3D intravital high-resolution photoacoustic tracing of meningeal lymphatic vessels

The discovery of meningeal lymphatic vessels changed understanding of immune privilege and metabolic waste clearance mechanisms in the central nervous system. These lymphatic vessels play a key role in regulating cerebrospinal fluid circulation, draining macromolecules, waste, and excess fluid from central nervous system to periphery by connecting cervical lymph nodes. Although research on meningeal lymphatic vessels is still in the exploration and learning stage, their importance for the removal of metabolic waste in the central nervous system has become increasingly prominent. Currently, there is a gap in the complete understanding of how meningeal lymphatics clear metabolic waste from the brain by regulating cerebrospinal fluid circulation. The morphological and functional differences of meningeal lymphatic vessels may be closely related to the onset and progression of some brain neurological diseases. Through high-resolution imaging of meningeal lymphatic vessels from a whole-brain perspective can deepen understanding of the complexity of brain’s immune function and provide important implications for the treatments of central nervous system-related diseases. However, in vivo visualization of meningeal lymphatic vessels requires not only high-resolution imaging, but also a large field of view covering the entire brain, which is a great challenge. 

Based on this, the research team proposed a technical method for three-dimensional high-resolution visualization of meningeal lymphatic vessels in vivo. This method achieves in vivo three-dimensional co-localization imaging of mouse meningeal lymphatic vessels and cerebral blood vessels by designing a dual-contrast functional photoacoustic microscope (Figure 1). The microscope scan range covers the entire mouse brain cross-section, providing dual-contrast lateral resolution of 8.9 μm and 6.1 μm. The research team took advantage of the drainage properties of macromolecular by meningeal lymphatic vessels and loaded the dye indocyanine green onto ovalbumin form a macromolecular tracer. After this tracer is injected into the cerebrospinal fluid, it is discharged through the drainage function of meningeal lymphatic vessels. The research team tracks and monitors this process to obtain in vivo photoacoustic three-dimensional high-resolution imaging results of brain lymph. At the same time, the endogenous contrast agent hemoglobin is used to image cerebral blood vessels. The microscope based on opto-acoustic confocal features has a depth imaging capability of 3.75 mm, which can realize depth stratification of photoacoustic three-dimensional imaging results and distinguish depth positions, thereby extracting images of meningeal lymphatic vessels located on the dura mater and glymphatic pathway located in the brain parenchyma (Figure 2).

Using this microscope, the research team carried out groundbreaking observation and studies on meningeal lymphatic vessels in mice. By observing the meningeal lymphatic vessels of Alzheimer's disease model mice and normal mice, they found that in the early course of Alzheimer's disease, the function of the meningeal lymphatic vessels has been significantly impaired, and the drainage volume is reduced by about 70%. As the disease progresses, meningeal lymphatic drainage continues to decrease. The results of the study showed that the reduction in drainage volume was related to the decrease in lymphatic function genes expression, which may be related to the deposition of amyloid, leading to the impairment of the function of meningeal lymphatic vessels (Figure 3). 

This study achieved high-resolution visualization of three-dimensional co-localization of meningeal lymphatic vessels and cerebrovascular vessels in vivo, successfully distinguish and extracted the glymphatic pathway and the meningeal lymphatic vessels in the three-dimensional imaging results, and solved the technical problem that it is impossible to image meningeal lymphatic vessels in wide field in the traditional technique. This study demonstrated that the system can dynamically monitor cerebrospinal fluid drainage while maintaining high resolution and found that the function of meningeal lymphatic vessels is damaged in the early stages of Alzheimer's disease (5-6 months). This phenomenon was discovered earlier than traditional immunofluorescence imaging methods (12-13 months), which advanced the detection window period for meningeal lymphatic vessels function. This discovery provides new directions and perspectives for the diagnosis and treatment of early Alzheimer's disease. In addition, the specific, high-resolution, and stereo morphologic and functional imaging achieved by this method in a large field of view is of great significance for the basic application research of meningeal lymphatic vessels in vivo. This technology also provides new tools for the diagnosis and treatment of meningeal lymphatic vessel-related diseases and opens up new horizons for understanding brain drainage mechanisms. 

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