When the Trash Builds Up and the Sink Doesn’t Drain: Lymphatic System Dysfunction Contributes to Migraine

A new cell and animal study highlights reduced lymphatic vessel permeability in the meninges as a novel mechanism underlying migraine.

Considering its many-faceted and important roles in health and disease, it’s surprising that the lymphatic system historically received very little fanfare in the world of science and medicine.

Separate from the vascular system of the blood, the lymphatic system is a network of lymph vessels, lymph nodes, and other structures that, among many functions, maintain fluid homeostasis in the spaces around cells, control the trafficking of immune cells, and regulate the growth and development of organs, where they play many specialized roles, such as the absorption of dietary lipids through lymph vessels in the intestine.

But recent times have seen a resurgence of interest in this area, with lymphatic system dysfunction now associated with a wide range of conditions that affect the brain. A new study now provides a wealth of evidence to show it’s time to add migraine to the list of diseases with important links to the lymphatic system.

Researchers led by Kathleen Caron, University of North Carolina, Chapel Hill, US, show that genetically deleting – specifically in lymphatic vessels – the calcitonin receptor-like receptor (Calcrl), the gene that encodes the calcitonin gene-related peptide (CGRP) receptor, alleviated pain, light avoidance, and anxiety-like behavior in a mouse model of chronic migraine. The group also documented changes in the gene expression profile – which genes are turned on or off – of the vessels.

CGRP signaling also affected the entry of a certain type of T cell into meningeal lymphatic vessels, revealing that lymphatic dysfunction during migraine has an immune component. Finally, the investigators also pinpointed reduced permeability of the vessels, and consequent reduced drainage of cerebrospinal fluid to lymph nodes, as an additional mechanism underlying the lymphatic system’s contribution to migraine.

“It’s a wonderful paper – a very interesting, exciting, and novel story from a fantastic lab that does really exceptional work on lymphatics,” said Jonathan Kipnis, a neuroscientist and immunologist at Washington University in St. Louis, US. About 10 years ago, Kipnis’ lab discovered the presence of a lymphatic system in the meninges, but he was not involved with the new research.

“This idea that lymphatic vessels are less permeable and so that’s how their function is impaired is a beautiful mechanism,” he added.

“This is awesome work that opens up the field to new targets for CGRP treatment or understanding how CGRP could work in migraine,” said Dan Levy, a headache researcher at Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, US, who also did not take part in the study.

The research appeared online ahead of print in the Journal of Clinical Investigation on May 14, 2024, and is scheduled to appear in the August 1, 2024, issue of the journal.

The brain has a sink
Caron’s lab has long studied the CGRP receptor, its associated proteins, and the role of G protein-coupled receptors (the receptor class to which the CGRP receptor belongs) in the development and function of the lymphatic system.

Considering the advent of migraine drugs targeting the CGRP system, Caron told Migraine Science Collaborative in an interview that it made sense for her lab to expand its research purview to migraine.

“It only seems natural that we would start to work on migraine, given all the therapeutics that are targeting the receptor for which we have so many genetic animal models,” she said. Here she was referring to mice her lab has genetically engineered to lack either the CGRP receptor or receptor activity-modifying proteins (RAMPs), which are accessory proteins for the CGRP receptor.

Previously, there was hardly any basic science research on the role of the meningeal lymphatic system in migraine. But many brain diseases, including Alzheimer’s disease, multiple sclerosis, and Parkinson’s disease, have links to dysfunction of this system.

Under normal circumstances, the lymphatic system effectively regulates the turnover of cerebrospinal fluid (CSF), a liquid that surrounds the brain and spinal cord. That’s because it is through lymphatic vessels that CSF and potentially dangerous waste products in it are removed – think of the lymphatic system as “the sink of the brain” or “the sewage pipes in your household,” as Kipnis put it.

But in Alzheimer’s disease, for example, a dysfunctional lymphatic system leads to a build-up of beta-amyloid, a peptide that is the main component of the plaques in the brains of people with the condition. Perhaps in migraine, the lymphatic system also works poorly, leading to problems with the drainage of CSF, or to detrimental effects on other known functions of the lymphatic system.

Implicating CGRP signaling in lymphatic vessels during migraine
To test the idea, the researchers genetically deleted the Calcrl gene specifically in lymphatic vessels from mice. They asked whether that would affect the behavior of the animals in response to multiple injections of nitroglycerin (NTG) into the peritoneal cavity, an animal model of chronic migraine.

Compared to control animals treated with NTG, mice missing Calcrl showed reduced facial expression of pain in response to NTG, as measured by the mouse grimace scale (just like humans, mice grimace in response to pain, as indicated by changes in the position of their ears, eyes, nose, and cheeks). Mice lacking Calcrl also showed less of the light avoidance and anxiety-like behavior usually seen in the NTG model.

These findings suggested that CGRP signaling in lymphatic vessels might contribute to migraine pain. Results from additional experiments with a different set of knockout mice missing Ramp1, one of three Ramp genes coding for the RAMP protein, supported that hypothesis.

“My favorite part of this study – and I think the one that I’m still just kind of in awe about – is that the lymphatics can impact pain, a behavioral phenotype in an animal,” Caron said. “This is taking cell biology function at a molecular level and translating it to the sensation of pain.”

A changing genetic profile
The researchers next asked whether there were changes in gene expression in meningeal lymphatic vessels in the NTG migraine model. The team identified 700 differentially expressed genes between the NTG mice and animals receiving saline as a control.

Those genes turned out to be associated with a number of biological processes. For instance, some of the genes play a role in Calcrl signaling in lymphatic vessels, while others code for serum biomarkers of migraine. Many genes related to immune responses were also differentially expressed. Evidence emerged that CGRP alone could affect the gene expression profile of the NTG-treated mice.

Of particular note, there was a change in the expression of a gene that encodes a protein, Pentraxin3, which is found in lymphatic capillaries and in lymphatic endothelial cells of lymph nodes. Previous work had shown that this protein is associated with dysfunction of endothelial cells during migraine as well as with vascular inflammation. In the current study, Pentraxin3 was at higher levels at the endpoints of meningeal lymphatic vessels in the NTG mice, compared to controls.

There were also changes in MADCAM1, a protein known as a cell adhesion molecule that enables the attachment of immune cells to endothelial cells.

Together, the gene expression findings provided evidence for interactions between the lymphatic system and the immune system in the NTG migraine model.

A rearrangement
Further experiments in the NTG model supported a role for a particular function of the lymphatic system, namely the exit of immune cells from one location and their entrance into another.

In this case, there was an increase in a type of T cell that can interact with endothelial cells containing MADCAM1. Specifically, a higher number of these T cells were present in the deep cervical lymph nodes (to which meningeal lymphatic vessels drain) of control mice receiving NTG, but not in knockout mice missing the CGRP receptor.

What about another important function of lymphatic vessels, namely, the regulation of CSF turnover and the exit of CSF from the central nervous system through lymphatic vessels? That mechanism also appeared to be at play.

To investigate this aspect, the researchers used cultured human lymphatic endothelial cells. In these lab dish experiments, they aimed to understand how CGRP might affect the junctions between endothelial cells.

They found that CGRP changed the physical arrangement of the junctions. Specifically, the junctions, which normally are separated from each other, became more linear and continuous. When cell junctions are arranged in that manner, cell permeability goes down, which could lead to the buildup of CSF in the lymphatic vessels. Additional cell experiments did show reduced permeability of lymphatic endothelial cells in response to CGRP.

Meningeal lymphatic vessels, responding to the migraine-associated pain peptide CGRP, govern the neuroinflammation, CSF drainage and pain of migraine in mice. This image depicts VE-cadherin staining (green) of a mouse meningeal lymphatic vessel (violet), highlighting the cellular junctions that reorganize in response to CGRP signaling. (VE-cadherin is an adhesion molecule found at junctions between endothelial cells. Staining for this molecule allows for the quantification of inter-endothelial cell junctions). Image Credit: Laszlo Balint and Nate Nelson-Maney.

Back to animals
Returning to the animal model of migraine, here, too, the researchers saw a higher proportion of continuous junctions between endothelial cells in meningeal lymphatic vessels in response to NTG. Consistent with that finding, the genetically engineered mice missing Ramp1 had fewer continuous junctions after they received NTG, compared to controls.

As in the cell studies, CGRP reduced the permeability of meningeal lymphatic vessels in the animals. Here, the researchers quantified how much of a traceable dye, containing either saline (as a control) or CGRP, the vessels were able to take up (a measure of permeability). They also measured the exit of the dye to the deep cervical lymph nodes.

In control experiments in mice receiving saline-containing dye injections into the cisterna magna (an anatomic space in the meninges filled with CSF), no difference in lymphatic vessel permeability was found between mice missing Calcrl in lymphatic endothelial cells and mice with normal levels of Calcrl.

However, the situation was different when the experimenters used dye containing CGRP instead of saline. In this case, there was decreased permeability in mice that had the usual presence of Calcrl. No such effect was seen in the mice missing the receptor.

Could reduced permeability in response to CGRP affect the exit of CSF from the lymphatic vessels and its entry into the deep cervical lymph nodes? Measuring uptake of the dye, which served as a secondary measure of CSF efflux, the researchers showed that including CGRP in the dye reduced the amount of dye in the lymph nodes, compared to saline.

“What is so impressive is the profound effect that this singular peptide has on the permeability of these cells. So when CGRP is elevated, when it acts on the receptors on these cells, they shut down – they don’t drain fluid anymore,” Caron said.

Based on all the findings, the researchers are proposing a model where two different mechanisms drive lymphatic dysfunction during migraine. First, CGRP signaling in lymphatic vessels reduces endothelial cell junction permeability, which in turn impairs the drainage of CSF to lymph nodes during migraine. That leads to pain.

Second, CGRP signaling in lymphatic endothelial cells containing MADCAM1 facilitates the entry of T cells into meningeal lymphatic vessels. From there, the T cells exit to the deep cervical lymph nodes.

You can’t deny it
Of the two lymphatic mechanisms in the meninges that appeared at play in the NTG migraine model, which one is more important? That’s an open question.

But the exact lymphatic mechanisms involved in disease likely differ depending on the condition, Kipnis said. For example, “Alzheimer’s disease may be more about waste clearance, and multiple sclerosis may be mediated mostly by immune system changes,” he said.

Exactly how lymphatic dysfunction produces pain in migraine is also unclear. If CSF fluid buildup is playing an important role, one possibility is that this will increase intracranial pressure, leading to headache pain.

“It would be interesting to measure intracranial pressure over time, with or without CGRP blocking,” Levy pointed out.

Regardless, Caron said the role of the lymphatic system in migraine pathophysiology can’t be denied, even if that system is different from the trigeminal nerve and other targets that people more commonly consider when they think about migraine disease.

“Our study doesn’t eliminate those other targets. They’re clearly very important. But I think our study highlights just how important the lymphatics are in this process, which really has not been appreciated at all.”

Kipnis expressed a similar sentiment.

“In Alzheimer’s disease, you clog the sink with beta-amyloid. In migraine, there are so many players. Whatever the mechanisms are for migraine, Alzheimer’s, or any other disease, I think they are probably substantially exacerbated by dysfunction of that final output of the fluid from the brain. But it’s not the mechanism,” Kipnis said.

Future work
In terms of future studies for her lab, Caron said she is curious about which cell types anti-CGRP drugs affect, and about the relative contribution of CGRP signaling to meningeal lymphatics versus other systems.

“We’ll be looking very curiously at the meningeal lymphatics to see just how much of elevated CGRP is influencing the meninges. We already know it’s influencing the vasculature. But how much and to what extent are the meninges and nearby immune cells being affected?” Caron said.

She’s also curious about how the CGRP receptor might be differentially regulated in women versus men, considering, she said, that lymphatic dysfunction, such as lymphedema (swelling of tissue with lymph fluid), is more prevalent in women.

Levy told MSC that it’s unclear if CGRP released in the meninges during neurogenic inflammation (a key migraine hypothesis) or generated by the central nervous system during migraine would have the same effects on the lymphatic system as in the NTG model.

Pointing to another remaining question, Kipnis said, “Which aspect of migraine is controlled by dysfunction of the lymphatic system – that’s a very interesting scientific question.”

He continued, “On the more clinical side, can we target the lymphatic vessels and then maybe alter migraine?”

It’s a plausible idea, considering that inhibitors of vascular endothelial growth factor (VEGF), a growth factor for lymphatic endothelial cells, or inhibitors of its receptor, are already approved for cancer treatment. Perhaps they could work in migraine as well.

Caron agreed that translating the basic science research into people is an important next step.

“We can, and we should, start to address whether meningeal lymphatics are playing a role in patients with migraine. This is a question that we can answer – we have the technology to do it and a patient population eager to participate in clinical trials and help advance research.”

Even with hopes for translation, Caron still emphasized that, to her, one take-home message of the research is that studies in animal models, with the experimental approaches used in those models, still matter, even in a world where there are safe and effective therapies that target CGRP.

“There are Lady Gaga commercials [for rimegepant], there’s a lot of fanfare, these anti-CGRP drugs are FDA approved, and people are taking these medications. And yet we’re still making foundational discoveries, using these sophisticated genetic tools, that are providing really important information about translational science, even for targets that are being drugged right now.”

Neil Andrews is a science journalist and executive editor of the Migraine Science Collaborative. Follow him on X (formerly Twitter) @NeilAndrews

Reference
Meningeal lymphatic CGRP signaling governs pain via cerebrospinal fluid efflux and neuroinflammation in migraine models.
Nelson-Maney et al.
J Clin Invest. 2024 May 14:e175616.