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

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.