Channeling immune cells from the skull to the brain

Within and outside of the field of neuroscience, scientists don’t tend to look at the skull as anything besides a brain helmet.  

Yet, Zeynep Ilgin Kolabas, a graduate student researcher at the Ludwig Maximilian University of Munich, and her colleagues recently revealed that channels between the skull and brain serve as a pathway to traffic immune cells (1). They also found skull-specific immune responses to stroke, Alzheimer’s disease, and multiple sclerosis that could serve as the basis for a new form of brain disease monitoring. 

“Now we’re seeing that it’s more than a physical barrier,” said Kolabas. “The cells within the [skull] bone marrow — immune cells that actually have a say in how the diseases progress or will exert themselves — are very active.”

Neuroscientist Ali Ertürk, Kolabas’s PhD supervisor and study coauthor, previously discovered the existence of the channels as short tube-like structures that connect the skull with the brain’s protective layer, the meninges (2). Ertürk had been developing a new protocol for studying neurons in mice, where they tagged neurons with a specially-designed fluorescent label and incubated their samples in various buffers that rendered surrounding tissues transparent. This allowed the researchers to image neurons throughout the entire mouse body, which had not been possible in previous protocols. “Keeping the meninges and the skull intact, we were able to basically see those channels for the first time,” said Kolabas. 

The channels were an unexpected discovery that left Kolabas and her team with several questions: “How many are there? Where are they? Are they homogenously distributed? Are they changing in size and density, depending on something specific?” said Kolabas.

Answering these questions with human samples was an arduous process; keeping the skull, meninges, and brain intact within the same sample proved to be their biggest challenge in the whole study. “These tissues are so, so different from each other,” Kolabas said. “Their water content, their hardness, everything is different about them.” Draining water from the tissues, a key step in the clearing protocol, only made them smaller and more easily detachable. Kolabas recounted feeling the pressure. “If we lose everything, it doesn’t make sense for us to clear [the tissues] because that's exactly what we want to see: how they're connected to each other,” she said.

After a number of failed attempts, the researchers were finally able to stabilize the samples in a corner of the incubation box to minimize movement and shrinkage. Even then, Kolabas still had doubts that they would see anything. “We're checking a very small portion of the brain — I'm speculating one fiftieth of the whole thing — so we would have to be lucky to run into those channels under the microscope.” The team was therefore very excited to not only find the channels coming from the skull but also to discover that they crossed deeper into the meninges than they had reported in the previous paper (2). “That might have been the best part of the whole process,” said Kolabas. “[It was] very, very mesmerizing to see that whole thing intact and real.” The channels were populated with myeloid cells, which encompass several cells in the immune system. This suggested that the channels facilitated immune cell trafficking between the skull and the meninges.

To see whether the channels were part of a larger skull-specific immune response, the team analyzed the skull’s RNA and protein content and compared it to various bones throughout the body in mice. They measured immune responses to brain injury by using a mouse model of stroke that inserts a filament into mice’s cerebral arteries and restricts blood flow into the brain. They found that the bone marrow within the skull had the most pronounced inflammatory response to the injury, driving up the production of immune cells that could potentially help the brain. It also had more unique biomarkers and differentially expressed genes than any other type of bone marrow. Most of these were in neutrophils, white blood cells that are considered the immune system’s first line of defense (3). In their stroke model, the neutrophils in the meninges more closely resembled those in the skull than in any other bone marrow type. This confirmed to Kolabas that the channels were shuttling immune cells between the skull bone marrow and the meninges. “I'm far from being able to say, ‘Okay, this specific gene does this,’” said Kolabas. “But we did find some interesting targets that should be further followed up.” 

“This paper is really unique and impactful, showing this specific role of the skull bone marrow in driving these neuroinflammatory responses to injury,” said Laura Fonken, a neuroimmunologist at the University of Texas at Austin who was not involved in the study. “It's identified a new area for further research.”

Finally, the team conducted PET scans in patients with various neurodegenerative diseases to find out whether their conditions were visibly reflected in the skull. They showed that patients with Alzheimer’s disease, stroke, and multiple sclerosis each had distinct patterns of inflammation within the skull that were specific to their condition. Follow up scans for patients with Alzheimer’s disease and stroke showed that these differences were sustained over time. The researchers’ work is the first to suggest that the skull could be used as a proxy for monitoring brain disease.  “It just expands the whole idea of what we can do and what we should check for in terms of how the brain functions,” said Kolabas. 

Kolabas and her colleagues are now investigating the biomarkers they identified in the hopes of gleaning further insights into the skull’s immune response. Ultimately, Kolabas hopes that the team’s work can lead to a more accessible non-invasive diagnostic and prognostic tool for neurodegenerative diseases. She also speculates that the skull may have specific responses to neurodevelopmental and psychiatric diseases as well, but that remains to be seen. “A lot more questions emerged after my paper, and there are, of course, many more questions to be answered in the upcoming years with more research,” she said.

References

1. Kolabas, Z.I. et al. Distinct molecular profiles of skull bone marrow in health and neurological disorders. Cell  186, 3706-3725.e29 (2023).
2. Cai, R. et al. Panoptic imaging of transparent mice reveals whole-body neuronal projections and skull–meninges connections. Nat Neurosci  22, 317-327 (2019).
3. Rosales, C. et al. Neutrophils: Their Role in Innate and Adaptive Immunity 2017 J Immunol Res  2017, 9748345 (2017).