Surgical Protocols for Deep Cervical Lymphovenous Anastomosis in a Rat Model: Lymph Node and Lymphatic Vessel Anastomoses
November 14th, 2025
Here, we present a protocol to clearly define the steps for anastomosing either the deep cervical lymph node or its afferent lymphatic vessel with the posterior facial vein in the rat, to minimize the influence of other variables and standardize the conduct of surgery-based research.
Our dcLVA technique shows promise for treating Alzheimer's, though the mechanisms remain unclear. Animal model are key to uncovering, though. A key challenge is the anatomical difference between rats and humans.
Standardizing surgical landmarks and techniques is crucial for reliable results. To begin, disinfect the midline of the anesthetized rat's neck three times, alternating between iodine tincture and 70%isopropyl alcohol to ensure a sterile surgical field. Then, make a midline incision approximately three centimeters in length using a surgical scalpel.
Take care not to damage the submaxillary gland and blood vessels during dissection. Retract the skin to expose the sternothyroid muscle, sternocleidomastoid muscle, and the omohyoid muscle. Identify the preauricular facial vein, a tributary of the external jugular vein, along the lateral margin of the sternocleidomastoid muscle.
At the bifurcation between the preauricular facial vein and the external jugular vein, occlude the preauricular facial vein using a microvascular clip. Continue to bluntly dissect the preauricular facial vein to obtain sufficient length, and clamp it at the distal end. Ensure the preauricular facial vein segment remains as loose as possible to allow for subsequent traction during anastomosis with the deep cervical lymph node.
Then, create a longitudinal incision parallel to the vessel axis using microvascular scissors. Make sure the preauricular facial vein incision is longitudinal, as the transverse incision can be enlarged and ruptured by traction. Irrigate the isolated vascular segment thoroughly with heparinized saline, prepared as 0.1%sodium heparin in 0.9%sodium chloride, until the blood erythrocytes are completely cleared.
Explore and fully expose the deep cervical lymph node and its afferent lymphatic vessel in the middle of the sternothyroid muscle and sternocleidomastoid muscle. Dissect the deep cervical lymph node gradually using a blunt technique. Carefully incise the fascia between the deep cervical lymph node and surrounding tissues, avoiding damage to both the node and its afferent lymphatic vessel.
Ensure that the preauricular facial vein and the deep cervical lymph node are close to each other to minimize tension after anastomosis. Next, create an opening at the distal end of the deep cervical lymph node by partially resecting the lymph node tissue. Ensure that the width of the opening is smaller than or similar to the diameter of the preauricular facial vein so that the blood flow in the vein is not significantly affected.
Using a 12-0 nylon suture, anastomose the proximal end of the preauricular facial vein incision to the proximal end of the deep cervical lymph node incision. Continue to expand the incision length of the preauricular facial vein to the distal end so that it is slightly larger than the length of the deep cervical lymph node incision. Then, pass the needle sequentially through the outer edge of the deep cervical lymph node transection and the inner edge of the preauricular facial vein incision, thereby pulling the lymph node transection into the lumen of the vein.
Perform a continuous suture from the inferior to the superior edge of the deep cervical lymph node transection. Irrigate the lumen with heparin sodium solution once more just prior to completing the final suture to prevent thrombus formation within the preauricular facial vein lumen. Maintain appropriate tension on the suture during the continuous closure to ensure a secure closure and prevent subsequent leakage.
Transect the afferent lymphatic vessel and preserve the surrounding tissue to serve as a suture anchor point. Using a 12-0 suture needle, insert it about two millimeters proximal to the preauricular facial vein incision, and advance it to exit through the opening of the vein incision. Enter the tissue near the stump of the afferent lymphatic vessel with the suture needle to catch the vessel.
Pass the needle into the preauricular facial vein incision and out through its proximal edge, then tie the suture to fix the afferent lymphatic vessel stump within the lumen of the vein. Completely insert the afferent lymphatic vessel stump into the lumen of the preauricular facial vein, orienting it toward the proximal direction to maintain physiological drainage. Finally, suture the surrounding tissue near the stump of the afferent lymphatic vessel to the inner edge of the preauricular facial vein incision to provide external fixation.
In deep cervical lymph node-vein anastomosis, the average hue angle of the post-anastomosis posterior facial vein was biased toward the blue tone at 278.2 degrees, compared to the adjacent anterior facial vein at 327.9 degrees, and showed a greater difference than the pre-anastomosis posterior facial vein at 24.8 degrees. For deep cervical lymphatic vessel-vein anastomosis, the average hue angle of post-anastomosis posterior facial vein was 282.4 degrees, showing a stronger blue tone than the adjacent anterior facial vein at 303.7 degrees, and the difference was more distinct than the pre-anastomosis posterior facial vein at 39.6 degrees. Our study provides specific references and a standardized framework of dcLVA, including dcLNVA and dcLAVA, supporting both specific research and clinical application.
We will investigate efficacy and mechanism of this surgical procedure in AD rat models in the future.
View the full transcript and gain access to thousands of scientific videos
Overview
This article presents a standardized protocol for performing deep cervical lymphovenous anastomosis (dcLVA) in rats, a surgical technique with potential therapeutic implications for Alzheimer’s disease (AD). The protocol details two distinct methods—deep cervical lymph node-vein anastomosis (dcLNVA) and deep cervical lymphatic vessel-vein anastomosis (dcLAVA)—and provides step-by-step guidance to facilitate reproducibility and minimize experimental variability in animal models.
Key Study Components
Area of Science
- Neuroscience
- Surgical techniques
- Animal modeling
Background
- Deep cervical lymphovenous anastomosis (dcLVA) is emerging as a promising intervention for Alzheimer’s disease.
- The mechanisms underlying dcLVA’s therapeutic effects remain unclear.
- Animal models are essential for investigating the efficacy and mechanism of dcLVA.
- Standardized surgical protocols are needed to ensure reproducibility and reduce confounding factors.
Purpose of Study
- To develop and describe a standardized protocol for creating dcLVA models in rats.
- To detail the surgical steps for both dcLNVA and dcLAVA procedures.
- To provide anatomical guidance and minimize inter-experimental variability.
Methods Used
- Preparation of a sterile surgical field on anesthetized rats.
- Midline neck incision and careful dissection to expose relevant muscles and vessels.
- Identification and preparation of the preauricular facial vein for anastomosis.
- Exposure and dissection of the deep cervical lymph node and its afferent lymphatic vessel.
- Microvascular anastomosis using 12-0 nylon sutures between the lymph node or lymphatic vessel and the preauricular facial vein.
- Assessment of post-anastomosis vascular changes using hue angle measurements.
Main Results
- Successful establishment of both dcLNVA and dcLAVA in rat models.
- Detailed procedural steps and anatomical landmarks provided for reproducibility.
- Post-anastomosis hue angle measurements indicated successful vascular changes, with a shift toward blue tones in the posterior facial vein.
- The protocol supports future research into the mechanisms and efficacy of dcLVA in AD models.
Conclusions
- The standardized dcLVA protocol enables reliable animal modeling for mechanistic and therapeutic studies in Alzheimer’s disease.
- Clear procedural guidance minimizes variability and supports both basic and translational research.
- This framework lays the groundwork for future investigations into the efficacy and mechanisms of lymphovenous anastomosis in neurodegenerative disease.
