and stereolithographic editing, it is now possible and inexpensive to rapidly produce detailed skeletal and soft tissue structures from X-ray CT data. Even in the early stages of development, the anatomical models produced by three-dimensional printing appeal to both educators and researchers who can utilize the technology to improve visualization proficiency. 3, 4 The real benefits of this method result from the tangible experience a researcher can have with data that cannot be adequately conveyed through a computer screen. The translation of pre-clinical 3D data to a physical object that is an exact copy of the test subject is a powerful tool for visualization and communication, especially for relating imaging research to students, or those in other fields. Here, we provide a detailed method for printing plastic models of bone and organ structures derived from X-ray CT scans utilizing an Albira X-ray CT system in conjunction with PMOD, ImageJ, Meshlab, Netfabb, and ReplicatorG software packages.

Using modern plastic extrusion and printing technologies, it is now possible to quickly and inexpensively produce physical models of X-ray CT data taken in a laboratory. The three -dimensional printing of tomographic data is a powerful visualization, research, and educational tool that may now be accessed by the preclinical imaging community.

Three-dimensional printing allows for the production of highly detailed objects through a process known as additive manufacturing.  Traditional, ...

Biology

3D Imaging of the Liver Extracellular Matrix in a Mouse Model of Non-Alcoholic Steatohepatitis

Non-alcoholic steatohepatitis (NASH) is the most common chronic liver disease in the United States, affecting more than 70 million Americans. NASH can progress to fibrosis and eventually to cirrhosis, a significant risk factor for hepatocellular carcinoma. The extracellular matrix (ECM) provides structural support and maintains liver homeostasis via matricellular signals. Liver fibrosis results from an imbalance in the dynamic ECM remodeling process and is characterized by excessive accumulation of structural elements and associated changes in glycosaminoglycans. The typical fibrosis pattern of NASH is called &#34;chicken wire,&#34; which usually consists of zone 3 perisinusoidal/pericellular fibrosis, based on features observed by Masson&#39;s trichrome stain and Picrosirius Red stains. However, these traditional thin two-dimensional (2D) tissue slide-based imaging techniques cannot demonstrate the detailed three-dimensional (3D) ECM structural changes, limiting the understanding of the dynamic ECM remodeling in liver fibrosis.
The current work optimized a fast and efficient protocol to image the native ECM structure in the liver via decellularization to address the above challenges. Mice were fed either with chow or fast-food diet for 14 weeks. Decellularization was performed after in situ portal vein perfusion, and the two-photon microscopy techniques were applied to image and analyze changes in the native ECM. The 3D images of the normal and NASH livers were reconstituted and analyzed. Performing in situ perfusion decellularization and analyzing the scaffold by two-photon microscopy provided a practical and reliable platform to visualize the dynamic ECM remodeling in the liver.

The present protocol optimizes the liver in situ perfusion/decellularization and two-photon microscopy methods to establish a reliable platform to visualize the dynamics of extracellular matrix (ECM) remodeling during non-alcoholic steatohepatitis (NASH).

Non-alcoholic steatohepatitis (NASH) is the most common chronic liver disease in the United States, affecting more than 70 million Americans. NASH can ...

Quantitative Analysis of Liver Stiffness Distribution

A Three-Dimensional Digital Model for Early Diagnosis of Hepatic Fibrosis Based on Magnetic Resonance Elastography

Hepatic fibrosis is an early stage of liver cirrhosis, and there are no better non-invasive and convenient methods for the detection and evaluation of the disease. Despite the good progress made with the liver stiffness map (LSM) based on magnetic resonance elastography (MRE), there are still some limitations that need to be overcome, including manual focus determination, manual selection of regions of interest (ROIs), and discontinuous LSM data without structural information, which makes it impossible to evaluate the liver as a whole. In this study, we propose a novel three-dimensional (3D) digital model for the early diagnosis of hepatic fibrosis based on MRE.
MRE is a non-invasive imaging technique that employs magnetic resonance imaging (MRI) to measure the liver stiffness at the scanning site through human-computer interaction. Studies have indicated a significant positive correlation between the LSM obtained through MRE and the degree of hepatic fibrosis. However, for clinical purposes, a comprehensive and precise quantification of the degree of hepatic fibrosis is necessary. To address this, the concept of Liver Stiffness Distribution (LSD) was proposed in this study, which refers to the 3D stiffness volume of each liver voxel obtained by the alignment of 3D liver tissue images and MRE indicators. This provides a more effective clinical tool for the diagnosis and treatment of hepatic fibrosis.

Author Spotlight: Advancing Hepatic Fibrosis Diagnosis Using Magnetic Resonance Elastography and AI 

The objective of this study was to develop a novel three-dimensional digital model for the early diagnosis of hepatic fibrosis, which includes the stiffness of each voxel in the patient's liver and can thus, be used to calculate the distribution ratio of the patient's liver at different fibrosis stages.

Hepatic fibrosis is an early stage of liver cirrhosis, and there are no better non-invasive and convenient methods for the detection and evaluation of the ...

Liver Transplantation in Mouse and Analyzing Allograft Patency

Successful Orthotopic Liver Transplantation in Mice Utilizing Microcomputed Tomography Angiography

Microcomputed tomography (microCT) angiography is an invaluable resource to researchers. New advances in this technology have allowed for high-quality images to be obtained of micro-vasculature and are high-fidelity tools in the field of organ transplantation. In this model of orthotopic liver transplantation (OLT) in mice, microCT affords the opportunity to evaluate allograft anastomosis in real time and has the added benefit of not having to sacrifice study animals. The choice of contrast, as well as image acquisition settings, create a high-definition image, which gives researchers invaluable information. This allows for evaluation of the technical aspects of the procedure as well as potentially evaluating different therapeutics over an extended duration of time. In this protocol, we detail an OLT model in mice in a stepwise fashion and finally describe a microCT protocol that can give high-quality images, which aid researchers in in-depth analysis of solid organ transplantation. We provide a step-by-step guide for liver transplantation in a mouse, as well as briefly discuss a protocol for evaluating the patency of the graft through microCT angiography.

Author Spotlight: Enhancing Transplantation Research Through MicroCT Angiography in Murine Models 

In this protocol, we discuss the implementation of a model of successful orthotopic liver transplantation (OLT) in mice. Additionally, adjuvants to further analyze allograft patency after successful OLT in a mouse are discussed as well, specifically utilizing microcomputed tomography (microCT) scans.

Microcomputed tomography (microCT) angiography is an invaluable resource to researchers. New advances in this technology have allowed for high-quality ...

A Comprehensive 3D-Liver Fat Function Distribution Model Using Dixon MRI

A 3D Quantification Technique for Liver Fat Fraction Distribution Analysis Using Dixon Magnetic Resonance Imaging

This study presents a 3D quantification methodology for the distribution of liver fat fraction (LFF) through the utilization of Dixon MRI image analysis. The central aim is to offer a highly accurate and non-invasive means of evaluating liver fat content. The process involves the acquisition of In-phase and Water-phase images from a Dixon sequence. LFF maps are then meticulously computed voxel by voxel by dividing the Lipid Phase images by the In-phase images. Simultaneously, 3D liver contours are extracted from the In-phase images. These crucial components are seamlessly integrated to construct a comprehensive 3D-LFF distribution model. This technique is not limited to healthy livers but extends to those afflicted by hepatic steatosis. The results obtained demonstrate the remarkable effectiveness of this approach in both visualizing and quantifying liver fat content. It distinctly discerns patterns that differentiate between normal and steatotic livers. By harnessing Dixon MRI to extract the 3D structure of the liver, this method offers precise LFF assessments spanning the entirety of the organ, thereby holding great promise for the diagnosis of hepatic steatosis with remarkable effectiveness.

Author Spotlight: A Non-Invasive Tool to Assess and Differentiate Fat Patterns in Liver Using 3D Dixon MRI 

This study introduces a unique 3D quantification method for liver fat fraction (LFF) distribution using Dixon Magnetic Resonance Imaging (Dixon MRI). LFF maps, derived from in-phase and water-phase images, are integrated with 3D liver contours to differentiate LFF patterns between normal and steatotic livers, enabling precise assessment of liver fat content.

This study presents a 3D quantification methodology for the distribution of liver fat fraction (LFF) through the utilization of Dixon MRI image analysis. ...

Novel In Vivo Micro-Computed Tomography Imaging Techniques for Assessing the Progression of Non-Alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease (NAFLD) is a growing global health issue, and the impact of NAFLD is compounded by the current lack of effective treatments. Considerable limiting factors hindering the timely and accurate diagnosis (including grading) and monitoring of NAFLD, as well as the development of potential therapies, are the current inadequacies in the characterization of the hepatic microenvironment structure and the scoring of the disease stage in a spatiotemporal and non-invasive manner. Using a diet-induced NAFLD mouse model, we investigated the use of in vivo micro-computed tomography (CT) imaging techniques as a non-invasive method to assess the progression stages of NAFLD, focusing predominantly on the hepatic vascular network due to its significant involvement in NAFLD-related hepatic dysregulation. This imaging methodology allows for longitudinal analysis of liver steatosis and functional tissue uptake, as well as the evaluation of the relative blood volume, portal vein diameter, and density of the vascular network. Understanding the adaptations of the hepatic vascular network during NAFLD progression and correlating this with other ways of characterizing the disease progression (steatosis, inflammation, fibrosis) using the proposed method can pave the way toward the establishment of new, more efficient, and reproducible approaches for NAFLD research in mice. This protocol is also expected to upgrade the value of preclinical animal models for investigating&#160;the development of novel therapies against disease progression.

Novel In Vivo Micro-Computed Tomography Imaging Techniques for Assessing the Progression of Non-Alcoholic Fatty Liver Disease

Using a diet-induced non-alcoholic fatty liver disease (NAFLD) mouse model, we describe the use of novel in vivo micro-computed tomography imaging techniques as a non-invasive method to assess the progression stages of NAFLD, focusing predominantly on the hepatic vascular network due to its significant involvement in NAFLD-related hepatic dysregulation.

DUCT: Double Resin Casting followed by Micro-Computed Tomography for 3D Liver Analysis

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