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

Summary

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.

Abstract

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.

Introduction

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 flow^1,2. In rodents, cannulation studies indicate that approximately 10%–20% of the total thoracic lymph flow originates from the liver^3,4. 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 chyli^5,6.

The hepatic lymphatic system is anatomically divided into portal, subcapsular, and sublobular networks^6,7. 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 processes^9,10. Biochemical analyses demonstrate that hepatic lymph contains albumin, lipoproteins, cytokines, and metabolites derived from hepatic parenchymal activity^3,4,6, 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 accumulation^9,12. 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 metastasis^13,14.

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.

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. Please click here to view a larger version of this figure.

Protocol

All experimental procedures involving animals were reviewed and approved by the Monash Institute of Pharmaceutical Sciences Animal Ethics Committee (Ethics ID #46283) and were conducted in strict accordance with the Australian and New Zealand Council for the Care of Animals in Research and Teaching (ANZCCART) guidelines and the Australian Code for the Care and Use of Animals for Scientific Purposes (National Health and Medical Research Council, Australia)¹⁵.

Animal models: Male or female outbred albino Rattus norvegicus, or Sprague–Dawley (SD) rats weighing 250–350 g were used. Animals were obtained from licensed laboratory animal suppliers (Monash Animal Research Platform) and housed in individually ventilated cages under controlled temperature (22 ± 2 °C) and a 12 h light–dark cycle, with free access to food and water prior to experimentation. Animals were acclimatized for at least one week before surgery to minimize stress-related physiological variation.

A hepatic lymph duct, carotid artery, and jugular vein cannulation model enabling simultaneous sampling of hepatic lymph and systemic blood is described. Optional terminal sampling of the hepatic artery and portal vein may be performed under anesthesia for comparative analysis of hepatic inflow and lymphatic outflow. The reagents and equipment used are listed in the Materials Table.

1. Preparations on the day before surgery

1. Fast the rat overnight if required for the specific experimental protocol. Ensure free access to water at all times.
2. Prepare sterile saline for intravenous infusion during lymph collection. Determine the required volume based on collection duration (typically 3–5 hours) and a suggested infusion rate of 1.5–3.0 mL/h to replace fluid loss due to lymph and blood collection as well as physiological losses such as urine output.
3. Prepare three polyethylene cannulas for hepatic lymph duct, carotid artery, and jugular vein cannulation as follows:
   1. Hepatic lymph duct
      1. Cut a 0.8 mm outer diameter (O.D.), 0.5 mm inner diameter (I.D.) cannula to 30–35 cm. Ensure the length allows collection without tension.
      2. Bevel the insertion tip at approximately 45° with a sterile surgical blade to ease insertion.
   2. Carotid artery
      1. Cut a 0.8 mm O.D., 0.5 mm I.D. cannula to 20–30 cm. Mark approximately 2–2.5 cm from the internal end to indicate insertion depth.
   3. Jugular vein
      1. Cut a 0.8 mm O.D., 0.5 mm I.D. or 0.96 mm O.D., 0.58 mm I.D. cannula to 50 cm or longer. Mark 1–1.5 cm from the internal end for insertion depth. Ensure the length can reach the infusion pump without tension.
4. Prepare an anticoagulant solution consisting of either 0.1% EDTA in sterile water or 20 IU/mL heparin in sterile saline.
5. Fill a 1 mL syringe with anticoagulant solution and attach a 25G needle to the 0.8 mm O.D. cannulas or a 23G needle to the 0.96 mm O.D. cannulas.
6. Flush each cannula with approximately three times its internal volume, ensuring no air bubbles remain.
7. Keep the cannulas filled with anticoagulant and plugged with the attached needle and syringe overnight. Pre-soaking with an anticoagulant minimizes clot formation.
8. Prepare collection tubes for blood and lymph. Add anticoagulant solution to achieve a final concentration of 1 mg/mL EDTA or 10 IU/mL heparin after sample collection.

2. Preparations immediately prior to surgery

1. Flush all cannulas with anticoagulant to confirm patency. Ensure no air bubbles are present.
2. Induce anesthesia in the rat with 5% isoflurane in oxygen at 1 L/min in an induction chamber.
3. Monitor respiration and ensure that the rate has slowed to approximately 1.5 breaths per second (approximately 90 breaths/min), indicating adequate anesthetic depth.
   CAUTION: Isoflurane is a volatile anesthetic and should be used within a properly ventilated anesthetic system or fume hood to minimize occupational exposure.
4. Transfer the rat to a heated surgical pad (37°C) and maintain anesthesia with 1–3% isoflurane in oxygen delivered at 0.4–0.6 L/min via a nose cone.
5. Confirm adequate anesthetic depth by the absence of corneal and toe-pinch reflexes. Maintain anesthesia throughout the surgical procedure and lymph collection period.
   NOTE: Continuous anesthesia is essential to prevent pain during surgery and prevent cannula dislodgment due to movement.
6. Shave surgical regions, including the right abdominal quadrant (midline to right flank, approximately 2 cm below the ribcage) and the right or left clavicular region.
7. Disinfect all surgical areas using 0.5% chlorhexidine followed by 70% ethanol.

3. Cannulation of the carotid artery

NOTE: The carotid artery cannulation may be performed before or after cannulation of the jugular vein. The same cervical incision may be used for both procedures.

1. Position the rat in dorsal recumbency with the neck extended. Make a 1–1.5 cm cervical incision to expose the surgical field.
2. Place a small support (e.g., 3 mL syringe) beneath the neck to elevate the carotid region.
   NOTE: This step is optional.
3. Separate the neck muscles to expose the carotid artery approximately 1 cm below the skin surface.
4. Isolate the artery using curved iris forceps. Identify and gently separate the adjacent vagus nerve to prevent damage to the nerve.
5. Place straight Graefe forceps under the artery to occlude blood flow and thread two 4-0 silk sutures beneath it. Tie the proximal (cranial) suture securely.
6. Make a small incision on the artery surface.
7. Gently lift the vessel flap with the tip of the lens forceps to stabilize the vessel lumen for cannula insertion.
8. Insert the cannula to the pre-marked 2–2.5 cm depth. Expect mild backflow; if excessive, re-occlude and clean before proceeding.
   CAUTION: Vascular puncture and cannulation involve sharp instruments and should be performed carefully to prevent accidental injury.
9. Confirm correct placement by gently withdrawing a small volume of blood and infusing anticoagulant solution back into the cannula without feeling resistance or observing subcutaneous swelling.
10. Secure the cannula with sutures and seal the external end with a plug.
11. The procedure may be paused briefly after carotid artery cannulation while maintaining anesthesia.
12. Close the wound on the neck with sterile sutures. An overview of carotid artery isolation and successful cannulation is shown in Figure 2.

Figure 2: Carotid artery cannulation. (A) The paired neck muscles flanking the carotid artery (orange arrows). The salivary gland (blue arrows) is tucked cranially. (B) The exposed carotid artery is approximately 1 cm beneath the skin surface. The salivary gland (blue arrows) is tucked cranially. (C) Surgical setup prior to cannula insertion, showing occlusion of blood flow with straight Graefe forceps and two silk sutures isolating the artery, with its superior end tied off. (D) Approximately 2 cm of the cannula is successfully inserted into the carotid artery (yellow arrows). The carotid artery cannula is indicated by the green arrows. (E) Successful cannulation of the carotid artery was confirmed by drawing blood into the cannula. (F) Three sutures (size 4-0) are tied around the carotid artery and cannula for securement (purple arrows). Please click here to view a larger version of this figure.

4. Cannulation of the jugular vein

1. Maintain the rat in dorsal recumbency with the neck extended and head turned slightly to the left.
2. Using the existing cervical incision, identify the jugular vein above the clavicle by its location and visible blood flow.
3. Bluntly dissect the subcutaneous tissue to expose the vein.
4. Separate the vein from the surrounding connective tissue using curved iris forceps. Confirm isolation by passing forceps beneath the vein.
5. Place two 4-0 silk sutures under the vein at each end of the exposed segment. Tie the cranial (superior) suture securely to occlude blood flow.
6. To prevent the backflow of blood, place straight Graefe forceps beneath the vein and rest the handles on a syringe or support.
7. Make a small (~1 mm) incision on the top of the vein one-third along the isolated segment using iris scissors.
8. Gently lift the vessel flap with the tip of the lens forceps to stabilize the vessel lumen for cannula insertion.
9. Insert the cannula tip into the incision using iris forceps and advance it to the pre-marked depth (1–1.5 cm).
10. Release the occlusion once the cannula is stabilized to allow blood flow.
    NOTE: Maintain a stable grip to prevent venous backflow. If bleeding occurs, re-occlude immediately and clean the field before proceeding.
11. Confirm correct placement by gently withdrawing a small volume of blood and infusing anticoagulant solution back into the cannula without feeling resistance or observing subcutaneous swelling.
12. Secure the cannula with sutures at both the proximal and distal ends.
13. Temporarily occlude the external tubing, remove the syringe, and seal the cannula with a plug.
14. Cover the incision with gauze moistened with warm saline until the carotid artery cannulation is complete. Representative images of jugular vein isolation and cannulation are shown in Figure 3.

Figure 3: Jugular vein cannulation. (A) The jugular vein (black dotted outline) is located just under the skin above the clavicle. The established carotid artery cannula can be observed proximate to the jugular vein (green arrows). (B) The jugular vein is isolated with two silk sutures; the superior end is tied off. (C) Approximately 1 cm of the cannula is successfully inserted into the jugular vein (yellow arrows). The jugular vein cannula is indicated by the blue arrows; the carotid artery cannula is indicated by the green arrows. (D) Three sutures (size 4-0) are tied around the jugular vein and cannula for securement (purple arrows). Successful cannulation of the jugular vein confirmed by smooth saline injection. Please click here to view a larger version of this figure.

5. Cannulation of the hepatic lymph duct

1. Position the rat in dorsal recumbency under a surgical microscope.
2. Make a 4–5 cm horizontal skin incision from the midline to the right flank, approximately 1.5–2 cm below the ribcage (Figure 4A, B).
3. Retract the skin and make matching incisions in the external and internal oblique muscles.
4. Cauterize or ligate any large blood vessels encountered to prevent excess blood loss.
5. Gently reposition organs using saline-moistened cotton swabs: move the liver toward the diaphragm, stomach to the left, and small intestine inferiorly.
6. Keep organs retracted with saline-soaked gauze. The incision site and organ positioning are illustrated in Figure 4C.
7. Identify the hepatic lymph duct (diameter ~0.6 mm) using established anatomical landmarks. The duct runs parallel to and slightly caudal to the hepatic artery, lying medial to the right kidney and adjacent to the inferior vena cava.
8. Observe the appearance of the hepatic lymph duct. It typically appears translucent and narrow.
9. Distinguish the hepatic lymph duct from the mesenteric lymph duct, which is larger and usually appears opaque or white due to chylomicron content. The mesenteric lymph duct can therefore serve as an anatomical landmark to assist in the identification of the hepatic lymph duct located cranially.
10. Remove connective tissue overlying the lymph duct and vena cava using straight Graefe forceps.
11. Create a passage under the vena cava by inserting straight Graefe forceps through the fat bed beneath the right kidney. Advance until the tips emerge on the opposite side beneath the vena cava, parallel to the hepatic lymph duct.
12. Grip the external end of the prepared cannula with the straight Graefe forceps and pull it through the tunnel created beneath the vena cava until the tip emerges on the opposite side.
13. Using the Halsted-Mosquito hemostat forceps, bend the tip of a 25G needle to approximately 90°. Using the bent needle, puncture the hepatic lymph duct on the right side (closest to the operator).
    CAUTION: Use care when handling sharp needles during lymph duct puncture to prevent accidental injury.
14. Ensure the cannula is filled with anticoagulant solution without air bubbles. Insert the bevelled end approximately 2 mm into the duct.
15. Observe the external end of the cannula for lymph flow (~ 100 µL/h). If no flow is observed, adjust or reinsert the cannula.
16. Once lymph flow has been confirmed, plug the cannula before applying a small drop of veterinary adhesive to secure the insertion site. Once dried, confirm lymph flow through the tubing.
17. Remove gauze and return organs to their original positions.
18. Place the collection tube containing anticoagulant solution below the cannula outlet. Confirm that lymph is flowing drop-wise into the tube.
19. Close the abdominal wall by suturing the internal oblique, external oblique, and skin layers sequentially. Identification and successful cannulation of the hepatic lymph duct are shown in Figure 5.
    NOTE: After successful hepatic lymph duct cannulation, the procedure may be paused briefly while anesthesia and hydration are maintained before initiating lymph collection.

Figure 4: Hepatic lymph duct exposure. (A) Anatomical landmark indicating 1.5–2 cm below the right ribcage for incision. (B) Incision through the skin and abdominal muscles, exposing the internal organs. (C) Organs repositioned to expose the hepatic lymph duct. The liver is shifted towards the diaphragm, the stomach to the left of the animal, and the small intestines are tucked inferiorly. The hepatic lymph duct is located between the hepatic artery (green arrow) and mesenteric artery (blue arrow). L: liver; SI: small intestine; IVC: inferior vena cava; RK: right kidney. Please click here to view a larger version of this figure.

Figure 5: Hepatic lymph duct identification and cannulation. (A) The lymphatic and arterial structures can be observed after repositioning organs and prior to removal of surrounding connective tissue. The hepatic artery (green arrows) is located cranially in relation to the adjacent hepatic lymph duct (yellow arrows). The mesenteric lymph duct (purple arrows) is located above the mesenteric artery (black dotted outline). These structures run perpendicular to the inferior vena cava. IVC: inferior vena cava; RK: right kidney. (B) The hepatic lymph duct (yellow arrows) can be visualized clearly after removal of the surrounding connective tissue. The hepatic lymph duct runs parallel to the hepatic artery (green dotted outline) and cranially in relation to the mesenteric lymph duct (purple arrows) and mesenteric artery (black dotted outline). (C) The cannula (white arrows) is inserted into the hepatic lymph duct (yellow arrows) via the small puncture made in the lymph duct (orange arrows). The mesenteric lymph duct (blue arrows) can be visualized. Please click here to view a larger version of this figure.

6. Post-surgical period

1. Immediately after successful hepatic lymph cannulation, initiate rehydration by infusing sterile saline at 1.0–2.0 mL/h through the jugular vein cannula.
2. Maintain infusion throughout lymph collection. Alternatively, rehydration can be performed via a gastric or duodenal cannula, allowing the jugular vein cannula to be reserved for venous blood sampling if required.
3. Keep the animal under anesthesia on a heated surgical pad (37°C) for the entire collection period.
4. Monitor respiratory rate and anesthetic depth throughout the experiment by observing breathing patterns and reflex responses while maintaining isoflurane anesthesia via a nose cone.
5. Hydration was maintained through continuous saline infusion (1.0–2.0 mL/h) via the jugular vein cannula.
6. To minimize physiological disturbance, the total volume of blood collected prior to terminal sampling was limited to <10% of the estimated circulating blood volume in accordance with institutional animal ethics guidelines.
7. Collect hepatic lymph continuously into anticoagulant-containing tubes. Replace collection tubes hourly or as needed.
8. Collect arterial blood samples at designated time points:
   1. Temporarily occlude the external tubing and attach an empty syringe.
   2. Withdraw blood until the tubing is filled.
   3. Replace the syringe with a fresh one for sample collection and withdraw approximately 100–200 µL of arterial blood at each designated sampling time point.
   4. Flush the cannula with anticoagulant solution after each collection.
   5. Seal the cannula with a plug.
9. After sample collection is complete, either proceed to terminal sampling of the hepatic artery and portal vein or humanely kill the rat with sodium pentobarbitone (>100 mg/kg) via the carotid or jugular cannula.

7. Terminal sampling of the hepatic artery and portal vein

1. At the completion of lymph and blood collection, maintain deep anesthesia and ensure stable respiration before proceeding with terminal vascular sampling.
2. Reopen the abdominal incision carefully to expose the upper abdominal cavity.
3. Retract the liver gently toward the diaphragm, stomach to the left, and small intestine inferiorly using saline-moistened gauze to reveal the cannulated hepatic lymph duct.
4. Identify the hepatic artery, which runs parallel and caudal to the hepatic lymph duct.
   NOTE: The artery appears smaller and paler than the portal vein and exhibits a more pulsatile flow.
5. Prepare a 0.61 mm O.D., 0.28 mm I.D. polyvinyl cannula, prefilled with anticoagulant solution and free of air bubbles.
6. Using curved iris forceps, isolate a short segment of the hepatic artery and place a 4-0 silk suture beneath it.
7. 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.
8. Using fine-point micro-scissors, make a small transverse incision (approximately 0.5 mm) in the artery wall between the sutures.
9. Gently lift the vessel flap with the tip of the Dumont forceps to stabilize the vessel lumen for cannula insertion.
10. Insert the bevelled end of the polyvinyl cannula into the opening and advance approximately 2–3 mm.
11. Secure the cannula by tying the proximal suture around both the vessel and cannula.
12. Confirm correct placement by observing pulsatile blood flow into the cannula or by gently aspirating a small amount of bright red arterial blood.
13. Collect 0.5–1 mL blood into a pre-labelled tube containing anticoagulant.
14. After collection, withdraw the cannula and achieve hemostasis by tightening both sutures. The hepatic artery exposure and cannulation procedure are illustrated in Figure 6.
15. Identify the portal vein, which runs medial and perpendicular to the hepatic artery. The portal vein has a larger diameter and darker coloration compared to the hepatic artery.
16. Fit a 23G or 25G needle to a 1 mL syringe pre-flushed with anticoagulant solution.
17. Insert the needle bevel-up into the portal vein at a shallow angle (approximately 20°–30°) to avoid vessel collapse or tearing.
18. Gently aspirate the required blood volume.
19. Because portal vein puncture is terminal, humanely kill the animal immediately following sample collection by administering >100 mg/kg sodium pentobarbitone via the carotid or jugular cannula while the animal remains under deep anesthesia. The portal vein location and terminal sampling method are shown in Figure 7.
    CAUTION: Sodium pentobarbitone is a controlled substance and should be handled and administered in accordance with institutional regulations and appropriate safety procedures.
20. Confirm death by cessation of respiration and cardiac activity prior to carcass disposal, in accordance with the Australian Code for the Care and Use of Animals for Scientific Purposes (NHMRC )¹⁵ and the ANZCCART guidelines.

Figure 6: Hepatic artery identification and cannulation. (A) Exposure of the hepatic artery (green arrows). The hepatic artery is located adjacent to the mesenteric lymph duct (black dotted outline) and perpendicular to the inferior vena cava. IVC: inferior vena cava; L: liver. (B) Isolation of the hepatic artery using curved forceps. (C) Successful insertion (purple arrows) of the 0.61 mm × 0.28 mm polyvinyl cannula (orange arrows) into the hepatic artery (green arrows), secured with 4-0 silk sutures for terminal arterial blood sampling. The mesenteric lymph duct (black dotted outline) can be visualized. PV: portal vein. (D) After the cannula is secured, the patency of the cannula is confirmed by drawing blood into the cannula. Please click here to view a larger version of this figure.

Figure 7: Portal vein identification and terminal sampling. (A) The portal vein (indicated by the 25G needle) lies between the hepatic artery (white dotted outline) and bile duct (black dotted outline). The portal vein appears larger and darker than the hepatic artery and is accessed under deep anesthesia for terminal blood collection. PV: portal vein; IVC: inferior vena cava; L: liver; SI: small intestine; RK: right kidney. (B) Successful insertion of the 25G needle into the portal vein (black dotted outline) for terminal venous blood sampling. Please click here to view a larger version of this figure.

Results

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/h^3,4,11. 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.

Lymphatic sites (conscious state)	Mean lymph flow rate (mL/h)
Thoracic lymph (conscious)	1.74 ± 0.33
Thoracic lymph (anesthetized)	0.65 ± 0.10
Mesenteric lymph (anesthetized)	0.45 ± 0.11
Hepatic lymph (anesthetized)	0.12 ± 0.02

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.

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. Please click here to view a larger version of this figure.

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.

	%wt/wt
Fractions	Phospholipid	Free cholesterol	Cholesteryl ester	Triglyceride
Mesenteric lymph
D-HDL	20 ± 3	1 ± 0	5 ± 1	16 ± 1
L-HDL	21 ± 4	2 ± 0	8 ± 2	23 ± 5
Hepatic lymph
D-HDL	24 ± 1	3 ± 0	12 ± 3	8 ± 1
L-HDL	23 ± 1	6 ± 1	13 ± 2	14 ± 2
Thoracic lymph
D-HDL	25 ± 1	1 ± 0	7 ± 0	11 ± 3
L-HDL	29 ± 1	2 ± 0	11 ± 1	11 ± 0
Plasma
D-HDL	29 ± 2	2 ± 0	20 ± 3	2 ± 0
L-HDL	36 ± 6	2 ± 0	18 ± 3	4 ± 1

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.

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. Please click here to view a larger version of this figure.

Discussion

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’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’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'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 conditions^17,18. 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 2–3 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 and 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 processes^12,20. 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.

Materials

List of materials used in this article

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