EVATAR- Building the female reproductive tract in a dish

EVATAR – a box of reproductive tissues

Researchers have made a hand-sized device with all the tissues of the female reproductive system (1). They call it EVATAR (Eve + Avatar – a digital human).

EVATAR can be used to test if a new medicine is likely to cause hormonal problems or affect fertility in women.

Up until now, such tests could only be performed on human beings.

The device itself is like a box with many compartments. Each compartment contains tissues from the female reproductive organs. These include the ovaries, fallopian tube, uterus, and cervix.

The compartments are connected – allowing them to share nutrients and talk to each other using chemical signals (1).

Special electromagnetic valves control the flow of nutrients between compartments and a computer program controls the amount of nutrient in each compartment (1,2).

Photo credit : Northwestern University, Woodruff Lab

Why do we need this device?

Animals are not the best models for human disease or for studying how people will respond to a new medicine (3,4).

So what do we do when a study/ trial cannot ethically be performed in a human?

Traditionally, a scientist would take out some bits of a tissue from the human body and keep it alive in the lab (5).

They would then use these for further tests and studies.

Many human tissues, however, die or loose their function outside the body.

In the EVATAR device, researchers have succeeded in keeping all the tissues from the female reproductive system alive and functional for the 28-day menstrual cycle (1,6).

 

Testing for the behavior of tissues

During the menstrual cycle, the ovary in a woman’s body produces a mature egg. This mature egg is either fertilized or removed from the body(6)

The reproductive system, brain, and pituitary gland work together to make this happen(6).

The pituitary gland secretes gonadotrophins (namely Follicle Stimulating Hormone and Luteinizing Hormone).

The ovaries in the device responded to an external supply of these hormones by making their own chemical signals/ hormones – oestradiol, progesterone, Inhibin A and B.

The ovaries also produced a mature egg within the device.

Other tissues of the reproductive tract responded to the ovarian hormones.

The lining of the uterus – the endometrium, made more receptors to the hormones progesterone and estrogen at end of the 28-day cycle. The cells of the endometrium also multiplied, as expected during the menstrual cycle.

The ectocervix is the bit of the cervix that is externally exposed. It maintained it’s skin-like appearance and structure through the cycle.

This tissue also made receptors for the hormone progesterone only on day 0 and not on day 14 of the cycle, possibly controlled by the secretions of the ovary.

In the fallopian tube, the hair-like projections or cilia, that nudge the egg from the ovary into the uterus, were beating and functional even after 21 days in the device (7).

Researchers can now use this device to study infections and hormonal problems, as well as the reproductive system itself.

Inclusion of liver tissue in the EVATAR device

Tissue from the liver was included in the EVATAR model in one of the compartments.

The liver is not directly a part of the female reproductive system or cycle. However,  it breaks down a new medicine in the body( 8).

After 28 days, the liver tissue looked normal and made a healthy amount of the protein, albumin, within the EVATAR device.

Researchers can now test a new medicine on the reproductive system and the liver simultaneously.

 

Want to know more about EVATAR and things mentioned in this article? Here are some links:

1. The study describing the construction of the EVATAR device – “A microfluidic culture model of the human reproductive tract and 28-day menstrual cycle” (https://www.nature.com/articles/ncomms14584)

2. Draper Labs on the technology behind EVATAR – http://www.draper.com/news/fighting-cancer-boosting-fertility-promise-first-female-reproductive-system-chip

3. “Man or mouse? Why drug research has taken the wrong turning”, October 2016, NewScientist. (https://www.newscientist.com/article/mg23230973-700-man-or-mouse/)

4. Why do we need animal models? https://speakingofresearch.com/facts/the-animal-model/

5. Tissue culture – http://www.biotechnologynotes.com/animals/animal-cell-culture-history-types-and-applications/671

6. Dr. Woodruff explains the menstrual cycle https://www.coursera.org/learn/reproductive-health (Lecture 2.2)

7. Video of the cilia of the fallopian tube beating – https://www.nature.com/article-assets/npg/ncomms/2017/170328/ncomms14584/extref/ncomms14584-s2.mov

8. How does the liver work? (https://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0072577/)

Thank you, Kelly McKinnon, Hunter Rogers (Dr. Woodruff’s lab) and Bernadette Sztojka for your feedback.

 

Can genetic variations define Ayurvedic Prakritis?

-A genome- wide study finds allelic differences between individuals that correlate with Ayurvedic body-types (Prakritis)

Snap-Shot of the study

Do the ayurvedic body types have genetic underpinnings? In a first step to answer this question, the authors evaluated differences between individuals whose body-type had been assigned by both Ayurvedic practitioners and a software. They found 52 variations across the genomes of 262 individuals which allowed them to be classified into ayurvedic Prakritis – Pitta, Kapha and Vata.

 Introduction to Ayurvedic prakritis

In Ayurveda, according to the ancient text Charaka Samhita –the body and mind must be brought together to lead a harmonious existence. People can be classified into Prakritis or types on the basis of relative contribution of the three constituents Pitta, Kapha and Vata (roughly translating to – arising from movement, digestion and accumulation – of toxic metabolites for instance) to their body. According to Ayurvedic philosophy- an understanding of this body type and the ability to maintain a diet and lifestyle suited to that body type translate to balance and health. Prakriti or Ayurvedic body-types which define a person’s intrinsic physical abilities, mental states and also have implications for their susceptibility to disease and response to drugs (1,2).

Background for this study

In a recent study (3), researchers have identified genetic variations associated with the traditional classification of people into Ayurvedic Prakritis – specifically if small differences (SNPs, Single nucleotide polymorphisms) throughout the human genome correlate with the Prakriti classification. All humans are genetically very similar to each other, differences between us (populations, races, ethnic groups etc.) are captured in variations of nucleotides which make up the DNA – these are called single nucleotide polymorphisms. Many studies have shown particular SNP or group of SNPs to be correlated with risk of disease, whether a person will develop resistance to therapy, etc. forming the basis for personalized medicine  (4,6).

 What did they do?

3416 individuals were classified for their prakritis by Ayurvedic practitioners as well as a software. Of these, DNA isolated from blood samples of 262 individuals (male, healthy, between 20-30 years), who were reliably classified as having a clear dominance of one of the three constituents (Vata, Pitta or Kapha) representing the “extreme” Prakritis were used for the genome-wide study. A microarray consisting of 1 million positions / SNPs was used to identify the genotype of these individuals.

 What did they find?

This study found 52 out of 1 million SNPs is sufficient to assign the Prakriti of individuals, irrespective of their ethnic background. One of the challenges in the study was that there was no control group – therefore each prakriti was compared to the other two. Subsequently, these 52 SNPs were able to cluster individuals into distinct groups by Principal Component Analysis. Interestingly, one of the SNPs in a PGM1 gene (Phosphoglucomutase 1), which codes for an important enzyme in sugar (glucose) metabolism, is significantly associated with Pitta dominant group that is known for efficient metabolism.

 Things to keep in mind about the study:

It is to be noted that the three categories compared and defined here represent extremes –and according to Ayurvedic principles most people are a composite of all three- Kapha, Vata and Pitta, with the dominant element defining their type. There are some previous studies, which have suggested that the Ayurvedic Prakriti classification may have a genetic or metabolic basis (2,5). They were conducted on fewer subjects and looked at fewer genes/ phenotypes compared to this study, which rigorously recruited a large number of subjects, used multiple methods of classifications (software and Ayurveda practitioners) and a genome wide approach. There is much work to be done in understanding the scientific basis of the Ayurvedic classification system and whether we can independently and reliably assign people (independent of race, gender and ancestry) to a type.

 What does this mean?

The 52 SNPs defined in this study can now be used independently in other populations and also provide a way of identifying new associations with metabolism and other phenotypes. For more than fifteen years now, we have been able to read our genome i.e – information in our genes but not been able to fully understand how it defines us as individuals. So on the one hand, this study by correlating phenotypes with genetic variations, helps us understand a little bit more about how genes make us who we are. On the other hand, by using modern genetic tools in the context of traditional knowledge, this study provides a rigorous way of assessing the framework of ayurvedic medicine.

Citations

1. Understanding personality from Ayurvedic perspective for psychological assessment: A caseS Shilpa and C. G. Venkatesha Murthy. Ayu. 2011 Jan-Mar; 32(1): 12–19.

2. Classification of human population based on HLA gene polymorphism and the conceptof Prakriti in Ayurveda. Bhushan P 1 , Kalpana J, Arvind C. J Altern Complement Med. 2005 Apr;11(2):349-53.

3. Genome-wide analysis correlates Ayurveda Prakriti. Govindaraj P, Nizamuddin S, Sharath A, Jyothi V, Rotti H, Raval R, Nayak J, Bhat BK, Prasanna BV, Shintre P, Sule M, Joshi KS, Dedge AP, Bharadwaj R, Gangadharan GG, Nair S, Gopinath PM, Patwardhan B, Kondaiah P, Satyamoorthy K, Valiathan MV, Thangaraj K. Sci Rep. 2015 Oct 29;5:15786. d

4. A database of humans SNPs and their recorded associations can be found here: http://www.snpedia.com/index.

5. Whole genome expression and biochemical correlates of extreme constitutional types defined in Ayurveda . Prasher B, Negi S, Aggarwal S, Mandal AK, Sethi TP, Deshmukh SR, Purohit SG, Sengupta S, Khanna S, Mohammad F, Garg G, Brahmachari SK; Indian Genome Variation Consortium, Mukerji M. J Transl Med. 2008 Sep 9;6:48.

6. http://www.scientificamerican.com/article/a-very-personal-problem/

Interview with Dr. K. Thangaraj

Q. What was the overlap between the prediction by the software, by different Ayurvedic practitioners? Have you tried to estimate if this classification is robust enough to suggest underlying genetic differences?

The first assessment was by Ayurvedic physicians and classified using their knowledge. To double check what they have done, we have used a computer software. The software is also based on various parameters specified by the Ayurveda physicians.There were many questions which the individual had to answer to be assigned a Prakriti. Across the three centres – Bangalore, Pune and Udupi, on average, 75% of the individuals were in concordance.

Q. Why not perform an enzyme profile or measure transcriptional differences for metabolic enzymes? Is there an advantage in taking an SNP approach?

The advantage is that the SNP does not changes, it is there from birth till the individual dies. Whereas transcription profiles may change at different times, depending on time of day, tissue type to tissue type etc. We have looked into that also. This is the very first step. We can extend this across countries, across ethnic groups and cluster them.

Q. Ayurvedic medicine believes in holistic changes including those of lifestyle, dietary along with medicines and does not rely overly on mechanistic explanations beyond the classification into types. There is less emphasis, if you will on dissection of cause-effect and more on restoring the overall balance. Do you agree with this? If yes, then what are the challenges with respect to the study design when you tried to apply the modern framework of science- which relies on a reductionist approach, finding a cause and targeting only that specifically, to the ayurvedic system?

I agree that there is a holistic approach, but the basic approach is to classify the individual. Based on the prakriti, they will make the changes to the diet, prescribe treatment etc., so this is a very important stage. The challenge is the following- I am a geneticist looking at diseases,looking at case-control studies is very easy – for every marker we can ask if the mutation is present or if the prevalence is higher in the patients versus the control. In this case all the individuals are normal and within the same age-group. The only difference is the prakiti. So we wanted to see how to differentiate these individuals- as there are three groups, not two. Then we decided to compare one prakriti against the other two types, and try to see if there is genetic variation between the groups. Then there was a lot of statistical analysis. We used 1 million markers, this has many advantages, the disadvantage is the robustness and having to come up with statistical analysis ourselves. (MT: So, by using 1 million regions, you may increase the chances of false associations, is that the worry?) Not, really false associations. For example we may not have information about a particular SNP in an individual. We need to use markers that are consistent between all the individuals. So, when data is not available, we need ways of retrieving the data. In that process we need to use genetic panel of markers which are Indian specific. We developed our own panel of markers – Dravidian, Indo-European and used as a reference and to extract what is the possible marker in a given position.

Q. Have you tried to validate your classification using the 52 SNP panel with an independent population? For instance, if you knew what a person’s SNP state is, how reliably can you assign their Prakriti? Would it be useful to perform a blind study in which both the SNP panel ayurvedic practitioners and the software performed the classification, with the aim of determining how often they match?

That is very interesting. What we did was, we has more than 300 samples analyzed for these 1 million genetic markers, from our initial studies on population genetics. We used some of those samples, as these are all populations specific – a very endogamous population. We tried to project some of those individuals into these three clusters, we did find that although the individuals have come from the same ethnic background (more homogeneous), they were falling in 2 or 3 different prakritis. The same ethinic background can be placed into different prakriti. This we tried to do with our own data, this is not as detailed as you suggest. Independent blind studies need to be done.

Q. You have excluded women from your study and restricted yourself to the Indian population, does this limit the applicability of your results?

Yes, of course. At this point we selected only males because we did not want any confounding effects, in the females there are many hormonal changes and so on.

Q. You have started a way of examining traditional medicine in the framework of modern science. What are the challenges and the future of this approach?

(The future of this approach) This has paved the way to do many more things. For example, the discovery of PGM1 has given the clue that you can take the phenotype of the particular prakriti and correlate it with the gene, this gene is involved in metabolism and individuals with the pitta prakirti have high metabolism. We can now use the characteristic feature of the prakriti and look into those genes in a detailed way. These are some of the futuristic aspects, one can take the study further with. We did try to look at the network of all the metabolic pathways genes, the problem is this will need transcription or metabolic profiles from tissues of these normal individuals.

 

Two wings that beat like one

Wing-wing and wing-haltere coordination in insect flight

 

Snap-shot

Even at incredibly high wing beat frequencies, small insects maintain precise control of relative wing movement. A recent study explores how this control is achieved (1).

Background for this work

Flight and mechanism of flight have captured human imagination since time immemorial. Ancient insects had four wings, two of these wings morphed into accessory flight organs, responsible for stability during flight, called halteres (2,3). In dipterans, a class of insects (4), it was the second pair or the hind wings that got modified into halteres.

People have noted for a long time that the two wings (on either side) move precisely in phase, while halteres and wings move anti-phase during flight, (i.e. pair of wings moves antiphase with the pair of halteres). How do insects manage this precise control?

There are at least two ways in which this can be achieved, neuronal connections which would instruct the flight muscles or a mechanical coupling of the wings/halteres themselves. The really fast movement of the wings and halteres in small insects ( about 100Hz or higher, which can be visualized using high speed videography) precludes neurons. If it were neuronal, it would be pretty close to the fastest connectivity known to us.

What did they do and find?

In this work, Deora et al., have examined these phenomena in soldier flies,  dipteran insects with easily tractable (relatively large and white -aiding contrast) halteres. They asked, how are the wings able to beat in unison and how are the halteres maintained so precisely in the opposite phase to wings? The first clues came from serendipitous observations. Dead flies were able to maintain the in-phase and anti-phase relationship of their wing -wing and wing- haltere movements. Suggesting that this connectivity was not a result of neuronal instructions and possibly resulted from mechanical linkages.

The researchers then examined which part of the exoskeleton was maintaining this connectivity by surgically removing different parts of the thoracic exoskeleton. They were able to identify a region on the thorax necessary for the near perfect co-ordination of the wings, when they removed this region, the flies were unable to maintain coordinated wing movements. Interestingly they were able to stick the piece of exoskeleton back again and restore coordinated wing beating. They also identified the region responsible for maintaining mechanical coupling between each wing and it’s haltere (on the same side).

The researchers then tested the nature and limits of this coupling. The decrease in length of wings results in increased frequency of wing beats, how long can the halteres keep up? It seems like the halteres keep up till about the time when the wing is 60% of its original length and then quite suddenly, the coupling between the haltere and wings break down.

Another unknown, which Deora et al., are trying to work out is how this mechanical coupling is switched on during flight and otherwise switched off. This question is posed by behaviours in flies for which only one wing is used. The authors suggest a gearbox and clutch mechanism and attempt to find this clutch that allows the connection of the wings during flight. As yet, the nature, position and exact mechanism of the clutch is not known.

Conclusions from this study

This work shows for the first time that the wing-wing in-phase motion is due to mechanical connections across the sides- left-right axis. The wing-haltere anti-phase relationship is maintained by mechanical connections along the body axis. But how does this mechanical connection maintain a precise phase-angle? This is still not known and remains an active area of investigation.

An interview with Dr. Sanjay Sane

Q. In your work you address the biomechanics of wing-wing coordination and wing-haltere coordination, during flight. What about insects lacking halteres altogether (like dragonflies and damselflies) and insects in which the forewings are modified into halteres?

Excellent question. Insects outside of the Order Diptera lack halteres. The only exception to this is the Order Strepsiptera, in which the forewings have been modified into halteres. In both these cases, halteres are thought to serve the function of gyroscopic sensors which are essential in informing the nervous system about the changes in body orientation during flight. They may also be sensors that provide feedback about timing which is very important for wing motion control. In larger and slower insects such as Dragonflies and damselflies, aerial control is thought to be exquisitely visually driven. Not surprisingly, almost all dragonflies and damselflies operate only under conditions of bright lights. One may ask then: what about insects that are nocturnal but lack halteres (such as moths and antlions), or else insects such as bees which typically operate under bright lights but with very high wing beat frequencies? We have experiments to show that for such insects the antennae serve a role that is similar to halteres. It is not clear presently whether the precise mechanism underlying their sensory function is the same as halteres (i.e. gyroscopic, or timing-related), and that is a major subject of investigation in my lab.

Q. Your work shows a precise control of relative wing angle and haltere angle? Why does this angle matter? Is it just to keep the halteres out of the wing’s path?

Again, an excellent question! It is not clear at all whether the precise control of wing/haltere relative angle matters or is just a results of the physics of coupled oscillators (which wings and halteres seem to be). In some flies, the phase difference is not 180 degrees, so there appears to be nothing sacrosanct about that value. Yet, in a majority of the flies, the phase difference is 180 degrees despite great variation in thoracic morphology – so that cannot be coincidental. What dies matter, as we have shown in the paper, is that whatever the characteristic phase difference is for the given insect, it stay true because the timing information is a crucial feature of the feedback that the haltere provides. Our current hypothesis is that the actual phase difference is just a consequence of the physics of the coupled oscillators that causes their motion to fall into fixed in-phase or out-of-phase modes.

Q. What inspired you to think about mechanical connectivity?

When we observed the maintenance of wing/wing and wing / haltere phase difference in dead flies, it was immediately evident that the nervous system was not involved. The only other possibility was mechanical connectivity – which was fantastic because it answered many other questions that we had about speed and precision.

Q. Tell us more about the clutch? If it is internal , how would you probe it?

Two ways to probe it. One is careful experiments in which we can manipulate individual components of the wing hinge. Not easy, but if we find the right kind of flies in plentiful supply, it may be possible. The second and more direct way is to use X-ray micro-tomography – except this is very difficult and you need to know where to look. Our old-fashioned experiments may help there.

Q. Did you think of using other insects with different wing sizes, instead of manipulating the same insect and making its wings shorter?

Whatever insects we use, we will need internal controls because various aspects of the thorax do not scale isometrically. Manipulating the same insect is a far better option under the circumstances because internal control is then guaranteed.

Q. How does your work impact the way we think about insect flight?

In a major way, I think. For one, it clearly demarcates which aspects of wing and haltere coordination are active vs. passive. This has not been clear at all in previous studies on this topic. The second is that it generates clear and predictive and minimal hypotheses to explain a diverse set of observations (such as bee wing warm-up etc.) in a vast number of insects. It also throws light on how insect flight coped with miniaturization of the body form, which is an important evolutionary question because it appears to underlie the spectacular evolutionary success of insects as a group.

Q. Can you reduce insects to mechanical machines? If yes, does this affect the way you view the world as well as investigate a question?

This is a philosophical question and my answer is going to be speculative. Also, it is a question that applies not only to insects, but to animals in general. I do not think animals can be reduced to mechanical machines, although the way we conduct our science is such that we cannot help but assume some reductionist scenario like that. Our approach is at least as old as and derived from Rene Descartes, who in his brilliant treatise “A discourse on method” argued that all animals are machines (except for humans, of course, because they “possess a soul” unlike the brutes). His ideas were influenced by Newton’s recent discovery of laws of mechanics, and William Harvey’s discovery of heart being merely a mechanical pump, etc. It must have been tempting to think that everything can be explained by laws of mechanics. In later years, this approach has included the function of nervous system which is also essentially driven by the physics of cable theory and electrical conduction and gained currency through Sherrington’s discovery of reflexes and the idea of “a chain of reflexes”. So, add electrical circuits to the mix – but keep the basic idea of a mechanical machine. Where this approach fails however, is that it is unable to explain how animals, including insects, generate new and “spontaneous” activity. If they did not generate spontaneous activity, one imagines a universe in which all animals were essentially fancier versions of wind-up toys – but we know from experience that they are not. How can humans or circus elephants learn to ride bicycles or mice their pin-wheels? How do insects learn to deal with scenarios such as electric shocks which they may have never encountered in their evolutionary history? Surely, the brain is capable of generating novelty activity that ensures that it does not produce simple, copy-book, discrete set of responses to challenges posed by a continuum world.

Q. Humans have historically been interested in all things that fly, with the vested interest of putting ourselves in the sky. Do you have a similar motivation for studying insect flight?

Not particularly. One has to choose one’s battles, and mine has been to understand the nature which is far more sophisticated and fascinating than anything I will be able to build, or have the expertise for. That said, I am in full admiration of anyone who tries to use these concepts for building things that fly. We collaborate with such engineers, and help them as much as we can, and sit back and watch with admiration when they achieve incredible feats of engineering. It makes us appreciate the intricacies of nature even more!

References

1.“Biomechanical basis of wing and haltere coordination in flies.” Tanvi Deora et al., Proc Natl Acad Sci U S A. 2015

2. “The Gyroscopic Mechanism of the Halteres of Diptera”, J. W. S. Pringle, Philosophical Transactions B., 1948

3. “The evolution of insect wings and their sensory apparatus.” Dickinson MH et al., Brain Behav Evol. 1997

4. Classification of Insects from the Royal Entomological Society

5. “The aerodynamics of insect flight.” Sanjay Sane, J Exp Biol. 2003

A novel pathway of Brain Drain

Two studies provide a new map of waste management systems in the mouse brain

 

Snap-shot of the study

How is the brain protected from pathogens or chemicals? For a long time, it was thought that the brain is protected by barriers, such that it is impermeable even to immune cells (that could protect it) and to most chemicals (including drugs that need to act in the brain) (1,2). More recently it has become clear that cells of our immune system do constantly survey the brain (3). How do these cells get in and out? In the July 2015 issues of Nature and JEM, two groups report the discovery of an extension of the lymphatic system into the brain.

Background

 If the circulatory system can be likened to the nutrient supply system of the body, the lymphatic system is somewhat like drainage system, collecting fluid, large molecules and white blood cells from tissues, thus recycling blood components. The lymphatic system includes the tonsils, spleen and lymph nodes and extends throughout the body. This system contributes to fluid clearance, pathogen recognition and mounting of an immune response to pathogens. So it is indeed surprising that there was an arm of this system in the brain that we had no idea about until about a month ago. Two recent papers describe a lymphatic vessel network in the membrane covering of the brain that carries the fluid in which the brain floats (the cerebrospinal fluid) and drains into the deep cervical lymph nodes (5,6).

What did they do and find?

In both of these studies using mainly imaging based techniques, the authors identify vessels in the mouse brain which they call the meningeal lymphatic vessels using molecular markers that tag lymphatic endothelial cells.

In the dura mater, the covering of the brain, T-cells (a kind of immune cells) are found scattered throughout. However, Louveau et al. noticed many T-cells lining the Superior Sagittal Sinus (SSS). Surprisingly, these cells were not seen in significant numbers in the lumen of (read, inside) the SSS. This suggested that these T-cells could be residing in a separate compartment like blood vessels or lymphatic vessels. The authors ruled out blood vessels and used specific markers (read, tags – namely Prox-1, Podoplanin and VEGFR3) to demonstrate that these were indeed lymphatic vessels containing lymphatic endothelial cells. The vessel containing the T-cells was positive for Lyve-1 (tags lymphatic vessel) and was clearly visible as a channel running close to the SSS. These results confirmed that this vessel was indeed a lymphatic endothelial compartment.

Last year, Aspelund et al. reported the surprising finding that the eye also contains a lymphatic-like vessel, the so-called Schlemm canal (4). While studying the eyes of mice whose lymphatic vessels were labelled with flourescent markers, they found the brain lymphatics in the adjacent tissues. The lymphatics were present along blood vessels in various parts of the dural membranes even at the base of the skull with several exit paths through the same openings used by cranial nerves. Further, they were also able to show a critical role for the VEGF receptor signalling pathway  in the formation of these lymphatic vessels. In the absence of these vessels, consistent with their role in clearance and management of waste, large molecules did not get cleared from the brain (6).

Both groups explored if these are functional lymphatic vessels by injecting intravenously with a fluorescent dyes or, special minute(nano) particles, to track the flow of fluids from the brain tissue through the different compartments. This experiment showed that the vessels carry the brain fluid (read, cerebrospinal fluid or CSF) into the deep cervical lymph nodes.

These two studies have important implications for conditions in which there is pathological aggregation of proteins in the brain (Alzheimer’s) and also on how the immune system keeps a check on the brain in health and disease.

An interview with Dr. K Alitalo, Dr. A Louveau

Q. How do you see this discovery will help to shed light on previously unanswered questions of our understanding of the central nervous system?

(Dr. K Alitalo) This is an important discovery that will enable scientists to look at neurological diseases from an entirely new standpoint. The vessels may be key in explaining some features of diseases that could not previously understood.

Q. We also came across a recent publication by Louveau et al., published in journal Nature which talks about a similar lymphatic system. Is this the same system that you show in your publication

( Dr. K Alitalo) It is indeed the same system. We did a more thorough job at characterizing it as we found out that the vessels extend to dural venous sinuses, meningeal arteries, cranial nerves and the cribriform plate. Louveau et al. used a methodology that only enabled the discovery of the vessels in the superior parts of the skull along dural venous sinuses.

Q. Do you think advances in the imaging techniques and the availability of well defined markers was the only reason for such a late discovery of this system? Are there any other possibilities that could have contributed to delay in this discovery?

( Dr. K Alitalo) Probably the greatest challenge for the visualization of meningeal lymphatic vessels has been the adjacent osseous bone. The need for decalcification is, in many respects, problematic for tissue analysis. Most importantly, I think both discoveries were enabled by advanced imaging technologies, which have transformed the world of traditional 2D tissue sections into 3D. In our study, the discovery was enabled by the previous generation of lymphatic-specific fluorescent reporter mice in which the lymphatic vessels can be visualized as they are in tissues by fluorescent microscopy, without the need for any processing.

Q. Do you think similar lymphatic vessel could be present in humans, what are the technical challenges you see in establishing it?

( Dr. K Alitalo) The techniques already exist, so it is just a matter of time when researches will perform this work. The only problem is that endothelial cells become autolytic soon after death.

Q. Your study clearly shows the role of lymphatic vessels in draining macromolecules out of the brain. What potential implications would this have on human health conditions like infectious diseases, disorders like Alzheimer’s and tumor metastasis?

( Dr. K Alitalo) The brain lymphatics may become structurally and functionally altered in infectious diseases. This needs to be evaluated in the context of pathogenesis of such diseases. – Alzheimer’s disease is characterized by the pathological accumulation of amyloid beta into the brain tissue. Proper meningeal lymphatic vessel function could perhaps protect against Alzheimer’s disease by clearing pathological amyloid beta from the brain parenchyma. Perhaps we can boost such clearance with our technology. Brain tumors rarely metastasize into cervical lymph nodes. However, when this happens, it has been unknown how the metastases arise. The meningeal lymphatic vessels are likely to represent the missing link.

Q. How do you see this discovery will help to shed light on previously unanswered questions of our understanding of the central nervous system?

(Dr. A Louveau) This discovery is changing an established paradigm described decades ago, that the brain is an immuno privileged organ because, at least in part, of the lack of classical lymphatic drainage. What we demonstrate is that the brain possesses a classical lymphatic drainage, but the brain remains immunologically unique. We will have to change the way we see the brain when we want to address diseases that have immunological factors.

Q. We also came across a recent publication by Aspelund et al., published in Journal of Experimental Medicine which talks about a similar lymphatic system. Is this the same system that you show in your publication?

(Dr. A Louveau) Yes, the study recently published by the group of Kari Alitalo confirmed our study and shows similar results to the one we published.

Q. Do you think advances in the imaging techniques and the availability of well defined markers was the only reason for such a late discovery of this system? Are there any other possibilities that could have contributed to delay in this discovery?

(Dr. A Louveau) The development of the imaging techniques and the fairly recent description of specific markers to identify lymphatic endothelial cells (early 2000) are certainly major factors in the late discovery of this system. One other reason is the unique location in the dura mater, a region of the meninges usually discarded when people study the brain (because this layer remains attached to the skull).

Q. In the paper you mentioned similar lymphatic vessel could be present in humans as well, what are the technical challenges you see in establishing this?

(Dr. A Louveau) We think that the challenge will not be technical but highly depend on the quality of the tissues collected. Most human tissue collection have been fixed for month to years rendering immunostaining challenging. Collecting more recently collected tissue should help, but lymphatic vasculature appear to be sensitive structure.

Q. What are the potential implications of this discovery, on human health conditions like infectious diseases, disorders like Alzheimer’s or Schizophrenia and tumor metastasis?

(Dr. A Louveau) We think that the meningeal lymphatic system might play a role in every neurological disease with a strong immune component, including the ones you are mentioning. In Alzheimer disease, the blockade of the meningeal lymphatic might be an initial event before the accumulation of unfolded protein in the brain. In the case of brain tumor, the meningeal lymphatic system might be used by the tumor to prevent its attack by the immune system. But all of this remains hypothetical and the function of the meningeal lymphatic system will have to be studied in all of those diseases.

Q. In one of your interviews you have mentioned that your discovery would require modifications in the textbooks. Could you elaborate more on the changes that you envisage?

(Dr. A Louveau) If you open a neuroscience textbook right now, you’ll read that the brain is devoid of a lymphatic system, therefore contributing to its immune privilege. Our discovery, more so if it extends to humans, proves that this statement is wrong and that the brain, like every other organ possesses a lymphatic system.

References

1. “The blood-brain barrier: an engineering perspective.” Andrew D. Wong et al., Front Neuroeng., 2013

2. “The gut immune barrier and the blood-brain barrier: are they so different?” Richard Daneman and Maria Rescigno, Immunity, 2009

3. “Immune surveillance in the central nervous system.”  Shalina S Ousman and Paul Kubes, Nat Neurosci., 2012

4. “The Schlemm’s canal is a VEGF-C/VEGFR-3-responsive lymphatic-like vessel.” Aleksanteri Aspelund et al., J Clin Invest. 2014

5. “Structural and functional features of central nervous system lymphatic vessels.” Antoine Louveau et al., Nature. 2015

6.“A dural lymphatic vascular system that drains brain interstitial fluid and macromolecules.” Aleksanteri Aspelund et al., J Exp Med., 2015