Endometriosis Foundation of America 2014 18
How valuable are animal models for endometriosis and what can they teach us?
- Asgi Fazleabas. Md, PhD
Thank you Bill and thank you Tamer for the invitation. This is a little bit different. I just want to share with you the work we have been doing for the last 15 years. In trying to develop model systems you heard a little bit about rodent models just now from Ali and what I would like to do is in the limited time is just give you a glimpse of what animal models could do, how they could help us to answer so many of the questions that have been raised throughout the day today. And then perhaps talk a little bit about, given the topic and the theme of the conference, a little bit about what our limitations are and why we would need more opportunities to move forward.
You have heard this, I do not need to tell you this over and over again we are still struggling as you have heard all day about trying to understand the underlying mechanisms. What is the spontaneous evolution and a question came up. Is it a defective inherent endometrium that gives rise to the disease or consequently does the presence of the lesions actually affect the endometrium? These are questions that you cannot ask in a human system as you well know and as you heard because of this component. By the time you see your patients by the time you get the tissue these patients have had disease for extended periods of time, well over seven years. So you cannot really take that tissue and ask the fundamental question where did the pathology begin? How did the pathophysiology start? This really led us to start thinking about how we could use animal models to try to answer these fundamental questions as it relates to trying to understand the basic pathophysiology and the etiology of this dreaded disease.
What is the advantage of a non-human primate model? First and foremost we use the baboon as a non-human primate model, rhesus macaques have been used – the baboon has been the most validated model. The biggest advantage we have is its reproductive physiology and anatomy is virtually identical to the human female.
These animals develop spontaneous disease at about the same rate as women do, in captivity this really accelerates and enhances the spontaneous disease in these animals. You can compare and contrast as I will show you an induce model with a spontaneous model and compare it directly with the human situation because they mimic the human situation. Our ability to induce the disease allows us to study the etiology of the disease from the very onset of when this disease occurs. We create this model as I will show you – we create peritoneal disease, so we are limited to peritoneal disease with this model. We cannot study endometriomas, we cannot study deep infiltrating disease.
Because they are long lived animals they are very strong and we are allowed to do multiple surgical procedures. We can under experiment controlled conditions begin to study the long term basis and see how the disease evolves in the same animal model from the time we induce the disease until the time of termination when these experiments are terminated.
The other big advantage is as you have heard a lot about proteomics, genomics and all of the omics is that the compatibility between this model system and our ability to use human platforms whether we are looking at microarrays, we are looking at antibodies, we are looking at therapies, the close phylogenetic relationship allows us to use various probes that I used in humans to ask the direct question whether these are relevant to the human situation. And again, this leads to treatment modalities because again you have to test them for long term efficacy and as advantageous as the rodent models are you cannot do the long term studies that are required to get these drugs to market.
That being said however there are disadvantages. These are highly outbred species, so in a sense it is great because they mimic the human situation but you also increase biological variation as you heard over and over again today about the limitations in treating our patients. We cannot selectively manipulate gene expression, well that is changing. You heard about the…monkeys that have come out in the last few weeks, the Chinese groups being able to selectively modulate gene expression in monkey models. Perhaps we will be at a point, provided we have the money, to be able to start thinking about manipulating gene expression. But that is way, way down the line. Limitations in being able to use these animals because of their complex social behaviour you need special housing, you need special husbandry and you are limited to where you can do these studies.
The big problem of course is the fertility evaluation, which is something we always struggle with because these animals, just like humans, have a very low fecundity and because they take a long time to breed the cost and the long gestation times there is cost nature to try to study the infertility component but we have tried to study it from a different angle. And there is certainly a cost to maintaining the colony.
So given this the question is why not rodent models? I think primarily of course is that they do not menstruate and they do not develop spontaneous disease. In trying to understand what is reality in terms of how we understand menstruation and how we understand endometriosis there are limitations. But as you saw from the previous talk from Ali they are very useful in a certain sense because you can study the early invasive components and the remodeling, we can do short term evaluation the genetic manipulation of these animals both in cells and tissue of choice allows us to ask very specific questions in terms of how various genes, how various pathways might actually play a role in this disease. And the opportunity to use immuno-compromised mice to do xenograft experiments also gives us a first pass to be able to study early events associated with the disease. If there are treatment modalities then there comes a point where we can actually begin to see where they are using the xenographic and do the first test and then pass it on to using large animal models, and eventually, hopefully, to human situations. So pluses and minuses in using animal models but I think they are very powerful in being able to allow us to study this very enigmatic disease.
That being said let me show you a little bit about how we go about this. But I just want to tell you how we utilize this model and what this video shows, about a two minute video. We first evaluate the animal to make sure she has no spontaneous disease. What we do is we basically mimic “Sampson’s Theory of Retrograde Menstruation”. On the second day of visible menses in these animals, these animals have visible menses, we aspirate menstrual tissue and menstrual fluid with no curetting just simply what is in the cavity using a unilateral pipelle. We can use all human instrumentation in these animals because of their size we do not have to modify any human instrumentation and we simply inoculate the peritoneal cavity of these animals. Literally, this is one month after we have done the second inoculation. We do two inoculations consecutively and nothing else and they have disease for life. You can begin to see again that within one month you can still see some of that tissue and debris still sitting in the peritoneal cavity and now you can begin to see the development of lesions that are identical to peritoneal lesions that you would see in humans. You can see IMR angiogenesis the beautiful blue and red lesions that you would normally begin to see through the laparoscope. This happens within one month after we inoculate these animals.
Six months later you can begin to see the disease continues. Now these animals have not been inoculated again since six months ago and you can begin to see that the lesions are starting to take forms you would normally see through the laparoscope as you are examining your patients. The chocolate, the scar tissue and all of these. So it tells you that we can create this disease that looks very similar to the human situation.
Here again is an example of a chart showing you what a human and baboon lesion looks like. The other advantage of this animal model is that now once we have inoculate these animals we can sample the animals every three months all the way out between 15 and 18 months is when we keep these animals again for cost and also because of the regulatory components that we can utilize these animals for.
Essentially what you can see is every three months you can take a sampling of these animals both eutopic and ectopic and ask how does the disease then progress from the time you inoculate the animals. The other advantage is that before we inoculate these animals we can go in and get eutopic endometrium from these animals so the same animal can serve as its own control. So you can ask the direct question what happens when you inoculate these animals, you create the disease, what are the changes you see in the ectopic endometrium associated in the same animal that before inoculation of menstrual tissue and after inoculation of menstrual tissue.
The next slide really summarizes much of our work and I do not have time to go into anything in detail but basically what we have shown over the years that the baboon recapitulates the peritoneal disease that you see in the human and new lesions evident at every time point when we go in and do the laparoscopic evaluation we see complete progression of the lesions and there is continuously seeding of new lesions even though the animals are not being inoculated. If you ovariectomize the animals and only treat them with estrogen, the disease will disappear. Basically you need the constant menstrual seeding to maintain the disease after you have inoculated these animals. What you see is a progression of early events associated with metastatic protein expression particularly in the lesions eventually with the development of proliferative apoptotic genes and Linda talked to you about nerve fibers. Again, in the human situation where you see the nerve fibers when the lesions are excised but what we have shown in the baboon model is that these lesions are progressive. So if you take lesions out at three months you do not see any nerve fibers. But by the time you go out 15 months the nerve fibers have already infiltrated. So there again is progression of development of nerve fibers into these lesions, which again when you are thinking of therapy intervention, pain treatment that these give us some ideas in terms of what you can do with these animals.
Linda showed you the changes in the eutopic endometrium and these are collaborations we have done with her. When we look at the eutopic endometrium one month after we induced the disease 40 percent of the genes that have been shown to be altered in the human eutopic endometrium are already altered in the eutopic endometrium of these baboons as early as one month after disease has been established. The progesterone resistance that we talked about is again a progressive component and about six months into the disease process is when these animals become resistant to progesterone.
The properties in the menstrual tissue do change and I think you are going to hear a little bit about this from Peter in the next talk. These are also altered by the presence of the lesions and the development of new lesions. We see similar observations in both the eutopic and ectopic tissues that has been observed in women and our baboons with this disease. Again, this gives us opportunities for targeting non-invasive and targeted non-invasive diagnosis and targeted therapy because you know when that disease was induced.
The disadvantage of course is that with an animal with only endometriosis you do not have the other complications that women have so you cannot differentiate that process like Linda talked about. But you can at least ask what happens if there is endometriosis in these animals and how is that reflected in terms of trying to develop non-invasive diagnosis and targeted therapy.
The big limitation is what most women suffer from is pain. It is very hard to diagnose pain in these animals. They are stoic. I can do a surgery on them in the morning and they will be turning cartwheels in their cage in the afternoon. So they are incredibly stoic. It is very difficult unless you have very strong observational components associated with good behavior biologist it is very difficult to assess pain. Maybe some of the things that Linda talked about this morning in using the very…devices maybe we can start to adapt some of these and see whether we can begin to see pain. But that is the one big, another big disadvantage of this.
So, what are the barriers? I have given you an idea and you can see that this is a wonderful model but there are barriers. I think that one of the biggest things that we are finding and this was just presented in Chicago is that there is tremendous opposition, an increasing opposition to the use of animals in research. And this is simply in terms of controversies and what we are talking about is simply that we as scientists have not made our case. We like to sit in our lab, talk about stuff we want, we do not go out and advocate. As you heard at the lunch seminar by Ms. Avery we need to advocate. As a result of all of the social media all of the people who are out there that are against animal research are out there making sure that that message gets across.
You can see in the last few years how dramatically the opposition has increased, particularly amongst women in terms of the use of animals for research. That compounded by the increasing regulatory components that we are associated with in maintaining these animals and all of the regulatory requirements that we have to go through makes it very, very expensive. Europe, primate studies are being carefully looked at. You are going to have very limited ability to do primate studies in Europe. As a result all of these regulatory burdens are going to minimize our ability to utilize these animal models to really try to understand things that are so critical to human disease and in particular the disease that we are talking about today.
The other problem again as Linda touched on is this dramatic decline in funding. The actual dollar amount of NIH’s funding has declined over 27 percent in real dollars in the last ten years. And she showed you a glimpse of how much goes towards endometriosis a measly $6 million from the NICHD budget. If you look at all of OB/GYN, which the majority of NICHD funding comes from we are right about the median. NICHD for the major institutes probably has the lowest pay line so as a result we again are not advocating for ourselves. We are not going out there to make sure like we heard about this afternoon to make sure that the money is there to be able to solve many of the issues that we are talking about.
Lone touched on this – I think this is what Endofound can do, we can do as scientists and others is “Ah, reproductive diseases – you don’t die from them! So who cares?” There is always this issue of why funnel money into something that is not life threatening and Lone touched on this this morning. Well, when you think about, talk about the devastation on women’s lives I think that is life threatening. When women cannot go to work, when they cannot have children, they cannot have a normal life, they cannot have normal social interactions I think that is tremendously devastating and I think the call that we got this afternoon during the lunch is really important. I think we do have to get out of our little ivory towers and begin to start advocating for what we need.
So with that I think really again I truly believe in this that I think we really need to make a point that at the end of the day when we are looking at women, healthy women and if you think about women who want to have children, plus healthy babies is a healthy life. I think that is really important in terms of us being able to get that message across.
I have always liked this quote from Francis Bacon, going back again 400 years that besides the scientific quest for enlightenment I think we really need to dream about our work and how we do it. To really relieve should not be man’s, I should change that to woman’s estate but of course this is 400 years ago so I guess it is excused.
And with that thank you very much.
Asgi Fazleabas MD, PhD - Medical Conference 2014
Endometriosis Foundation of America 2014 18