Surgical Protocols for Deep Cervical Lymphovenous Anastomosis in a Rat Model: Lymph Node and Lymphatic Vessel Anastomoses

Summary

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.

Abstract

Deep cervical lymphovenous anastomosis (dcLVA) has become a promising treatment strategy for Alzheimer's disease (AD), offering significant improvements compared to the current limited treatment options. However, the underlying mechanism of dcLVA remains unclear. Although clinical trials are ongoing, animal models simulating dcLVA provide a valuable tool for exploring its mechanism. This study aims to develop a standardized protocol for creating dcLVA models in animals to facilitate basic research and address clinical challenges. We describe the surgical procedures and key steps for anastomosing the deep cervical lymph node (dcLN) and its afferent lymphatic vessel (ALV) with the posterior facial vein (PFV), detailing these two distinct surgical methods: deep cervical lymph node-vein anastomosis (dcLnVA) and deep cervical lymphatic vessel-vein anastomosis (dcLaVA). Beyond promoting lymphatic drainage, the comparison of these two surgical methods also helps us to understand and explore the role of the deep cervical lymph node and deep cervical lymphatic system in the pathogenesis of AD. Overall, the complete and clear presentation of the surgical procedure and key anatomical landmarks in the rat helps to standardize the surgical protocol, which can minimize confounding factors and reduce inter-experimental variability, thus laying a solid methodological foundation for a deeper understanding of the mechanisms involved.

Introduction

Alzheimer's disease (AD) is a gradually progressive neurodegenerative disorder that often results in cognitive decline. The distinctive pathological features of AD are amyloid-beta (Aβ) plaques and neurofibrillary tangles, which are formed by abnormally phosphorylated tau proteins. Recent research on the brain lymphatic system has opened new therapeutic avenues for treating AD. This system facilitates the drainage of Aβ from the interstitial fluid (ISF) into the cerebrospinal fluid (CSF) and subsequently to the deep cervical lymph nodes via the meningeal lymphatic vessels (mLVs)¹. In 2024, Professor Xie's team reported the first case of cognitive improvement in an AD patient following deep cervical lymphovenous anastomosis (dcLVA) and subsequently initiated a clinical trial (NCT06530732)².

Although dcLVA, as a novel therapeutic approach for AD, offers promising prospects for patients, the precise mechanisms underlying its therapeutic effects require further elucidation³. Animal models provide an indispensable platform for investigating these mechanisms. A comprehensive understanding of the therapeutic mechanisms of dcLVA will not only enhance our understanding of AD pathogenesis but also optimize surgical techniques, clarify treatment indications, and ultimately deliver greater clinical benefits to patients.

Professor Xie's team recently reported a dcLVA model, demonstrating the feasibility of connecting tributaries of the external jugular vein with the deep cervical lymph node⁴. However, the description of the model is still not sufficient. Firstly, the description of the deep cervical lymph node (dcLN) and its surrounding anatomy is inadequate. Secondly, there is no objective, quantitative assessment of postoperative patency. More importantly, there is a lack of detailed descriptions of the steps for anastomosis of dcLN and veins, as well as clear and intuitive pictures and videos that are convenient for teaching.

Given that this technique involves intricate surgical manipulation and considering the anatomical differences between rats and humans, variations in surgical procedures and technical details in rat models may lead to significant discrepancies in experimental outcomes. Therefore, it is both critical and urgent to clearly illustrate the relevant anatomical landmarks in rats and provide detailed technical descriptions of the anastomosis procedure. Such efforts are essential to enhance the reproducibility and standardization of the surgical protocol, thereby supporting broader application by researchers and advancing related scientific investigations. Furthermore, given the diversity of clinical surgical techniques, such as lymph node-to-vein anastomosis and lymphaticovenous anastomosis, and the potential for varying outcomes depending on the surgical approach, comparative studies are warranted to evaluate their relative efficacy. In response to these challenges, we present two highly reproducible dcLVA methods: deep cervical lymph node-to-vein anastomosis (dcLnVA) and deep cervical lymphatic vessel-vein anastomosis (dcLaVA). These methods aim to standardize the model creation process and facilitate subsequent basic research.

Protocol

This study was conducted with the approval of the Institutional Animal Care and Use Committee (IACUC) of Harbin Medical University (Approval No. SYDW2025-037). The subjects were male Sprague-Dawley (SD) rats, weighing 400-500 g and aged 12-14 weeks, provided by the university's animal center. The center maintains a Specific Pathogen-Free (SPF) facility and complies with national breeding standards.

NOTE: This study primarily investigates two methods of anastomosis between the deep cervical lymph and veins. Establishing a surgical connection between the dcLN and the posterior facial vein (PFV); this technique is referred to as dcLnVA. Creating an anatomical connection between the deep cervical lymphatic vessel (dcLaV) and the PFV; this technique is referred to as dcLaVA. We describe both microsurgical procedures in detail, including their operative methodologies and post-anastomosis evaluation.

1. Preoperative preparation

1. Anesthetize the rat with isoflurane (3–5% at a flow rate of 1–2 L/min) for induction.  Once the animal loses its righting reflex, reduce to 1.5–2.5% for maintenance of a surgical plane of anesthesia. Monitor the rat's responses closely. Proceed only when the pedal reflex is completely absent.
2. Using a blunt-tip needle, slowly administer Evans blue (EB) solution (approximately 60-100 µL/kg , 5%)⁵ into the nasal mucosa of a unilateral nasal cavity approximately 5 min before the start of the next step. Position the rat in lateral recumbency with the EB-injected side down to facilitate gravity-assisted drainage of the dye toward the deep cervical lymph nodes, while preventing EB from entering the contralateral nostril or respiratory tract.
3. Shave the hair on the rat's neck to ensure a clear surgical field. Apply Vaseline to both eyes to prevent dryness or corneal injury during surgery.

2. Surgical incision

1. Disinfect the midline of the rat's neck three times, alternating between iodine tincture and 70% isopropyl alcohol, to ensure a sterile surgical field.
2. Make a midline incision approximately 3 cm in length using a scalpel.
   NOTE: Take care not to damage the submaxillary gland (SMG) and blood vessels during dissection.
3. Retract the skin to expose the sternothyroid muscle (STM), sternocleidomastoid muscle (SCM), and the omohyoid muscle (OHM).

3. Preparation of the PFV

1. Identify the PFV, a tributary of the external jugular vein (EJV), along the lateral margin of the SCM.
2. At the bifurcation between the PFV and the EJV, occlude the PFV using a microvascular clip.
3. Continue to bluntly dissect the PFV to obtain sufficient length, and clamp it at the distal end. Ensure the PFV segment remains as loose as possible to allow for subsequent traction during anastomosis with the dcLN.
4. Create a longitudinal incision parallel to the vessel axis using microvascular scissors.
   NOTE: Make sure the PFV incision is longitudinal, as the transverse incision can be enlarged and ruptured by traction.
5. Irrigate the isolated vascular segment thoroughly with heparinized saline (0.1% sodium heparin in 0.9% NaCl) until the blood are completely cleared.

4. Lymph node-vein anastomosis

1. Explore and fully expose dcLN and its afferent lymphatic vessel (ALV) in the middle of the STM and SCM⁴.
2. Dissect the dcLN gradually using a blunt technique. Carefully incise the fascia between the dcLN and surrounding tissues, avoiding damage to both the dcLN and its ALV.
   NOTE: Ensure that PFV and dcLN are close to each other to minimize tension after anastomosis.
3. Create an opening at the distal end of the dcLN by partially resecting the lymph node tissue. Ensure that the width of the opening is smaller than or similar to the diameter of the PFV, so that the PFV blood flow will not be significantly affected.
4. Using a 12-0 nylon suture, anastomose the proximal end of the PFV incision to the proximal end of the dcLN incision.
5. Continue to expand the incision length of the PFV to the distal end so that it is slightly larger than the length of the dcLN incision.
6. Pass the needle sequentially through the outer edge of the dcLN transection and the inner edge of the PFV incision, thereby pulling the dcLN transection into the lumen of PFV.
7. Perform a continuous suture from the inferior to the superior edge of the dcLN transection. Irrigate the lumen with heparin sodium solution once more just prior to completing the final suture to prevent thrombus formation within the PFV lumen. Maintain appropriate tension on the suture during the continuous closure to ensure a secure closure and prevent subsequent leakage.
8. Place interrupted sutures wherever the continuous anastomosis is loose.

5. Lymphatic vessel-vein anastomosis

1. Transect the ALV and preserve the surrounding tissue to serve as a suture anchor point.
2. Using a 12-0 suture needle, insert it about 2 mm proximal to the PFV incision and advance it to exit through the opening of the PFV incision.
3. Enter the tissue near the stump of ALV with the suture needle to catch the ALV.
4. Pass the needle into the PFV incision and out through its proximal edge, then tie the suture to fixation the ALV stump within the lumen of PFV.
   NOTE: Completely insert the ALV stump into the lumen of PFV , orienting it toward the proximal direction to maintain physiological drainage.
5. Suture the surrounding tissue near the stump of ALV to the inner edge of the PFV incision to provide external fixation. Irrigate the lumen with heparin sodium solution once more just prior to completing the final suture to prevent thrombus formation within the PFV lumen..

6. Suture and postoperative care

1. Perform continuous suturing for the subcutaneous fascia and interrupted suturing for the skin.
2. Disinfect the surgical area with iodine tincture. Then apply erythromycin ointment uniformly around the incision to prevent infection.
3. Postoperatively, provide analgesia by subcutaneous injection of meloxicam (1–2 mg/kg) once daily for up to 72 h, in accordance with institutional animal care guidelines. Inject into the loose subcutaneous tissue over the neck or flank.

7. Validation of anastomosis

1. Identify and dissect the anterior facial vein (AFV), a tributary of the EJV, lateral to the PFV.
2. Verify the success of the anastomosis by observing significantly more intense staining in the PFV compared to the adjacent AFV.
3. Quantify color changes objectively using the standardized CIE LAB color space. Define and extract Regions of Interest (ROIs) in Fiji to obtain the mean CIE L*a*b* values, then calculate the hue angle (h°) in degrees using the formula:
   h° = atan2(b, a) × (180/π)^6,7.

Results

The surgical procedures and postoperative outcomes of dcLnVA and dcLaVA are illustrated with one representative case each. In the dcLnVA case, PFV (2 mm in diameter) was identified and dissociated from the lateral border of the SCM (Figure 1A, Figure S1A). In the gap between the SCM and STM, approximately 4 mm medial to the PFV, a long oval dcLN (5 mm × 4 mm × 2 mm) was found and finely separated (Figure 1B,C, Figure S1B-E

Discussion

In 2012, ILif et al. first described the glymphatic system⁸. In 2015, Louveau et al. identified T-cell aggregates in the lumen of lymphatic vessels and termed these structures meningeal lymphatics. They further demonstrated that meningeal lymphatics are connected to both the glymphatic system and dcLN. In summary, under the regulation of the brain lymphatic system, including both the glymphatic system and meningeal lymphatics, Aβ in the interstitial fluid is drained into the cerebrospinal flu...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.9% Sterile Saline Solution	Shandong Qidu Pharmaceutical Co., Ltd.	R9824041307
Erythromycin Ointment	Sanqi Pharmaceutical Co., Ltd.	H34020307
Evans Blue Dye 5% (50mL)	LEAGENE	DK0053
Heparin Sodium Solution 0.1%	BIOSCO	BAC006
Micro forceps	ANLIXIN	AT-15
Microvascular clamp	Tigergene	TG-AC-18-Z
Penicillin Sodium	Hebei Yuanzheng Hemu Pharmaceutical Co., Ltd	JFT230602
White Vaseline	Tiancheng	20250202