Updated June 4, 2015: Discovery of meningeal lymphatic vessels in the mouse’s brain by Jonathan Kipnis’ lab at the University of Virginia, USA. Study published online on June 1, 2015 in Nature:
Page on nature.com
UVA news report about the study:
Missing link found between brain, immune system — with profound disease implications
Goose bumps, I’m that excited about this discovery! Hopefully, the final nail in the coffin for the idea that brain has Immune privilege.
Let’s examine the premise first. Should there be lymph nodes in the brain? After all, other solid organs such as the heart and kidneys don’t have lymph nodes inside them either. Even so, the brain has a totally different relationship to lymphatics. Unlike those other solid organs, the brain and spinal cord don’t have lymphatics. This is a difference in kind. Over much of the past century, this has been a central feature of the Blood-Brain Barrier (BBB) narrative.
What is the Blood-Brain Barrier (BBB)?
When a dye or tracer is injected into the blood, eventually, it extravasates through the blood capillaries into tissues. Extent of this extravasation reveals the degree of capillary permeability. Our blood capillaries are not all the same. For example, capillaries in certain regions of the liver are much more permeable than those anywhere else. OTOH, blood capillaries in the brain are more impermeable. How do we know this? Since the 19th century, dye and tracer injection studies have suggested this.
- In 1900, Max Lewandowsky saw that direct, but not intravenous, injection into the brain ventricles of strychnine or sodium ferrocyanide produced pharmacological effects.
- In 1913, Edwin Goldmann found that the dye Trypan blue injected into the CSF (Cerebrospinal fluid) of dogs stained brain tissue and also appeared in the deep cervical lymph nodes. In other words, a direct link between brain and lymphatics (1).
- Though many scientists have wrongly attributed to Max Lewandowsky the coinage of the term BBB, more recent careful research in the history of science shows that the Latvian scientist Lina Stern first coined the term barrier for flow of material into and out of the CNS (Central Nervous System) in her paper with R. Gautier, ‘Le passage dans le liquide céphalo-rachidien de substances introduites dans la circulation et leur action sur le système nerveux central chez les différentes espèces animales (The barrier which opposes the movement into the CSF of substances in the blood shows notable differences in different animal species) (2).
- Here are brief bulleted biographies of these three remarkable scientists, Max Lewandowsky, Edwin Goldmann, Lina Stern.
Lina Stern‘s in particular is soap operatic in terms of its remarkable ups and downs, ranging from winning the Stalin Prize and becoming the first woman admitted to the USSR Academy of Sciences to utter downfall a few years later, sentenced to imprisonment and exile in a Stalinist Putsch. These brief bulleted nuggets suggest not merely a groundbreaking scientist but also a woman of phenomenal fortitude and resilience. Her life story deserves a full-length biography and wider public knowledge.
Proof positive for the BBB came in the form of this groundbreaking 1967 paper by Reese and Karnovsky (3).
- In this mouse model study, they intravenously injected the 40 kDa (kilodalton) glycoprotein enzyme HRP (Horseradish peroxidase).
- Endothelial capillaries have basement membranes at the base of each cell and tight junction proteins between cells.
- More gaps in basement membrane and tight junctions, more permeable the endothelial capillary.
- Reese and Karnovsky’s technological innovation was to use electron microscopy to show definitively that the BBB is at the brain endothelial capillary level.
- They showed that HRP couldn’t penetrate beyond the first inter-endothelial tight junctions in brain capillaries.
- Thus BBB is a physical barrier that makes it more difficult for blood circulation materials to get into brain tissue.
- Since the barrier consists of additional cells such as astrocytes, pericytes, microglia and even neurons, in modern terms the BBB is more accurately described as a ‘neurovascular unit‘ (4, 5).
Is it just as difficult for extracellular brain fluids to get out?
Over the course of the 20th century, many neuro-immuno-physiologists arrived at a problematic and erroneous extrapolation of the BBB, namely, that CNS remained relatively impregnable to immune cells and functions because of the impregnable BBB.
Though erroneously assumed to be so for many decades, the brilliant brain lymphatics researcher Helen Cserr was decades ahead when she definitively showed that extracellular brain fluids indeed access lymphatics, albeit in a unique manner. Building upon the pioneering 1960s studies of the German lymphologist Michael Foldi, she and her team used mouse, rat and sheep models in the 1980s and 1990s to show brain fluid circulation patterns accessed local extra-cranial lymphatics.
Why then do scientific reviews tend to continue to refer to brain as immune privileged, i.e. not/less accessible to the immune system? Unfortunately, Helen Cserr died in 1994 of a brain tumor at the relatively (for a scientist) young age of 57, and scientists tend to stick to the conventional script, even after it’s been convincingly disproved. Paradigm shifts take time, sometimes decades, often even generations.
However, in the past 10 years, the tide has begun to turn and now it’s increasingly accepted that CNS accesses local lymphatic drainage in normal physiology.
- Helen Cserr’s studies in the 1980s and 1990s were key in helping overturn the conventional view that brain fluids did not reach local lymphatics.
- If CSF drains into local extra-cranial head and neck CLN (cervical lymph nodes), and now we know it does, we can no longer consider the CNS as being an immune privileged site.
- Immune privilege was a notion developed by Nobel Prize-winner Peter Medawar in the 1950s.
- Immune privilege implies certain tissue sites such as the CNS remain relatively ignored by/inaccessible to the immune system.
- Medawar and his colleagues, Rupert E. Billingham and Leslie Baruch Brent, arrived at the notion of immune privilege based on the observation that allogeneic (genetically distinct) tissue grafts in the same animals were accepted at ‘immune privileged’ sites such as the CNS while they were speedily rejected from other sites, such as the skin.
- Today we better understand that acceptance of allogeneic grafts at sites such as CNS is not due to ‘absence of immune response’ but rather due to ‘presence of different kinds of immune responses‘, responses that do not lead to graft rejection.
The data that brain fluids reach local lymphatics
What happens to dyes and tracers injected into the CSF or brain tissue? Experiments done since the late 19th century have shown intra-cranial dyes and tracers make their way to extra-cranial head and neck lymphatics and lymph nodes at/near the cribiform plate, in mice, rats (6, 7, 8), rabbits, sheep (9, 10), pigs, monkeys (11).
Thus, an abundance of observational data from 1869 until 2005 prove CSF-Lymphatics link (11).
- Studies consisted of material injected into CSF and recovered elsewhere including from surrounding lymphatics such as CLN (Cervical Lymph Nodes) in the pharynx and nasal lymphatics.
- Dyes and tracers injected into the subarachnoid space enter lymphatics close to the olfactory nerves at the extra-cranial surface of the cribiform plate.
- Wide range of materials: Dyes (Berlin blue, Evan’s blue, India ink, etc), Tracers (RISA, Microfil, etc).
- Wide range of mammals: Cat, Dog, Guinea Pig, Human, Monkey, Mouse, Sheep, Rabbit, Rat.
- Wide range of researchers, experimental design, materials and species used strengthen CSF-lymphatics link.
Abundance of functional data also suggests that CSF drains into lymphatic circulation because
- Cervical lymph flow increases when intracranial pressure increases (12, 13).
- Experimental studies that block CSF flow to lymphatics through the cribiform plate decrease CSF absorption (14, 15).
Why do our tissues and organs need lymphatics?
Blood capillaries don’t absorb all the proteins, solutes, water, tissue debris and other products secreted by the cells in tissues and organs. The tissue’s lymphatics clear the leftovers, thereby ensuring optimal fluid and pressure balance of tissues and organs. The lymphatic system is our second circulatory system, functioning as an adjunct to the blood circulatory system. Thus in addition to blood vessels, most of our body’s organs and tissues have lymphatics, with brain as the main exception. Why is that? Unique anatomically imposed constraints.
The skull imposes strict restrictions on intracranial pressure. In turn, the brain has developed the unique and tightly coupled system of cerebrospinal and interstitial fluids to ensure proper fluid drainage of brain tissue. Thus the debate is not about brain lymph nodes but rather has been and still is on whether and how much these brain-intrinsic fluid drainage systems connect with extra-cranial head and neck lymphatic systems, namely the cervical lymph nodes. For more than a century the BBB has driven understanding of CNS-immune system interface. Today, a more accurate understanding of the physiology shows that, rather than avoid or minimize immune system interactions, its unique anatomical constraint has driven equally unique approaches for the CNS to connect with the immune system.
What are brain fluids and how do they drain?
Brain ISF (Interstitial Fluid)
Brain ISF sources are capillary endothelium, cell metabolism and recycled CSF (Cerebrospinal Fluid).
Two opposing forces are in play within blood capillaries. Hydrostatic pressure of the capillary wall and colloid osmotic pressure of the plasma proteins. The former favors filtration while the latter opposes it. In most tissues, capillary wall composition favors filtration such that interstitial fluid (ISF) is forced into the lymphatics. The brain capillaries are different in kind with extremely tight intercellular junctions that create a tight barrier to proteins, the BBB. Brain vasculature filtration is thus very low. As a result, brain ISF has little protein compared to other organs. Thus, the reverse of normal capillaries in that the colloid osmotic pressure of the plasma proteins dominates the capillary wall hydrostatic pressure. The low ISF protein content also means that CNS does not have true lymph, negating its need for a true lymphatic system.
Brain CSF (Cerebrospinal Fluid)
Two separate fluid compartments, the subarachnoid spaces and the ventricles, surround the brain. Studies in rat suggest approximately 10% fluid leakage from the ISF to the CSF (16). To all intents and purposes, brain ISF and CSF appear to be separate with distinct production, drainage and function, though this issue is still the subject of active debate and research. CSF flows through the foramina of the cribiform plate, present along the subarachnoid space surrounding the olfactory nerves. From there, CSF is easily absorbed by lymphatic vessels associated with the olfactory nerve roots.
Thus, current understanding of brain lymphatic drainage goes something like this.
A. CSF drains from sub-arachnoid space through cribriform plate.
B. It then travels via nasal lymphatics to
C. CLNs (Cervical Lymph Nodes).
D. Nasal lymphatics are large enough to accommodate immunocytes such as lymphocytes and monocytes.
E. CSF also drains into blood via arachnoid granulations and villi
F. Into vascular endothelium, though it’s now actively debated if such drainage is even important.
G. ISF drains from brain tissue along cerebral capillary and artery walls.
H. This ISF drainage occurs along the basement membranes surrounding the arterial smooth muscle cells.
I. ISF also drains along internal carotid artery walls in the neck, also to the CLNs.
- Goldmann, Edwin Ellen. Vitalfärbung am Zentralnervensystem: Beitrag zur Physio-Pathologie des Plexus chorioideus und der Hirnhäute. No. 1. Königl. Akademie der Wissenschaften, 1913.
- From Saunders, Norman R., et al. “The rights and wrongs of blood-brain barrier permeability studies: a walk through 100 years of history.” Frontiers in neuroscience 8 (2014). The rights and wrongs of blood-brain barrier permeability studies: a walk through 100 years of hi…
- Reese, T. S., and Morris J. Karnovsky. “Fine structural localization of a blood-brain barrier to exogenous peroxidase.” The Journal of cell biology 34.1 (1967): 207-217. FINE STRUCTURAL LOCALIZATION OF A BLOOD-BRAIN BARRIER TO EXOGENOUS PEROXIDASE
- Cohen, Z. V. I., et al. “Serotonin in the regulation of brain microcirculation.” Progress in neurobiology 50.4 (1996): 335-362.
- Neuwelt, Edward A. “Mechanisms of disease: the blood-brain barrier.” Neurosurgery 54.1 (2004): 131-142.
- Zhang, E. T., et al. “Directional and compartmentalised drainage of interstitial fluid and cerebrospinal fluid from the rat brain.” Acta neuropathologica 83.3 (1992): 233-239.
- Kida, S., A. W. R. O. Pantazis, and R. O. Weller. “CSF drains directly from the subarachnoid space into nasal lymphatics in the rat. Anatomy, histology and immunological significance.” Neuropathology and applied neurobiology 19.6 (1993): 480-488.
- Kida, S., et al. “Anatomical pathways for lymphatic drainage of the brain and their pathological significance.” Neuropathology and applied neurobiology 21.3 (1995): 181-184.
- Zakharov, Andrei, Christina Papaiconomou, and Miles Johnston. “Lymphatic vessels gain access to cerebrospinal fluid through unique association with olfactory nerves.” Lymphatic research and biology 2.3 (2004): 139-146.
- Zakharov, A., et al. “Integrating the roles of extracranial lymphatics and intracranial veins in cerebrospinal fluid absorption in sheep.” Microvascular research 67.1 (2004): 96-104.
- Koh, Lena, Andrei Zakharov, and Miles Johnston. “Integration of the subarachnoid space and lymphatics: is it time to embrace a new concept of cerebrospinal fluid absorption.” Cerebrospinal Fluid Res 2.6 (2005): 8454-2. Page on fluidsbarrierscns.com
- Boulton, M., et al. “Raised intracranial pressure increases CSF drainage through arachnoid villi and extracranial lymphatics.” American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 275.3 (1998): R889-R896. Page on physiology.org
- Silver, I., et al. “Relationship between intracranial pressure and cervical lymphatic pressure and flow rates in sheep.” American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 277.6 (1999): R1712-R1717. Page on physiology.org
- Casley-Smith, J. R., E. Földi-Börsök, and M. Földi. “The prelymphatic pathways of the brain as revealed by cervical lymphatic obstruction and the passage of particles.” British journal of experimental pathology 57.2 (1976): 179. The prelymphatic pathways of the brain as revealed by cervical lymphatic obstruction and the passage of particles.
- Casley‐Smith, J. R., et al. “The effects of chronic cervical lymphostasis on regions drained by lymphatics and by prelymphatics.” The Journal of pathology 124.1 (1978): 13-17.
- Szentistvanyi, I. S. T. V. A. N., et al. “Drainage of interstitial fluid from different regions of rat brain.” American Journal of Physiology-Renal Physiology 246.6 (1984): F835-F844.