Colic Researchers: Tight Junctions Crucial in Gut Function
Colic remains a preeminent focus among equine researchers, and a North Carolina State University research team is breaking it down to the nitty gritty by examining how infectious disease and inflammation impact cells in the horse’s gut. Specifically, they are focusing on the “tight junctions,” or the spaces between microvilli–microscopic cellular membrane protrusions–in the intestinal mucosal lining.

“Sessions on tight junctions at the (human) medical conferences are packed,” said Anthony Blikslager, DVM, PhD, Dipl. ACVS, professor of equine surgery and gastroenterology at the university’s College of Veterinary Medicine. It’s only appropriate that veterinarians examine the same structures in horses. Blikslager described his team’s progress at the Ninth International Conference on Equine Infectious Diseases, held October 2012 in Lexington, Ky.

Blikslager uses a tool designed originally in Denmark by Professor Hans Ussing to study all types of epithelial tissue, or any barrier that transports electrolytes, by measuring electrical current and voltage. At the cellular level substances cross epithelium two ways: by passing through intracellular spaces between cells (paracellular transport) and by traveling through the cells themselves (trancellular transport). He and his colleagues have noticed “resistance” in the intracellular spaces in the normally functioning gut, but these spaces tend to become “pretty leaky” in the damaged mucosal barrier–more so in the small intestine than the colon, due to its role in food breakdown and absorption–no matter the cause of the insult.

“Most of the injury to mucosa is fairly similar whether you’re dealing with infectious disease or strangulation,” Blikslager noted.

He and his colleagues have focused on neutrophils in their research, which are white blood cells that engulf and destroy pathogens and cell debris. In cases of injured mucosa, such as strangulated intestine or infected intestine, the body signals neutrophils to cross the mucosal barrier and attack microorganisms capable of secreting toxins or breaking down to release toxins such as endotoxin. (Gram-negative bacteria, which live in the horse’s gut to break down fibrous feed, relinquish portions of their cell wall when they multiply and then die, releasing endotoxin.)

A scanning electron microscopic image of enteropathogenic E. coli.
These toxins damage the barrier and can cause the deadly disease endotoxemia, but the attacking neutrophils are also adept at “destroying the mucosa and causing a much larger leak,” said Blikslager. “They’re going to do this whether you want them to or not. They’re going to go off to the organisms once they’re signaled to, and unfortunately once they do that they destroy the mucosa. During strangulating obstruction the initial damage is the most important, but inflammation exacerbates damage just as the mucosa is attempting to heal. During infectious disease, infection is the initial cause of damage, such as in salmonellosis, but inflammation is what makes this type of infection so deadly.

“Neutrophils are very helpful and needed,” he added, “but you don’t want the level of damage.”

Besides neutrophils and endotoxins, other important damaging substances also cross these mucosal barriers. “We think that horses are particularly sensitive to endotoxin, and that’s really what we need to go after, but that’s really not the case,” he pointed out. “Any toxins can get across here–both Gram-negative and positive.”

He showed a scanning electron microscopic image of enteropathogenic (causes disease in the gastrointestinal tract) Escherichia coli, considered rare in horses, and how it gains access through the mucosal barrier via tight junctions. The research team is using these organisms to figure out how they “open up” a tight junction.

Therapeutic Approaches

All talk about damaging microbes aside; veterinarians could probably therapeutically target tight junctions to benefit horses. For example, there are medications on the human market that are already known to do this and have been patented by Blikslager’s lab for this very purpose. Blikslager sees a number of severe inflammatory colics come through the university’s hallways, and he’s always looking for better ways to treat pain and speed up repair so that horses can be discharged earlier with fewer long-term complications. “In the case of inflammation, this can also be more appropriately treated,” he said. “For instance, this is currently approached with non-steroidal anti-inflammatory drugs, but these have been shown not to reduce inflammation and to reduce repair, leading to studies on alternative medications for pain and inflammation.”

Seromuscular inflammation (red-stained cells) shown in a colitis case.
“Horses are very stoic (when they’re in pain),” he noted. “We will pain-score the horses on the ICU floor. In the worst-case scenario the horse, weanling foal or adult, is standing at back of stall with head down, ears forward, not really paying much attention. That’s one of the easiest things to ignore, but we need to treat for pain. When we do treat for pain, we will use opiates, systemic local anesthetics, and would like to use selective COX-2 inhibitors.”

Blikslager relayed that he and colleagues successfully used the local anesthetic lidocaine to address pain from gut inflammation. “We’re not sure why lidocaine is anti-inflammatory,” he said, “but it probably has something to do with the nervous system.”

The veterinary community has a long way to go in understanding exactly how cells function in all types of colic, but Blikslager sees a lot of promise with the current in vitro (in the laboratory) research methods. He summarized, “The future? Epithelium taken out of a dead horse–we have grown these up into organoids containing stem cells” for an even more accurate way to determine gut damage at the cellular level.