Breathing Problems in Babies Whether it involves studying respiratory muscles or brain-stem cells controlling breathing, Heritage Scientist Dr. John Greer’s research has one theme: understanding why some babies can’t breathe properly. The diaphragm: an essential muscle for breathing “My interest is in understanding the basis for breathing problems in babies; all of my research centres around that theme,” explains Dr. Greer, a University of Alberta physiology professor. “I study one muscle in particular that plays a central role in breathing: the diaphragm. Some of my research focuses on a devastating condition called congenital diaphragmatic hernia or CDH, which many people have never even heard of. Essentially, babies with CDH develop holes in their diaphragms while still in the womb. These babies have lungs that don’t properly develop, and they often die in their first few days of life. We are now beginning to scratch the surface of what’s really going on with CDH.” CDH CDH occurs in one out of every 2,500 births, an incidence similar to that of cystic fibrosis. Dr. Greer is intent on finding out what occurs during development to cause this condition. The diaphragm is a sheet of muscle that contracts and relaxes to help expand and contract the rib cage, forcing air in and out of the lungs during breathing. It also has a second function, which many people take for granted: it separates the organs within the rib cage from the lower abdominal organs such as the liver and the intestines. If there’s a hole in the diaphragm, the lower abdominal organs can travel through that hole and compress the lungs, stalling their development. Dr. Greer and colleagues have now determined that CDH occurs during the first stage of diaphragm development, around the fourth week of pregnancy. They have performed some basic scientific research in rats that has yielded very interesting results. “If we expose pregnant female rats to a chemical called nitrofen, we find their offspring develop the exact same holes in their diaphragms as the human fetus,” explains Dr. Greer. “We’ve studied each part of development in the rat, and what we’ve discovered is that nitrofen perturbs the vitamin A pathway. Pregnant female rats fed a diet deficient in vitamin A, again, have offspring with the same holes. We’re now using animal models, with genes controlled by the vitamin A pathway knocked out, to further our understanding of the cause of CDH.” Vitamin A Dr. Greer stresses that the lack of vitamin A in fetuses that develop CDH likely has nothing to do with how much vitamin A mothers have in their diets. “It’s not that mothers aren’t getting enough vitamin A, it’s that these babies can’t utilize or access it. There may be a problem with transfer from mother to fetus. These infants may have a genetic defect which causes the holes; that’s something we still have to find out. Expectant mothers should not be taking high doses of vitamin A because it does not prevent CDH and too much can cause birth defects.” Currently, there is no way to prevent CDH. “Of course our long-term goal is to prevent it,” says Dr. Greer, “but we’re still at the stage of trying to understand what’s going on.” Dr. Greer and University of Alberta neonatologists Dr. Bernard Thébaud (a Heritage Clinical Investigator) and Dr. Thierry Lacaze are establishing a multi-centre study examining vitamin A levels in babies born in Alberta, Ontario, Quebec, and the Netherlands. The developing brain and breathing Another area of research in Dr. Greer’s lab is neural control of infant breathing. Along with Heritage Senior Scholar Dr. Gregory Funk and Heritage Scientist Dr. Klaus Ballanyi, Dr. Greer studies the brain stem, where breathing is controlled. This internationally recognized research group is interested in how this part of the brain develops and how it functions before birth and immediately following birth. “This area of the brain can shut off after a child goes to sleep, causing the child to stop breathing for fifteen to twenty seconds,” says Dr. Greer, noting that when this happens in an adult, we know it as sleep apnea. “During sleep, a set of nerve cells in the brain stem may not be excited enough, or their activity may actually be depressed, and breathing ceases. The airway may even collapse, because when these nerve cells are on, they release neurotransmitters to keep the airway open. These repeated bouts of lack of oxygen are of concern for the proper development of babies.” Difficulty The difficulty in studying how nerve cells in the brain control breathing lies in the fact that the brain stem has many different types of nerve cells mingling with one another, each responsible for different functions in the body. As few as 700 nerve cells may be responsible for breathing, a small number of the total nerve cells in the brain stem. The challenge was to separate the nerve cells responsible for breathing from the rest. Silvia Pagliardini, an AHFMR-funded Ph.D. student in Dr. Greer’s laboratory, took advantage of the fact that the nerve cells responsible for breathing have a receptor on their surface called substance P, which is not found on any other type of cell in the brain stem. She then used a special tag that recognizes substance P and binds to it. When viewed under a fluorescent microscope, the tag glows and those cells with substance P light up and can be isolated. Dr. Greer is excited about this new methodology. “This is a substantial development for researchers who study breathing and development. Now that we can visualize and isolate these cells, it will completely change the way we study problems like apnea and devise ways to stimulate breathing with drugs.” A rewarding career Dr. Greer became involved in research in his teens, participating in summer research programs. He’s been hooked ever since. “I immediately took a liking to research and have never had any doubts that working at a university and participating in medical research is the ideal profession for me. One of the greatest pleasures in this line of work is interacting with young trainees. I believe that much of my laboratory’s success over the past twelve years is the result of exceptional students and post-doctoral fellows. Although the laboratory has made fundamental advances toward understanding what is necessary for newborns to breathe properly, I feel that the best is yet to come.” AHFMR Scientist Dr. John Greer is an associate professor in the Department of Physiology in the Centre for Neuroscience at the University of Alberta. Dr. Greer’s work is also supported by the Canadian Institutes of Health Research (CIHR), the March of Dimes, and the Alberta Lung Association. Selected Publications Babiuk RP, Thébaud B, Greer JJ. Reductions in the incidence of nitrofen-induced diaphragmatic hernia by vitamin A and retinoic acid. American Journal of Physiology – Lung Cellular and Molecular Physiology 2004 Jan16;286(5):L970-973. Ba F, Ren J, Greer JJ. Brain-derived neurotrophic factor release with neuronal activity in fetal rats. NeuroReport 2005 Feb 8;16(2):141-144.