IBD: Exploring the role of neurons in gut motility
Cutting-edge techniques to image neurons in living tissue are being developed to track the effects of inflammation in the gastrointestinal tract. These studies eventually could mean improved therapies for patients with inflammatory bowel disease (IBD), such as Crohn's disease and ulcerative colitis.
Early every morning, David R. Linden, Ph.D., plunges into a pool to revive the competitive swimming edge he enjoyed in college. In truth, the competitive edge never left. It simply metamorphosed into a desire for a more altruistic edge — that of discovery. Dr. Linden also has a penchant for diving deep into detail, a trait that he honed in his hobby of exploring the lives of his predecessors and linking them with contemporary history. It is a trait that is serving him well as he pursues his hypothesis that the loss of, and damage to, nerve cells in one part of the intestine cause changes in function in other parts of the intestine. His goal is to relieve the debilitating and painful symptoms that accompany a diagnosis of inflammatory bowel disease.
A preeminent group
Dr. Linden is a member of Mayo Clinic's Enteric NeuroScience Program (ENSP), which is internationally recognized as one of the best of its kind in the world. The group's four laboratory scientists — Dr. Linden, Joseph H. Szurszewski, Ph.D., Gianrico Farrugia, M.D., and Tamas Ordog, M.D., — share resources, lab meetings and ideas, and collaborate closely with the program's large group of clinical researchers. ENSP research is geared toward grasping the molecular, genetic, cellular and physiological intricacies that regulate gut motility and sensation. ENSP is well funded by the National Institutes of Health (NIH). Direct NIH funding totals about $10 million, including a large program project grant for which Dr. Szurszewski is the principal investigator. The knowledge the group is gaining is used to develop new medical and surgical therapies and diagnostic tools for patients with gastrointestinal disorders, such as irritable bowel syndrome, nonulcer dyspepsia, pseudo-obstruction, gastroparesis, constipation and IBD. There are more than 20 clinical trials being conducted on all three of Mayo Clinic's campuses based on the discoveries generated by ENSP.
A breakthrough in technique: Watching nerve activity in the living gut
Dr. Linden is paving the way for a much deeper understanding of how the gut's intrinsic nervous system functions in health and disease by developing a new method to actually observe nerve cells in the intestines of living animals. Dr. Linden's lab uses mice that are genetically engineered to express fluorescent proteins in nerve cells and injects them with another fluorescent dye that labels immune cells with a different color. During live cell imaging, a small portion of the intact large intestine of an anesthetized mouse is withdrawn and placed under a confocal microscope.
"The confocal microscope can illuminate a very thin layer of tissue, so we can clearly see the cells that we've lit up," Dr. Linden says. "Imaging nerve cells and immune cells of the intestine at the same time, and in real time, in intact living tissue hasn't been done before."
In studies conducted during his postdoctoral fellowship at the University of Vermont, Dr. Linden was surprised to find that inflammation in the gut leads to a loss of about 20 percent of the neurons that reside within the gut wall (Neurogastroenterology and Motility, 2005). Neurons that are located in the wall of the gut make up the enteric nervous system, which controls all aspects of the digestive system, including nutrient absorption, muscle contraction and maintaining an immunological barrier. Apart from the central nervous system, the enteric nervous system is the only substantial grouping of nerve cells that form circuits capable of autonomous reflex activity.
Dr. Linden's lab has shown that before neurons disappear (between 12 and 24 hours after artificially inducing inflammation), cells of the immune system enter the regions where enteric neurons reside. These experiments used fixed time points to stop the experiment and image the tissue. But much like a series of photographs miss what happens between images, these experiments were missing a lot of information. The next series of experiments are seeking to capture images more like a movie so that significant events aren't missed.
With the new method only recently launched, Dr. Linden has begun studies to pinpoint the window of time when nerve cell loss occurs. In mice, the lab has found it to be sometime between 24 and 48 hours. That's a later — and longer — window than the lab found in previous studies in another animal species. The lab is now sharpening to a more manageable imaging span in mouse studies.
"If we know the neurons are lost between, say 28 and 30 hours, we will be able to image in that narrower window and observe whether immune cells directly remove the neurons or if they are causing signals to have the neurons self-destruct," Dr. Linden says.
The discoveries that are possible as the lab finesses the experiments have the potential of leading to new treatments.
"We plan to intervene in the destructive neuroimmune processes with drugs or genetically engineered animal models — such as mice bred to lack particular types of immune cells, or proteins expressed in the nerve or immune cells — in an effort to find drug targets that are involved in the process," Dr. Linden explains. "Then we will experiment with methods that block the mechanisms that contribute to neuron loss. For example, if a process is attracting immune cells into the system, I'll look for the signals that attract them and try to block them."
Electrical activity and gut inflammation?
While neuroinflammation and cutting-edge imaging methods are important new aspects of his research, Dr. Linden's forte is electrophysiology — studying how nerve cells communicate by electrical impulses. He knows that when things go wrong with the electrical impulses, patients can end up with the uncomfortable and sometimes debilitating symptoms of constipation, diarrhea and abdominal cramps — all problems of gut motility. When scientists refer to gut motility, they mean peristalsis. Peristalsis occurs when the circular and longitudinal muscle fibers of the gut wall contract and relax in waves. The stretching and squeezing of the muscles propel the contents of the intestine onward. The strength of the muscle squeeze and how often and how quickly the wave occurs are all regulated by the enteric nervous system.
Using a technique that measures voltages within nerve cells of the enteric nervous system and muscle cells of the gut wall, Dr. Linden studies how the properties of neurons and muscle cells change, which, in turn, contributes to changes in cellular function.
"Most IBD specialists are interested in immunology," Dr. Linden says. "Cutting off inflammation caused by infiltrating immune cells certainly works — Mayo played a huge role in the development of Remicade and other members of the new class of biological therapeutics that has revolutionized IBD therapy. But even though all measurements of inflammation subside after treatment, many patients still go through periods when they have the same wretched symptoms even though there's no inflammation, and I think this is a long-lasting effect of the initial inflammation acting on the nervous system to change the way the gut functions."
Dr. Linden is particularly interested in a small but important subset of neurons in the enteric nervous system called intestinofugal neurons — nerve cells that have their cell body between muscle layers of the gut wall and axons that grow outside of the gut wall, extending to and connecting with masses of nerve cell bodies, called ganglia, near the aorta. The cells with which they connect then send axons back to all regions of the gut, changing intrinsic reflex activity in the enteric nervous system.
"Intestinofugal neurons are the start of nerve conduction pathways, called reflex arcs," Dr. Linden says. "These reflexes coordinate activity in large portions of the gastrointestinal tract."
Dr. Linden's lab has found that inflammation causes a loss of intestinofugal neurons and is followed by distinct changes within the cells with which they make connections, mostly making it easier for them to fire action potentials. Action potentials are rapid changes in cell voltage that propagate down an axon.
"Action potentials are the framework by which all nerve cells communicate and accomplish complex functions," Dr. Linden explains. "So, when inflammation in the large intestine hyperexcites nerve cells within this reflex arc, I think that message is being sent to regions far away from where the original inflammation occurred, causing dysfunction in other regions of the bowel where there is no inflammation."
New technologies in genetically engineered mice are making it possible to combine Dr. Linden's major approaches. Mice that express molecules that report nerve activity, or action potentials, through fluorescence, provide the potential to simultaneously image immune cell infiltration with enteric nerve cell excitability — an intersection Dr. Linden is eagerly anticipating.
With his research going this well, Dr. Linden looks forward to the day his hypotheses translate into treatments that help people with inflammatory bowel disease.
"I am surrounded by clinicians who inform me about digestive diseases in their clinical practice while I keep them abreast of advances in gastrointestinal physiology," Dr. Linden says. "Mayo has excellent resources — most importantly, the generosity of its patients in giving specimens for research purposes. It's an ideal place to do translational research."
— November 2011