Running Up and Down Your Spine: Imaging the elusive vertebral lymphatic network

Image courtesy of Alice Tirard

Between our cells lies a concealed, interwoven tapestry of vessels that plays a central role in transporting material all over our bodies. The primary function of these lymphatic vessels is to drain our tissues of fluid that leaks out from the systemic circulatory system, helping us to get rid of excess substances, toxic metabolic waste, and potentially harmful pathogens. Lymphatic vessels can be found almost everywhere––from our skulls to the vertebral column. But visualizing vertebral lymphatic vessels is not always as easy as visualizing blood vessels, for example. The fact that lymph—the material that lymphatic vessels carry around—is transparent, combined with the microscopic space through which it travels, confers this network an almost invisible quality. The task becomes even more complicated in the spaces that surround the spinal cord, where the minuscule vertebral lymphatic vessels (vLVs) are so expertly hidden within the bone chambers of the vertebral column that, despite the fact that the scientific community has been aware of their existence for a few years, no one had known exactly how they were organized in three dimensions.

However, earlier this year, this barrier in scientific imaging was finally broken by a collaborative effort between researchers at Yale and institutions in France and Finland. Led by Jean-Léon Thomas, an associate professor of neurology at the Yale School of Medicine, and Laurent Jacob, a postdoctoral researcher at the Brain and Spine Institute (ICM) the Université Pierre et Marie Curie in Paris, the team innovatively combined a series of advanced imaging methods, including iDISCO+ tissue clearing and light-sheet fluorescence microscopy to reconstruct––in three dimensions––the anatomy of the lymphatic vasculature within the spinal cavity of mice for the first time. Their reconstruction reveals the projections of lymphatic vessels as they extend from spinal nerve roots to lymph nodes in the rest of the body and to the thoracic duct, the body’s largest lymphatic duct. “We wanted to better localize all of the connections between the vertebral lymphatic vessels and the lymph nodes, as well as their connections with the periphery,” Jacob said. “We were able to precisely describe this network all along the spinal cord.”

Intricate details call for intricate methods

The intricate microscopic architecture of the vLV network meant that advanced immunohistology, complex imaging methods, and other state-of-the-art techniques had to be combined. After sectioning the vertebral column into segments, the tissues were subjected to iDISCO+ (immunolabeling-enabled three-dimensional imaging of solvent-cleared organs), a protocol in which tissues were decalcified and treated with organic solvents. This process removes fat molecules from the tissues to make them more transparent and easier to microscopically examine in greater detail. Next, whole mount immunolabeling was performed, whereby the lymphatic endothelial cells were stained using two antibodies that bound to two lymphatic endothelium-specific markers, LYVE-1 and PROX1. These antibodies worked as labels, enabling the researchers to the track and monitor these lymphatic endothelial cells around the spinal cord and inside the vertebral canal. They then turned to light-sheet fluorescent microscopy, which uses a sheet of light to visualize sections of tissue deep within the sample. Light-sheet microscopy can scan large surfaces of tissues very quickly at high resolution. The group then pieced all of this information together using a 3D software called Imaris to produce a comprehensive final set of images and videos. Importantly, their imaging approach preserves the surrounding anatomical structures of the spinal cord, framing this vertebral lymphatic system within the context of surrounding muscle and bone tissue, lymph nodes, and nerve cells.

Being able to visualize the three-dimensional vLV network is crucial to understand its function more than other systems in the body. “You need to see the connections between the vessels and the lymph nodes because, essentially, the [lymphatic] vessels are like pipes,” Thomas said. “If you don’t know where the pipes are connected––that is, where they are draining and where they are collecting fluids––you don’t have all of the information.” Truly comprehending the macroscopic arrangement of this network, therefore, also involves thinking about how it relates to lymph nodes, which serve as collection points for lymph throughout the body. With these connections in hand, the images provide essential information for the vertebral lymphatic system to be studied mechanistically, structurally, and even functionally.

A step beyond imaging: the function of vLVs

While the initial objective of this paper was to report images that revealed the three-dimensional arrangement of the vertebral lymphatic vasculature, the researchers now found themselves with many opportunities to study its function. For example, some scientists hypothesized that vLVs could be implicated in immune responses that help the body repair areas where spinal cord lesions have been locally sustained. To look into that question, the researchers injected a chemical known to damage spinal cord cells into adult mice and measured the extent of vLV networking after one week. According to the data they acquired, increased inflammation in the spinal cord in response to the injury led to an increase in the size of vLVs, confirming a previous report that indicated that lymphatic vessels regulate the immune surveillance of tissues in the central nervous system.

Moreover, the group also explored the functionality of this vertebral lymphatic network as a drainage system. To do so, they injected molecular tracers into specific locations on the vertebral column, and then analyzed their distribution in the surroundings of the injection site after fifteen to forty-five minutes. Using their immunolabeling approach, they were able to observe that those tracers were, in fact, present in the fluid collected by the lymph nodes locally connected to the vertebral lymphatic vessels. This finding substantiates the theory that the vLV network is involved in the absorption of molecules and the draining of tissues (pooling the fluid into lymph nodes), thereby validating the possibility that this vertebral lymphatic network could play an essential role in circulating immune cells between the central nervous system and the lymph nodes around the spinal cord. In addition, the researchers also found that the vLV system is organized into segmented regions that connect to local lymph nodes, suggesting that lymph is drained at the level of each vertebra, rather than as a continuous stream down the spine.

Looking into the unknown: other roles of vLVs

Knowing that the vLV network could help in immune surveillance of the central nervous system,  others can now study the transport mechanisms leading to the development and progression of inflammation, infections, and pathologies in the central nervous system. “We can think differently now that we know that there are lymphatic vessels around the spinal cord and…how they are arranged,” Jacob said. “We can ask new questions, such as what their role in a neuropathological context could be, or in the context of spinal cord lesions, or in the context of neurodegenerative diseases, for example.”

Another potential function of vLVs is in cancer metastasis, that is, the process in which cancer cells are disseminated away from the site of the primary tumor. “It is possible that this network could be important for transporting and propagating all kinds of pathogens or metastatic cells from the periphery of the body towards tissues in the CNS, as well as tissues that surround it, which are the meninges, the vertebral bones and the skull,” Thomas said. As many as seventy percent of cancer patients develop spinal metastases. In that context, the vLV architecture could provide a roadmap to understand how metastatic cells move along the spinal cord, possibly abetted by the vLV network.

By shedding light onto a biological arrangement that had not been previously studied, Thomas, Jacob, and their collaborators have given the scientific community a valuable resource. With this three-dimensional structure in hand, scientists can now work towards a deeper, more structurally driven understanding of the vertebral lymphatic system, and through it, explore new theories surrounding the spread of infections, central nervous system immune functions, and the development of metastatic tumors.