Male Speaker: I want to welcome up our next speaker Dr. Bob Bartlett. Dr. Bartlett is an MD graduated Magna Cum Laude from the University of South Alabama School of Medicine. He is an attending physician in hyperbaric medicine at St. Luke’s Medical Center, Milwaukee which has the largest hyperbaric center and facilities in North America. Dr. Bartlett currently serves as Chief Medical Officer for RestorixHealth and he is the past president of the American College of Hyperbaric Medicine. And he is going to talk to us today about the role of oxygen in wound healing. So thank you very much.

Dr. Robert Bartlett: Okay. I'd like to thanks Stan Kalish, Dr. Frykberg, Dr. Rogers, and the other organizers for this opportunity to spend a few minutes with you addressing the role of oxygen in healing. Specific objectives here, is to understand the phases of healing specifically as relate to oxygen. I think many people intuitively understand oxygen is good. You hear a lot about hyperbaric oxygen but there’s a lot more to the oxygen story than just giving a lot of metabolic substrate. When I was in medicine school, I was basically told oxygen does two things. Number one it creates energy. Number two, it's a building block for a number of key components; collagen being one of them. But today we realized that oxygen does far more than we could ever imagine. In fact, if you’ve been out of medical school, nursing school for more than 10 years, it's sobering to realize that half of everything you were taught was probably incorrect, incomplete, or just flat out wrong. That is how fast the world is changing today. So hopefully in this presentation, you'll realize that oxygen does many more things than you perhaps have been told when you were in formal training. I’d like to begin this presentation with a quote from Dr. Dunphy, a pioneer in wound healing. “Surgeons have sought for centuries ways to accelerate healing. But in the end they have merely restored it to nature's normal period.” The point being is that hyperbaric oxygen as a therapy does not transform patients into super healers. What it does do is to compensate, correct and normalize healing. And we certainly don't give this therapy to normal people. We're giving it to people with handicaps and deficits. And then in the end if we simply achieve nature’s normal period, that in itself is sufficient. The bottom line is that we are already optimized to heal as fast as possible and when we don't, it's simply due to a myriad of abnormalities. One of which of course is related to oxygen. So when you look at the classical phases of healing, the hemostatic, inflammatory, proliferative and you view it from the perspective of oxygen is quite revealing. The Hemostatic phase, what’s that all about? Well that's about the weekly debridements we know. We're removing biofilm. We're removing various epithelial cell migration and we get bleeding. The bleeding is a good thing because the platelets will aggregate and release growth factors. But if the platelet is to aggregate, we pay a price. A partial occlusion of the micro circulation. The Inflammatory phase, neutrophils, macrophages populate the wound bed increasing the demand for oxygen. Proliferative phase. Collagen, absolute requirement for oxygen. Conversion of procollagen to collagen requires the uptake and incorporation of oxygen to create collagen. So it becomes readily apparent that oxygen indeed is the right limiting step and for some it is the lynchpin that governs success and failure entirely. So what I really want to provide for you in this session is sort of a 360 analysis of everything we know about oxygen and how it is affecting all of the various phases of healing. And I think the best way to do this is to really talk about the mechanisms of action. Because when most providers understand the science and the mechanism, they can almost intuitively decide, you know this is a patient who may benefit from hyperbaric therapy. And then you let the people who do that kind of thing figure it out. You know, is it a value or not?

[05:07]

So in terms of mechanism. The first one of course would be hyperoxygenation. But there are others. We enhanced granulocyte clearance. You heard more than a few presentations about bacteria. We enhance fibroblast activity, collagen deposition, VGF. And yes we are even manipulating stem cells. There’s some exciting new work in that area. So let's begin to this first one, oxygenation. Now I am going to share with you one technical term which is ATA's or Atmospheres Absolute. So how do you dose hyperbaric oxygen? I mean most drugs we dose in milligrams, in CCs and so on. The way that we dose hyperbaric oxygen is with pressure. And the way we measure pressure is what’s called Atmospheres Absolute. In this room, sea level, we are at one ATA. If we double the pressure the room we’re at 2 ATA. We triple it we’re at 3 ATA. Now this is the only technical term I'm going to share with you. But we couldn't have a coherent conversation about hyperbaric therapy without understanding the dosing. So ATAs is how you dose your drug. So let’s focus on this topic of oxygenation. A normal blood gas is a PO2 of a 100. At 3 ATA a patient will have a PO2 an excess of 2000 millimeters. Now think about what that means. That's not twice normal, 5, 8, 10. That's 20 times normal. And when you move to that level, you’re truly having a pharmacologic effect. So with that mount of oxygen, what can one do with that? Well if you look at the normal diffusion distance of oxygen, it's about 64 microns from the capillary. You give a sense of scale, a red cell is about 5 microns. So we are going some 10 to 15 red cell diameters outside of the capillaries. Now if this is the capillary bed of a diabetic whose foot just got injured, run over by a car. Diabetic foot that just got infected. You know that foot swollen the next day. And what you’re going to find is that these capillaries now have separated in space. Creating this ischemic water shed zones, these hypoxic zones. Under hyperbaric conditions we can easily bridge those and support that tissue in the transitional phase. Preventing unneeded necrosis, ulceration, breakdown and so on. Now of course it’s typically in the hyperbaric chamber, about an hour and a half. And if you measure the blood gas say 30 minutes later, it would be normal. So more important question, relevant question is to actually say, “Well what’s the PO2 in the skin and the muscle? In that location it's not nearly as high. But look at this. The duration is considerably longer. Your patient is receiving benefit for some four to five to six hours after leaving that chamber. And this is related to a concept called oxygen half-life. Time doesn't permit me to go into it but you certainly be aware that the oxygen will persist in the tissue for many, many hours after leaving that chamber. The next mechanism of action relates to enhancement of white cell function. I think earlier you just heard a presentation on hyperchlorous acid. I was listening to a part of it, the discovery of that and the benefits. And clearly, we understand that one of the reasons that a wound becomes chronic is that it's stalled in the inflammatory phase. Well why it’s stalled in the inflammatory phase? One of the key elements for that inflammation is the presence of foreign protein specifically bacteria. And in other words all wounds are infected. It's just a matter of degree from simple contamination to paying sepsis. But the price we pay is the foreign protein that elicits the host's response. So anything we can do to enhance the clearance of bacteria is of course in our best interest. And the killing capacity of the granulocyte is directly related to the amount of oxygen available. Now there have been virtually hundreds of papers on this. I'm only going to share with you too. This first one is a paper that looked at the killing capacity of a normal leukocyte and compared it with a CGD leukocyte. You say what's a CGD? Well that's a patient with Chronic Granulomas Disease. It was actually the CGD leukocyte that allowed us to understand what the white cell was doing with the oxygen. This is a very rare double recessive disorder but for individuals who have a CGD defect. Most of them will not survive into adulthood without the routine use of antibiotics. So Honing College postulated that if you deprive a white cell of oxygen it would be no better than a white cell that cannot use oxygen. And you can see that the graph intersects. So what is the increase in kill capacity? About 50% and that is a difference between life and death.

[10:12]

So this is not of academic interest and this is huge. The other study that I'll just share with you, is one with a rather clever title Oxygen Dash And Antibiotic. In this study what they did is they injected bacteria on the backs of guinea pigs, looked at the amount of necrosis. Divided the pigs into three groups, hypoxic, normoxic, oxygen rich. And you can see again the clear difference between the hypoxic group, meaning 12% oxygen, normoxic, 21% and the supplemental oxygen. You know the Inca's live high in the Andes, altitudes above 10,000 feet. Understood a thousand years ago that when members of their tribe would not heal, if they simply took them down to the Amazon rain forests. The mysterious powers of nature would heal them. Well there's little mystery today. At 10,000 feet you have a PO2 of 50, at sea level it’s twice that, and for some it was simply oxygen that made the difference. The next area or mechanism I’d like to look at or highlight is the role of oxygen in fibroblast activity and collagen production. Both of which are absolute prerequisites for neovascularlization. Clearly neovascularlization is a cornerstone of any wound healing program. You got to grow new blood vessels. So a very simple model of healing would be the following. You make an incision in the tissue. There are some key steps that must occur. First and foremost there must be an elevation of PO2 on the wound edge. This is usually achieved by histamine, bradykinins and it’s a characteristic red flare we see around all of our wounds. With the elevated PO2 comes an activation of fibroblast. The fibroblast in turn begin to produce collagen. And then and only then do we get neovascularlization. When I began my education, I thought it would all be about the blood vessels. Oh contraire, it's about the collagen. No collagen, no angiogenesis. With angiogenesis done, of course we get healing and repair. So clearly manipulating oxygen specifically through hyperbarics will activate and proliferate the fibroblast. This promotion of collagen which in turn leads to angiogenesis and repair. Now I’m often times asked, “Well if oxygen is valuable why don't we just put people on oxygen masks?” I mean I know it's not going to be as potent perhaps as HBO. But surely it has some benefit. Well the reality is it does not. So here is a study that actually addresses that very issue. These are standardized wounds in animals. The endpoint is VDE vascular density equivalent. Here's a group of animals on air. A group of animals on oxygen. No differences. Only the hyperbaric group, and look at the difference. Nine-fold greater for angiogenesis. So this is truly a hyperbaric effect. It cannot be achieved through normal baric methods. Much of our understanding of wound healing today can really be I guess attributed to T.K Hunt in the pioneering work that they did at the University of San Francisco and continue. That’s a simple but profound question. What starts healing? What stops healing? I'm sure all of you could probably list four or five possibilities. And to better understand this, they’ve developed what remains one of the most powerful windows into understanding nature's design. It's called a rabbit ear chamber model. What you do is you wound a rabbit's ear. You clamp it front and back. And then you watch the blood vessels as they migrate in to repair the wound. At the center of that chamber was a membrane. They could either leave it opened or closed to the atmosphere. In the first scenario, they left the membrane closed. The center of the wound is hypoxic. This of course is the normal state of affairs. They’ve established a healing curve. They then moved on to a second group of animals where they opened the central membrane allowed oxygen to rise at the center of the wound. Now under this condition, scenario two here, many of the investigators predicted surely it will heal faster. Now you have an abundance of oxygen. But much to their surprise, healing came to a halt. And you can see this when you actually measure the distance the blood vessels were moving into these chambers. Here we have the hypoxic center. You see your x-axis. Steady distance over time. But with the hyperoxic center it's totally flat. No migration, no movement, no healing, no repair. They then moved on to a third group where they left the central membrane closed and induced a state of peripheral hyperoxia. Now this is what's happening under hyperbaric conditions.

[15:10]

And what they found here is there is suddenly a burst in angiogenesis. So what does the data really telling us? What it's telling us, it's not just oxygen. It's the oxygen gradient that governs angiogenesis and drives repair. Sometimes when I'm giving a longer presentation, I pose a question is there such a thing as cellular intelligence? And I would suggest yes, there is. Any intelligent system has the ability to survey its environment and make decisions based on the changing environment. So cells clearly do that. You cut your finger, take it for granted that your cells somehow know there is something wrong. And they automatically begin to repair it. So that's an intelligent system. You don't have to send a memo or text message to your finger “Please heal yourself today.” So the way the cell knows is you begin to think about this. Is how does it know? It knows through chemical sensing. Cells don't have eyes they don't have ears. What they do is they sense. They're sensing a chemical imbalance. And this really takes you back to medical school, the universal principle of homeostasis. All systems trend back to a mean. So take a step further. If I'm a cell and I realized there is an imbalance. How would I correct that? Well I supposed you could do electrolysis to split water and hydrogen and oxygen. I have oxygen or let's grow blood vessels. And that's exactly what Hunt has discovered. Others of course have reaffirmed this. Here’s a similar model where they implanted acrylic chambers in the backs of animals. One group gets hyperbaric oxygen. The other does not. They’re measuring the size of the buds, the angiogenesis and the number buds. And you can see that the HBO groups had larger buds more numerous buds. So is it oxygen pressure or content that really drives this process. Here's a study by Joslin that looked at that very issue. They’re measuring oxygen pressure versus collagen deposition. You can see as the oxygen pressure increases, you get more collagen. Same thing is true when you look at perfusions score. But when you look at hematocrit or content, there is no change. So really is the gas pressure. And that's what we are manipulating in the chamber. As I said it can go up 20 fold. Well do we know what the ideal PO2 is for maximum production? [Cough]. Well this study again from Hunt and colleagues give us some insight. Animals are divided into three different groups. And we see as we increase the PO2, that we get an enormous jump in collagen production. But content per se increases by less than a fraction of 1%. So it's really not content. It's gas pressure that makes the difference. I'm going to skip the discussion here because I'm watching my timer at the bottom give me a little pressure. Fibroblast production. Again this study is done with a human fibroblast taken from diabetic patients, normal patients. Your line at the bottom is gas pressure. You could translate that as ATAs of oxygen. You can see that the diabetic fibroblast continues to proliferate faster and faster and faster as we increase the PO2 with hyperbaric oxygen. VEGF, Vascular Endothelial Growth Factor, we realize this is a very important growth factor in terms of healing. And sometimes a question's raised would HBO perhaps suppress VEGF? After all a rational person would suggest that VEGF is released in response to hypoxia. If I eliminate hypoxia then maybe I don't get VEGF. Well quite to the contrary, that's not the case. So this study actually looks at the relationship of HBO to VEGF as well as lactate levels. And what they found in this is there is no change in lactate because lactate is a powerful modulator of healing. So we do not suppress lactate production. They also found that VEGF is not suppressed. Quite to the contrary it's actually enhanced. And they find 50% higher VEGF levels. So we have to sort of restructure our notion our model of what stimulates the release of VEGF. And it's actually not hypoxia that's controlling it. The mechanism is well established now. It's actually the result of reaction, reactive oxygen species. If you care to read some of the work from Chandan Sen Ohio State. Again time doesn’t permit to go into that.

[20:02]

Here's another interesting model where the authors are looking at a classical study of ears on mice. You take one ear. You ligate it. You make it ischemic and you use contralateral ears to control. You'll notice the control group over there on your right, the ischemic ear takes several days longer, 14 days versus 11 days. But the HBO group the ischemic ear heals as if there is no ischemia. Normalizing natures intended period in the words of Dr. Dunphy. The last two areas here my remaining minute or so. Stem cells and receptor signaling. I think this is really, really exciting stuff here. There is now evidence to show that HBO is not just providing oxygen the classical concept. But we’re actually manipulating a force of nature. And that force of nature are stem cells. So today we are really arriving on a whole new concept of oxygen more than a metabolite. You know it's only been a few years since two American scientists were awarded the Noble Prize for the discovery of nitric oxide. And what was so profound about that discovery it was the first time in science that have ever been shown that a gas could be a cell signal. And today we realize that oxygen is not just a metabolite. It is a cell signal. You say “Well what are some of the evidence for that?” Well here are some examples. Here's some work by [Indecipherable] [21:32] colleagues up regulations to PGDF receptors. There is nothing metabolic about a receptor. Expression of VGEF, nothing metabolic about VGEF. Up regulation on nitric oxide synthase. Work by restro. Expression of multiple receptors, EGF receptors, TDGF receptors. And more recently there has been evidence showing sustained effect. Tompacks, 72 hours of fibroblast proliferation. A metabolic model simply cannot explain this. Haydenberger - single dose of HBO, 24 hours of mitosis. These speak to genomic mechanisms. And the more recent model by [Indecipherable] [22:19] and colleagues of stem cell mobilization. I remember being in the summit conference in Dallas commenting that in the future we will still be healing people with HBO, but it will be for entirely different reasons than we believe today. I'm fortunate to live long enough to see that come true. So here is what happens when you put a person in a hyperbaric chamber. You'll notice that following every treatment stem cell count rises higher and higher and higher. So the reality today is that yes, we are correcting hypoxia. But for many people what we are doing are driving stem cells. And there are two kinds of angiogenesis. There is local angiogenesis, neovascularlization. And then there is angiogenesis driven by the bone marrow stem cells which is the mechanism I was never taught in medical school. Much of the healing I believe in a diabetic is due to the stem cell migration and correcting some of the defects that we know exists in the diabetic. So today, we really arrive at a whole new paradigm that cells on the wound view oxygen pressure as a trigger or signal indicating that the environment is ripe and proceed to healing. As a regular reinforcement that maintains that momentum for repair. So to summarize here what I've covered in this brief section is that the key steps to healing and repair are clearly linked to oxygen. Oxygen is the rate limiting stuff. The notion of just correcting hypoxia although correct there is much more to this than a metabolic story. It's really a story today of cell signaling and stem cell enhancement and migration. So again I would like to thank the organizers here for the opportunity to present. Thank you.