Name: A Comprehensive Rat Model For Assessing Hepatic Transport Pathways Through Simultaneous Lymphatic And Blood Vascular Sampling (Video) | JoVE Journal
Uploaded: 2026-05-15T13:20:00.000+00:00
Duration: 7 min 56 s
Description: Learn a surgical rat model for simultaneous hepatic lymph and blood vascular sampling to study hepatic transport, drug metabolism, and immune signaling. This method enables continuous lymph collection alongside arterial and venous blood sampling.

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The authors thank Preeti Yadav and Enyuan Cao for their contributions to the hepatic and mesenteric lymph collections used in the studies by Yadav et al. (2018) and Gracia et al. (2020), which provided foundational data referenced in this work. This work was supported by an Advanced Research Projects Agency for Health (ARPA-H) LIGHT program award. The views and conclusions contained in this document are those of the authors and should not be interpreted as representing the official policies, either expressed or implied, of the U.S. Government.

<ol>
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M. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+lipoprotein+of+density+less+than+1.006+g/ml+from+rat+hepatic+lymph.">Isolation and characterization of lipoprotein of density less than 1.006 g/ml from rat hepatic lymph.</a> J Lipid Res. 24 (7), 810-820 (1983).</li><li>Jeong, J., Tanaka, M., Iwakiri, Y. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+lymphatic+vascular+system+in+health+and+disease.">Hepatic lymphatic vascular system in health and disease.</a> J Hepatol. 77 (1), 206-218 (2022).</li><li>Tamburini, B. A. J., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+liver+disease+in+humans+causes+expansion+and+differentiation+of+liver+lymphatic+endothelial+cells.">Chronic liver disease in humans causes expansion and differentiation of liver lymphatic endothelial cells.</a> Front Immunol. 10, 1036 (2019).</li><li>Burchill, M. A., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidized+low-density+lipoprotein+drives+dysfunction+of+the+liver+lymphatic+system.">Oxidized low-density lipoprotein drives dysfunction of the liver lymphatic system.</a> Cell Mol Gastroenterol Hepatol. 11 (2), 573-595 (2021).</li><li>Gogineni, A., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+VEGF-C+modulates+distal+lymphatic+remodeling+and+secondary+metastasis.">Inhibition of VEGF-C modulates distal lymphatic remodeling and secondary metastasis.</a> PLoS One. 8 (7), e68755 (2013).</li><li>Trevaskis, N. 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(97), e52389 (2015).</li><li>Dahan, A., Mendelman, A., Amsili, S., Ezov, N., Hoffman, A. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+general+anesthesia+on+the+intestinal+lymphatic+transport+of+lipophilic+drugs.">The effect of general anesthesia on the intestinal lymphatic transport of lipophilic drugs.</a> Eur J Pharm Sci. 32 (4-5), 367-374 (2007).</li><li>Tanaka, M., Iwakiri, Y. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hepatic+lymphatic+vascular+system:+structure,+function,+markers,+and+lymphangiogenesis.">The hepatic lymphatic vascular system: structure, function, markers, and lymphangiogenesis.</a> Cell Mol Gastroenterol Hepatol. 2 (6), 733-749 (2016).</li></ol>

The authors declare no competing financial interests or personal relationships that could have influenced the work reported in this article.

Hepatic lymph duct cannulation is a technically difficult procedure, even for experienced microsurgeons. Several critical steps can improve the likelihood of preserving lymph duct integrity and achieving stable lymph flow. When identifying and isolating the hepatic lymph duct (Protocol steps 5.7-5.10), the initial step is to remove the connective tissue and fat overlying the mesenteric lymph duct and vena cava near the hepatic lymph duct. The hepatic lymph duct is translucent and fragile compared with the thicker and more opaque mesenteric lymph duct and can be easily ruptured if handled directly. The mesenteric lymph duct serves as a useful anatomical landmark, as clearance of its overlying tissue reveals the hepatic lymph duct located superiorly. Because the hepatic lymph duct often exhibits kinks or curvature, the operator should select a cannulation point along a straight segment and orient the cannula parallel to the duct&#8217;s natural course to minimize the risk of occlusion or tearing (step 5.11-5.14).
The cannulation itself is the most critical determinant of success. After puncturing the duct (step 5.13), intraluminal access can be confirmed visually by inserting the tip of the forceps into the opening and ensuring that only one vessel wall overlies the instrument. The appearance of two opposing walls indicates a through-and-through puncture. Once intraluminal access is confirmed, the cannula can be advanced smoothly along the duct at the same angle. Care should be taken to avoid damaging adjacent vasculature, as bleeding into the field can introduce blood into the lymph collection cannula, contaminating the sample and substantially increasing the risk of clot formation. After establishing lymph flow (step 5.15), the collecting end of the cannula should be capped, and veterinary adhesive should be applied carefully to prevent it from entering the lumen. The cannula must also be checked for air bubbles, as trapped air increases back pressure and can impede lymph flow. Because hepatic lymph is generated under low driving pressure, the collecting end of the cannula should always be positioned below the level of the surgical site to facilitate drainage by gravity.
Successful hepatic lymph duct cannulation is indicated by the immediate onset of lymph flow. Once established, hepatic lymph typically flows at approximately 0.1 mL/h, with stable output for several hours. The flow rate may vary depending on the animal&#8217;s anatomy, physiology, and experimental conditions. For example, lymph was collected under isoflurane anesthesia in this protocol. Isoflurane anesthesia may influence physiological parameters relevant to lymphatic function, and experimental conditions involving anesthesia should therefore be considered when interpreting lymph formation rates16. Hepatic lymph duct cannulation requires laparotomy and delicate microsurgical manipulation of abdominal lymphatic vessels, and therefore, lymph collection from conscious rats is not feasible in this model. Previous studies comparing lymph transport under anesthetized and conscious conditions or using different anesthetic regimens have shown that anesthesia may modestly influence absolute lymph flow rates but does not substantially alter overall lymphatic transport patterns4. Therefore, lymph flow values obtained in this model should be interpreted within the context of anesthetized experimental conditions.
A gradual reduction or cessation of flow during collection is most often caused by clot formation within the cannula, resulting from blood contamination or fibrin deposition. Flow can sometimes be restored by localizing and gently pinching the cannula with fine forceps to mechanically fragment the clot, or by gently aspirating through the cannula&#39;s collecting end to dislodge the obstruction. If the blockage is confined to the external portion, the cannula may be cut proximal to the clot and reconnected to fresh tubing using a sterile silicone connector. If the clot forms at the cannulation site, re-cannulation is usually required. In such cases, removal of the adhesive may be attempted with great care to avoid rupture of the duct or adjacent blood vessels. Hemostasis must be achieved before reattempting cannulation, as even minor blood leakage into the cannula markedly promotes clotting within the low-pressure hepatic lymph system.
Physiological factors also influence lymph flow and patency. Hepatic lymph flow in rats is inherently slower than mesenteric or thoracic duct flow, even under basal conditions17,18. Moreover, general anesthesia can reduce lymph flow in other lymphatic circuits compared with conscious, freely moving rats19. Because hepatic lymph cannulation requires anesthesia and laparotomy, these factors likely contribute to the relatively low flow rates and increased tendency to clot observed during prolonged collections. Maintaining hydration through the jugular vein or gastrointestinal cannula during collection helps to stabilize lymph flow and reduce clot formation. In this model, stable hepatic lymph flow is typically achievable for 2&#8211;3 hours, after which flow can deteriorate and clotting is more likely. However, under optimal experimental conditions, lymph collection can be maintained for longer periods, with a maximum duration of up to ~8 hours reported4.
The inclusion of hepatic artery and portal vein sampling (Figure 6&#160;and&#160;Figure 7) expands the applicability of the model by allowing direct comparison between hepatic inflow, lymphatic efflux, and systemic circulation. This terminal sampling step enables simultaneous assessment of hepatic microvascular transport and interstitial clearance within a single animal. Such comparisons provide valuable insights into the distribution of solutes between the hepatic vascular and lymphatic compartments, particularly for large macromolecules or lipophilic compounds that are dependent on interstitial diffusion.
Despite these challenges, the hepatic lymph cannulation model remains a valuable and informative technique. The liver contributes a significant proportion of total body lymph flow, and hepatic lymph contains proteins and other components that originate from hepatic interstitial fluid. By sampling lymph draining directly from the liver, this model enables analysis of molecules and cells leaving the liver before they are diluted by mesenteric and systemic lymph in the thoracic duct. Biochemical analysis of hepatic lymph provides insight into hepatic interstitial metabolism and lipid transport processes12,20. This complements information obtained from the portal or systemic blood, which integrates the effects of hepatic metabolism and extrahepatic exchange. Importantly, this configuration, combining hepatic lymph, carotid arterial, and jugular venous cannulations, permits dynamic assessment of solute gradients between hepatic efflux and vascular inflow, providing a more comprehensive view of hepatic transport and clearance.
Previous studies using this model demonstrate that hepatic lymph sampling enables evaluation of lymphatic recovery of macromolecular therapeutics and comparison of solute transport between hepatic lymph and systemic circulation. Together, hepatic lymph cannulation, concurrent vascular sampling, and biochemical profiling provide a robust experimental platform for studying hepatic microvascular and interstitial dynamics. The model enables simultaneous evaluation of hepatic transport, metabolism, lipid processing, and immune signalling, and can be adapted to investigate changes under pathophysiological conditions such as MASLD or hepatic inflammation.
In conclusion, this refined surgical model, integrating hepatic lymphatic, hepatic arterial, and portal venous access, offers a comprehensive and reproducible approach for examining hepatic transport pathways. It allows direct comparison of vascular inflow, interstitial efflux, and lymphatic drainage, which supports studies that provide mechanistic insight into liver-specific transport, metabolism, and pharmacokinetic processes. In addition, the ability to sample hepatic lymph provides a unique opportunity to study immune cell and antigen trafficking from the liver, offering valuable insights into hepatic immune surveillance, inflammation, and tolerance mechanisms.

The liver is a central metabolic and immunologic organ responsible for nutrient processing, xenobiotic detoxification, and immune surveillance. The liver is the largest lymph-producing organ in the body. Studies in large mammals suggest that it contributes 25%–50% of total lymph flow1,2. In rodents, cannulation studies indicate that approximately 10%–20% of the total thoracic lymph flow originates from the liver3,4. Hepatic lymph originates from plasma filtered across the fenestrated endothelium of the sinusoidal capillaries within the liver parenchyma. This filtrate collects in the space of Disse, an interstitial compartment between hepatocytes and sinusoidal endothelial cells. The filtrate then passes through the space of Mall into portal lymphatic capillaries that converge into collecting vessels draining toward the hepatic hilum and cisterna chyli5,6.
The hepatic lymphatic system is anatomically divided into portal, subcapsular, and sublobular networks6,7. Recent volumetric imaging studies demonstrate that hepatic lymphatics form a highly ramified Vascular Endothelial Growth Factor C (VEGF-C)–dependent network, largely confined to the portal tracts. This network is composed predominantly of lymphatic capillaries that drain retrogradely toward the portal vein. On the other hand, the subcapsular and sublobular lymphatics, distributed along the hepatic surface and in the central venous regions, respectively, are additional drainage pathways. Human postmortem imaging has further revealed a dense lymphatic vessel network along hepatic veins, indicating that periportal and hepatic venous lymphatics jointly regulate hepatic lymph outflow8.
Hepatic lymph represents a plasma-derived ultrafiltrate that reflects both metabolic and immune processes9,10. Biochemical analyses demonstrate that hepatic lymph contains albumin, lipoproteins, cytokines, and metabolites derived from hepatic parenchymal activity3,4,6, consistent with the general proteomic profile of lymphatic fluid reported in other tissues10. Early studies demonstrated that hepatic lymph contains distinct lipoprotein classes, with high-density lipoproteins (HDL) representing a dominant fraction, consistent with the liver’s central role in HDL synthesis and remodeling11. Notably, more recent work showed that hepatic lymph is enriched in HDL and cholesterol esters compared with mesenteric lymph, highlighting its distinct contribution to lipid trafficking and reverse cholesterol transport3. Moreover, hepatic lymph serves as an immunological conduit, carrying immune mediators, antigen-presenting cells, and extracellular vesicles that link hepatic microenvironments with systemic immunity9.
Alterations in hepatic lymph formation and drainage are key features of liver disease. In cirrhosis and portal hypertension, elevated sinusoidal pressure drives a several-fold increase in lymph production, leading to lymphangiectasia, fibrosis, and ascites once drainage capacity is exceeded. Chronic liver injury also promotes VEGF-C/VEGFR-3–dependent lymphangiogenesis, with excessive or aberrant lymphatic remodeling contributing to hepatic inflammation, edema, and lipid accumulation9,12. Furthermore, in metabolic dysfunction–associated steatotic liver disease (MASLD) and hepatocellular carcinoma, increased hepatic lymphatic density has been shown to correlate with disease progression and metastasis13,14.
Despite hepatic lymph's central physiological and clinical role, it remains understudied and underexplored in experimental research, primarily because of technical challenges in isolating pure hepatic lymph. Thoracic and mesenteric lymph collection models have been more widely described, but these provide only indirect insight into hepatic transport mechanisms. A limitation in the field was addressed by developing a rat hepatic lymph cannulation model, which enabled direct collection of hepatic lymph4. Using this approach, it was shown that most lymphatic uptake of intravenously administered therapeutic proteins occurs in the liver and mesentery. These findings highlight hepatic lymph as a key route for macromolecular exchange between the blood and lymphatic systems, with important implications for the pharmacokinetics and distribution of biologic therapeutics4.
To bridge remaining gaps, the present study describes a comprehensive rat model enabling simultaneous sampling of hepatic lymph and systemic blood via cannulation of the hepatic lymph duct, carotid artery, and jugular vein. This approach allows real-time assessment of solute transport across and exchange between hepatic interstitial and systemic compartments, providing a robust experimental platform for investigating hepatic transport, metabolism, immune signalling, and drug disposition under physiological and pathological conditions. The model also encompasses optional terminal sampling of the hepatic artery and portal vein to enable direct comparison of hepatic inflow, outflow, and lymphatic drainage.
<img alt="Rodent cardiovascular anatomy diagram; blood and lymphatic sampling sites for experimental infusion." class="xfigimg" src="/files/ftp_upload/70986/70986fig1v2.jpg" data-altupdatedat="1787025648000" data-alt="Figure 1"> 
Figure 1. Schematic of the multi-cannulation model for simultaneous lymphatic and blood sampling. A comprehensive cannulation model illustrating simultaneous sampling from the carotid artery, jugular vein, hepatic artery, hepatic lymph duct, and portal vein. (A) The carotid artery is cannulated for intermittent blood sampling. (B) The external jugular vein is cannulated for continuous saline infusion to maintain hydration and compensate for fluid loss during sampling. (C) The common hepatic artery, which runs parallel to the hepatic lymph duct, is cannulated for terminal blood sampling. (D) The hepatic lymph duct is cannulated for continuous lymph collection. (E) The portal vein is accessed via needle puncture for terminal blood sampling. Procedures shown in (A), (B), and (D) are performed prior to terminal sampling, followed by (C) hepatic artery sampling and subsequently (E) portal vein sampling. Abbreviations: EJV, external jugular vein; CA, carotid artery; SVC, superior vena cava; IVC, inferior vena cava; Ao, aorta; CHA, common hepatic artery; HL, hepatic lymph duct; ML, mesenteric lymph duct; SMA, superior mesenteric artery; PV, portal vein. <a href="https://www.jove.com/files/ftp_upload/70986/70986fig1largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>Anticoagulant solution (EDTA 0.1%)</td><td>Invitrogen</td><td>AM9261</td><td>Prevents clot formation in cannulas and collected samples</td></tr><tr><td>Anticoagulant solution (Heparin 20 IU/mL)</td><td>Pfizer</td><td>00409-2723-01</td><td>Prevents clot formation in cannulas and collected samples</td></tr><tr><td>Chlorhexidine (0.5%)</td><td>McFarlane Medical</td><td>12120-OR</td><td>Used for surgical site disinfection</td></tr><tr><td>Collection tubes (microcentrifuge)</td><td>Greiner Bio-One</td><td>616201</td><td>Used for lymph and blood collection</td></tr><tr><td>Cotton gauze</td><td>Winc Australia</td><td>18874125</td><td>Used for organ manipulation and moisture maintenance</td></tr><tr><td>Cotton swabs</td><td>Pacific Laboratory</td><td>1005199</td><td>Used for organ repositioning during surgery</td></tr><tr><td>Curved iris forceps</td><td>Macquarie Medical Systems</td><td>TTP5002-5</td><td>Used for vessel isolation and tissue manipulation</td></tr><tr><td>Dumont forceps</td><td>Fine Science Tools</td><td>11251-35</td><td>Used to open and stabilize the vessel lumen of the hepatic artery for cannula insertion</td></tr><tr><td>Ethanol (70%)</td><td>Ajax</td><td>AJA214-2.5PL</td><td>Used for surgical site disinfection</td></tr><tr><td>Halsted-Mosquito hemostat forceps</td><td>Fine Science Tools</td><td>13009-12</td><td>Used to bend the 25G needle for hepatic lymph duct puncture</td></tr><tr><td>Heated surgical pad (37 &amp;deg;C)</td><td>Ratek</td><td>IC-WT1</td><td>Maintains body temperature during surgery</td></tr><tr><td>Induction chamber</td><td>Darvall</td><td>1859</td><td>Used for initial anesthesia induction</td></tr><tr><td>Infusion pump</td><td>Adelab Scientific</td><td>NE-300</td><td>Provides controlled saline infusion via cannula</td></tr><tr><td>Iris scissors</td><td>Stark Medical</td><td>E-AC985-11</td><td>Used for skin and muscle incisions</td></tr><tr><td>Isoflurane</td><td>Provet Pty Ltd</td><td>ISOF07</td><td>Inhalation anesthetic used for induction and maintenance of anesthesia</td></tr><tr><td>Kelman-McPherson lens forceps</td><td>ProSciTech</td><td>TY-1301</td><td>Used to open and stabilize the vessel lumen of the jugular vein and carotid artery for cannula insertion</td></tr><tr><td>Needle (23G &amp;times; 1&quot;)</td><td>Livingstone</td><td>DN23GX1.0LV</td><td>Used for portal vein puncture and blood sampling</td></tr><tr><td>Needle (25G &amp;times; 1&quot;)</td><td>Livingstone</td><td>DN25GX1.0LV</td><td>Used for cannula flushing and hepatic lymph duct puncture</td></tr><tr><td>Nose cone anesthesia system</td><td>Darvall</td><td>7885</td><td>Maintains anesthesia during surgery</td></tr><tr><td>Oxygen supply with flowmeter</td><td>BOC</td><td>400NE</td><td>Carrier gas for isoflurane anesthesia</td></tr><tr><td>Polyethylene cannula (0.8 mm O.D., 0.5 mm I.D.)</td><td>Microtube Extrusions</td><td>PE8050</td><td>Used for carotid artery, jugular vein, and hepatic lymph duct cannulation</td></tr><tr><td>Polyvinyl cannula (0.61 mm O.D., 0.28 mm I.D.)</td><td>Microtube Extrusions</td><td>PIV6128</td><td>Used for hepatic artery cannulation</td></tr><tr><td>Silk sutures (4-0)</td><td>SMI AG</td><td>SM8015</td><td>Used to secure vessels and cannulas</td></tr><tr><td>Sodium pentobarbitone</td><td>Lyppard Australia Pty Ltd</td><td>LETH250</td><td>Used for euthanasia (&gt;100 mg/kg)</td></tr><tr><td>Sterile saline (0.9% NaCl)</td><td>FreeFlex</td><td>K690531</td><td>Used for rehydration and flushing</td></tr><tr><td>Straight Graefe forceps</td><td>Fine Science Tools</td><td>11150-10</td><td>Used for delicate vessel isolation</td></tr><tr><td>Surgical microscope</td><td>Zeiss</td><td>Stemi 2000-CS</td><td>Used for visualization during hepatic lymph duct cannulation</td></tr><tr><td>Surgical sterile blades (for scalpel use)</td><td>Livingstone</td><td>SBLDCL15</td><td>Used for bevelling cannula tips during preparation</td></tr><tr><td>Syringe (1 mL)</td><td>Terumo</td><td>19032-TE</td><td>Used for flushing cannulas</td></tr><tr><td>Syringe (3 mL)</td><td>Terumo</td><td>19048-TE</td><td>Used for blood sampling</td></tr><tr><td>Syringe (30 mL)</td><td>Terumo</td><td>19034-TE</td><td>Used for saline infusion</td></tr><tr><td>Vannas scissors</td><td>Fine Science Tools</td><td>91500-09</td><td>Used for vascular incisions</td></tr><tr><td>Veterinary tissue adhesive</td><td>3M</td><td>1469SB</td><td>Used to secure hepatic lymph duct cannula</td></tr></tbody></table>

a comprehensive rat model for assessing hepatic transport pathways through simultaneous lymphatic and blood vascular sampling

A Comprehensive Rat Model For Assessing Hepatic Transport Pathways Through Simultaneous Lymphatic And Blood Vascular Sampling

The liver is a central metabolic and immunologic hub that processes nutrients, xenobiotics, and circulating immune mediators. Substances reach the hepatocytes and other hepatic cells via the sinusoidal capillaries that receive blood from the intestine via the portal vein and from the systemic circulation via the hepatic artery. Plasma filtrate from the sinusoidal capillaries drains into the space of Disse, where it can undergo metabolic transformation, participate in immune surveillance, or flow into lymphatic vessels to form hepatic lymph. Sampling of hepatic lymph and blood can help enable detailed analysis of hepatic transport, metabolic processes, and immune activity. A surgical rat model allowing simultaneous sampling of hepatic lymph outflow and systemic blood through three distinct cannulation sites is described: hepatic lymph duct, carotid artery, and jugular vein. Hepatic lymph and arterial cannulations enable continuous sampling, whereas the jugular vein cannula can be used for continuous hydration and fluid support or for venous blood collection, allowing comparison between arterial and venous blood. Successful cannulation yields hepatic lymph flow rates of approximately 0.1 mL/h in anesthetized rats, enabling continuous lymph sampling of up to 8 hours while simultaneous arterial and venous blood sampling is performed. This setup allows direct comparison between systemic arterial blood and hepatic lymphatic outflow, enabling comprehensive examination of hepatic transport and clearance, as well as metabolic and immune signaling processes. To further extend the model, terminal sampling of the hepatic artery and portal vein can be performed at the end of the procedure to enable direct comparison of hepatic inflow, outflow, and lymphatic drainage. Ultimately, these techniques provide a versatile platform for evaluating hepatic processes, including drug pharmacokinetics, nutrient flux and metabolism, and liver-specific responses, under various pathophysiological conditions.

The surgical approach described in the Protocol section allows clear identification of the hepatic lymph duct, located medial to the right kidney and running parallel to the celiac and hepatic arteries, as previously reported4. Representative anatomy and cannulation are shown in Figure 5. Successful cannulation is confirmed by the immediate appearance of clear to slightly turbid lymph fluid, free of milky chylomicron contamination, suggesting minimal mixing with mesenteric lymph. This observation is consistent with the reported characteristics of hepatic lymph3. Identification of hepatic lymph was based on its anatomical position relative to the hepatic artery and inferior vena cava, as well as its characteristic clear appearance, in contrast to the typically opaque or milky appearance of mesenteric lymph.
Hepatic lymph flow should remain stable throughout the collection period, with an average rate of approximately 0.1 mL/h3,4,11. Hepatic lymph flow typically remains most stable for approximately 2–3 h following cannulation under anesthetized conditions, although longer collection periods (up to ~6–8 h) may be achievable under optimal experimental conditions. Representative lymph flow values reported in previous studies4 are summarized in Table 1. Hepatic lymph flow is generally lower than that reported for mesenteric and thoracic lymph duct flow rates in anesthetised rats.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Lymphatic sites (conscious state)</td>
			<td>Mean lymph flow rate (mL/h)</td>
		</tr>
		<tr>
			<td>Thoracic lymph (conscious)</td>
			<td>1.74 ± 0.33</td>
		</tr>
		<tr>
			<td>Thoracic lymph (anesthetized)</td>
			<td>0.65 ± 0.10</td>
		</tr>
		<tr>
			<td>Mesenteric lymph (anesthetized)</td>
			<td>0.45 ± 0.11</td>
		</tr>
		<tr>
			<td>Hepatic lymph (anesthetized)</td>
			<td>0.12 ± 0.02</td>
		</tr>
	</tbody>
</table>
Table 1: Mean lymph flow rates (mL/h) through thoracic, mesenteric, and hepatic lymph duct cannulations in conscious and anesthetized rats. Data are presented as mean ± SEM for n = 3–5 rats. Adapted from Table 3 in Yadav et al. (2018)4.
Representative outcomes illustrating lymphatic transport of macromolecules are shown in Figure 8. These examples demonstrate how the model can be used to evaluate lymphatic recovery following intravenous administration of therapeutic macromolecules, including approximately 60 kDa pegylated interferon α2a (IFN-PEG40), approximately 31 kDa pegylated interferon α2b (IFN-PEG12), and the monoclonal antibody trastuzumab, suggesting that the liver is a major site of extravasation and lymphatic access for macromolecular therapeutics4.
<img alt="Bar chart; Trastuzumab, IFN-PEG40, IFN-PEG12 dose recovery, thoracic, hepatic, mesenteric regions." class="xfigimg" src="/files/ftp_upload/70986/70986fig8.jpg" data-altupdatedat="1787025648000" data-alt="Figure 8"> 
Figure 8: Lymphatic recovery of intravenously administered macromolecular therapeutics across thoracic, mesenteric, and hepatic lymph. Total percentage of dose recovered in thoracic, mesenteric, and hepatic lymph following intravenous administration of PEGylated interferons of different molecular sizes (IFN-PEG12 and IFN-PEG40) and a monoclonal antibody (trastuzumab). Data represent mean ± SEM of dose recovery over 8 hours. Adapted from Yadav et al. (2018)4. <a href="https://www.jove.com/files/ftp_upload/70986/70986fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The model also enables detailed lipoprotein profiling of hepatic lymph3. Representative analyses of lymph lipid composition are shown in Figure 9. Compared with thoracic and mesenteric lymph, hepatic lymph exhibits a markedly lower total lipid content but protein concentrations comparable to those of other lymph sources. Lipoprotein analysis of hepatic lymph highlights the liver’s unique role in lipid trafficking and reverse cholesterol transport. Representative lipid composition data are summarized in Table 2. Hepatic lymph HDL is enriched in cholesteryl esters, mesenteric lymph HDL is predominantly triglyceride-rich, and thoracic lymph HDL contains relatively higher phospholipid content. These compositional differences indicate distinct roles of each lymphatic source in lipid metabolism and transport.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="4">%wt/wt</td>
		</tr>
		<tr>
			<td>Fractions</td>
			<td>Phospholipid</td>
			<td>Free cholesterol</td>
			<td>Cholesteryl ester</td>
			<td>Triglyceride</td>
		</tr>
		<tr>
			<td>Mesenteric lymph</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>D-HDL</td>
			<td>20 ± 3</td>
			<td>1 ± 0</td>
			<td>5 ± 1</td>
			<td>16 ± 1</td>
		</tr>
		<tr>
			<td>L-HDL</td>
			<td>21 ± 4</td>
			<td>2 ± 0</td>
			<td>8 ± 2</td>
			<td>23 ± 5</td>
		</tr>
		<tr>
			<td>Hepatic lymph</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>D-HDL</td>
			<td>24 ± 1</td>
			<td>3 ± 0</td>
			<td>12 ± 3</td>
			<td>8 ± 1</td>
		</tr>
		<tr>
			<td>L-HDL</td>
			<td>23 ± 1</td>
			<td>6 ± 1</td>
			<td>13 ± 2</td>
			<td>14 ± 2</td>
		</tr>
		<tr>
			<td>Thoracic lymph</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>D-HDL</td>
			<td>25 ± 1</td>
			<td>1 ± 0</td>
			<td>7 ± 0</td>
			<td>11 ± 3</td>
		</tr>
		<tr>
			<td>L-HDL</td>
			<td>29 ± 1</td>
			<td>2 ± 0</td>
			<td>11 ± 1</td>
			<td>11 ± 0</td>
		</tr>
		<tr>
			<td>Plasma</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>D-HDL</td>
			<td>29 ± 2</td>
			<td>2 ± 0</td>
			<td>20 ± 3</td>
			<td>2 ± 0</td>
		</tr>
		<tr>
			<td>L-HDL</td>
			<td>36 ± 6</td>
			<td>2 ± 0</td>
			<td>18 ± 3</td>
			<td>4 ± 1</td>
		</tr>
	</tbody>
</table>
Table 2: Lipid composition of density (D-HDL) and light (L-HDL) high-density lipoprotein fractions isolated from mesenteric, hepatic, and thoracic lymph, and plasma. Values represent the percentage by weight (% wt/wt) of phospholipid, free cholesterol, cholesteryl ester, and triglyceride relative to the total lipid fraction. Data are presented as mean ± SEM. Adapted from Table 1 in Gracia et al. (2020)3.
<img alt="Protein and lipid proportions in lymph; three pie charts depicting mesenteric, hepatic, thoracic." class="xfigimg" src="/files/ftp_upload/70986/70986fig9.jpg" data-altupdatedat="1787025648000" data-alt="Figure 9"> 
Figure 9: Protein and lipid composition of mesenteric, hepatic, and thoracic lymph. Proportional content of protein and total lipid in mesenteric, hepatic, and thoracic lymph. Lipid content includes phospholipids (PL), triglycerides (TG), cholesteryl esters (CE), and free cholesterol (FC), expressed as the percentage of lipid weight relative to the total weight of protein and lipid. Data are presented as mean ± SEM. Adapted from Gracia et al. (2020)3. <a href="https://www.jove.com/files/ftp_upload/70986/70986fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

This experimental protocol describes a hepatic lymph duct-cannulated rat model that enables direct and quantitative assessment of hepatic lymphatic and vascular drainage. The model allows simultaneous sampling of hepatic lymph and systemic blood to investigate hepatic transport, metabolism, and immune signaling, supporting studies of drug pharmacokinetics and liver-specific biology.

The liver is a central metabolic and immunologic hub that processes nutrients, xenobiotics, and circulating immune mediators. Substances reach the ...

Our lab studies the role of the lymphatic system in inflammatory and cardiometabolic diseases and develops drug delivery strategies to target the lymphatics to treat these diseases. This protocol enables evaluation of hepatic processes, including drug metabolism, nutrient flux, immune signaling and liver response under diverse conditions. To begin, place an anesthetized male Sprague-Dawley rat weighing approximately 250-300 grams that has been successfully cannulated via the carotid artery and jugular vein in dorsal recumbency under a surgical microscope.Make a 4-5 centimeter horizontal skin incision from the midline to the right flank, approximately 1.5-2 centimeters below the ribcage. Retract the skin and make matching incisions in the external and internal oblique muscles. Cauterize or ligate any large blood vessels encountered during the procedure.Use saline-moistened cotton swabs to move the liver toward the diaphragm, the stomach to the left, and the small intestine inferiorly. Keep the organs retracted with saline-soaked gauze. Identify the hepatic lymph duct running parallel to and slightly caudal to the hepatic artery, medial to the right kidney and adjacent to the inferior vena cava.Use the larger opaque or white mesenteric lymph duct as an anatomical landmark to identify the translucent and narrow hepatic lymph duct located caudally. Use straight Graefe forceps to remove the connective tissue overlying the lymph duct and vena cava. Insert the forceps through the fat bed beneath the right kidney to create a tunnel under the vena cava.Advance the forceps until the tips emerge parallel to the hepatic lymph duct on the opposite side of the vena cava. Use the forceps to grasp the external end of the pre-prepared cannula and pull the cannula through the tunnel until its tip emerges on the opposite side. Use the Halsted Mosquito Hemostat forceps to bend the tip of a 25-gauge needle to approximately 90 degrees.Using the bent needle, carefully puncture the hepatic lymph duct on the right side closest to the operator. Check the cannula pre-filled with anticoagulant solution and remove air bubbles, if any. Insert the beveled end approximately 2 millimeters into the duct.Confirm lymph flow at the external end of the cannula at approximately 100 microliters per hour. Adjust or reinsert the cannula if no flow is observed. Plug the cannula at the collecting end, then apply a small drop of veterinary adhesive to secure the insertion site.Once the adhesive is dry, confirm lymph flow through the tubing. Afterward, remove the gauze and return the organs to their original positions. Place the collection tube containing the anticoagulant solution below the cannula outlet and confirm that lymph is flowing drop-wise into the tube.Then close the abdominal wall by suturing the internal oblique, external oblique and skin layers sequentially. Immediately after the hepatic lymph cannulation, initiate rehydration by infusing sterile saline at 1.5-3.0 milliliters per hour through the jugular vein cannula. Monitor breathing and reflexes while maintaining anesthesia via a nose cone.After therapeutic dosing via the jugular vein cannula, collect hepatic lymph continuously into tubes containing anticoagulant solution. Replace the collection tubes hourly or as needed. Collect carotid artery blood samples at designated time points, keeping total volume below 10%of the estimated circulating blood volume before terminal sampling.Reopen the abdominal incision to expose the upper abdominal cavity while maintaining deep anesthesia and stable respiration. Use saline-moistened gauze to gently retract the liver toward the diaphragm, the stomach to the left, and the small intestine inferiorly. Identify the hepatic artery running parallel to the hepatic lymph duct situated adjacent to the mesenteric lymph duct.Obtain a polyvinyl cannula with an outer diameter of 0.61 millimeters and an inner diameter of 0.28 millimeters pre-filled with an anticoagulant solution and remove any air bubbles. Using curved Iris forceps, isolate a short segment of the hepatic artery. Place a 4-0 silk suture beneath the artery.Gently lift the artery and place a second suture distal to the first. Tie the distal suture to control backflow while leaving the proximal suture untied for later closure. Using fine point micro scissors, make a small transverse incision approximately 0.5 millimeters in the artery wall between the sutures.Use the tip of the Dumont forceps to lift the vessel flap to stabilize the vessel lumen for cannula insertion. Insert the beveled end of the polyvinyl cannula into the opening and advance the cannula approximately 2-3 millimeters. Secure the cannula by tying the proximal suture around both the vessel and cannula.Confirm correct placement by gently aspirating a small amount of bright red arterial blood. Collect 0.5-1 milliliter of blood into a pre-labeled tube containing anticoagulant. Lymphatic recovery of intravenously administered macromolecular therapeutics across thoracic, hepatic and mesenteric lymph demonstrates the model's utility for evaluating lymphatic transport.Hepatic lymph has a markedly lower total lipid content than thoracic and mesenteric lymph, while its protein concentration is comparable to that of the other lymph sources. This protocol enables simultaneous measurements of hepatic lymph and blood to evaluate transport, metabolism and clearance of molecules including drugs, nutrients and signals. Precise cannulation of the hepatic lymph duct is technically challenging and requires careful anatomical identification and surgical technique.Future studies can use this technique to map hepatic transport, clearance and metabolism, advancing understanding of liver diseases and targeted therapies.

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JoVE Journal

Immunology and Infection

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