Lymphovenous Canada: New Research Developments

NEW RESEARCH DEVELOPMENTS IN UNDERSTANDING LYMPHOVENOUS DISORDERS

About ten years ago, I gave a presentation to the Surgery Department. A prominent member of the Department got up and essentially challenged the whole concept that lymph was important clinically. He did not seem to believe that lymph existed. I was absolutely stunned by this. The problem goes even further. About two weeks ago, I was able to give second year medical students their first presentation entirely on lymphatics. This again is astonishing, because lymphatics are a very important part of the circulatory system.

If you were to add up the number of publications on blood vessels every week, there might be hundreds of them. If you look through the literature and you try to find an article on lymphatics, chances are you will not find one. In the course of a year, probably half a dozen to a dozen key articles might be produced on lymphatics.

Part of the problem is that there are very few groups that actually study these vessels. Worldwide, I think we can count serious groups on two hands. In the United States, there are perhaps five or six groups that study these vessels. In Europe, there are a few. Now if you were to compare that to the numbers of groups that study blood vessels or cancer, in Toronto alone there are probably hundreds of scientists and physicians. In addition, no resources are available specifically for lymphatic research.

Of course, we have to acknowledge that lymphatics are very difficult to study. The medical students can dissect cadavers and never once find any lymphatic vessels, save possibly the thoracic duct (in the neck). Lymphatics are essentially invisible to them and to us, unless you develop very specialized techniques. This is why the lymphatic vessels of sheep are very useful for lymphatic research. Sheep are passive animals and they have very large lymphatic vessels, much like humans or cattle. A particular species of bat is also very useful in studying lymphatics.

Much of what we know of lymphedema was obtained through research many years ago using only the microscope. That is a problem because, when we look at the lymphatics through a microscope, it really provides us with little information on the changes that are occurring in the tissues. I hope to show you in a few minutes some of the other approaches that we can use.

The Mystery of Lymph Formation

If I were to ask any one of you how blood circulates in the body, I am sure you could tell me. We have a four-chambered heart that contracts and pumps blood under pressure through all your blood vessels. However, if I were to ask any of you how lymph is formed, you could not tell me and I do not know the answer.

Naturally then, if we take lymphedema as one example, how can we possibly develop an effective therapy against a disorder in which we do not even know how lymph is formed in the first place? This is one of the major deficits in our knowledge and, until such time as we can develop an experiment to study this, it will remain a mystery.

Last, I thought I would just make a point about some of the lymphology societies. They have had a tremendously negative impact on the field. I do not mean societies such as yours. I mean some of the pseudo-scientific societies that exist primarily in Europe and the United States. They have been infiltrated by a lot of individuals who make money out of lymphedema patients and they are a discredit to both the patients who are afflicted with these disorders and to the scientific discipline of lymphology. Right now, serious scientists in the field are members of the Microcirculatory Society and the various physiological societies around the world.

The Role of Lymphatics In Our Circulatory System

Enough gloom and doom. Let us try to make this a bit more upbeat. Lymphatics are part of a remarkable circulatory system and I want to give you some idea what roles they play in the body. I think you might be surprised at some of these.

First of all, one could not have a cardiovascular system without lymphatics. You may not be aware of it, but your blood vessels are very leaky and every day a volume equivalent to your total plasma volume leaks into the tissue spaces. So in a typical 60 kg male or female that might represent three litres of fluid. If you were without lymphatics, your blood vessels would just collapse. There would be nothing to pump around. Clearly, they are remarkably important; without them we could not have a closed cardiovascular system.

Second, the dissemination of immunological memory and antibodies is dependent on a lymphatic circulation. If I were to inject an antigen into my foot and divert the lymph from my popliteal lymphatic, essentially no secondary immunity to that antigen would be present. That is because antibody molecules are large and they cannot get directly into the blood. They have to enter the blood stream by traversing the complex network of lymphatic vessels. This is one reason why patients with lymphedema are so susceptible to infection from a cut. The antigen cannot transport or be transported to the local lymph node where a suitable immunoresponse can be generated. Lymphatics play a key role in fluid homeostasis and also facilitate immunological reactions as well.

You may not be aware that essentially all lipids (fat) are absorbed by lymphatics in the bowel. In fact, if I were able to show you a sample of intestinal lymph, it would look like milk because of the lipid molecules that are absorbed from the bowel.

I am often told that there are no lymphatic diseases. But nothing could be further from the truth. There is an individual in Hawaii who claims that one-fourth of the entire population on Earth is infested with a parasite, a nematode that obstructs lymphatic channels and ultimately causes filariasis and lymphedema.

Last, when people think of lymphatic vessels, they think rather negatively because there are certain tumours that metastasize selectively via the lymphatic circulatory system. We do not understand why this is so, but clearly lymphatics play a role in disseminating various cancer cells around the body.

Current Research Theories

Now let us turn our attention to what the problem might be in lymphedema. First of all, if we consider lymphatics, we have to consider that they are part of a system. Any time we want to understand lymphedema, it is not just the lymphatics that are important, we have to consider also the blood vessels and the tissue space that surrounds the cells, the interstitial matrix. We have to view lymphedema in a comprehensive sense.

If we were to obstruct the lymphatics, we would get swelling or edema. But if we were to increase the filtration from the blood stream and overwhelm the lymphatics, that would induce edema as well. Or if we were to change the properties of the tissue spaces, we could also produce edema. In fact, I am going to propose a hypothesis later on in my talk that lymphedema may not be a lymphatic disorder at all.

It may be a disorder of the tissue spaces or matrix, and you may have to change the name of your society to the Matrix Venous Society!

Because we know so little, I think you have to have an open mind that a lymphatic disorder could be caused by defects in any one or all of these elements.

Overwhelmed Lymphatic Drainage Capacity

The lymphatics have a finite ability to deal with fluid and remember that the protein and liquid in your blood stream is leaking continuously from the blood vessels into the tissue spaces and getting into the lymphatics. So presumably every time you have enhanced leakage, you are likely to develop edema or swelling. A good example of this is in the burn patient. Burn patients have massive changes in the permeability of their blood vessels and, because the blood vessels start to break down, proteins and fluid leak into the tissue spaces resulting in massive edema. This process may overwhelm lymphatic transport.

With congestive heart failure venous return is diminished and patients have an elevated capillary pressure. Because of the higher pressure, more liquid is driven out of the blood vessels because it is a pressure-driven phenomenon. This increased fluid filtration can overwhelm the lymphatic transport capacity and ultimately edema occurs. These are not necessarily called lymphedemas because the defect is thought not to be at the lymphatic site, but rather at the blood vessel. It is another type of edema that certainly involves the lymphatic circulation.

Physical Obstruction

It seems obvious that if you were to obstruct a lymphatic vessel, you would have marked edema. The interesting issue is that, with post-surgical lymphedema, for example after a radical mastectomy, there does not appear to be any obvious obstruction. Another interesting observation is that in many cases, the lymphatic pathways seem to be intact.

Destruction of Lymphatics

What about the destruction of lymphatic vessels or inhibition of lymphatic vessel regeneration? If we want to study lymphatic vessels and their impact on disease, you would think that it would be a relatively simple matter to sever the lymphatic vessels and then study the implications of this procedure. Over the long run, it is very difficult to do this because the lymphatic vessels regenerate very rapidly.

A scientist in the United States studies heart transplants and why they do not work very well when they are immediately put back into the recipient patient. One of the reasons they do not work effectively is the lymphatics have not yet regenerated. Until they do, the cardiac muscle receives a vascular supply, but there are no lymphatics to drain tissue fluid away. The heart becomes very edematous and stiff and this investigator could chronicle the changes in cardiac function that occur and how they improve as the lymphatic vessels regenerate.

Lymphatics have a remarkable capacity to regenerate, but there are many things that may inhibit this process, for example, radiation therapy. Radiation can cause tissues to become fibrotic. One would imagine that a fibrotic lymphatic vessel may not transport fluid very well. This could possibly be part of the pathogenesis of lymphedema, particularly in cancer patients who are undergoing vigorous radiotherapy.

Impaired Lymphatic Pumping Activity

One of the most interesting possibilities, and one of the issues I have spent about twenty years studying in my career, is impaired lymphatic pumping activity. I am not certain if you are familiar with the fact that lymphatic vessels are really not vessels at all. Lymphatic vessels can be modeled along the lines of your heart. They are not passive conduits; they contract, and generate the majority of the energy required to propel lymph.

A scientist from Poland cannulated (inserted a plastic tube into) a tiny popliteal lymphatic vessel in humans and measured the pressure in that lymphatic after obstructing the outflow. The pressure was able to build to 100 millimetres of mercury. Think about it. That is the mean systemic arterial pressure in most humans. The contractile properties of the lymphatic vessel are very important.

What I would like to do now is talk a little bit more about these lymph pumps and how they can be inhibited because this may provide some clues in terms of the pathogenesis of lymphedema. The first thing I want to draw your attention to is the fact that the lymph flow is pulsatile because it flows only when the lymphatic vessel contracts. The interesting thing about this is that the lymphatics continue to contract for up to an hour after an animal dies. This is strong evidence that the lymphatic vessel is responsible for transporting lymph via this active contraction process.

We do not understand how fluid enters the initial lymphatic vessel. One of the theories is that it relates to the contractile properties of the lymphatic. The theory goes like this. Normally, the pressure in the space around the cell is lower than the pressure in the lymphatic. This was a great problem in trying to understand how fluid could move from the extracellular space into the initial lymphatic vessel because fluid cannot move uphill; it cannot go against the pressure gradient. But several scientists were able to demonstrate that these lymphatics contract and, during relaxation, they created a suction force and fluid was drawn into the initial lymphatic duct. Unfortunately, these vessels are very difficult to study. This is why lymph formation remains to this day a mysterious process.

What we can do is study the larger collecting ducts of an animal, because we can see them. Lymphatics, at least in the larger collecting ducts, are not passive tubes. They are muscular structures with tremendous contractile properties. We can cut them out and put them in a tissue bath to study their mechanical properties.

My introduction to this field occurred when I was in England doing a post-doctoral fellowship. One of the other post-docs was studying a ring of arterial tissue from a pig. He had placed this ring in an organ bath and was monitoring its mechanical activity. I decided to take a tiny lymphatic vessel that was just a fraction of the weight and put it in his organ bath, just to see if it would contract. The most remarkable thing was that that tiny lymphatic vessel contracted, in some ways, better than that muscular segment from the aorta. This observation absolutely astonished me.

The other thing that you should be aware of is that lymphatic circulation is very complex. Lymphatics are a series of hearts, thousands of individual hearts that are arranged in series. Each of these hearts is called a lymphangion. With analogy to the cardiac system, each lymphangion has an inflow valve and an outflow valve. One of the great mysteries is how all of these lymphangions are coordinated to generate the energy to move lymph.

Two of my graduate students have constructed an organ bath to study lymphatic ducts. I have to remind you that, unlike working with blood vessels where you can order a standard organ bath from a catalogue, nothing like that is available for lymphatics. If you want to study lymphatic vessels, you have to make the bath yourself. My students perfected the design of this bath, which allowed them to quantitate the pumping activity of a small lymphatic vessel outside the animal.

Every hour, these vessels can pump about a tenth of a litre, totally on their own. The valves prevent the flow from going backward. This may be one of the problems in lymphedema. The valves may become ineffective, or they may rupture, or they may have been damaged in some way. So when the lymphatic vessels contract, the fluid does not get pumped back to the blood stream.

We can study the pharmacological properties of these ducts. When you pressurize the lymphatics, which is the same as increasing the fluid load, they start to pump better until a point is reached when the output starts to decline. The lymphatics start to beat so rapidly that there is not enough filling time, much like when your heart starts to fibrillate.

To my knowledge, no one has ever tested lymphatics from patients with lymphedema to determine whether the biomechanical properties of the lymphatics are impaired relative to individuals who do not have this condition. This is one of the areas that I think might be a fruitful area of investigation in the future.

My graduate students have developed a system to measure the pressure and the volume inside one of these single lymph hearts. They defined the contraction cycle exactly like the contraction cycle of the heart, with a systolic phase and a diastolic phase. Systolic pressure occurs during that period when the heart is contracting; diastolic pressure occurs during that period when the heart is relaxing.

We can alter the contractile activity of these lymph hearts. There are molecules that are produced in all of us that can markedly affect the lymph pump. For example, look what happens when a certain type of prostaglandin meets a lymphatic vessel at high concentrations. Each one of these spikes represents one contraction of the vessel and at high concentrations the duct goes into spasm. This molecule can completely abolish effective contractions of lymphatic vessels and who is to say that these molecules are not produced in lymphedema patients. Again, nobody has studied this.

When Blood Enters The Lymphatic System

To take this one step further, one of the most interesting inhibitors of lymphatic pumping is oxyhemoglobin, a component of your red blood cells. Hemoglobin is responsible for transporting oxygen around your body. But when you have a tissue injury, your blood vessels rupture and red blood cells flow into the tissue spaces. When red blood cells rupture, the hemoglobin is released and it enters lymphatic vessels. When hemoglobin hits a lymphatic, it has a profound effect on its pumping activity. It inhibits it. This is one reason why we think a tissue becomes edematous after injury.

You know this yourself from your own experience. If you have a gardening accident and you hurt your finger, you see evidence of a blood vessel rupturing and it swells up. One reason it swells, of course, is that the vessel has been damaged and more fluid leaks out. But at the same time, that lymphatic vessel which should be pumping fluid away may be unable to transport the fluid effectively.

We hypothesize that in an inflammatory reaction, edema occurs not just because of elevated movement of fluid from the capillaries, but also because lymphatic pumping is inhibited. This is a rather complex concept that involves many other factors. However, I bring it up now because it illustrates that we are beginning to understand some of the intricate details of how fluid accumulation relates to altered lymphatic contractile activity and therein may be one of the clues to lymphedema. We, and others, have catalogued dozens of molecules and how they affect these tiny lymph hearts. Some of this is likely to have relevance to the lymphedema problem.

Elevated Outflow Pressure

Remember that lymph is absorbed ultimately from the tissues, pumped through lymph nodes back into the blood stream via the thoracic duct, which is the major lymph vessel at the base of the neck. If you increase blood pressure, the lymphatics are forced to pump against this pressure. In essence then, any kind of partial or complete down-stream obstruction makes it much more difficult for lymphatics to pump fluid back into the blood.

The Tissue Spaces As A Sponge

What if the lymphedema has really nothing to do with lymphatics at all? There is one possibility that I think needs to be entertained as a result of some experiments that were performed in Scandinavia.

The tissue space between blood vessels and lymphatics (matrix) acts much like a sponge. Imagine that I have a sponge in my hand and I compress it. Then, I put my hand in a bucket of water, then lift it out and release the sponge. I think you would imagine that it would not have much water in it. But what would happen if I compressed the hand holding the sponge, put my hand in the bucket of water and relaxed it, and then lifted out the sponge? You would imagine that the sponge would have much more water. That is what seems to happen in certain pathophysiologic conditions.

Hyaluronic acid is a unique molecule because it holds water like a sponge. There are a group of molecules that bind hyaluronic acid in various connective tissue cells and hold the matrix in a compressed configuration. During an inflammatory reaction, these bonds may be broken and the gel of the matrix may swell. This could have a number of implications. When you cause the matrix to swell, it creates a suction force and pressure drops in the matrix. Now, if the blood pressure has remained the same, you have a much bigger pressure gradient and so more fluid moves out of the blood vessel. This may contribute to edema.

Whether this has anything to do with lymphedema, I do not know, but it is something that I think scientists in this area should take into consideration. It is an entirely new way of viewing edema. In some lymphedema patients there may be congenital or acquired defects in how the interstitial matrix is regulated.

Lymphatic Fluid In The Cranial Cavity

When individuals have hydrocephalus, which is fluid accumulating in the cranial cavity, doctors treat the condition by shunting fluid from the head into the peritoneal cavity where the lymphatics drain the liquid to the blood stream. When these unfortunate kids get an obstruction of this tube, many things happen. Cranial pressure starts to rise and they get sick. One of their complaints can be nasal congestion.

This is interesting because one of the major routes for cerebrospinal fluid (CSF) drainage is believed to be through the nasal submucosa. Through this space, the cerebrospinal fluid passes out of the cranial vault into the lymphatic vessels. In addition, cerebrospinal fluid seems to move by pressure gradients along many nerves and, ultimately, is taken up by the lymphatic vessels in various parts of the body, but mainly in the nasal area.

A scientist at the University of Rochester and one of my Ph.D. students have devised a mathematical model based on knowing the concentrations of certain radioactive proteins in various compartments. My student has done a series of experiments over three years that have enabled him to come up with some interesting information. For example, 50% of all cerebrospinal fluid drains out of the cranium into lymphatic vessels. The other 50% goes out to specialized structures termed arachnoid villi.

So the issue is: Is there lymphedema of the brain? In hydrocephalus, meningitis and other conditions, we hypothesize that molecules from the brain may transport to those lymphatics in the neck and block absorption through this pathway. This may lead to elevations in intracranial pressure.

How Brain Hemorrhages Might Effect Cranial Lymphatic Drainage

When red blood cells are in the cerebrospinal fluid, they are very fragile. Many of them rupture and we believe that hemoglobin transports to the cervical lymphatics.

We know from our other studies that hemoglobin can impair the tiny lymph hearts. So one hypothesis that we are trying to investigate now is that, with (subarachnoid) hemorrhage, the lymphatic component to drainage is impaired. This may contribute to the raised pressures observed in some patients with an intracranial bleed. Even though lymphatic vessels do not exist within the cranial vault, I think they may play a very important role in the clearance of cerebrospinal fluid.

What Does The Future Hold?

At a recent meeting in the United States, several of my colleagues and I discussed the possibility of setting up a research group to investigate lymphedema in greater detail. Clearly there is recognition that this is an important clinical problem and that greater efforts are required from a scientific perspective in order to develop new therapies to treat this disorder.

From these deliberations, I hope that scientists in this area will be able to initiate studies in the near future that will provide a foundation on which to build a consensus on the causes and treatments of the various types of lymphedemas.

Much of the information I have communicated to you today does not relate directly to lymphedema, but clearly some of the basic concepts are likely to be relevant to the pathogenesis of this family of disorders. Despite huge biomedical ignorance concerning lymphatics, we are beginning to understand some basic principles, issues that integrate the blood vessels, the interstitial matrix and the lymphatic circulatory system.

Perhaps together, we can make the whole lymphatic system a little less mysterious.