Developments in Understanding Laminitis
For those who follow laminitis research to any degree, the name of Chris Pollitt, BVSc, PhD, is synonymous with advanced research on the subject. At the Second International Equine Conference on Laminitis and Diseases of the Foot, he reinforced that reputation with a discussion of the latest work at the Australian Equine Laminitis Research Unit, the mission of which is: To discover the mechanism of laminitis and make it a preventable disease.
"We focus primarily on the cause, pathophysiology, and developmental stages," Pollitt stated.
He began his presentation by stating that laminitis is, "a dynamic, molecular process superimposed on normal biology. Many of its features are normal processes appearing at the wrong time and place. We need to know more about the genes and proteins of the laminar region--what is the genetic control of the attachments between hoof wall and connective tissue? What turns these factors on and off? We need a better understanding of some of these key activities and processes to shed light on how laminitis might occur."
Hoof Wall Growth
While hoof wall growth might seem at first glance to be unrelated to the laminitis process, Pollitt doesn't agree. The first research he presented dealt with the mechanism of hoof growth, since growth and laminitis share some of the same processes, albeit at very different levels.
Pollitt described the growth mechanism of cells proliferating at the coronet in which the hoof wall grows down toward the ground (the growth pattern with which most of us are familiar), and he also discussed how some cell proliferation occurs in the proximal (upper) epidermal laminae (those located closest to the coronet). The middle and distal laminae don't proliferate much at all, he said. (The laminar attachment consists of thousands of interlocking leaf-like projections, the hoof laminae. They project from the innner hoof wall, or epidermal side, and interlock with the dermal or connective tissue laminae that are attached to the distal phalanx or coffin bone.)
In fact, their research has shown that there is a 20-fold difference in cell proliferation between the proximal and mid-laminar regions. However, proliferation does recommence in the terminal papilla zone of the hoof (nearest the ground). Thus, "The majority of the laminae 'concentrate' on maintaining the suspensory apparatus," he said.
The question linking growth and laminitis is that if the hoof wall grows down to the ground, and if all of the laminae don't grow and move downward at the same rate, then how do the cells of the dermal and epidermal laminae break their bonds and reattach during downward growth of the wall in the normal horse? The dermal laminae don't move, bound as they are to the stationary third phalanx, or P3.
Pollitt described this activity as "remodeling of the laminar epidermis, involving the controlled release of activated matrix metalloproteinases (MMPs, a type of enzyme) and their subsequent inhibition by tissue inhibitors of metalloproteinases (TIMPs). MMPs exist in laminar hoof, and their uncontrolled activation is proposed as a mechanism for the pathogenesis of laminitis.
Structures called hemidesmosomes attach the basal cells of the laminae to the basement membrane that both sets of laminae adhere to, Pollitt explained. These structures are very important in human skin disease, he added.
"These structures (hemidesmosomes and basement membranes) are targeted by MMPs, which can dissolve the attachments between them," Pollitt said. "MMPs are highly concentrated in epidermal laminae. The laminae are constantly responding to the stresses and strains of growth and locomotion by releasing MMPs and TIMPs for cellular reorganization. Inadvertent or uncontrolled laminar MMP activation could trigger laminitis. So -- is laminitis a normal process gone wrong?"
Part of the answer might be figuring out how those MMPs become abnormally activated.
Why Does Colitis Trigger Laminitis?
Pollitt's next focus was on fructans in pasture grasses. Fructans are fructose chain molecules that function as reserve carbohydrates for grasses and are stored in different areas of the plant, he said. They tend to be higher in stressed pasture than in lush pasture, he said (see "Cutting Down on Carbs," article #4777, for more information).
"Horses seek out plants with high amounts of fructans--they love them!" he said. Also, he said, they are easy to come by because many plant breeders strive to increase fructan levels in plants. Fructan makes the plants more tolerant of winter and makes them grow faster in spring, and improves weight gain and milk production in sheep, cattle, and deer. But in horses, fructans are much more dangerous--they are frequently used in laboratory settings to induce laminitis, because they do so very reliably.
Pollitt reported that under some conditions, fructan in grass stems can reach up to 50% of the plant's dry matter content. Even if a plant only has 30% fructans, a horse eating about 15 kg of pasture dry matter per day would eat three to four kilograms of fructans per day. The problem with this is that fructans aren't digested in the small intestine; instead, they are fermented by the microflora (bacteria) of the colon, which results in a significant change in the bacteria population there and a drop in pH (increase in acidity).
"Fructan in the hindgut (colon) causes massive overgrowth of Streptococcus at the expense of Enterobacter species," he said. The Streptococcus damage the lining of this area of the gut; once this happens, toxins from the bacteria can get out of the gut and into the bloodstream. From there, they travel into the hooves, where they can activate MMPs and thus cause laminitis 40-48 hours after the fructan consumption, he explained.
Controlling grazing, then, might be advisable to reduce a horse's risk of fructan-induced laminitis, especially for at-risk horses. "Grazing muzzles, electric fencing, etc.--we need to do these things," Pollitt recommended.
For those horses for which it's too late to prevent fructan intake, Pollitt's team evaluated a way to prevent or minimize the delivery of these toxins to the feet--using cryotherapy (cold therapy).
Cryotherapy to Prevent Laminitis
Pollitt theorized that cryotherapy's hypometabolic (metabolism-slowing) and vasoconstrictive (blood vessel-constricting) effects might help prevent the development of laminitis. Six Standardbreds had one forelimb placed in an ice bath (50% water, 50% ice) to just below the knee for two days following induction of laminitis. The cold foot was thus maintained at around 5° Celsius, while the other limb ranged from about 13-32° Celsius. The horses were evaluated for lameness, their laminar tissues were sampled and graded, and MMP-2 expression was evaluated to define laminitis severity.
"The horses didn't mind this (having one leg in the ice bath) one bit," Pollitt reported. "This is not surprising when you consider what they go through in winter."
The results? No lameness was observed in the cooled limbs (lameness was seen in uncooled limbs), and tissue scores and MMP-2 expression were both significantly lower (more normal; p<0.05) than in the untreated feet. Thus, cryotherapy was deemed effective in preventing acute laminitis development.
"This is only working in the developmental stage," Pollitt cautioned. These horses had not yet shown any lameness or other signs of laminitis prior to the cryotherapy. "There is no evidence yet that it is beneficial in acute or later stages. It may be effective then, but we just don't know that yet."
Anecdotally, other clinicians at the conference discussed cases where "at-risk" horses, such as those in hospitals for surgical colic or retained placenta, had been treated with cryotherapy as a preventive measure and did not develop laminitis.
Laminitis on the Cellular Level
Pollitt then delved further into the microscopic process of laminitis, describing a study in which explants of laminar tissue were cultured either without glucose (depriving cells of the energy to function) or with the MMP activator p-amino-phenol-mercuric acetate, or APMA. The explants were then subjected to tension and prepared for electron microscopy.
The result of both treatments was that the explants separated at the dermo-epidermal junction, he said. However, the reasons were different.
"Lack of glucose reduced hemidesmosome numbers (that attach laminae to the basement membrane) until they disappeared and the basal cell cytoskeleton collapsed," he reported. "Anchoring filaments (that the hemidesmosomes use to attach basal cells to the basement membrane) were unaffected, although they failed under tension."
In contrast, APMA activation of MMPs didn't affect the hemidesmosomes, but caused the anchoring filaments to disappear.
"Natural laminitis may occur in situations where glucose uptake by laminar basal cells is compromised (e.g., equine Cushing's disease, obesity, hyperlipidemia or abnormally high concentrations of fats in the blood plasma, ischemia or low oxygen from a lack of blood supply, and septicemia or infection/toxins in the blood) or when laminar MMPs are activated (as with gastrointestinal carbohydrate overload)," he summarized. "The change in hemidesmosomes is treatment-specific; a lack of glucose causes one lesion and MMP activation causes another. Both are present in fructan-induced laminitis, suggesting that at least two mechanisms are in play. Therapies designed to facilitate peripheral glucose uptake and inhibit laminar MMP activation may prevent or ameliorate laminitis."
To further describe what happens when laminae separate, Pollitt investigated the substances involved in hemidesmosome failure, cytoskeleton damage, and basement membrane failure using immunofluorescence microscopy.
Without going too deep into the results, the overall conclusion was that in laminitis, a protein called laminin-5 (L5) is cleaved or split, allowing failure of the anchoring filaments attaching basal cells and the basement membrane.
"Suppressing or inhibiting MMP activity may prevent L5 cleavage," Pollitt said.
Overall, Pollitt concluded: "The biological basis of laminitis has become molecular, the discipline of molecular biology is being focused on laminitis, and a coherent body of knowledge will soon emerge that will demystify laminitis."
About the Author
Christy West has a BS in Equine Science from the University of Kentucky, and an MS in Agricultural Journalism from the University of Wisconsin-Madison.
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