Pathophysiology of Laminitis

Christopher C. Pollitt

A tough, flexible, connective tissue suspensory apparatus suspends the distal phalanx (DP) to the inside of the hoof wall. The surface of the inner hoof wall is folded into leaflike lamellae (laminae) to increase the surface area of attachment between the hoof and the DP. A horse has laminitis when this attachment apparatus is compromised. Without the DP properly attached to the inside of the hoof, the weight of the horse and the forces of locomotion drive the DP down and away from the hoof wall. Arteries, veins, and nerves are sheared and crushed, and secondary to the lamellar pathology, the corium of the coronet and sole is damaged. Eventually even the DP itself may suffer deterioration and lysis of its dorsal margin. Unrelenting pain in the feet (bilateral forelimb usually) and a characteristic lameness occur. In acute laminitis the lamellar tissue suspending the DP from the inner hoof wall is affected at the junction between the connective tissue of the dermis or corium (the bone side) and the basal cell layer of the epidermal lamellae (the hoof side). This junction, the basement membrane zone or dermal/epidermal interface, appears to be the weak link in an otherwise robust and reliable structure. Wholesale epidermal cell detachment from and lysis of the lamellar basement membrane may occur,^1,2 leading to disruption of lamellar anatomy and ultimately compromise (to varying degrees) of the suspensory attachment between the DP and hoof. Associated with basement membrane lysis and dysadhesion are changes to lamellar epidermal cell morphology that result in lamellar stretching and attenuation. Thus whether by loss of basement membrane attachment or by lamellar stretching (or both), the DP draws away from the inner hoof wall and sinks downward. The degree of separation and sinking varies between horses affected with laminitis, ranging from barely detectable (mild) to catastrophically severe (“sinkers”). Ponies and small horses are usually less severely affected than their heavier cousins. A good correlation exists between the severity, as seen with the microscope (histopathology), and the degree of lameness (using the Obel grading system³) shown by the horse.¹ When a horse first shows laminitic pain, the anatomy of the hoof wall lamellae is already compromised. The higher the lameness grade, the more severe the microscopic damage. Any activity that places stress on an already weakened lamellar attachment apparatus (such as forced exercise) exacerbates damage and is contraindicated. The use of nerve blocks to eliminate pain encourages locomotion and does more damage. Favorable outcomes are associated with effective preventive and early therapeutic strategies.

Histopathology of Acute Laminitis

Most descriptions of laminitis histopathology derive from laminitis induced experimentally with a single large dose of carbohydrate of either grain starch or oligofructose.^3,16,17 The sequences of microscopic events that lead to clinical laminitis follow a consistent temporal pattern,¹⁸ and the stages of histological laminitis can be identified by the degree of severity of these changes. Making the lamellar basement membrane clearly visible is important and requires staining lamellar tissues with periodic acid–Schiff (PAS) stain or with immunohistochemical methods using basement membrane–specific antibodies.^1,2

Normal lamellar anatomical characteristics, assessed before allocating a laminitis grade to a section of lamellar hoof tissue, are as follows (Figure 34-1):

• The tips of the secondary epidermal lamellae are always rounded (club-shaped) and never tapered or pointed.

• The basal cell nucleus is oval, with the long axis of the oval perpendicular to the long axis of the secondary epidermal lamellae. These parameters can be satisfactorily assessed using routine hematoxylin and eosin staining of sections.

• The basement membrane penetrates deeply into the crypts between the secondary epidermal lamellae and outlines the wafer-thin, but connective tissue–filled, secondary dermal lamellae.

• The basement membrane tightly adheres to the basal cells of each secondary epidermal lamella. The PAS or immunohistochemical stains show this best.

Fig. 34-1 Micrograph of normal hoof lamellae stained to highlight the basement membrane. The basement membrane (arrows) of each secondary epidermal lamella (SEL) is a magenta (black in the figure) line closely adherent to the SEL basal cells. At secondary dermal lamellae (SDL) tips, between the bases of each SEL, the basement membrane (BM) penetrates deeply (arrowheads) and is close to the anuclear, keratinized, primary epidermal lamella (PEL). The SEL tips are rounded (club-shaped). The basal cell nuclei are oval in shape (stars) and positioned away from the BM at the apex of each cell. The long axis of each basal cell nucleus is at right angles to the long axis of the SEL. The SDL are filled with connective tissue even at their very tips, between the SEL bases. These parameters of hoof lamellar anatomy form the basis of the histological grading system of laminitis histopathology. Stain, Periodic acid–Schiff; bar, 10 µm.

Grade 1 Histopathology

The earliest change attributable to laminitis is loss of shape and normal arrangement of the lamellar basal and parabasal cells. The basal cell nuclei become rounded instead of oval and take an abnormal position in the cytoplasm of the cell. The secondary epidermal lamellae become stretched, long, and thin, with tapering instead of club-shaped tips. These changes were present at 12 hours in serial lamellar biopsies taken after oligofructose dosing.¹⁸ First noticeable at the tips of the secondary epidermal lamellae, teat-shaped bubbles of loose basement membrane form. PAS staining shows this best (Figure 34-2).

Fig. 34-2 Grade 1 histological laminitis (periodic acid–Schiff stain). Micrograph showing hoof lamellar tissues stained to highlight the basement membrane. The basement membrane (arrows) is stained dark magenta (black in the figure). At the now-tapered tips of the secondary epidermal lamellae (SELs), the basement membrane has lifted away (stars) from the underlying basal cells. Between the SEL bases the basement membrane (BM) is in its normal position, close to the primary epidermal lamellae; bar, 10 µm.

Examination of laminitis tissues with the electron microscope confirms lysis and separation of the lamellar basement membrane.¹⁹ Importantly, the greater magnification shows widespread loss of basal cell adhesion plaques (hemidesmosomes) and contraction of the basal cell cytoskeleton away from the inner cell surface. Electron microscopy shows why the basement membrane separates from the feet of the basal cells. The filaments that anchor hemidesmosomes to the lamina densa of the basement membrane no longer bridge the dermal/epidermal interface (Figure 34-3).¹³

Fig. 34-3 Electron micrograph of hoof lamellar tip developing laminitis. The lamina densa (LD) of the basement membrane is separating from the plasmalemma of the lamellar basal cell (EBC). Some hemidesmosomes (black arrow) appear undamaged, but others (white arrow) have faded, are losing their anchoring filament attachments, and are drawing away from the basement membrane. Normally anchoring filaments (arrowhead) bridge the lamina densa firmly to the EBC. D, Dermis.

Grade 2 Histopathology

Because the basement membrane is no longer completely tethered to the basal cells, it slips farther away with each cycle of weight bearing by the horse. Portions of the lamellar basement membrane are lysed initially between the bases of the secondary epidermal lamellae (see Figure 34-2). The basement membrane retracts from the tips of secondary epidermal lamellae, taking the dermal connective tissue with it. The basement membrane–free epidermal cells appear not to be undergoing necrosis, at least initially, and clump together to form amorphous, basement membrane–free masses on either side of the lamellar axis.¹

Grade 3 Histopathology

In laminitis the worst-case scenario is a rapid and near-total basement membrane separation from all the epidermal lamellae of the hoof toe, quarters, heel, and bars. Sheets of basement membrane peel away to form aggregations of loose, isolated basement membrane in the connective tissue adjoining the lamellae. The epidermal lamellar cells are left as isolated columns with little viable connection to the dermal connective tissue still attached to the DP. The hoof lamellar tips slide away from the basement membrane connective tissue attachments, at first microscopically, but as the degree of separation increases, the distance between hoof and DP becomes measurable in millimeters (Figure 34-4) and can be detected radiologically.²⁰ This manifests clinically as a sinker. Because the basement membrane is the key structure bridging the epidermis of the hoof to the connective tissue of the DP, wholesale loss and disorganization of the lamellar basement membrane follow and inexorably lead to the pathology of hoof and bone that characterizes the chronic stage of laminitis.

Fig. 34-4 Grade 3 histological laminitis (immunostain). The basement membrane of a lamellar tip is highlighted by type IV collagen immunostaining. The tip of the primary epidermal lamella (PEL) has completely detached from its basement membrane. The PEL basal cells are now an unattached, amorphous mass. Collapsed tubes of basement membrane, now empty of epidermal cells, are still attached to connective tissue (arrowheads). The PEL has already moved 0.03 mm from its dermal compartment, and soon the distance will be measured, using a tape measure, on a radiograph. The inset shows a normal lamellar tip immunostained the same way. Type IV collagen immunostain; bars, 10 µm.

The laminitis process also affects the lamellar capillaries. As the basement membrane and the connective tissue between the secondary epidermal lamellae disappear, so do the capillaries. They become obliterated, compressed against the edges of the primary dermal lamellae. Without a full complement of capillaries in the lamellar circulation, blood probably bypasses the capillary bed through dilated arteriovenous shunts,²¹ changing the nature of the foot circulation. A bounding pulse becomes detectable by finger palpation of the digital arteries. Furthermore, epidermal cell necrosis, intravascular coagulation, and edema are not universally present in sections made from tissues in the early stages of laminitis. The vessels in the primary dermal lamella, even the smallest, are predominantly open, without evidence of microvascular thrombi. The gross anatomical appearance of freshly dissected laminitis tissue is dryness. Sometimes the lamellae just peel apart.

Leukocytes, Inflammation, and Laminitis

It is rare to detect leukocytes in the lamellar tissues of normal horses.¹⁴ However, extravasation of leukocytes into the perivascular lamellar dermis occurs in carbohydrate-,¹ Black walnut extract–,^14,22 and hyperinsulinemia-induced¹³ forms of laminitis. Because leukocytic infiltration of tissues is associated with inflammation, the discovery that leukocytic infiltration is common to most, if not all, forms of laminitis has reemphasized an inflammatory pathway to laminitis development. There is molecular evidence that inflammatory mediators may activate many of the processes known to damage the lamellar interface.^23,24 Polymorphonuclear leukocytes are rich in MMP-9, and their presence within lamellar epidermal compartments in grade 3 histopathology¹ suggests that this basement membrane–degrading enzyme may have a pathological role in disease development. Also neutrophils produce reactive oxygen species and proinflammatory cytokines that probably contribute to cellular damage within the lamellar milieu.¹⁵ Lamellar damage and leukocyte infiltration are readily detected by calprotectin immunostaining,^14,25 and because leukocyte infiltration precedes the expression of calprotectin in the lamellar epidermis, a role for leukocytes in initiating lamellar pathology has been suggested.^22,23 However, carbohydrate-induced laminitis studied at the 12-hour postdosing time point shows basement membrane degradation occurring in advance of leukocyte infiltration, thus downplaying an initiating role for leukocytes in lesion development.²⁵

Diagnosis of Laminitis

Sue J. Dyson

Laminitis is characterized by an acute-onset lameness of variable severity involving one or more feet. Most often both front feet are affected, with or without the hind feet, but unilateral laminitis does occur, usually caused by excessive load bearing because of severe contralateral limb lameness. Occasionally the hind feet are affected, without involvement of the front feet. The horse may be extremely reluctant to move and, if persuaded to move, tends to land heel first, with a short, pottery gait, with the hindlimbs placed unusually far underneath the body. There is a marked shortening of the caudal phase of the stride at the walk. Lameness may be accentuated as the horse turns. Lameness is worse on hard ground than on soft ground. Although many horses show severe lameness, in those with milder lameness the lameness may be less typical, although suggestive of foot pain. When standing still the horse may position the hindlimbs unusually far underneath the body and may constantly shift weight between the limbs. A horse with severe primary lameness that has developed laminitis in the contralateral limb may start to load the originally lame limb, shifting weight between the two limbs.

Usually, but not invariably, a clinically significant increase in digital pulse amplitude occurs; however, in a thick-skinned cob-type horse this may not be palpable. In the acute phase the affected feet may be hot. Pressure or percussion applied to the feet, especially in the toe region, usually causes pain, but if the horn is excessively hard, the horse may not react. Careful palpation around the coronary band may reveal an unusual depression associated with sinking of the distal phalanx (DP). An area of unusual softness may herald infection tracking proximally in association with laminitis complicated by submural abscessation.

Clinical signs are usually diagnostic, except in less severely affected horses or those with involvement of only the hind feet, when the characteristics of the lameness may suggest foot pain, but not necessarily pathognomonic for laminitis. The response to perineural analgesia varies and is not associated necessarily with the degree of pain and lameness. Apparent desensitization of the foot with palmar (abaxial sesamoid) nerve blocks may have absolutely no effect on the lameness in some horses, although some improvement may occur in others.

Because laminitis frequently develops secondarily to a primary disease process, it is critical to evaluate the entire horse and to identify any predisposing factors that require treatment, such as endotoxemia, septic metritis, equine metabolic syndrome, or equine Cushing’s disease. If equine metabolic syndrome is suspected, serum insulin concentrations and the response to an intravenous glucose tolerance test should be assessed. It has been suggested that the presence of three or more of the following may increase the risk of laminitis development tenfold: insulin resistance, compensatory β-cell response, hypertriglyceridemia, and obesity (body condition score, >6/9).¹ Recent administration of corticosteroids may also be a risk factor; in a survey of 36 European sports horse practitioners, 12 had experienced the development of severe laminitis within 7 to 10 days of administration of corticosteroids in 22 horses.² Corticosteroids incriminated included dexamethasone, triamcinolone, and betamethasone administered either systemically or intraarticularly for treatment of recurrent airway obstruction, joint disease, or back pain, at what would be considered to be normal dose rates. The majority, but not all, of the horses were considered overweight. Approximately 50% of affected horses were humanely destroyed because of uncontrollable sinking of the DPs.

Radiographic examination is critical for establishing a treatment protocol and prognosis. Although rotation of the DP often can be managed successfully, sinking warrants an extremely guarded prognosis. Lateromedial images help to determine whether the condition is acute or an exacerbation of a more chronic problem. Abnormal thickness of the dorsal hoof wall, with or without modeling of the toe of the DP, implies previous disease. Lateromedial images are also important to establish the baseline position of the DP within the hoof capsule. Dorsopalmar images may be useful for assessing mediolateral balance in horses with chronic, unstable laminitis and to delect radiolucent lines indicative of lamellar separation (Figure 34-6, B). If the foot is grossly misshapen, trimming it first is preferable; otherwise, a false impression of severe rotation of the DP, which merely reflects the abnormal hoof wall growth and the development of a lamellar wedge, may occur.