Name: Cervical Lymph Duct-Cannulated Rat Model for Assessing Lymphatic Transport from the Head and Brain (Video) | JoVE Journal
Uploaded: 2026-03-10T13:10:00.000+00:00
Duration: 8 min 3 s
Description: Learn cervical lymph duct cannulation in rats to directly sample and quantify brain-derived lymphatic transport. This method enables measurement of CNS clearance rates and supports research into neurodegenerative disease mechanisms.

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5 minutes

The authors sincerely thank Mr. Zhenglin Hao and Ms. Jie Zhao at the Animal Experimental Centre of China Pharmaceutical University for their support in conducting animal experiments.

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<li>Khang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intrathecal+delivery+of+nanoparticle+PARP+inhibitor+to+the+cerebrospinal+fluid+for+the+treatment+of+metastatic+medulloblastoma%5BTitle%5D)+AND+%22Sci+Transl+Med%22%5BJournal%5D)">Intrathecal delivery of nanoparticle PARP inhibitor to the cerebrospinal fluid for the treatment of metastatic medulloblastoma.</a> Sci Transl Med. 15 (720), eadi1617(2023).</li>
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<li>Yağmurlu, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anatomical+features+of+the+deep+cervical+lymphatic+system+and+intrajugular+lymphatic+vessels+in+humans%5BTitle%5D)+AND+%22Brain+Sci%22%5BJournal%5D)">Anatomical features of the deep cervical lymphatic system and intrajugular lymphatic vessels in humans.</a> Brain Sci. 10 (12), 953(2020).</li>
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<li>Zawieja, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lymphatic+cannulation+for+lymph+sampling+and+molecular+delivery%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">Lymphatic cannulation for lymph sampling and molecular delivery.</a> J Immunol. 203 (8), 2339-2350 (2019).</li>
<li>Hoang, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantifying+the+lymphatic+transport+of+model+therapeutics+from+the+brain+in+rats%5BTitle%5D)+AND+%22Mol+Pharm%22%5BJournal%5D)">Quantifying the lymphatic transport of model therapeutics from the brain in rats.</a> Mol Pharm. 21 (5), 2473-2483 (2024).</li>
<li>Hoang, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Acute+neuroinflammation+alters+the+transport+of+a+model+therapeutic+protein+from+the+brain+into+lymph+and+blood%5BTitle%5D)+AND+%22Mol+Pharm%22%5BJournal%5D)">Acute neuroinflammation alters the transport of a model therapeutic protein from the brain into lymph and blood.</a> Mol Pharm. 21 (10), 5138-5149 (2024).</li>
<li>Maloveska, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamics+of+Evans+blue+clearance+from+cerebrospinal+fluid+into+meningeal+lymphatic+vessels+and+deep+cervical+lymph+nodes%5BTitle%5D)+AND+%22Neurol+Res%22%5BJournal%5D)">Dynamics of Evans blue clearance from cerebrospinal fluid into meningeal lymphatic vessels and deep cervical lymph nodes.</a> Neurol Res. 40 (5), 372-380 (2018).</li>
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<li>Miao, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Brain+clearance+is+reduced+during+sleep+and+anesthesia%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">Brain clearance is reduced during sleep and anesthesia.</a> Nat Neurosci. 27 (6), 1046-1050 (2024).</li>
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</ol>

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article.

This protocol establishes a reproducible method for cannulating the cervical lymph duct in rats, enabling continuous lymph collection over extended periods. With adequate microsurgical training, the procedure can be performed efficiently and yields consistent lymph samples under stable physiological conditions, maintained by continuous anesthesia, making it suitable for routine experimental use. Once mastered, the surgical procedure typically requires approximately 40 to 50 min to complete, enabling the processing of mul...

The lymphatic system, which is distributed throughout the body, is essential for maintaining fluid homeostasis, regulating immune responses, and facilitating the transport of nutrients and signaling molecules1. The peripheral lymphatics begin in the lymphatic capillaries, which collect interstitial fluid and solutes and drain into larger collecting lymphatic vessels (LVs) that possess a surrounding smooth muscle layer; these vessels then deliver lymph to local lymph nodes for immune processing2,3. The lymphatic network draining the head region is important for the homeostasis of both intracranial (e.g., brain) and extracranial (e.g., nasal and oral cavities) organs. The structure and organization of the extracranial lymphatic network are believed to resemble those of peripheral lymphatics throughout the body, whereas the intracranial lymphatic system exhibits unique anatomical and functional features (see Figure 1 for a detailed schematic representation in rat models)4,5. Although typical LVs are absent within the brain parenchyma, lymphatic-like functions are mediated by the specialized glial-dependent lymphatic transport (glymphatic) system that facilitates the exchange between cerebrospinal fluid (CSF) and interstitial fluid (ISF)6. While the glymphatic system enables the mixing of ISF within the brain parenchyma with CSF, the meningeal, perivascular, and nasopharyngeal LVs transport solutes from the CSF away from the cranial cavity. Specifically, the meningeal lymphatic pathway directs CSF from the subarachnoid space into meningeal LVs, linking intracranial fluid dynamics with peripheral lymphatic clearance7. In parallel, perineural pathways enable the efflux of CSF and associated solutes along cranial and spinal nerve sheaths, most prominently via the olfactory nerves into the nasopharyngeal lymphatic plexus5,8. Concurrent work of these systems maintains fluid homeostasis within the brain6,7,8. Recent advances in understanding the CNS-draining lymphatic system have provided new insights into brain physiology and pathophysiology. These findings may offer new avenues for therapeutic intervention in CNS diseases9.
<img alt="Glymphatic pathway diagram; brain lymphatic system in rodents showing veins, arteries, lymph flow." class="xfigimg" src="/files/ftp_upload/70217/70217fig1.jpg" data-altupdatedat="1780372819000" data-alt="Figure 1"> 
Figure 1: Schematic representation of CNS lymphatic clearance pathways. These pathways consist of (A) the glymphatic route via perivascular spaces; (B) the nasopharyngeal lymphatic route, and (C) the meningeal LVs. <a href="https://www.jove.com/files/ftp_upload/70217/70217fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The lymphatic networks draining from the head and CNS converge into the cervical lymph ducts located in the neck, where lymph is filtered through the cervical lymph nodes before entering the systemic circulation. The cervical lymph nodes are classified into superficial (sCLNs) and deep cervical lymph nodes (dCLNs) according to their anatomical locations within the neck. Alterations in the composition of lymph fluid within the cervical lymph ducts are thought to reflect underlying pathophysiological variations in the brain, with Alzheimer's disease (AD), Parkinson's disease (PD), traumatic brain injury (TBI), and post-stroke cognitive impairment (PSCI) being representative examples9,10,11,12,13. Notably, in AD, impaired drainage of amyloid-β (Aβ) and phosphorylated tau proteins (P-tau) through these lymphatic pathways has been implicated as a contributing factor in disease progression9,10,14. By directly connecting lymph ducts to adjacent veins to reroute lymphatic drainage, lymphatic venous anastomosis (LVA) is being investigated in both animals and humans as a therapeutic strategy to enhance lymphatic outflow from the CNS, thereby facilitating Aβ clearance from the brain and potentially delaying the progression of AD6,15,16. The importance of this pathway is further illustrated by observations in mice subjected to surgical removal of the cervical lymph nodes (lymphadenectomy), resulting in reduced cervical lymphatic clearance. Impaired clearance leads to toxic accumulation of Aβ and P-tau, with subsequent activation of pathological kinase signaling pathways. These changes are associated with a progressive worsening of AD-like tauopathy and an increased risk of anxiety- and depression-like behaviours14,16. Furthermore, CSF-based studies suggest that CNS tumors, such as brain lymphomas, can physically obstruct lymphatic clearance through the deep cervical lymph ducts while concurrently elevating oncoprotein concentrations in the CSF9,17,18. Evidence from CNS inflammation-based studies further indicates that cells originating in the meninges can migrate via the meningeal LVs to the cervical lymph nodes8,19. These cells consist primarily of lymphocytes, with smaller populations of dendritic cells, macrophages, and endothelial cells19,20. During inflammation or following CNS injury or surgery, lymphatic immune cell trafficking becomes evidently enhanced, accompanied by elevated levels of immune biomarkers such as cytokines, chemokines, and other inflammatory mediators21.
In addition to its patho-physiological roles, the cervical lymphatic network is increasingly acknowledged as an important conduit for drug disposition. Previous animal studies have demonstrated that several drug formulations designed for CNS administration can undergo clearance from the brain via the cervical lymph ducts and lymph nodes (to varying extents), exemplified by intracerebrally administered nanoparticle-based agents (e.g., lamotrigine, talazoparib) and biologics (e.g., monoclonal antibodies, radiolabeled tracer proteins)22,23,24,25. In humans, MRI imaging studies have demonstrated that certain molecules, such as gadobutrol, undergo active transport from the glymphatic system to the cervical lymph ducts26.
As aforementioned, cervical lymph nodes, both sCLNs and dCLNs, drain lymph from distinct regions of the head; however, the specific cranial regions drained by each node remain unclear27,28. Overlapping drainage areas, inter-individual anatomical variability, and potential interspecies structural differences further complicate the description of cervical lymphatic drainage patterns in both animals and humans29. Within the complex cervical lymphatic network, the deep cervical lymph ducts have been identified as major outflow routes for CSF, located downstream of the nasopharyngeal and meningeal LVs8. Compared with the superficial cervical lymph ducts, which form fine and variable networks in the subcutaneous tissue and differ greatly in size and branching between individual rats, the deep cervical lymph duct offers a more accessible and practical route for collecting CNS-derived lymph in small animal models20,30. The current protocol is therefore focused on establishing a reliable method for collecting lymph from the deep cervical lymph duct.
Only a limited number of studies conducted in the past decade have described the collection of cervical lymph fluids to study CNS diseases or analyze drug distribution. A previous study described measurement of drug concentrations in cervical lymph by flushing or squeezing fluid from isolated tissue sections (such as those adjacent to a single dCLN)31. However, experimental protocols for the continuous collection of lymph from the cervical lymph ducts in small laboratory animals, particularly rats, remain underdeveloped. This protocol establishes a readily accessible and reproducible method for cannulating the (rat) cervical lymph duct to enable continuous lymph collection and to investigate CNS lymphatic transport, immune responses, and pharmacokinetics. Its key advantage lies in the ability to quantify lymph flow and composition over defined time intervals, thereby enabling the quantitative measurement of drugs, lipids, fatty acids, and cellular constituents. Particularly, this model captures lymphatic transport processes that are not adequately represented by plasma/CSF sampling or imaging techniques, offering novel insights into CNS drug disposition, lipid and nutrient transport, and immune signalling20.

<table><tbody><tr><td>30 gauge needle</td><td>Yushou Health</td><td></td><td>Used for connecting the proximal end of cannula (0503) with the heparin syringe</td></tr><tr><td>Anaesthesia dispenser with vaporiser</td><td>YYUN Instrument Company</td><td></td><td></td></tr><tr><td>Blunt forceps</td><td>RWD Life Science</td><td>F22002-10&amp;nbsp;</td><td></td></tr><tr><td>Cotton swabs</td><td>Qingdao Shultz Biotechnology</td><td>MQ-02</td><td>Used to hold muscle tissue in place temporarily</td></tr><tr><td>Heated surgical pad</td><td>Shanghai Yiheng Instruments</td><td></td><td>Dimension 15 &amp;times; 20 cm</td></tr><tr><td>Heparin sodium</td><td>Shanghai Aladdin Biochemical Technology</td><td>H123383</td><td>Dilution required before use</td></tr><tr><td>Insulin syringe (1 mL)</td><td>Yushou Health</td><td>U100-1ML</td><td>Used for heparin pre-coating only</td></tr><tr><td>Isoflurane solution for inhalation</td><td>RWD Life Science</td><td>R510-22-10</td><td>Used for anesthesia through vaporiser</td></tr><tr><td>Marker pens</td><td>Any brand</td><td>N/A</td><td></td></tr><tr><td>Micro-centrifuge tube (1.5 mL)</td><td>Nantong Hairui Experimental Equipment</td><td>HR10003</td><td>Used for collection of lymph</td></tr><tr><td>Micro-scissors</td><td>RWD Life Science</td><td>GD234BA0007925</td><td></td></tr><tr><td>Nylon surgical suture&amp;nbsp;</td><td>Shanghai Yiheng Instruments</td><td>4-0 35CM</td><td></td></tr><tr><td>Pipette (volume adjustable, measuring range 5 to 50 &amp;mu;L)</td><td>Dragon Laboratory Instruments Limited</td><td>7030301006</td><td>Used for measuring tissue adhesive only</td></tr><tr><td>Pipette tips&amp;nbsp;</td><td>Biosharp Life Sciences</td><td>BS-10-T</td><td>Used for transferring tissue adhesive only, no specific required volume</td></tr><tr><td>Polyeyhylene (PE) cannula 0.5 mm OD, 0.3 mm ID (0503)</td><td>AniLab Scientific Instruments(Ningbo)</td><td>PE-0503</td><td></td></tr><tr><td>Polyeyhylene (PE) cannula 0.6 mm OD, 0.5 mm ID (0605)</td><td>AniLab Scientific Instruments(Ningbo)</td><td>PE-0605</td><td></td></tr><tr><td>Razor blades</td><td>Gillette Shanghai</td><td>Q31/0115000301C004</td><td>Used for fur shaving only</td></tr><tr><td>Rubber band</td><td>Any brand</td><td>N/A</td><td>Used for restraining forelimbs of rats</td></tr><tr><td>Ruler</td><td>Any brand</td><td>N/A</td><td></td></tr><tr><td>Scapel blades (#23)</td><td>RWD Life Science</td><td>S31010-01</td><td></td></tr><tr><td>Sodium chloride (0.9%) solution for infusion</td><td>Shijiazhuang No. 4 Pharmaceutical</td><td>H20066533 (approval number)</td><td>Used for fur rinsing only</td></tr><tr><td>Sprague-Dawley Rats</td><td>Beijing Vital River Laboratory Animal Technology</td><td>101</td><td>Weight 220 to 250 grams, no age requirement</td></tr><tr><td>Surgical light</td><td>Beijing Heo Bio-Tech</td><td>XHA011-1</td><td></td></tr><tr><td>Surgical microscope</td><td>Shanghai Cewei Optoelectronic Technology</td><td>SMZ755T</td><td>Dissecting microscope without a mechanical stage, can be equipped with a retractable stand</td></tr><tr><td>Tissue adhesive</td><td>3M Vetbond</td><td>1469SB</td><td>Main ingredient cyanoacrylate&amp;nbsp;</td></tr><tr><td>Tissue expander (Colibri)</td><td>RWD Life Science</td><td>R22029-04</td><td>Expansion range 20mm/4cm</td></tr></tbody></table>

cervical lymph duct-cannulated rat model for assessing lymphatic transport from the head and brain

Cervical Lymph Duct-Cannulated Rat Model for Assessing Lymphatic Transport from the Head and Brain

Lymph flows from the central nervous system (CNS) via a series of lymphatic vessels (LVs) and lymph nodes that converge at the cervical lymph ducts. In addition to maintaining fluid balance, these lymphatic ducts play a key role in transporting a wide array of substances, including endogenous metabolites, signaling molecules, immune cells, and small and macromolecular drugs. This is critical for physiological homeostasis and immune function in both intracranial (e.g., brain) and extracranial regions (e.g., nasal and oral cavities). Impaired lymphatic drainage from the brain has increasingly been linked to a range of neurological and neurodegenerative disorders. Cervical lymph duct cannulation enables the collection of lymph draining the head and brain, allowing the measurement of the concentration and transport rate of various substances via the lymphatic system. Changes in these factors in response to different challenges (e.g., drugs, stress, trauma) and diseases (e.g., stroke, infection, Alzheimer's disease) can also be determined. Here, we describe an anesthetized, deep cervical lymph duct cannulated rat model that enables lymph collection for several hours following surgery. The method may be combined with imaging and multi-omics technologies for the measurement of a wide range of parameters of interest in the lymph. This facilitates fundamental physiological and pathophysiological research of the head and neck region, as well as pharmacokinetic/pharmacodynamic studies of drugs, particularly for the treatment of CNS diseases.

A representative dataset of hourly lymph flow rates (mL/h) was obtained using the cervical lymph cannulation model (refer to Figure 7 for the corresponding chart). In this experiment involving three (n = 3) study rats, lymph was collected continuously into microcentrifuge tubes, which were replaced hourly over a 6 h period.
<img alt="Lymph flow rate vs. time graph, plotting mL/h over 8h, shows flow rate declining post-peak." class=...

This experimental protocol demonstrates a deep cervical lymph duct-cannulated rat model that enables direct and quantitative assessment of lymphatic drainage from the head region. The model facilitates detailed analysis of central nervous system (CNS)-derived solutes, immune cell populations, and the distribution and clearance of CNS-targeted therapeutics.

Lymph flows from the central nervous system (CNS) via a series of lymphatic vessels (LVs) and lymph nodes that converge at the cervical lymph ducts. In ...

Our project examines how brain lymphatics relate to CNS diseases, such as Alzheimer's, stroke, and neuroinflammation, by identifying substances that drain from the brain. Existing methods lacked a protocol and reliable approach to theoretically sample and quantify CNS-derived lymphatic outflow which is enabled by this protocol. To begin, preload approximately 0.7 milliliters of dilute anticoagulant solution into a one-milliliter sterile insulin syringe with a 30-gauge needle for coating the inner surface of the polyethylene cannula.Store the preloaded syringe under sterile conditions until use. To prepare cannulas, use tubing with 0.5-millimeter outer diameter and 0.3-millimeter inner diameter for both terminal sections and 0.6-millimeter outer diameter and 0.5-millimeter inner diameter tubing for the middle section. Using scissors, cut the cannula segments to lengths of two centimeters for the proximal portion connecting to the syringe needle, 40 centimeters for the middle section, and eight centimeters for the distal portion intended for cannulation.Assemble the cannula in a head-to-tail configuration. Then cut and bevel the distal end of the cannula assembly at an angle of approximately 45 degrees to facilitate smooth insertion into the cervical lymph duct. Verify the patency of each cannula by connecting the preloaded syringe to the proximal end of the assembled cannula.Gently release a small volume of solution to flush the cannula. Ensure the solution exits smoothly from the distal end without evidence of leakage. Position an anesthetized rat supine on a heated surgical pad maintained at 37 degrees Celsius.Tilt the head and extend the neck using a small support pillow, if necessary, to increase exposure of the cervical region. Gently restrain the forelimbs using rubber bands or surgical tapes to minimize movement. Rinse the fur over the cervical region with sterile normal saline to facilitate shaving.Then thoroughly shave the entire surgical site to ensure complete removal of fur prior to incision. Mark the surgical site by drawing a 2.5-centimeter dotted line to confirm the length of the initial incision. Place the upper end of the incision approximately one to 1.5 centimeters below the lower lip.After confirming the anesthetic depth by toe pinch reflex, carefully incise the superficial layer of the cervical musculature along the marked line. Then gently separate the remaining muscle layers, primarily the superficial cervical fascia, to expose the cervical artery. Retract the sternomastoid muscle and submandibular gland, and tilt them away from the surgical field to facilitate identification of the cervical lymph duct.Use a tissue expander to maintain the surgical window. With a cotton swab, temporarily hold aside muscle tissue while locating the lymph node and associated lymph duct. Locate the opaque colorless cervical lymph duct running parallel to the carotid artery.Using blunt forceps, gently pass through the separated connective tissues and maintain the forceps in position. Carefully isolate the cervical lymph duct from surrounding connective and adipose tissues by blunt dissection. Then create a small incision in the upper half of the exposed vessel.A small secondary skin puncture approximately 1.5 centimeters from the surgical window may be created to serve as an additional fixation point for the cannula. Insert the cannula through the second opening, grasp its distal end with forceps, and ensure its beveled face is directed upward. Then, gently insert the distal end approximately two millimeters into the lymph duct through the incision, orienting it toward the head.Monitor the proximal end of the cannula for one to two minutes. Confirm successful cannulation by observing a slow and steady outflow of lymph fluid. Secure the cannula by applying a small drop of cyanoacrylate tissue adhesive with a pipette at the incision site and the secondary fixation hole.Observe lymph flow for several minutes to confirm steady outflow. Then carefully remove the tissue expander, and reposition the submandibular gland to its original location. Attach a pre-labeled, anticoagulant-coated microcentrifuge tube to the distal end of the cannula.Then reposition all muscle layers to their original locations, and close the skin incision with sutures and/or tissue adhesive. Collect lymph continuously into anticoagulant-coated microcentrifuge tubes, replacing the tubes at predetermined intervals, typically hourly. Ensure the rat remains anesthetized throughout the collection period.Store the collected samples with ice packs in a cooler box. Hourly lymph flow rates measured over a six-hour collection period in successfully cannulated rats ranged from 0.09 to 0.13 milliliters per hour. In healthy rats, cumulative lymphatic transport of intrastriatally-administered, tritium-labeled albumin remained negligible during the first two hours and increased substantially thereafter.Comparison of tritium-labeled albumin concentrations between lymph and plasma demonstrated different concentration profiles over time in healthy rats. Neuroinflammation induced by interperitoneal administration of Escherichia coli lipopolysaccharide was associated with a slightly reduced overall lymph flow rate over eight hours and a nearly twofold reduction in the proportion of total dose transported into lymph. The lymph-to-plasma concentration ratio showed no significant difference between healthy and neuroinflamed rats, indicating similar cervical lymphatic clearance profiles.This protocol enabled direct biological sampling of brain-derived lymph, bridging indirect external imaging or resinous method with measurable central nervous system outflow. The protocol enables studying centrally-administered drug clearance through lymphatic pathways, allowing measurement of drainage routes and clearance rates. Future studies can examine how CNS disease states alert to lymphatic clearance and use this knowledge to optimize drugs to restore brain lymphatic function for treatment.

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Research

JoVE Journal

Immunology and Infection

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Learn cervical lymph duct cannulation in rats to directly sample and quantify brain-derived lymphatic transport. This method enables measurement of CNS clearance rates and supports research into neurodegenerative disease mechanisms.

Cervical Lymph Duct-Cannulated Rat Model for Assessing Lymphatic Transport from the Head and Brain

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