The newly discovered lymphatic system in the brain, and a possible brain microbiome

November 11th, 2015 by Amy Proal

The brain has long been considered to be a sterile organ, an “immunoprivileged” body site that microbes cannot directly enter and infect. Most medical textbooks still contend that microbes cannot enter the healthy brain due to a layer of endothelial cells that separate the brain from the body. This layer of cells, called the blood-brain barrier, is believed to separate circulating blood in the brain from fluid in the central nervous system and the rest of the body. Current dogma also dictates that bacteria are too large in shape and size to penetrate this barrier.

Despite this blood-brain “barrier”, researchers have regularly detected at least some pathogens in the brain: Borrelia, Group B streptococci, and Treponema pallidum – to name a just few. Such microbes’ presence is generally explained by the hypothesis that the blood-brain barrier becomes more permeable under conditions of inflammation. Certain pathogens, especially those able to survive inside of the cells of the immune system, are then considered capable of crossing into the brain’s circulatory system.

Yet just a few months ago, two new papers were published that delineate another major pathway by which microbes in the blood and central nervous system can easily penetrate the brain. The research teams behind these papers demonstrate the existence of previously undiscovered lymphatic vessels that carry both fluid and immune cells from the cerebrospinal fluid and cervical lymph nodes directly into the brain.

Lymphatic vessels form an important part of the immune and circulatory systems. Blood vessels throughout the human body run parallel to lymphatic vessels. Toxins, waste, and in some cases infected cells, are continually filtered from the blood into these vessels. The vessels contain a clear fluid called lymph. As lymphatic fluid shuttles toxins towards the liver and kidneys, the lymphatic system effectively detoxifies the blood.

Until very recently however, the lymphatic system was believed to traverse the body but stop short of the central nervous system. That was until researchers at the University of Helsinki began studying what appeared to be a lymphatic-like vessel in the eye. Surprised that the eye, also traditionally regarded as “immunoprivileged”, would contain this vessel, the team began to look for similar structures elsewhere in the central nervous system.

Maps of the lymphatic system: old (left) and updated to reflect the new discovery.

Maps of the lymphatic system: old (left) and updated to reflect the new discovery.

The researchers turned to mice, where they began studying layers of brain tissue called meninges. The meninges cover the brain and contain blood vessels and cerebrospinal fluid. Using a technique called immunofluorescence, the team was able to cause structures associated with lymphatic vessels to light up with fluorescent dye. They were then able to analyze and photograph these vessels under a microscope. Overall, their data shows that lymphatic vessels are present in the outer meninges of the central nervous system and drain out of the skull via a structure called the foramina. This happens at the base of the skull, alongside arteries, veins, and cranial nerves.

As far as I can tell, at the same time that the Helsinki team was conducting the above research, a team at the University of Virginia School of Medicine also stumbled upon the same lymphatic system in their research lab. This second team also began by studying the vessels in mice, and also used fluorescence-based techniques. In one study, they tracked fluorescent dye as it traversed the newly discovered cerebrospinal lymphatic vessels. Again, the vessels were found to carry fluid and immune cells from the cerebrospinal fluid, along veins in the sinuses, and into the deep cervical lymph nodes.

In this image, mouse mouse meninges lymphatic vessels (red) are shown along side the blood vasculature (green). (Antoine Louveau)

Mouse meninges lymphatic vessels (red) are shown along side the blood vasculature (green). (Antoine Louveau)

According to Cosmos magazine, The University of Virginia team has proceeded to examine autopsy specimens of human meninges. They believe they have found structures similar to the mouse central nervous system lymphatic vessels in the human tissue.

How was the existence of these vessels missed until 2015? Both research teams contend that the vessels exist in difficult-to-image areas of the brain shadowed by major blood vessels. Because the newly discovered lymphatic vessels run so closely alongside blood vessels, it was apparently hard to tell the two systems apart.

Researchers from both the Helsinki and Virginia research teams are excited that their findings might shed light on how the brain drains unwanted waste products in patients with various neurological conditions. A central nervous system lymphatic system can also better explain how immune cells such as T cells enter the brain and combat pathogens. However, if immune cells can easily enter the brain via the blood and cerebrospinal fluid, then so can a host of microbes. Thus, the findings certainly shed light on how the brain might support microbial populations, even in the absence of inflammation.

Indeed, the ability of microbes in the cerebrospinal fluid and blood to directly enter the brain is supported by an increasing number of studies showing the presence of persistent pathogens in brain tissue. One such study, conducted by researchers at the University of Alberta, recently detected an ecosystem of chronic microbes in the autopsied brains of patients with HIV and a range of other medical conditions associated with severe neurological dysfunction. The team used deep sequencing techniques to search for bacteria, viruses and bacteriophages in their brain samples.  They detected 173 bacteria and bacteriophage-derived samples. Many of these microbes were identified inside macrophages, astrocytes, microglia, and other cells of the immune system.

Various bacterial classes detected in the brains studied. (Branton et al.)

Various bacterial classes detected in the brains studied. (Branton et al.)

Entire communities of microbes – a microbiome – were found in each brain studied. The microbiomes of most human body sites tend to be dominated by microbes from the phyla Firmicutes and Bacteroidetes, often in concert with colonization by Actinobacteria and Proteobacteria. However, all the brains examined by the Canadian team were dominated by the phylum Alphaproteobacteria, a highly diverse class of bacteria comprised of dozens of species. To be specific, Alphaproteobacteria represented about 70% of bacteria identified in the brain samples, while the other 30% of bacteria identified belonged to a wide range of other classes.

Several microbes identified in the brains were pathogens associated with specific human diseases. They included Delfia acidovorans, a pathogen implicated in endocarditis, bacteremia, and urinary tract infections. Delfia acidovorans has even been identified as part of the bacterial community in the arterial wall of patients who have aortic aneurysms. Viruses (including various herpes viruses) were also identified in many of the brain samples.

Bacteria detected in the brain samples after staining for peptidoglycan

Bacteria detected in the brain samples after staining for peptidoglycan. Some microbes were found inside the cells of the immune system.

A great deal of the University of Alberta study is devoted to describing techniques employed by the research team to help convince the reader that the brains under study were not contaminated during the research process. In other words, they tried to show that identified microbes really were derived from the brain tissue and not, say, from the hands of someone in the lab. After autopsy, the brains were immediately frozen in liquid nitrogen. The team also used different batches of reagents, in different labs, at different times, when examining the brains – yet their results remained consistent despite these variables.

One arm of the study also transferred solution from several of the diseased brains into the brains of immunocompromised mice with apparently sterile brains. However, in half the mice, the solution was heated to kill any bacteria present. The brains of mice who received “live” brain solution became colonized by many of the microbes in the original diseased brains. The brains of mice who received the dead microbes remained sterile.

Several of the brains were stained with antibodies to peptidoglycan, a component of bacterial cell walls. These stains cause peptidoglycan to turn a different color, causing microbes in the brain samples to become visible under an electron microscope. The team was thus able to directly generate images of several of the microbes under study.

Keep in mind that the brains in this study were taken from very ill individuals. It remains to be seen what type of microbiome might persist in the healthy human brain, and if a healthy brain microbiome would also be dominated by Alphaproteobacteria. But certainly the University of Alberta study dispels the idea that the brain is sterile and “immunoprivileged.”

journal.pone.0054673.g001I think it’s likely that a microbiome will be identified in the healthy human brain. But I doubt it will be dominated by Alphaproteobacteria. That is because the University of Alberta study also examines brain tissue taken from two patients with epilepsy during a medical procedure. These patients were alive at the time the samples were taken. As you can see in the figure above, the microbial populations in the epilepsy brains were more diverse than those identified in the brains removed after autopsy. This suggests that Alphaproteobacteria may be a phylum of microbes that takes over the brain microbiome when patients are extremely ill and immunocompromised (near death). Patients who are less ill, or people who are more healthy, might then be expected to harbor more diverse brain microbiome populations. I look forward to future studies that might better explore this possibility.

Last but not least, the Canadian team spends time in their discussion wondering how the microbes in their brain samples were able to cross the “blood-brain barrier.” It’s interesting to think that their research was conducted around the same time that the brain’s lymphatic system was being discovered. Now, moving forward, the Canadian team can interpret their findings in light of the new information.

I have more thoughts on this last study about microbes in the brain. Listen to this audio recording to hear them:

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