The Australian Take on Laminitis

The second most-common killer of our horses after colic is laminitis. The disease is a crisis, and it is often chronic and life-altering. It can be caused by illness unrelated to the foot, such as a retained placenta or grain overload. It can be caused by trauma to the foot. It can be caused by bearing too much weight when another limb is injured. Unfortunately, researchers have found that by the time a horse shows any lameness because of laminitis, it is too late to prevent damage; you can only try to stop additional destruction of the foot. How, then, can you fight this disease as a horse owner?

The mission of the Australian Equine Laminitis Research Unit (AELRU) is: To discover the mechanism of laminitis and make it a preventable disease. This is what owners are looking for.

For those who follow laminitis research to any degree, the name of AELRU director 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 and the 2003 American Association of Equine Practitioners (AAEP) annual convention in November 2003, he reinforced that reputation with discussions of the latest work from AELRU.

Dr. Christopher Pollitt
Christopher Pollitt, BVSc, PhD, Director of the Australian Equine Laminitis Research Unit.
Images from Dr. Pollitt's Research

"We focus primarily on the cause, pathophysiology, and developmental stages," Pollitt states. Researching laminitis has been, as he calls it, his life's work; at the AAEP convention, he regaled his educated audience with nearly four consecutive hours of research findings and high-tech imagery, for which he received a standing ovation. He also authored or co-authored no less than nine studies published in the Laminitis Special Issue of the Equine Veterinary Journal (April 2004).

It would take several textbooks to describe in detail all of Pollitt's research and contributions to our understanding of laminitis. In this article, we'll focus on his most recent published work. For more background information on laminitis, see the Laminitis category under Lameness (Hoof) and Shoeing at www.TheHorse.com.

The Devastation of Laminitis

Although researchers such as Pollitt continue to reveal new information about the triggers and microscopic events that occur during laminitis, the basic problem has been understood for a long time--the attachment between the hoof and the coffin bone breaks down to some degree in affected hooves. This results in varying levels of pain and lameness, and can escalate to the horse's coffin bone sinking to penetrate the sole or the entire hoof capsule sloughing.

Our understanding of laminitis (which Pollitt notes is the second most common killer of horses after colic) has been complicated by the fact that its signs arise from many different apparent causes--grain overload, mechanical overload, severe illness such as a retained placenta, reactions to medication, or even no apparent cause at all. What do they all have in common to cause this devastating disease of the feet?

To explain, first we have to understand normal hoof function on the microscopic level to see how normal processes are affected by the myriad causes of laminitis. Pollitt says 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 describes 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 discussed how some cell proliferation occurs in the proximal (upper) epidermal laminae (those located closest to the coronet). The middle and distal (closest to the ground) laminae don't proliferate much at all, he says. (The laminar attachment consists of thousands of interlocking leaf-like structures--the hoof laminae. They project from the inner 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, Pollitt's research has shown that there's 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 says.

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 describes this activity as "remodeling of the laminar epidermis, involving the controlled release of activated matrix metalloproteinases (MMP, an enzyme) and their subsequent inhibition by tissue inhibitors of metalloproteinases (TIMPs) in order to accommodate stresses, strains, and growth. 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, to which both sets of laminae adhere. Pollitt explains: "The basement membrane is like a piece of shrink wrap completely lining the inside of the hoof wall, extending into every nook and cranny." Hemidesmosomes and basement membranes are intensively researched in human skin and dental disease, he adds.

"These structures (hemidesmosomes and basement membranes) are targeted by MMPs, which can dissolve the attachments between them," Pollitt says. "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?" he queried his audience.

One study Pollitt co-authored evaluated the levels of MMP-2 in normal vs. laminitic horses. The researchers found that laminar tissue in laminitic hooves contained roughly twice as much MMP-2 as normal hooves (see graph below).

"Increased transcription and activation of MMP has been implicated in various human cancers," the study noted. "Remarkably, the increase in MMP-2 expression (in study horses) occurred in the 48 hours between administration of a carbohydrate alimentary overload and the development of the first clinical signs of lameness. Our findings represent a warning to clinicians that the basement membrane lesion of laminitis is insidious and well under way before any clinical signs are apparent. Any preventive strategies must be in place before overt foot pain develops if horses are to experience the developmental phase of laminitis without significant laminar damage."

The study also noted that "Real-time PCR analysis of laminar MMP-2 accurately monitors laminitis development at the molecular level and can be used diagnostically and for testing preventive strategies."

Why Does Colitis Trigger Laminitis?

Another big focus for Pollitt is 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 says. They tend to be higher in stressed pasture than in lush pasture (see "Cutting Down on Carbs," www.TheHorse.com/ViewArticle.aspx?ID=4777, for more information). 

"Horses seek out plants with high levels of fructans--they love them!" he says. Also, he continues, 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, 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 reports 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 is that fructans aren't digested in the small intestine; 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 says. 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 explains.

Another study Pollitt co-authored investigating the link between the dosage of oligofructose (one type of fructan) and laminar damage found a dose-dependent relationship--i.e., the higher the dose, the worse the damage. The damage consisted of a loss of the hemidesmosomes we discussed earlier, as well as separation of the basement membrane, cytoskeleton failure (breakdown of the basal cells' physical structure), and alteration in the shape of basal cell nuclei. These characteristics were seen even at the lowest dosage of 7.5 grams of oligofructose per kilogram of body weight (3,750 grams for a 500-kg horse).

Controlling grazing might help 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 recommends.

For horses for which it's too late to prevent fructan intake, Pollitt's team has evaluated a way to prevent or minimize the delivery of these toxins to the feet--using cryotherapy (cold therapy).

Cryotherapy to Prevent Laminitis

Cold therapy (cryotherapy) has long been used to minimize the inflammation of injury in many species. How many times have you iced a sprain? Another example, Pollitt adds, is the "cold caps" that human cancer patients often wear during chemotherapy to decrease delivery of the drugs to the hair follicles, minimizing hair loss.

Pollitt theorized that cryotherapy's hypometabolic (metabolism-slowing) and vasoconstrictive (blood vessel-constricting) effects might help prevent the development of laminitis in horses which have already undergone some sort of insult likely to result in the disease (such as a known foray into the feed room, retained placenta, colic surgery, colitis, etc.).

Six Standardbreds had one forelimb put into 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 41°F (5°C), while the other limb ranged from about 55.4-89.6°F (13-32°C). 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 reports. "This is not surprising when you consider what they go through in winter."

The results? No lameness was observed in the cooled limbs, but lameness was seen in uncooled limbs. 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 cautions. 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, he also discussed "at-risk" horses, such as those in hospitals for colitis, surgical colic, or retained placenta, who were treated with cryotherapy as a preventive measure and did not develop laminitis.

See cryotherapy images here.

Healing Hooves

While it's nice to understand why and how laminitis occurs, many horse owners and veterinarians--especially those who own or treat laminitic horses--are more concerned with healing after the insult. Pollitt has found that the strength and healing environment in laminitic hooves are very different from those of normal hooves.

"The laminae (tiny, interlocking, fingerlike projections inward from the inside of the hoof wall and outward from the surface of the coffin bone) provide the ultimate internal shock absorber (for weight bearing)," says Pollitt. "Under normal (healthy) circumstances, the laminae will tear off bits of the coffin bone before they separate from each other." However, laminitis obviously greatly degrades the laminar attachment (between the epidermal or outer laminae and dermal or inner laminae), causing them to separate much more easily than normal.

With respect to injury healing, he adds, healthy hooves are very good at healing injured areas. Even if there is injury to an area where laminae don't proliferate as much, the cell proliferation process will "switch on" to provide new cells to repair the defect. One example is a wall stripping study, in which a vertical strip of wall is removed (and the hoof supported) to evaluate the healing process (see photos here).

"Even when physically tearing out this piece of the wall, the basement membrane remains virtually intact, providing a template for rebuilding the wall," he reports. "But with laminitis, the template (basement membrane) is severely damaged, and the rebuilt laminar architecture is distorted and weak." This explains why recovering laminitic horses often have slightly to severely compromised laminar architecture following laminitis episodes.

Laminitis on the Cellular Level

Pollitt also 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 results of both APMA and glucose deprivation treatments were that the explants separated at the dermo-epidermal junction, he reports. However, the reasons for the separation were different.

"Lack of glucose reduced hemidesmosome numbers (attaching laminae to basement membrane) until they disappeared and the basal cell cytoskeleton collapsed," he reports. "Anchoring filaments (that 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 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 summarizes. "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."

Delving Deeper

To further describe what happens when laminae separate, Pollitt has 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 says.

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."

Editor's note: Dr. Pollitt would like to thank the Animal Health Foundation for their support of AELRU research. Click here for more information about the Foundation, including donation information.


FURTHER READING

Australian Equine Laminitis Research Unit: www.uq.edu.au/~apcpolli/VE411/lamini/template.htm.

Background information on laminitis: See the Laminitis category under Lameness (Hoof) and Shoeing at www.TheHorse.com.


A SIGNIFICANT PROBLEM

"The condition of acute laminitis continues to be one of the most commonly presented and frustrating conditions to manage in equine medicine, causing pain and debilitation for a significant percentage of the domestic equine population at any one time (Anon 2000)...

"Acute laminitis may be considered as a systemic disease which is only finally manifested as a condition of the foot (Hood 1999a); and that clinical signs only become apparent once lamellar tissues have already been subjected to extensive activation of metabolic, inflammatory, and degenerative cellular changes, making effective treatment so difficult (Hood et al. 1993)."--S.R. Bailey, BVMS, PhD, MRCVS, Equine Veterinary Journal (2004) 36 (3), 199-203.


AN UNUSUAL CASE: A Foal With Laminitis?

While laminitis is extremely rare in foals, Chris Pollitt, BVSc, PhD, Director of the Australian Equine Laminitis Research Unit, co-authored a report of a Quarter Horse colt with bilateral forelimb (in both front feet) laminitis in the 2004 Laminitis Special Issue of the Equine Veterinary Journal.

"Foals are often afflicted with conditions that commonly trigger laminitis, yet they rarely develop the disease themselves," the authors wrote. However, this foal showed clinical signs typical of a laminitic horse (supporting more weight on the hindquarters than usual, with the front feet extended forward) the day after he was born.

At about four weeks of age, the foal was still lame and had a discharge from the soles of both front feet. When he was 45 days old, Pollitt examined him and found feet of normal size and shape, but they had dark red-brown rings in the upper half of the hooves that were closer together at the toe than at the heel. Also, each forefoot had dead sole at the toe and the sole coriums had prolapsed from this area (the foal was understandably sore to palpation here).

Following humane euthanasia and study, it was concluded that the foal had a genetic defect that prevented expression of a protein called plectin, which is a part of the hemidesmosome adhesion complex (hemidesmosomes attach the basal cells of the laminae to the basement membrane, to which both sets of laminae adhere) and a component of muscle tissue. This defect caused some signs similar to junctional epidermolysis bullosa, sometimes seen in Belgians.

See images here.

"This foal demonstrated that laminitis can result from disruption or damage to a single molecule of the hemidesmosome adhesion of the inner hoof wall laminae," authors said. "Thus it is immaterial whether the hemidesmosome defect results from an inherited gene 'knockout' or a metabolic crisis in adulthood, dermo-epidermal separation within the hoof laminae, the signature lesion of laminitis, still occurs."

The study also noted the role of weight-bearing in the development of laminitic lesions; even though the defect was present in the hind feet as well, they showed no laminitis pathology.

About the Author

Christy M. West

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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