Abstract:

BACKGROUND: Meningeal lymphatic vessels can drain cerebral spinal fluid and amyloid β-protein, promoting T lymphocyte to transport and home to deep cervical lymph nodes. A simple, quick and definite method of dural separation and accurate localization of deep cervical lymph nodes can provide strong support for the study of neurodegenerative diseases.
OBJECTIVE: To establish a convenient and practical method for exfoliating dural and deep cervical lymph nodes.
METHODS: ICR mice, 3 months old, were taken, anesthetized and injected with Evans blue and tracer in the occipital pool for localizing deep cervical lymph nodes. A midline incision of about 3 cm in length was made about 5 mm above the clavicle, the superficial fat and fascia were bluntly separated, and the lateral sternocleidomastoid muscle was pulled to expose the deep cervical lymph nodes, which were removed under a stereomicroscope and frozen at -80°C. Subsequently, the mouse head was cut and the skin and muscles of the head were separated to expose the entire skull structure. The skull and brain tissue were separated from the foramen magnum along the lower parietal bone with scissors, and the complete skull top was obtained. The skull was sequentially fixed in 40 g/L paraformaldehyde solution for 24 hours, 120 g/L paraformaldehyde for 24 hours, and 120 g/L paraformaldehyde for 10, 20, 30, and 40 minutes, and the dural structure was stripped. The drainage capacity of meningeal lymphatic vessels and deep cervical lymphatic vessels was verified by tracer, and the meningeal lymphatic vessels were identified by the lymphatic vessel endothelial hyaluronan receptor 1 using the immunofluorescence method.
RESULTS AND CONCLUSION: (1) Obvious blue staining was observed in deep cervical lymph nodes 15 minutes after Evans blue staining. (2) The skull was sampled and fixed in 120 g/L paraformaldehyde for 24 hours, resulting in a less tight connection between the dura mater and the skull, and easier stripping of the dural structures with an intact shape. The dura mater fixed at 120 g/L concentration was more resilient and remained more intact during peeling compared with the conventional 40 g/L concentration; 120 g/L paraformaldehyde fixed meninges for a short time, and 30-40 minutes was preferred. (3) The frozen section of deep cervical lymph nodes showed the presence of the tracer, complete meningeal lymphatic vessels were visible in the dura mater, and the tracer was observed at the tail of lymphatic vessels. Immunofluorescence staining for endothelial hyaluronan receptor 1 was positive in the deep cervical lymph nodes and dural lymphatics. In summary, the best peeling concentration and time is 120 g/L paraformaldehyde fixed for 24 hours. At this concentration, the dura mater has a stretched morphology, a better toughness, and is more intact after peeling, which is conducive to later use. Verified by Evans blue, tracers and immunofluorescence, deep cervical lymph nodes are located accurately, which can be used as a basis for the study of various neurodegenerative diseases.

Key words: dura mater, deep cervical lymph nodes, meningeal lymphatic vessels, cerebral lymphatic drainage, Alzheimer’s disease, neurodegenerative disease, engineered tissue construction