- Email: [email protected]
The Digital Pathologies of Chronic Laminitis
LAMINITIS
0749-0739/99 $8.00
+ .00
THE DIGITAL PATHOLOGIES OF CHRONIC LAMINITIS Deborah A. Grosenbaugh, DVM, PhD, Sherry J. Morgan, DVM, PhD, and David M. Hood, DVM, PhD
The equine hoof has evolved as a structure hard enough to withstand enormous mechanical forces yet resilient enough to resist permanent deformation. 3 In addition, the foot must be capable of both withstanding extreme environmental challenges and, at the same time, regenerating itself. The adaptations necessary to meet these demands originate as specializations of its metabolic, growth, and circulatory physiology as well as in its anatomy and conformation. Ultimately, however, the foot must be viewed as a highly integrated complex organ in which the survival and function of the whole depend on all of its components working as an integrated unit. This complexity places the foot at an increased risk of disease. Simply put, the high level of specialization and adaptation increases the probability of dysfunction and enhances the seriousness of problems when pathologies such as laminitis occur. Because of the foot's integrated functions, a single problem has the potential to induce a pathologic cascade, eventually involving pathologies of the foot's vascular supply, metabolism, and growth dysplasias. 21 The challenge in chronic laminitis is to recognize, diagnose, and subsequently treat all the pathologies present rather than focusing on a single problem. This process is perhaps simplified by categorizing the pathologies as (1) mechanical collapse, (2) metabolic and growth dysplasias, (3) vascular compromise, and (4) sepsis. 22,25 Ultimately, it is the combined presence and severity of these various pathologies that determines the patient's clinical status and the extent to which rehabilitation is possible, This article focuses on the metabolic and growth dysplasias, the acquired vascular pathologies, and the problem of digital sepsis as they occur in chronic laminitis. Mechanical failure is dealt with
From the Department of Animal Health Research and Development, Abbott Laboratories, North Chicago, Illinois (DAG); the Department of Anatomic Pathology, Drug Safety Evaluation, Abbott Laboratories, Abbott Park, Illinois (SJM); and the Department of Veterinary Physiology and Pharmacology; College of Veterinary Medicine, Texas A&M University, College Station, Texas (DMH)
VETERINARY CLINICS OF NORTH AMERICA: EQUINE PRACTICE VOLUME 15 • NUMBER 2 • AUGUST 1999
419
420
GROSENBAUGH et al
separately in the article on the mechanisms and consequences of structural failure of the foot in this issue. ALTERATIONS IN HOOF WALL GROWTH DURING LAMINITIS
How the normal hoof wall grows and replaces itself is a challenging question and one that is not fully answered as yet. There is little question that the bulk of the wall is the product of the reproducing coronary band basal epithelial cells which differentiate as they move distally toward the ground surface. 16 Unanswered is how and where this fully formed epidermal layer attaches to the deeper epidermal! dermal components of the foot. The "sterile bed"30 and "sliding contact"4 hypotheses of hoof wall growth both seek to explain the processes and to define how much, if any, the laminar epithelium contributes to the inner surface of the wall. There is little question that this normal growth pattern is variably disrupted in chronic laminitis. Although some investigators have suggested that the normal laminar epithelium is sterile and unproductive, it is invariably stated that these basal cells have the potential to become proliferative in disease states, giving rise to the hyperproliferative submural tissues. 27, 39 Likewise, the frequent reshoeings and divergence of founder rings reflect an increased and irregular rate of wall growth in these patients. The growth alterations lead to deformations of the wall and influence the hoof's ability to function biomechanically. Sustaining a normal pattern of hoof growth logically depends on two factors: the rate at which the basal epithelial cells reproduce and the architectural orientation of the basal cells relative to the surface of the foot. The hoof wall, sole, bulbs, frog, and white line are all produced by the epithelium covering the papillary and interpapillary dermis of the coronary band, sole, and terminal laminae. In these regions, the basal epithelial cells are primarily oriented and organized so that proliferation induces growth toward the ground surface. At the laminar interface, however, the basal epithelial cells cover the folded and refolded primary and secondary laminar dermis and laminar papillae and are largely oriented outward toward the stratum medium of the wall. These different cell orientations predict the consequences of increasing the rate of basal cell reproduction. An increased rate occurring at the coronary band results in an increased rate of wall growth, that is, as long as the orientation of the coronary band epithelium is not altered, the conformation of the wall can be normal. This is not the case when a hyperplastic response occurs in the laminar basal epithelial cells. In this instance, because of the orientation of the cells, the enhanced rate of reproduction tends to produce a thicker wall. This does not happen in the normal foot, because the reproduction rate of the laminar epithelium is timed so that the rate of laminar epithelial cell replication is coordinated with that of the coronet and sole. The changes that occur in the growth pattern during laminitis can be conceived as resulting from (1) an altered rate of basal cell reproduction, (2) a lack of coordination between coronary and laminar hyperplasia and cornification, and (3) a reaction secondary to a reorientation of the epithelial cells, or some combination of these factors. In addition, the altered mechanics of the foot incurred during digital collapse has the potential to alter conformation of the wall after it is formed. Together, these factors act to produce the changes in the appearance of the wall that are seen in affected horses. It is stated that the rate of dorsal wall growth is slowed by the foot's collapse in laminitis.B The proposed underlying mechanism is ischemia induced
THE DIGITAL PATHOLOGIES OF CHRONIC LAMINITIS
421
by compressive forces created as the dorsal wall is pushed proximally by the loading forces. The data seem to support that a generalized hyperplastic response is characteristic of the foundered horse. 4o The normal rate of wall growth has been reported to range from 0.14 to 0.24 mm/d (mean, 0.19 mm/d), with most of the variance related to environmental conditions. In addition, the rate of toe and heel growth is fairly uniform in the normal horse. In laminitis, the growth rate increases to a mean of 0.25 mm/ d. In these horses, it was noted that heel growth was significantly increased relative to that of the toe. This unbalanced growth rate contributes to the predisposition for the dished conformation of the dorsal wall and the long heels noted in the untrimmed and chronically foundered foot. The gross and histopathologic descriptions also reflect a hyperproliferative response in the laminar epithelium.27,39 It is unknown if the laminar hyperplasia is of the same magnitude as that present at the coronary band or if it is greater. Epidermal hyperplasia in response to injury is usually considered to be a locally regulated event associated with loss of contact inhibition or altered growth factor regulation. '4 If this holds true for the foot, it is logical that a loss of the necessary coordination between coronary band and laminar growth rates is expected. The mechanism responsible for this increased growth necessarily includes an increased reproductive rate of the basal cells. This, in tum, is associated with loss of regulatory control occurring at the cellular level. Loss of epidermal growth factor (EGF) control has been demonstrated in chronic laminitis!7 and, as described below, could playa significant role in lack of coordination between cellular proliferation and cornification. The consequences of mechanical failure of the foot can dramatically affect the foot's growth pattern. As discussed in the article on the mechanisms and consequences of structural failure of the foot in this issue, the architecture of the coronary band and laminar terminal papillae can be severely altered by the mechanical forces encountered during digital collapse. The coronary band and terminal papillae regions are significantly widened following collapse, resulting in a thickened wall as seen on radiographs and the widened white line seen on the solar surface. In addition, the orientation of the coronary papillae can be redirected by digital collapse. 12 Instead of being generally directed toward the long axis of the wall, they are displaced peripherally, especially in the dorsal part of the foot. As the wall is necessarily going to grow along the orientation of the papillae, this change results in the severe wall deformation frequently encountered in the chronically affected horse. Changes in the foot's growth pattern have diagnostic and prognostic significance. In the completely compensated and rehabilitated horse, the only physical evidence of previous disease is often a wider than normal white line and radiographic evidence of a vertically displaced distal phalanx with an increased distance from the bone's parietal surface to the exterior surface of the wall. From a prognostic standpOint, it should be noted that changes in the architecture of the coronary band are untreatable at this time, Thus, the presence of coronary band papillae reorientation denotes patients in which complete rehabilitation is unlikely. METABOLIC DYSPLASIAS IN LAMINITIS
In 1948, Nils Obel concluded his classic work with the statement "The problem presented by laminitis will probably only be reached through studies
422
GROSENBAUGH et al
of the biochemistry of the process of cornification in laminitis."35 Although this may be an oversimplification, the concept holds true that hoof wall cornification must be understood in order to manage the disease process. Cornification is the term applied to the defined series of biochemical steps by which the soft, flexible, and relatively fragile regenerating basal epithelial cells are transformed into the rigid cells of the hoof wall. Regardless of the hoof region being considered, the steps of cornification consist of (1) keratinization, (2) formation of the cell envelope, and (3) deposition of the extracellular matrix (Table 1). This process is dynamic, involving a finely tuned balance between cellular proliferation and differentiation and the exquisitely regulated sequential expression of structural and matrix molecules. The normal physiology of the hoof wall cornification process is briefly reviewed below. Keratinization
Hoof wall keratinization begins as the reproducing basal epithelial cells form keratinocytes. In this step, keratin polypeptides are synthesized from amino acids and then organized into an internal cytoskeletal structure with the assistance of intermediate filament-associated proteins (IFAPs).42,45 This skeleton can be likened to an interconnecting lattice attaching both to itself and to the internal surface of the cell wall, giving it tensile strength. Keratins do not make the keratinocyte rigid; most of the keratins are formed in basal and spinous cells of the soft and flexible secondary epidermal laminae, Keratins belong to a group of intermediate filaments, low sulfur proteins (40-70 kd), which are found in virtually all epithelial cells, The keratin polypeptides from various regions of the equine hoof have been characterized with respect to electrophoretic mobility on polyacrylamide gel and reactivity with monoclonal antibodies. 15, 16, 18, 46 Differences in keratin expression have been noted for different areas of the hoof wall. These compositional differences are consistent with the tendency toward the expression of higher molecular weight keratins as differentiation progresses?' 41, 51 Likewise, there are few data indicating that the keratin concentration of the soft epidermis is different from that of the hoof walL This implies that the toughness of the hoof wall is potentially derived from other biochemical sources. Once synthesized, the keratin filaments are assembled into four-chain complexes containing two pairs of coiled-coil keratin molecules that are stabilized Table 1. BASIC STEPS IN HOOF WALL CORNIFICATION Cornification Step
Cellular Location
Keratinization Intracellular (filamentous low-sulfur proteins containing methionine) Cell envelope formation Intracellular (polymerized high-sulfur proteins containing cysteine) Extracellular matrix deposition (lipids, principally thought to be phospholipids)
Cells Involved
Function
Basal and spinous cells Cell strength
Transitional cells of the Cell rigidity primary and secondary epidermal lamina Extracellular Transitional cells of the Cell adhesion primary epidermal lamina
TIlE DIGITAL PATIIOL(X:;IES OF CHRONIC LAMINITIS
423
by hydrophobic interactions and disulfide bonds between sulfur-containing amino acids. The coiled-coil pair is composed of two keratin molecules, one of each of the two subtypes (AE1 and AE3).6 It is this ornate architecture that lends strength to the molecule (Fig. 1). The successful formation of the complex coiled-coil structures of keratin requires their interaction with a second class of proteins, IFAPs.45 These globular proteins (10-30 kd) are enriched with the sulfur-containing amino acids cysteine, threonine, and tyrosine as opposed to the keratins that contain more methionine.B, 9, 15
Formation of the Cell Envelope
The second step in the cornification process is the formation of the cell envelope. This structure acts as a chemically resistant and physically tough protein shell for each cell. Formation of the cell envelope involves chemical cross-linking of soluble cell envelope proteins (CEPs) on the cytoplasmic side of the cell wall into an insoluble layer. 38 Like the IFAPs, CEPs are high in sulfur, being rich in cysteine.43 Cross-linking of the CEP precursors is a calcium-dependent transglutaminase reaction which may have implications regarding final hoof wall strength.16, 38 The formation of the cell envelope has several important consequences. Physically, it greatly increases the stiffness, or rigidity, of the keratinocyte,38 which is essential to the wall's hardness. In addition, its appearance coincides with what is referred to as "terminal cornification," in which the cell wall loses its structural integrity, becomes porous to water and small crystalloids, and ultimately dies.
'JYpe 1
'JYpe 2
Coiled Coil .
Keratin Dimer ·
Keratin Tetramer
Figure 1. Keratin molecule. Keratins contain methionine, but are classified as low-sulfur, intermediate filament proteins. Each molecule is composed of four peptide strands arranged as coiled-coil, first paired into dimers, then into tetramer structure. Intermediate filament associated proteins (IFAP) are high-sulfur proteins essential to proper tetramer alignment.
424
GROSENBAUGH et al
Deposition of the Extracellular Matrix
During the final maturation process, keratinocytes secrete lipid into the extracellular spaces.u Using histochemical staining techniques, we have observed that the equine hoof matrix contains phospholipids (Fig. 2). This is in contrast to the stratum cornium, which is a soft and flexible epithelium, where the extracellular matrix contains significant amounts of neutrallipids. ll Electron opaque deposits similar to lipid have been noted to be associated with some of the intercellular junctions of the equine hoof wall.30-32 The functions of the extracellular matrix include a cementing of individual epidermal cells of the hoof into a solid structure. Although the basal epithelial cells are anchored to the basement membrane and spinous epidermal cells by hemidesmosomes and desmosomes, the majority of the cells of the stratum medium lack this attachment; it is the matrix substances that perform this function. In addition, the extracellular matrix adds further rigidity to the wall and serves to inhibit epidermal desquamation. Finally, this matrix contributes to the insolubility of the wall in its normal aqueous environment. . Control of Normal Hoof Wall Growth and Metabolism
Regulation of the cornification process in the foot is complex. The proliferation and differentiation rates of the basal epithelial cells must be coordinated with the supply of nutritive substrates, which, in tum, requires an adequate circulation. In addition, the identified steps of cornification are timed and occur at different places in the foot. Work in our laboratory has shown that cultured laminar explants incubated with 355-cysteine are labeled preferentially in the partially keratinized primary epidermal laminae and zona alba, where the uptake of 355-methionine is principally restricted to basal and spinous cells of the secondary laminae (Fig. 3). Further studies indicate that the 3ss-methionine is incorporated into keratins, whereas the 355-cysteine is incorporated into either
Figure 2. Wall tubule located in axial regions of zona alba. Note thick barrier between keratinocytes composed of cell envelope proteins, cell membrane, and extracellular matrix. Matrix stains positive for lipids.
THE DIGITAL PATHOLOGIES OF CHRONIC LAMINmS
425
Figure 3. Laminar explant cultured with radiolabeled cysteine (A) and methionine (B). Cysteine is preferentially concentrated in primary epidermal lamina where methionine is localized in secondary epidermal lamina. Data indicates that cysteine and methionine are incorporated into cell envelope membrane proteins and keratins, respectively (see text).
the IFAPs or the CEPs. This clearly indicates that the primary epidermal laminae are transitional cells that have yet to conclude cornification and that the different steps in the cornification process occur at different sites in the wall. As with any tissue, the regulation of this process is under the control of a number of signal transduction systems that orchestrate growth and differentiation of the organ system. The control of this process is complex, involving the interaction of many factors, not all of which are understood. A list of these factors is found in the box on p 426.
Metabolic Derangements Associated with Chronic Laminitis
The earliest studies 35 noted changes consistent with the inhibition of keratinocyte differentiation. This notion was supported in later work, in which thickening of the laminar layer resulting from hyperplasia and irregular growth of the epithelium was noted in hoof tissue taken from horses affected by chronic laminitis.27• 39 There are many areas in which the hoof wall cornification process can go awry. We are only beginning to identify those areas that appear to play a role in the pathology of chronic laminitis. Several specific abnormalities of
426
GROSENBAUGH et al
Role of various factors regulating cornification Enhanced keratinocyte proliferation Epidermal growth factor Transforming growth factor-alpha Cholera toxin Epinephrine Enhanced keratinocyte differentiation Hydrocortisone Calcium Vitamin D Integrity of the barrier function
Una(erc aCI'd Increased desquamation Arachidonic acid deficiency Hypocholesterolemic drugs
cornification, including epidermal hyperproliferation, altered keratinization, and failure to complete cornification, have been identified in horses with chronic laminitis. One of the hallmarks of chronic laminitis is the presence of a hyperproliferative wedge of tissue that occupies, or attempts to fill, the submural space created by the displaced distal phalanx. Most commonly, this tissue is a mixture of dermal and epidermal elements, in which the latter predominates. Both gross and histologic examination reveals that the nature of this proliferative healing response varies significantly among horses (Fig. 4). In some, rapidly proliferating and poorly cornified epidermal cells predominate; in others, the tissue appears to be partially or fully cornified. This difference is significant, as the poorly cornified hyperproliferative state allows a greatly weakened and mechanically unstable foot characteristic of a clinically uncompensated patient. Defining what allows the proliferative laminae from one horse to halt the cornification process is an important question. Intuitively, there are two metabolic dysplasias that can result in this condition: (1) a basal cell hyperplasia and (2) a lack of cell differentiation. Both reflect an altered regulation of the cornification process. Work in our laboratory14 and elsewhere 10 has demonstrated the presence of receptors for EGF in hoof wall laminar tissue. EGF is a polypeptide that has been shown to affect the differentiation of epidermal tissue under both normal and abnormal conditions.5 When EGF receptor preparations from normal and laminitic tissue are compared, there is a loss of high-affinity binding sites for EGF in tissue from horses affected by chronic laminitisP It has been suggested that the loss of high-affinity binding sites may remove cells in laminitic tissue from normal EGF-mediated control. Furthermore, autoradiographic localization of EGF receptors revealed an altered receptor distribution pattern with disease. In normal laminar tissue, receptors are densest over basal cells and decrease with the degree of keratinization. In chronic laminitis, the EGF receptors are less organized and evenly distributed over regions of hyperproliferative tissue. It has long been proposed that disturbances in the keratinization step of cornification occur in laminitis, which leads to a weakened hoof wall. Larsson et al's29 in vivo radiolabeling studies indicated that cysteine uptake was markedly
THE DIGITAL PATHOLOGIES OF CHRONIC LAMINmS
427
Figure 4. Laminar explant from a horse with chronic laminitis cultured with radio labeled cysteine (A) and methionine (8). Note incorporation is more intense than in Figure 3, but the distribution of label is same.
reduced in horses affected by acute laminitis. Both Larsson et aP9 and Kameya et aPB have analyzed the amino acid composition of normal and laminitisaffected hooves and demonstrated elevated methionine and decreased cysteine in the laminae of affected feet compared with the stratum medium or stratum externum of either control or affected feet. One can speculate that these changes derive from an altered ability of the damaged circulation to provide the necessary nutrition, an altered ability of cells to incorporate the substrates into normal keratins, or a change in the epidermal cell type present. Today, the increased understanding of the cornification process allows the interpretation of these data to suggest that the increased laminar methionine levels reflect a hyperproliferation of poorly differentiated basal cells and spinous cell layers as well as a decreased, or delayed, formation of the cell envelope during terminal cornification. In vitro autoradiographic labeling of laminar explants that indicates enhanced incorporation of 35S-methionine into the hyperproliferative regions of laminitis-affected hooves and of 355-cysteine into the cornified regions (see Fig. 4) is consistent with this interpretation. The expression of specific keratins has been shown to depend on epithelial cell type, state of differentiation, disease state, and cellular environmental conditions. 34, 42 One such keratin identified as cytokeratin 14 (CK14), although present in all cell layers, only reacts to in situ immunostaining in the cells of the stratum basale. 51 Its expression in suprabasal cell layers has been associated with
428
GROSENBAUGH et al
pathologic hyperproliferative states. 19• 48. 49 In normal lamina, CK14 is confined to the basal cells of the secondary epidermal laminae. Similarly, in horses in which proliferation and cornification are occurring, CK14 staining is confined to the basal cells, regardless of the architectural dysplasias present (Fig. 5). When the proliferation is characterized by poor cornification, however, CK14 staining extends into suprabasal layers, including the primary epidermal laminae. Thus, CK14 localization has potential value as a diagnostic tool to separate laminitis patients on the basis of their metabolic healing response.
Figure 5. Cytokeratin 14 (CK14) immunostaining from a normal horse (A), and two horses with chronic laminitis (B and C). CK14 preferentially stains basal cells that are relatively nonrigid. A, Note only the single layer of basal cells are stained (arrows). B, Demonstrates a healing response. Epidermal differentiation is occurring normally and only one layer of basal cells (arrows) are present even though laminar dysplasia is evident.
THE DIGITAL PATHOLOGIES OF CHRONIC LAMINITIS
429
Figure 5 (Continued). C, Depicts a basal cell hyperplasia that is nondifferentiating and noncornifying. Entire epidermal lamina stains positive for CK14 (area between arrows). Basal cell hyperplasia in (C) contributes to digital instability.
Basement Membrane/Metalioproteinases
The basement membrane in humans has recently received considerable attention in the literature. 33 Invisible under light microscopy with conventional hematoxylin and eosin staining, it was considered to be an amorphous layer separating the parenchymal cells of various tissues (in this case, the laminar epithelium) from the underlying connective tissue (Fig. 6).44 Recent work has assigned an important role to the basement membrane as being critical in organizing the cytoskeletal framework of the epidermal cells and influencing the exchange of nutrients and growth regulating factors.l Structural examination has implicated the basement membrane as playing a major role in maintaining the integrity of the attachment of parenchymal tissue to underlying dermal tissue. 33 The major component of the basement membrane, the lamina densa, is a network of anastomosing chords composed primarily of type IV collagen reinforced by the glycoprotein laminin and heparin sulfate proteoglycan. A dynamic equilibrium between degradation of the synthesis of collagen during physiologic remodeling is controlled by the activation of the metalloproteinases. 47• 50 The nature and density of the connecting structures are tissue specific and correlate well with the strength of attachment. Recently, the nature of the basement membrane of the equine hoof wall dermal-epidermal junction has been studied. 36 It is analogous to other epithelial basement membranes but contains an increased number of anchoring fibrils and lamina densa extensions, suggesting increased strength of attachment in comparison to that of basement membranes of other tissues. Electron microscopic examination of the coronary and terminal papillae has revealed that the basement membrane folds into numerous ridges parallel to the long axis of the papilla. This suggests an ancillary role for the basement membrane as forming channels that direct the orientation of keratinocytes as they differentiate in the
430
GROSENBAUGH et al
Figure 6. Normal lamina stained for presence of the laminar basement membrane. This (arrows) is essential for attachment of the basal epithelial cells to the underlying dermis.
proximodistal direction at the epidermal-dermal interface. Epithelial tissue is characterized by proliferation of a basal cell layer (stratum basale), whose cells undergo terminal differentiation as they progress to the outer (distal) layers. Loss of basement membrane integrity and degradation of its attachment to basal cells have been shown to be early events in acute laminitis. 37 It therefore follows that the loss of basement membrane/basal cell integrity contributes to the pathology of the chronic disease. This notion is supported by studies indicating that the extracellular matrix metalloproteinase activity is substantially greater in connective tissue of hoof wall laminae from laminitis-affected horses than in that of controls. 26 Inappropriate expression or regulation of the matrix metalloproteinases in chronic laminitis may contribute to the loss of structural integrity of the dermal-epidermal interface; this suggests a point at which the progression of the disease may be modified.26 In addition, if the suggestion is true that the basement membrane acts to form channels that direct the orientation of keratinocytes,37 maintenance of basement membrane integrity may be critical to promoting a stable architecture for optimizing redeveloping laminar epithelium orientation during healing. Supplementation with the sulfur-containing amino acid methionine has been advocated in the promotion of hoof wall growth ever since it was suggested that methionine is essential to proper keratin synthesis. 29 Although methionine is an essential component of keratins, cysteine appears to play a major role in the final structural integrity of the keratinocyte itself. Given that the predominant
THE DIGITAL PATHOLOGIES OF CHRONIC LAMINmS
431
end-stage metabolic lesion appears to be a defect in the signaling process that controls the proliferation and keratinization of the laminar epidermal cells, supplementation with these precursors alone may not be palliative. VASCULAR PATHOLOGIES OF CHRONIC LAMINITIS
There is ample evidence that vascular pathologies contribute to the clinical status of the chronic laminitis patient. Angiographic evaluation often reveals a markedly decreased and irregular vascular supply along with apparently avascular regions within the fooU Near-infrared spectroscopic evaluation indicates a decreased oxygen store in the digits of these patients, confirming the suspected vascular compromise. 20 Categorically, this compromise involves either malperfusions or vascular hyperreactivity. Malperfusion implies that the foot's circulation is either totally or regionally deficient in or oversupplied with blood. Both conditions have significant clinical implications for the patient. Descriptions of perfusion defects in affected horses have been demonstrated using vascular perfusion casts. 24 Deficient malperfusions often occur in the dorsal submural tissues, coronary bed, or solar weight-bearing portions (Fig. 7). The vascular insufficiencies of the dorsal parietal surface are assumed to be secondary to either (1) traumatic tearing occurring during the displacement or instability of the distal phalanx or (2) pressures created by sepsis or edema in the digit. Those on the solar surface of the bone are intuitively related to compression of the vasculature between the descending phalanx and the inner surface of the sole. The mechanical basis for this is discussed in the article on the mechanisms and consequences of structural failure of the foot in this issue.
Figure 7. Vascular cast from a normal (A), and chronically affected horse (B). Note vascular supply to weight-bearing region of solar surface (arrows) is absent, whereas same region in the normal horse is covered with vascular papillae. Perfusion defect results from compression of dermis between distal phalanx and sole.
432
GROSENBAUGH et al
Regardless of its origin, the consequence of deficient malperfusion is that it interferes with or precludes the ability of the foot to heal. Patients with a mild chronic vascular insufficiency may demonstrate a poorly growing hoof wall associated with a marginally inadequate nutrient supply. Elevating the blood level of nutrients in these patients via oral supplementation is sometimes effective, reputedly through the increased diffusion gradients for substrates. As the severity of the vascular insufficiency increases, a point is reached at which the nutrient supply cannot meet the demands made by the healing hyperplastic epithelium. This predisposes the hoof to focal regions of ischemic necrosis and altered cornification. In its most severe form, vascular insufficiency can lead to a gangrenous foot. The second type of malperfusion is that in which the circulation is a part of the hyperplastic response of the chronic disease. The rapidly proliferative epidermis makes metabolic demands on the digital circulation, which responds by angiogenesis. Simply put, the circulation proliferates to keep up with the demand. The problem here is that this type of proliferative response consists of both dermal and hyperplastic epidermal components. These tissues are characteristically soft, elastic, and flexible; thus, they create significant instability at the laminar interface. The presence of significant angiogenesis is potentially a diagnostic aid to discern the type of laminar response occurring in the healing foot. The third digital circulatory problem in laminitis is the appearance of a digital vascular hyperreactivity or hypersensitivity. The prevalence and severity of this problem seem to vary with the severity and duration of the hyperactivity or hypersensitivity. It is represented by an overreaction of the foot's circulation to vasoactive agents such that transitory ischemic episodes may occur. Like Raynaud's syndrome in humans, there is evidence that the sensitivity of the digital circulation increases when the foot is exposed to cold temperatures.23 SEPSIS IN CHRONIC LAMINITIS
Digital sepsis poses a diagnostic challenge and significant risk to the chronic laminitis patient. The clinical significance and need for therapeutic intervention are related to its location. It can be described as being restricted to regions of (1) the fully cornified epithelium of the wall, (2) the viable epidermal or dermal components, or (3) the components of the axial skeleton within the fooU2 Given its environment, it would be difficult to conceive of a foot totally free of microbial contamination. The macro- and microarchitectural changes that occur in chronic laminitis allow surface contamination access to varying depths of the stratum medium. The risk that this contamination poses is that it can serve as a source of infection for the deeper and less cornified layers of the epithelium. One mechanism by which this can occur includes progression of the primary pathology to such a point that defects such as perforation of the sole or coronary shear pathologies allow internal contamination or infection. It has also been proposed that some organisms can attack and destroy the cornified epithelium and thus can invade the deeper and more viable regions of the foot. Frequently, depending on the therapeutic approach, sepsis is iatrogenically introduced as a consequence of wall and sole resections or excessive trimming. Diagnostically, contamination of the external fully cornified hoof is largely ignored, except when it appears in the dysplastic laminar (Fig. 8) and white line tissues. During the acute and early chronic phases of laminitis, hemorrhage and necrotic epidermal cells are trapped as the laminar interface undergoes hyperpla-
THE DIGITAL PATHOLOGIES OF CHRONIC LAMINITIS
Figure 8. Histologic section showing a neutrophilic epidermal layers of dysplastic interface in a horse contain significant amounts of cytokines that serve when loss of basement membrane integrity occurs tion x 100).
433
infiltrate (arrows) between dermal and with chronic laminitis. Epidermal cells to stimulate an inflammatory response (hematoxylin-eosin, original magnifica-
sia and healing. As these tissues grow distally, they appear at the solar surface and are referred to as "seedy toe." The decreased structural integrity and altered degree of cornification in these tissues variably alter their material properties. This, in turn, increases the risk of introducing sepsis to the deeper layers. Diagnostically, the challenge is to assess ·if the contaminated tissues should be debrided, potentially causing a further weakening and predisposing to spread of infection. It is when microbial agents obtain access to the basal and spinous layers or dermis that treatment becomes necessary. These cells are less cornified and more reactive to the invading agents. When they are damaged or stimulated, epidermal cells can potentially result in activation of the metalloproteinase enzyme systems, resulting in the loss of basal cell-to-basement membrane integrity and a further collapse of the foot. Also, the epidermal cells contain large amounts of cytokines, including interleukin-l. These induce an inflammatory response when they gain access to the dermal tissues as the basement membrane integrity is lost. The resulting inflammatory response leads to local increases in the submural pressure, which, in turn, are a source of pain, necrosis, and abscess formation in these patients. Finally, sepsis in this region predisposes the foot to pedal osteitis.
434
GROSENBAUGH et al
When sepsis occurs in elements of the axial skeleton, it poses an even more significant clinical problem. In the chronic laminitis patient, the distal phalanx often has avascular and devitalized regions that are predisposed to infection. When sepsis enters into the skeletal element, it is difficult to diagnose, and treatment often requires surgery. Rarely, digital sepsis can spread and become a local or systemic problem. Gangrene of the foot can occur as a consequence of laminitis, and as it does, it causes a local cellulitis visible proximal to the hoof wall. Submural abscesses can cause a similar swelling, with the primary difference being the pain that the patient displays. SUMMARY
This review indicates that the patient-to-patient uniqueness commonly seen in chronic laminitis represents the variable presence of the digital pathologies. Although some degree of mechanical failure is always present, the secondary metabolic and growth dysplasias, vascular pathologies, and sepsis mayor may not be evident. The presence and severity of these pathologies appear to have a more significant impact on the prognosis of individual cases than does the displacement of the distal phalanx. It should be reiterated that it is often the combined presence of these individual pathologies that gives rise to the patient that is totally refractory to treatment. In the absence of these pathologies, many horses with significant displacement of the distal phalanx are not in pain and are not in need of treatment. It thus follows that a key to the improved rehabilitation of difficult patients is focusing research on the physiopathology and diagnosis of these nonmechanical problems. References 1. Abrahamson DR: Recent studies on the structure and pathology of basement membranes. J PathoI149:257, 1986 2. Ackerman N, Gamer HE, Coffman JR, et al: Angiographic appearance of the normal equine foot and alterations in chronic laminitis. JAVMA 166:58, 1975 3. Bertram JE, Gosline JM: Fracture toughness design in horse hoof keratin. J Exp BioI 125:29, 1986 4. Budras KD, Hullinger RL, Sack WO: Light and electron microscopy of keratinization of the laminar epidermis of the equine foot with reference to laminitis. Am J Vet Res 50:1150, 1989 5. Deuel TF: Polypeptide growth factors: Roles in normal and abnormal cell growth. Annu Rev Cell BioI 3:443, 1987 6. Eckert RL: Structure, function and differentiation of the keratinocyte. Physiol Rev 69:1316, 1989 7. Eichner R, Bonitz P, Sun IT: Classification of epidermal keratins according to their immunoreactivity, isoelectric point, and mode of expression. J Cell BioI 95:1388, 1984 8. Ekfa1ck A: Amino acids in different layers of the matrix of the normal equine hoof. Possible importance of the amino acid pattern for research on laminitis. Zentralbl Veterinarmed 37[B):1, 1990 9. Ekflack A, Appelgren LE, Funkquist B, et al: Distribution of labeled cysteine and methionine in the matrix of. the stratum medium of the wall and in the laminar layer of the equine hoof. Zentralbl Veterinarmed 37[A):481, 1990 10. Ekfa1ck A, Funkquist B, Jones B, et al: Presence of receptors for epidermal growth factor (EGF) in the matrix of the bovine hoof-A possible new approach to the laminitis problem. Zentralbl Veterinarmed 35[A):321, 1988
THE DIGITAL PAlHOLOGIES OF CHRONIC LAMINITIS
435
11. Elias PM: Epidermal lipids, barrier function and desquamation. J Invest Dermatol 80(suppl):44S, 1983 12. Goetz T: Anatomic, hoof, and shoeing considerations for the treatment of laminitis in horses. JAVMA 190:1323, 1987 13. Goetz TE: The treatment of laminitis in horses. Vet Clin North Am Equine Pract 5:1989 14. Grosenbaugn DA: The Role of Epidermal Growth Factor as a Mediator of Hyperplasia in Equine Laminitis [dissertation]. College Station, TX, Texas A&M University, 1989 15. Grosenbaugh DA, Hood DM: Keratin and associated proteins of the equine hoof wall. Am J Vet Res 53:1859, 1992 16. Grosenbaugh DA, Hood DM: Practical equine hoof wall biochemistry. Equine Pract 15:8,1993 17. Grosenbaugh DA, Hood DM, Amoss MS, et al: Characterization and distribution of epidermal growth factor receptors in equine hoof wall laminar tissue: Comparison of normal horses and horses affected with chronic laminitis. Equine Vet J 23:201, 1991 18. Hamada M, Oyamada T Yoshikawa H, et al: Keratin expression in equine normal epidermis and cutaneous papillomas using monoclonal antibodies. J Comp Pathol 102:405, 1990 19. Heyden A, Huitfeldt HS, Koppang HS, et al: Cytokeratins as epithelial differentiation markers in premalignant and malignant oral lesions. J Oral Pathol Med 21:7, 1992 20. Hinclkey KA, Fearn S, Howard BR, et al: Near-infrared spectroscopy of pedal haemodynamics and oxygenation in normal and laminitic horses. Equine Vet J 27:465, 1995 21. Hood DM: Perspectives on chronic laminitis. In Hood DM, Wagner IP, Jacobson AC (eds): Proceedings of The Hoof Project. College Station, TX, Private Publisher, 1997, pp 21-34 22. Hood DM: Pathophysiologic mechanisms and treatment of chronic laminitis. In Proceedings of the International Conference on Equine Laminitis, Stoneleigh, 1998 23. Hood DM, Amoss MS, Grosenbaugh DA: Equine laminitis: A potential model of Raynaud's phenomenon. Angiology 41:270,1990 24. Hood DM, Slater MR, Grosenbaugh DA: Vascular perfusion in the horse with chronic laminitis. Equine Vet J 26:191, 1994 25. Hood DM, Grosenbaugh DA, Chaffin MK, et al: Pathophysiology of chronic laminitis. In Proceedings of the American Association of Equine Practitioners 39th Annual Meeting, San Antonio, 1993, p 199 26. Johnson PJ, Tyagi SC, Katwa LC, et al: Activation of extracellular matrix metalloproteinases in equine laminitis. Vet Rec 142:392, 1998 27. Kameya T, Kiryu K, Kaneko M: Histopathogenesis of thickening of the hoof wall laminae in equine laminitis. Jpn J Vet Sci 42:361, 1980 28. Kameya T, Kiryu K, Kaneko M, et al: Chemical composition of equine hooves affected with laminitis. Experimental Reproduction Equine Health Laboratory, Japan Racing Association, 16:1, 1979 29. Larsson B, Obel N, Aberg B: On the biochemistry of keratinization in the matrix of the horse's hoof in normal conditions and in laminitis. Nord Vet Med 8:761, 1956 30. Leach D, Oliphant LW: Ultrastructure of the equine hoof wall secondary epidermal lamellae. Am J Vet Res 44:1561, 1983 31. Leach DH: Structural changes in intercellular junctions during keratinization of the stratum medium of the equine hoof wall. Acta Anat (Basel) 147:45, 1993 32. Leach DH, Oliphant L: Annular gap junctions of the equine hoof wall. Acta Anat (Basel) 116:1, 1983 33. Leblond C, Inoue S: Structure, composition, and assembly of basement membrane. Am J Anat 185:367, 1989 34. Moll R, Frand WW, Schiller DL, et al: The catalog of human cytokeratins: Patterns of expression in normal epithelia, tumors and cultured cells. Cells 31:11, 1982 35. Obel N: Studies on the Histopathology of Acute Laminitis, [dissertation]. Uppsala, Almqvist & Wiksells Boltryckeri AB, 1948 36. Pollitt CC: The basement membrane at the hoof dermal epidermal junction. Equine Vet J 26:399, 1994 37. Pollitt CC: Basement membrane pathology: A feature of acute equine laminitis. Equine Vet J 28:38, 1996
436
GROSENBAUGH et al
38. Rice RH, Green H: The cornified envelope of terminally differential human epidermal keratinocytes consists of cross-linked protein. Cell 11:417, 1977 39. Roberts ED, Ochoa R, Haynes PF: Correlation of dermal-epidermal laminar lesions of equine hoof with various disease conditions. Vet Pathol 17:656, 1980 40. Ryan TP: Hoof growth in normal and laminitic horses. Forge 5:11,1989 41. Sun IT, Eichner R, Nelson WG, et al: Keratin classes: Molecular markers for different types of epithelial differentiation. J Invest Dermatol 81(suppl):109S, 1983 42. Sun IT, Tseng SCG, Huang AJ-W, et al: Monoclonal antibody studies of mammalian epithelial keratins: A review. Ann NY Acad Sci 455:307 1985 43. Tezuka T, Takahashi M: The cystine-rich envelope protein from human epidermal stratum corneum cells. J Invest Dermatol: 88:47, 1987 44. Vracko R: Basal lamina scaffold-anatomy and significance for maintenance of orderly tissue structure. Am J Pathol 77:314, 1974 45. Wang E: Intermediate filament associated proteins. Ann NY Acad Sci 45:32, 1985 46. Wattle 0: Cytokeratins of the equine hoof wall, chestnut and skin: Bio- and immunohisto-chemistry. Equine Vet J 66:(suppl): 139, 1998 47. Weber KT, Sun Y, Tyaci SC, et al: Collagen network of the myocardium: Function, structural remodeling and regulatory mechanisms. J Mol Cell Cardiol 26:279, 1994 48. Weiss RA, Eichner R, Sun TT: Monoclonal antibody analysis of keratin expression in epidermal diseases: A 48- and 56-k dalton keratin as molecular markers for hyperproliferative keratinocytes. J Cell BioI 98:1397, 1984 49. Wetzels RH, Kuijpers HI, Lane EB, et al: Basal cell-specific and hyperproliferationrelated keratins in human breast cancer. Am J Pathol 138:751, 1991 50. Woessner FJ: Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J 5:2145, 1991 51. Woodcock-Mitchell I, Eichner R, Nelson WG, et al: Immunolocalization of keratin polypeptides in human epidermis using monoclonal antibodies. J Cell BioI 98:580, 1982
Address reprint requests to Deborah A. Grosenbaugh, DVM, PhD Abbott Laboratories, Department 32p Building Al-2 1401 Sheridan Road North Chicago, IL 60048
0749-0739/99 $8.00
+ .00
THE DIGITAL PATHOLOGIES OF CHRONIC LAMINITIS Deborah A. Grosenbaugh, DVM, PhD, Sherry J. Morgan, DVM, PhD, and David M. Hood, DVM, PhD
The equine hoof has evolved as a structure hard enough to withstand enormous mechanical forces yet resilient enough to resist permanent deformation. 3 In addition, the foot must be capable of both withstanding extreme environmental challenges and, at the same time, regenerating itself. The adaptations necessary to meet these demands originate as specializations of its metabolic, growth, and circulatory physiology as well as in its anatomy and conformation. Ultimately, however, the foot must be viewed as a highly integrated complex organ in which the survival and function of the whole depend on all of its components working as an integrated unit. This complexity places the foot at an increased risk of disease. Simply put, the high level of specialization and adaptation increases the probability of dysfunction and enhances the seriousness of problems when pathologies such as laminitis occur. Because of the foot's integrated functions, a single problem has the potential to induce a pathologic cascade, eventually involving pathologies of the foot's vascular supply, metabolism, and growth dysplasias. 21 The challenge in chronic laminitis is to recognize, diagnose, and subsequently treat all the pathologies present rather than focusing on a single problem. This process is perhaps simplified by categorizing the pathologies as (1) mechanical collapse, (2) metabolic and growth dysplasias, (3) vascular compromise, and (4) sepsis. 22,25 Ultimately, it is the combined presence and severity of these various pathologies that determines the patient's clinical status and the extent to which rehabilitation is possible, This article focuses on the metabolic and growth dysplasias, the acquired vascular pathologies, and the problem of digital sepsis as they occur in chronic laminitis. Mechanical failure is dealt with
From the Department of Animal Health Research and Development, Abbott Laboratories, North Chicago, Illinois (DAG); the Department of Anatomic Pathology, Drug Safety Evaluation, Abbott Laboratories, Abbott Park, Illinois (SJM); and the Department of Veterinary Physiology and Pharmacology; College of Veterinary Medicine, Texas A&M University, College Station, Texas (DMH)
VETERINARY CLINICS OF NORTH AMERICA: EQUINE PRACTICE VOLUME 15 • NUMBER 2 • AUGUST 1999
419
420
GROSENBAUGH et al
separately in the article on the mechanisms and consequences of structural failure of the foot in this issue. ALTERATIONS IN HOOF WALL GROWTH DURING LAMINITIS
How the normal hoof wall grows and replaces itself is a challenging question and one that is not fully answered as yet. There is little question that the bulk of the wall is the product of the reproducing coronary band basal epithelial cells which differentiate as they move distally toward the ground surface. 16 Unanswered is how and where this fully formed epidermal layer attaches to the deeper epidermal! dermal components of the foot. The "sterile bed"30 and "sliding contact"4 hypotheses of hoof wall growth both seek to explain the processes and to define how much, if any, the laminar epithelium contributes to the inner surface of the wall. There is little question that this normal growth pattern is variably disrupted in chronic laminitis. Although some investigators have suggested that the normal laminar epithelium is sterile and unproductive, it is invariably stated that these basal cells have the potential to become proliferative in disease states, giving rise to the hyperproliferative submural tissues. 27, 39 Likewise, the frequent reshoeings and divergence of founder rings reflect an increased and irregular rate of wall growth in these patients. The growth alterations lead to deformations of the wall and influence the hoof's ability to function biomechanically. Sustaining a normal pattern of hoof growth logically depends on two factors: the rate at which the basal epithelial cells reproduce and the architectural orientation of the basal cells relative to the surface of the foot. The hoof wall, sole, bulbs, frog, and white line are all produced by the epithelium covering the papillary and interpapillary dermis of the coronary band, sole, and terminal laminae. In these regions, the basal epithelial cells are primarily oriented and organized so that proliferation induces growth toward the ground surface. At the laminar interface, however, the basal epithelial cells cover the folded and refolded primary and secondary laminar dermis and laminar papillae and are largely oriented outward toward the stratum medium of the wall. These different cell orientations predict the consequences of increasing the rate of basal cell reproduction. An increased rate occurring at the coronary band results in an increased rate of wall growth, that is, as long as the orientation of the coronary band epithelium is not altered, the conformation of the wall can be normal. This is not the case when a hyperplastic response occurs in the laminar basal epithelial cells. In this instance, because of the orientation of the cells, the enhanced rate of reproduction tends to produce a thicker wall. This does not happen in the normal foot, because the reproduction rate of the laminar epithelium is timed so that the rate of laminar epithelial cell replication is coordinated with that of the coronet and sole. The changes that occur in the growth pattern during laminitis can be conceived as resulting from (1) an altered rate of basal cell reproduction, (2) a lack of coordination between coronary and laminar hyperplasia and cornification, and (3) a reaction secondary to a reorientation of the epithelial cells, or some combination of these factors. In addition, the altered mechanics of the foot incurred during digital collapse has the potential to alter conformation of the wall after it is formed. Together, these factors act to produce the changes in the appearance of the wall that are seen in affected horses. It is stated that the rate of dorsal wall growth is slowed by the foot's collapse in laminitis.B The proposed underlying mechanism is ischemia induced
THE DIGITAL PATHOLOGIES OF CHRONIC LAMINITIS
421
by compressive forces created as the dorsal wall is pushed proximally by the loading forces. The data seem to support that a generalized hyperplastic response is characteristic of the foundered horse. 4o The normal rate of wall growth has been reported to range from 0.14 to 0.24 mm/d (mean, 0.19 mm/d), with most of the variance related to environmental conditions. In addition, the rate of toe and heel growth is fairly uniform in the normal horse. In laminitis, the growth rate increases to a mean of 0.25 mm/ d. In these horses, it was noted that heel growth was significantly increased relative to that of the toe. This unbalanced growth rate contributes to the predisposition for the dished conformation of the dorsal wall and the long heels noted in the untrimmed and chronically foundered foot. The gross and histopathologic descriptions also reflect a hyperproliferative response in the laminar epithelium.27,39 It is unknown if the laminar hyperplasia is of the same magnitude as that present at the coronary band or if it is greater. Epidermal hyperplasia in response to injury is usually considered to be a locally regulated event associated with loss of contact inhibition or altered growth factor regulation. '4 If this holds true for the foot, it is logical that a loss of the necessary coordination between coronary band and laminar growth rates is expected. The mechanism responsible for this increased growth necessarily includes an increased reproductive rate of the basal cells. This, in tum, is associated with loss of regulatory control occurring at the cellular level. Loss of epidermal growth factor (EGF) control has been demonstrated in chronic laminitis!7 and, as described below, could playa significant role in lack of coordination between cellular proliferation and cornification. The consequences of mechanical failure of the foot can dramatically affect the foot's growth pattern. As discussed in the article on the mechanisms and consequences of structural failure of the foot in this issue, the architecture of the coronary band and laminar terminal papillae can be severely altered by the mechanical forces encountered during digital collapse. The coronary band and terminal papillae regions are significantly widened following collapse, resulting in a thickened wall as seen on radiographs and the widened white line seen on the solar surface. In addition, the orientation of the coronary papillae can be redirected by digital collapse. 12 Instead of being generally directed toward the long axis of the wall, they are displaced peripherally, especially in the dorsal part of the foot. As the wall is necessarily going to grow along the orientation of the papillae, this change results in the severe wall deformation frequently encountered in the chronically affected horse. Changes in the foot's growth pattern have diagnostic and prognostic significance. In the completely compensated and rehabilitated horse, the only physical evidence of previous disease is often a wider than normal white line and radiographic evidence of a vertically displaced distal phalanx with an increased distance from the bone's parietal surface to the exterior surface of the wall. From a prognostic standpOint, it should be noted that changes in the architecture of the coronary band are untreatable at this time, Thus, the presence of coronary band papillae reorientation denotes patients in which complete rehabilitation is unlikely. METABOLIC DYSPLASIAS IN LAMINITIS
In 1948, Nils Obel concluded his classic work with the statement "The problem presented by laminitis will probably only be reached through studies
422
GROSENBAUGH et al
of the biochemistry of the process of cornification in laminitis."35 Although this may be an oversimplification, the concept holds true that hoof wall cornification must be understood in order to manage the disease process. Cornification is the term applied to the defined series of biochemical steps by which the soft, flexible, and relatively fragile regenerating basal epithelial cells are transformed into the rigid cells of the hoof wall. Regardless of the hoof region being considered, the steps of cornification consist of (1) keratinization, (2) formation of the cell envelope, and (3) deposition of the extracellular matrix (Table 1). This process is dynamic, involving a finely tuned balance between cellular proliferation and differentiation and the exquisitely regulated sequential expression of structural and matrix molecules. The normal physiology of the hoof wall cornification process is briefly reviewed below. Keratinization
Hoof wall keratinization begins as the reproducing basal epithelial cells form keratinocytes. In this step, keratin polypeptides are synthesized from amino acids and then organized into an internal cytoskeletal structure with the assistance of intermediate filament-associated proteins (IFAPs).42,45 This skeleton can be likened to an interconnecting lattice attaching both to itself and to the internal surface of the cell wall, giving it tensile strength. Keratins do not make the keratinocyte rigid; most of the keratins are formed in basal and spinous cells of the soft and flexible secondary epidermal laminae, Keratins belong to a group of intermediate filaments, low sulfur proteins (40-70 kd), which are found in virtually all epithelial cells, The keratin polypeptides from various regions of the equine hoof have been characterized with respect to electrophoretic mobility on polyacrylamide gel and reactivity with monoclonal antibodies. 15, 16, 18, 46 Differences in keratin expression have been noted for different areas of the hoof wall. These compositional differences are consistent with the tendency toward the expression of higher molecular weight keratins as differentiation progresses?' 41, 51 Likewise, there are few data indicating that the keratin concentration of the soft epidermis is different from that of the hoof walL This implies that the toughness of the hoof wall is potentially derived from other biochemical sources. Once synthesized, the keratin filaments are assembled into four-chain complexes containing two pairs of coiled-coil keratin molecules that are stabilized Table 1. BASIC STEPS IN HOOF WALL CORNIFICATION Cornification Step
Cellular Location
Keratinization Intracellular (filamentous low-sulfur proteins containing methionine) Cell envelope formation Intracellular (polymerized high-sulfur proteins containing cysteine) Extracellular matrix deposition (lipids, principally thought to be phospholipids)
Cells Involved
Function
Basal and spinous cells Cell strength
Transitional cells of the Cell rigidity primary and secondary epidermal lamina Extracellular Transitional cells of the Cell adhesion primary epidermal lamina
TIlE DIGITAL PATIIOL(X:;IES OF CHRONIC LAMINITIS
423
by hydrophobic interactions and disulfide bonds between sulfur-containing amino acids. The coiled-coil pair is composed of two keratin molecules, one of each of the two subtypes (AE1 and AE3).6 It is this ornate architecture that lends strength to the molecule (Fig. 1). The successful formation of the complex coiled-coil structures of keratin requires their interaction with a second class of proteins, IFAPs.45 These globular proteins (10-30 kd) are enriched with the sulfur-containing amino acids cysteine, threonine, and tyrosine as opposed to the keratins that contain more methionine.B, 9, 15
Formation of the Cell Envelope
The second step in the cornification process is the formation of the cell envelope. This structure acts as a chemically resistant and physically tough protein shell for each cell. Formation of the cell envelope involves chemical cross-linking of soluble cell envelope proteins (CEPs) on the cytoplasmic side of the cell wall into an insoluble layer. 38 Like the IFAPs, CEPs are high in sulfur, being rich in cysteine.43 Cross-linking of the CEP precursors is a calcium-dependent transglutaminase reaction which may have implications regarding final hoof wall strength.16, 38 The formation of the cell envelope has several important consequences. Physically, it greatly increases the stiffness, or rigidity, of the keratinocyte,38 which is essential to the wall's hardness. In addition, its appearance coincides with what is referred to as "terminal cornification," in which the cell wall loses its structural integrity, becomes porous to water and small crystalloids, and ultimately dies.
'JYpe 1
'JYpe 2
Coiled Coil .
Keratin Dimer ·
Keratin Tetramer
Figure 1. Keratin molecule. Keratins contain methionine, but are classified as low-sulfur, intermediate filament proteins. Each molecule is composed of four peptide strands arranged as coiled-coil, first paired into dimers, then into tetramer structure. Intermediate filament associated proteins (IFAP) are high-sulfur proteins essential to proper tetramer alignment.
424
GROSENBAUGH et al
Deposition of the Extracellular Matrix
During the final maturation process, keratinocytes secrete lipid into the extracellular spaces.u Using histochemical staining techniques, we have observed that the equine hoof matrix contains phospholipids (Fig. 2). This is in contrast to the stratum cornium, which is a soft and flexible epithelium, where the extracellular matrix contains significant amounts of neutrallipids. ll Electron opaque deposits similar to lipid have been noted to be associated with some of the intercellular junctions of the equine hoof wall.30-32 The functions of the extracellular matrix include a cementing of individual epidermal cells of the hoof into a solid structure. Although the basal epithelial cells are anchored to the basement membrane and spinous epidermal cells by hemidesmosomes and desmosomes, the majority of the cells of the stratum medium lack this attachment; it is the matrix substances that perform this function. In addition, the extracellular matrix adds further rigidity to the wall and serves to inhibit epidermal desquamation. Finally, this matrix contributes to the insolubility of the wall in its normal aqueous environment. . Control of Normal Hoof Wall Growth and Metabolism
Regulation of the cornification process in the foot is complex. The proliferation and differentiation rates of the basal epithelial cells must be coordinated with the supply of nutritive substrates, which, in tum, requires an adequate circulation. In addition, the identified steps of cornification are timed and occur at different places in the foot. Work in our laboratory has shown that cultured laminar explants incubated with 355-cysteine are labeled preferentially in the partially keratinized primary epidermal laminae and zona alba, where the uptake of 355-methionine is principally restricted to basal and spinous cells of the secondary laminae (Fig. 3). Further studies indicate that the 3ss-methionine is incorporated into keratins, whereas the 355-cysteine is incorporated into either
Figure 2. Wall tubule located in axial regions of zona alba. Note thick barrier between keratinocytes composed of cell envelope proteins, cell membrane, and extracellular matrix. Matrix stains positive for lipids.
THE DIGITAL PATHOLOGIES OF CHRONIC LAMINmS
425
Figure 3. Laminar explant cultured with radiolabeled cysteine (A) and methionine (B). Cysteine is preferentially concentrated in primary epidermal lamina where methionine is localized in secondary epidermal lamina. Data indicates that cysteine and methionine are incorporated into cell envelope membrane proteins and keratins, respectively (see text).
the IFAPs or the CEPs. This clearly indicates that the primary epidermal laminae are transitional cells that have yet to conclude cornification and that the different steps in the cornification process occur at different sites in the wall. As with any tissue, the regulation of this process is under the control of a number of signal transduction systems that orchestrate growth and differentiation of the organ system. The control of this process is complex, involving the interaction of many factors, not all of which are understood. A list of these factors is found in the box on p 426.
Metabolic Derangements Associated with Chronic Laminitis
The earliest studies 35 noted changes consistent with the inhibition of keratinocyte differentiation. This notion was supported in later work, in which thickening of the laminar layer resulting from hyperplasia and irregular growth of the epithelium was noted in hoof tissue taken from horses affected by chronic laminitis.27• 39 There are many areas in which the hoof wall cornification process can go awry. We are only beginning to identify those areas that appear to play a role in the pathology of chronic laminitis. Several specific abnormalities of
426
GROSENBAUGH et al
Role of various factors regulating cornification Enhanced keratinocyte proliferation Epidermal growth factor Transforming growth factor-alpha Cholera toxin Epinephrine Enhanced keratinocyte differentiation Hydrocortisone Calcium Vitamin D Integrity of the barrier function
Una(erc aCI'd Increased desquamation Arachidonic acid deficiency Hypocholesterolemic drugs
cornification, including epidermal hyperproliferation, altered keratinization, and failure to complete cornification, have been identified in horses with chronic laminitis. One of the hallmarks of chronic laminitis is the presence of a hyperproliferative wedge of tissue that occupies, or attempts to fill, the submural space created by the displaced distal phalanx. Most commonly, this tissue is a mixture of dermal and epidermal elements, in which the latter predominates. Both gross and histologic examination reveals that the nature of this proliferative healing response varies significantly among horses (Fig. 4). In some, rapidly proliferating and poorly cornified epidermal cells predominate; in others, the tissue appears to be partially or fully cornified. This difference is significant, as the poorly cornified hyperproliferative state allows a greatly weakened and mechanically unstable foot characteristic of a clinically uncompensated patient. Defining what allows the proliferative laminae from one horse to halt the cornification process is an important question. Intuitively, there are two metabolic dysplasias that can result in this condition: (1) a basal cell hyperplasia and (2) a lack of cell differentiation. Both reflect an altered regulation of the cornification process. Work in our laboratory14 and elsewhere 10 has demonstrated the presence of receptors for EGF in hoof wall laminar tissue. EGF is a polypeptide that has been shown to affect the differentiation of epidermal tissue under both normal and abnormal conditions.5 When EGF receptor preparations from normal and laminitic tissue are compared, there is a loss of high-affinity binding sites for EGF in tissue from horses affected by chronic laminitisP It has been suggested that the loss of high-affinity binding sites may remove cells in laminitic tissue from normal EGF-mediated control. Furthermore, autoradiographic localization of EGF receptors revealed an altered receptor distribution pattern with disease. In normal laminar tissue, receptors are densest over basal cells and decrease with the degree of keratinization. In chronic laminitis, the EGF receptors are less organized and evenly distributed over regions of hyperproliferative tissue. It has long been proposed that disturbances in the keratinization step of cornification occur in laminitis, which leads to a weakened hoof wall. Larsson et al's29 in vivo radiolabeling studies indicated that cysteine uptake was markedly
THE DIGITAL PATHOLOGIES OF CHRONIC LAMINmS
427
Figure 4. Laminar explant from a horse with chronic laminitis cultured with radio labeled cysteine (A) and methionine (8). Note incorporation is more intense than in Figure 3, but the distribution of label is same.
reduced in horses affected by acute laminitis. Both Larsson et aP9 and Kameya et aPB have analyzed the amino acid composition of normal and laminitisaffected hooves and demonstrated elevated methionine and decreased cysteine in the laminae of affected feet compared with the stratum medium or stratum externum of either control or affected feet. One can speculate that these changes derive from an altered ability of the damaged circulation to provide the necessary nutrition, an altered ability of cells to incorporate the substrates into normal keratins, or a change in the epidermal cell type present. Today, the increased understanding of the cornification process allows the interpretation of these data to suggest that the increased laminar methionine levels reflect a hyperproliferation of poorly differentiated basal cells and spinous cell layers as well as a decreased, or delayed, formation of the cell envelope during terminal cornification. In vitro autoradiographic labeling of laminar explants that indicates enhanced incorporation of 35S-methionine into the hyperproliferative regions of laminitis-affected hooves and of 355-cysteine into the cornified regions (see Fig. 4) is consistent with this interpretation. The expression of specific keratins has been shown to depend on epithelial cell type, state of differentiation, disease state, and cellular environmental conditions. 34, 42 One such keratin identified as cytokeratin 14 (CK14), although present in all cell layers, only reacts to in situ immunostaining in the cells of the stratum basale. 51 Its expression in suprabasal cell layers has been associated with
428
GROSENBAUGH et al
pathologic hyperproliferative states. 19• 48. 49 In normal lamina, CK14 is confined to the basal cells of the secondary epidermal laminae. Similarly, in horses in which proliferation and cornification are occurring, CK14 staining is confined to the basal cells, regardless of the architectural dysplasias present (Fig. 5). When the proliferation is characterized by poor cornification, however, CK14 staining extends into suprabasal layers, including the primary epidermal laminae. Thus, CK14 localization has potential value as a diagnostic tool to separate laminitis patients on the basis of their metabolic healing response.
Figure 5. Cytokeratin 14 (CK14) immunostaining from a normal horse (A), and two horses with chronic laminitis (B and C). CK14 preferentially stains basal cells that are relatively nonrigid. A, Note only the single layer of basal cells are stained (arrows). B, Demonstrates a healing response. Epidermal differentiation is occurring normally and only one layer of basal cells (arrows) are present even though laminar dysplasia is evident.
THE DIGITAL PATHOLOGIES OF CHRONIC LAMINITIS
429
Figure 5 (Continued). C, Depicts a basal cell hyperplasia that is nondifferentiating and noncornifying. Entire epidermal lamina stains positive for CK14 (area between arrows). Basal cell hyperplasia in (C) contributes to digital instability.
Basement Membrane/Metalioproteinases
The basement membrane in humans has recently received considerable attention in the literature. 33 Invisible under light microscopy with conventional hematoxylin and eosin staining, it was considered to be an amorphous layer separating the parenchymal cells of various tissues (in this case, the laminar epithelium) from the underlying connective tissue (Fig. 6).44 Recent work has assigned an important role to the basement membrane as being critical in organizing the cytoskeletal framework of the epidermal cells and influencing the exchange of nutrients and growth regulating factors.l Structural examination has implicated the basement membrane as playing a major role in maintaining the integrity of the attachment of parenchymal tissue to underlying dermal tissue. 33 The major component of the basement membrane, the lamina densa, is a network of anastomosing chords composed primarily of type IV collagen reinforced by the glycoprotein laminin and heparin sulfate proteoglycan. A dynamic equilibrium between degradation of the synthesis of collagen during physiologic remodeling is controlled by the activation of the metalloproteinases. 47• 50 The nature and density of the connecting structures are tissue specific and correlate well with the strength of attachment. Recently, the nature of the basement membrane of the equine hoof wall dermal-epidermal junction has been studied. 36 It is analogous to other epithelial basement membranes but contains an increased number of anchoring fibrils and lamina densa extensions, suggesting increased strength of attachment in comparison to that of basement membranes of other tissues. Electron microscopic examination of the coronary and terminal papillae has revealed that the basement membrane folds into numerous ridges parallel to the long axis of the papilla. This suggests an ancillary role for the basement membrane as forming channels that direct the orientation of keratinocytes as they differentiate in the
430
GROSENBAUGH et al
Figure 6. Normal lamina stained for presence of the laminar basement membrane. This (arrows) is essential for attachment of the basal epithelial cells to the underlying dermis.
proximodistal direction at the epidermal-dermal interface. Epithelial tissue is characterized by proliferation of a basal cell layer (stratum basale), whose cells undergo terminal differentiation as they progress to the outer (distal) layers. Loss of basement membrane integrity and degradation of its attachment to basal cells have been shown to be early events in acute laminitis. 37 It therefore follows that the loss of basement membrane/basal cell integrity contributes to the pathology of the chronic disease. This notion is supported by studies indicating that the extracellular matrix metalloproteinase activity is substantially greater in connective tissue of hoof wall laminae from laminitis-affected horses than in that of controls. 26 Inappropriate expression or regulation of the matrix metalloproteinases in chronic laminitis may contribute to the loss of structural integrity of the dermal-epidermal interface; this suggests a point at which the progression of the disease may be modified.26 In addition, if the suggestion is true that the basement membrane acts to form channels that direct the orientation of keratinocytes,37 maintenance of basement membrane integrity may be critical to promoting a stable architecture for optimizing redeveloping laminar epithelium orientation during healing. Supplementation with the sulfur-containing amino acid methionine has been advocated in the promotion of hoof wall growth ever since it was suggested that methionine is essential to proper keratin synthesis. 29 Although methionine is an essential component of keratins, cysteine appears to play a major role in the final structural integrity of the keratinocyte itself. Given that the predominant
THE DIGITAL PATHOLOGIES OF CHRONIC LAMINmS
431
end-stage metabolic lesion appears to be a defect in the signaling process that controls the proliferation and keratinization of the laminar epidermal cells, supplementation with these precursors alone may not be palliative. VASCULAR PATHOLOGIES OF CHRONIC LAMINITIS
There is ample evidence that vascular pathologies contribute to the clinical status of the chronic laminitis patient. Angiographic evaluation often reveals a markedly decreased and irregular vascular supply along with apparently avascular regions within the fooU Near-infrared spectroscopic evaluation indicates a decreased oxygen store in the digits of these patients, confirming the suspected vascular compromise. 20 Categorically, this compromise involves either malperfusions or vascular hyperreactivity. Malperfusion implies that the foot's circulation is either totally or regionally deficient in or oversupplied with blood. Both conditions have significant clinical implications for the patient. Descriptions of perfusion defects in affected horses have been demonstrated using vascular perfusion casts. 24 Deficient malperfusions often occur in the dorsal submural tissues, coronary bed, or solar weight-bearing portions (Fig. 7). The vascular insufficiencies of the dorsal parietal surface are assumed to be secondary to either (1) traumatic tearing occurring during the displacement or instability of the distal phalanx or (2) pressures created by sepsis or edema in the digit. Those on the solar surface of the bone are intuitively related to compression of the vasculature between the descending phalanx and the inner surface of the sole. The mechanical basis for this is discussed in the article on the mechanisms and consequences of structural failure of the foot in this issue.
Figure 7. Vascular cast from a normal (A), and chronically affected horse (B). Note vascular supply to weight-bearing region of solar surface (arrows) is absent, whereas same region in the normal horse is covered with vascular papillae. Perfusion defect results from compression of dermis between distal phalanx and sole.
432
GROSENBAUGH et al
Regardless of its origin, the consequence of deficient malperfusion is that it interferes with or precludes the ability of the foot to heal. Patients with a mild chronic vascular insufficiency may demonstrate a poorly growing hoof wall associated with a marginally inadequate nutrient supply. Elevating the blood level of nutrients in these patients via oral supplementation is sometimes effective, reputedly through the increased diffusion gradients for substrates. As the severity of the vascular insufficiency increases, a point is reached at which the nutrient supply cannot meet the demands made by the healing hyperplastic epithelium. This predisposes the hoof to focal regions of ischemic necrosis and altered cornification. In its most severe form, vascular insufficiency can lead to a gangrenous foot. The second type of malperfusion is that in which the circulation is a part of the hyperplastic response of the chronic disease. The rapidly proliferative epidermis makes metabolic demands on the digital circulation, which responds by angiogenesis. Simply put, the circulation proliferates to keep up with the demand. The problem here is that this type of proliferative response consists of both dermal and hyperplastic epidermal components. These tissues are characteristically soft, elastic, and flexible; thus, they create significant instability at the laminar interface. The presence of significant angiogenesis is potentially a diagnostic aid to discern the type of laminar response occurring in the healing foot. The third digital circulatory problem in laminitis is the appearance of a digital vascular hyperreactivity or hypersensitivity. The prevalence and severity of this problem seem to vary with the severity and duration of the hyperactivity or hypersensitivity. It is represented by an overreaction of the foot's circulation to vasoactive agents such that transitory ischemic episodes may occur. Like Raynaud's syndrome in humans, there is evidence that the sensitivity of the digital circulation increases when the foot is exposed to cold temperatures.23 SEPSIS IN CHRONIC LAMINITIS
Digital sepsis poses a diagnostic challenge and significant risk to the chronic laminitis patient. The clinical significance and need for therapeutic intervention are related to its location. It can be described as being restricted to regions of (1) the fully cornified epithelium of the wall, (2) the viable epidermal or dermal components, or (3) the components of the axial skeleton within the fooU2 Given its environment, it would be difficult to conceive of a foot totally free of microbial contamination. The macro- and microarchitectural changes that occur in chronic laminitis allow surface contamination access to varying depths of the stratum medium. The risk that this contamination poses is that it can serve as a source of infection for the deeper and less cornified layers of the epithelium. One mechanism by which this can occur includes progression of the primary pathology to such a point that defects such as perforation of the sole or coronary shear pathologies allow internal contamination or infection. It has also been proposed that some organisms can attack and destroy the cornified epithelium and thus can invade the deeper and more viable regions of the foot. Frequently, depending on the therapeutic approach, sepsis is iatrogenically introduced as a consequence of wall and sole resections or excessive trimming. Diagnostically, contamination of the external fully cornified hoof is largely ignored, except when it appears in the dysplastic laminar (Fig. 8) and white line tissues. During the acute and early chronic phases of laminitis, hemorrhage and necrotic epidermal cells are trapped as the laminar interface undergoes hyperpla-
THE DIGITAL PATHOLOGIES OF CHRONIC LAMINITIS
Figure 8. Histologic section showing a neutrophilic epidermal layers of dysplastic interface in a horse contain significant amounts of cytokines that serve when loss of basement membrane integrity occurs tion x 100).
433
infiltrate (arrows) between dermal and with chronic laminitis. Epidermal cells to stimulate an inflammatory response (hematoxylin-eosin, original magnifica-
sia and healing. As these tissues grow distally, they appear at the solar surface and are referred to as "seedy toe." The decreased structural integrity and altered degree of cornification in these tissues variably alter their material properties. This, in turn, increases the risk of introducing sepsis to the deeper layers. Diagnostically, the challenge is to assess ·if the contaminated tissues should be debrided, potentially causing a further weakening and predisposing to spread of infection. It is when microbial agents obtain access to the basal and spinous layers or dermis that treatment becomes necessary. These cells are less cornified and more reactive to the invading agents. When they are damaged or stimulated, epidermal cells can potentially result in activation of the metalloproteinase enzyme systems, resulting in the loss of basal cell-to-basement membrane integrity and a further collapse of the foot. Also, the epidermal cells contain large amounts of cytokines, including interleukin-l. These induce an inflammatory response when they gain access to the dermal tissues as the basement membrane integrity is lost. The resulting inflammatory response leads to local increases in the submural pressure, which, in turn, are a source of pain, necrosis, and abscess formation in these patients. Finally, sepsis in this region predisposes the foot to pedal osteitis.
434
GROSENBAUGH et al
When sepsis occurs in elements of the axial skeleton, it poses an even more significant clinical problem. In the chronic laminitis patient, the distal phalanx often has avascular and devitalized regions that are predisposed to infection. When sepsis enters into the skeletal element, it is difficult to diagnose, and treatment often requires surgery. Rarely, digital sepsis can spread and become a local or systemic problem. Gangrene of the foot can occur as a consequence of laminitis, and as it does, it causes a local cellulitis visible proximal to the hoof wall. Submural abscesses can cause a similar swelling, with the primary difference being the pain that the patient displays. SUMMARY
This review indicates that the patient-to-patient uniqueness commonly seen in chronic laminitis represents the variable presence of the digital pathologies. Although some degree of mechanical failure is always present, the secondary metabolic and growth dysplasias, vascular pathologies, and sepsis mayor may not be evident. The presence and severity of these pathologies appear to have a more significant impact on the prognosis of individual cases than does the displacement of the distal phalanx. It should be reiterated that it is often the combined presence of these individual pathologies that gives rise to the patient that is totally refractory to treatment. In the absence of these pathologies, many horses with significant displacement of the distal phalanx are not in pain and are not in need of treatment. It thus follows that a key to the improved rehabilitation of difficult patients is focusing research on the physiopathology and diagnosis of these nonmechanical problems. References 1. Abrahamson DR: Recent studies on the structure and pathology of basement membranes. J PathoI149:257, 1986 2. Ackerman N, Gamer HE, Coffman JR, et al: Angiographic appearance of the normal equine foot and alterations in chronic laminitis. JAVMA 166:58, 1975 3. Bertram JE, Gosline JM: Fracture toughness design in horse hoof keratin. J Exp BioI 125:29, 1986 4. Budras KD, Hullinger RL, Sack WO: Light and electron microscopy of keratinization of the laminar epidermis of the equine foot with reference to laminitis. Am J Vet Res 50:1150, 1989 5. Deuel TF: Polypeptide growth factors: Roles in normal and abnormal cell growth. Annu Rev Cell BioI 3:443, 1987 6. Eckert RL: Structure, function and differentiation of the keratinocyte. Physiol Rev 69:1316, 1989 7. Eichner R, Bonitz P, Sun IT: Classification of epidermal keratins according to their immunoreactivity, isoelectric point, and mode of expression. J Cell BioI 95:1388, 1984 8. Ekfa1ck A: Amino acids in different layers of the matrix of the normal equine hoof. Possible importance of the amino acid pattern for research on laminitis. Zentralbl Veterinarmed 37[B):1, 1990 9. Ekflack A, Appelgren LE, Funkquist B, et al: Distribution of labeled cysteine and methionine in the matrix of. the stratum medium of the wall and in the laminar layer of the equine hoof. Zentralbl Veterinarmed 37[A):481, 1990 10. Ekfa1ck A, Funkquist B, Jones B, et al: Presence of receptors for epidermal growth factor (EGF) in the matrix of the bovine hoof-A possible new approach to the laminitis problem. Zentralbl Veterinarmed 35[A):321, 1988
THE DIGITAL PAlHOLOGIES OF CHRONIC LAMINITIS
435
11. Elias PM: Epidermal lipids, barrier function and desquamation. J Invest Dermatol 80(suppl):44S, 1983 12. Goetz T: Anatomic, hoof, and shoeing considerations for the treatment of laminitis in horses. JAVMA 190:1323, 1987 13. Goetz TE: The treatment of laminitis in horses. Vet Clin North Am Equine Pract 5:1989 14. Grosenbaugn DA: The Role of Epidermal Growth Factor as a Mediator of Hyperplasia in Equine Laminitis [dissertation]. College Station, TX, Texas A&M University, 1989 15. Grosenbaugh DA, Hood DM: Keratin and associated proteins of the equine hoof wall. Am J Vet Res 53:1859, 1992 16. Grosenbaugh DA, Hood DM: Practical equine hoof wall biochemistry. Equine Pract 15:8,1993 17. Grosenbaugh DA, Hood DM, Amoss MS, et al: Characterization and distribution of epidermal growth factor receptors in equine hoof wall laminar tissue: Comparison of normal horses and horses affected with chronic laminitis. Equine Vet J 23:201, 1991 18. Hamada M, Oyamada T Yoshikawa H, et al: Keratin expression in equine normal epidermis and cutaneous papillomas using monoclonal antibodies. J Comp Pathol 102:405, 1990 19. Heyden A, Huitfeldt HS, Koppang HS, et al: Cytokeratins as epithelial differentiation markers in premalignant and malignant oral lesions. J Oral Pathol Med 21:7, 1992 20. Hinclkey KA, Fearn S, Howard BR, et al: Near-infrared spectroscopy of pedal haemodynamics and oxygenation in normal and laminitic horses. Equine Vet J 27:465, 1995 21. Hood DM: Perspectives on chronic laminitis. In Hood DM, Wagner IP, Jacobson AC (eds): Proceedings of The Hoof Project. College Station, TX, Private Publisher, 1997, pp 21-34 22. Hood DM: Pathophysiologic mechanisms and treatment of chronic laminitis. In Proceedings of the International Conference on Equine Laminitis, Stoneleigh, 1998 23. Hood DM, Amoss MS, Grosenbaugh DA: Equine laminitis: A potential model of Raynaud's phenomenon. Angiology 41:270,1990 24. Hood DM, Slater MR, Grosenbaugh DA: Vascular perfusion in the horse with chronic laminitis. Equine Vet J 26:191, 1994 25. Hood DM, Grosenbaugh DA, Chaffin MK, et al: Pathophysiology of chronic laminitis. In Proceedings of the American Association of Equine Practitioners 39th Annual Meeting, San Antonio, 1993, p 199 26. Johnson PJ, Tyagi SC, Katwa LC, et al: Activation of extracellular matrix metalloproteinases in equine laminitis. Vet Rec 142:392, 1998 27. Kameya T, Kiryu K, Kaneko M: Histopathogenesis of thickening of the hoof wall laminae in equine laminitis. Jpn J Vet Sci 42:361, 1980 28. Kameya T, Kiryu K, Kaneko M, et al: Chemical composition of equine hooves affected with laminitis. Experimental Reproduction Equine Health Laboratory, Japan Racing Association, 16:1, 1979 29. Larsson B, Obel N, Aberg B: On the biochemistry of keratinization in the matrix of the horse's hoof in normal conditions and in laminitis. Nord Vet Med 8:761, 1956 30. Leach D, Oliphant LW: Ultrastructure of the equine hoof wall secondary epidermal lamellae. Am J Vet Res 44:1561, 1983 31. Leach DH: Structural changes in intercellular junctions during keratinization of the stratum medium of the equine hoof wall. Acta Anat (Basel) 147:45, 1993 32. Leach DH, Oliphant L: Annular gap junctions of the equine hoof wall. Acta Anat (Basel) 116:1, 1983 33. Leblond C, Inoue S: Structure, composition, and assembly of basement membrane. Am J Anat 185:367, 1989 34. Moll R, Frand WW, Schiller DL, et al: The catalog of human cytokeratins: Patterns of expression in normal epithelia, tumors and cultured cells. Cells 31:11, 1982 35. Obel N: Studies on the Histopathology of Acute Laminitis, [dissertation]. Uppsala, Almqvist & Wiksells Boltryckeri AB, 1948 36. Pollitt CC: The basement membrane at the hoof dermal epidermal junction. Equine Vet J 26:399, 1994 37. Pollitt CC: Basement membrane pathology: A feature of acute equine laminitis. Equine Vet J 28:38, 1996
436
GROSENBAUGH et al
38. Rice RH, Green H: The cornified envelope of terminally differential human epidermal keratinocytes consists of cross-linked protein. Cell 11:417, 1977 39. Roberts ED, Ochoa R, Haynes PF: Correlation of dermal-epidermal laminar lesions of equine hoof with various disease conditions. Vet Pathol 17:656, 1980 40. Ryan TP: Hoof growth in normal and laminitic horses. Forge 5:11,1989 41. Sun IT, Eichner R, Nelson WG, et al: Keratin classes: Molecular markers for different types of epithelial differentiation. J Invest Dermatol 81(suppl):109S, 1983 42. Sun IT, Tseng SCG, Huang AJ-W, et al: Monoclonal antibody studies of mammalian epithelial keratins: A review. Ann NY Acad Sci 455:307 1985 43. Tezuka T, Takahashi M: The cystine-rich envelope protein from human epidermal stratum corneum cells. J Invest Dermatol: 88:47, 1987 44. Vracko R: Basal lamina scaffold-anatomy and significance for maintenance of orderly tissue structure. Am J Pathol 77:314, 1974 45. Wang E: Intermediate filament associated proteins. Ann NY Acad Sci 45:32, 1985 46. Wattle 0: Cytokeratins of the equine hoof wall, chestnut and skin: Bio- and immunohisto-chemistry. Equine Vet J 66:(suppl): 139, 1998 47. Weber KT, Sun Y, Tyaci SC, et al: Collagen network of the myocardium: Function, structural remodeling and regulatory mechanisms. J Mol Cell Cardiol 26:279, 1994 48. Weiss RA, Eichner R, Sun TT: Monoclonal antibody analysis of keratin expression in epidermal diseases: A 48- and 56-k dalton keratin as molecular markers for hyperproliferative keratinocytes. J Cell BioI 98:1397, 1984 49. Wetzels RH, Kuijpers HI, Lane EB, et al: Basal cell-specific and hyperproliferationrelated keratins in human breast cancer. Am J Pathol 138:751, 1991 50. Woessner FJ: Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J 5:2145, 1991 51. Woodcock-Mitchell I, Eichner R, Nelson WG, et al: Immunolocalization of keratin polypeptides in human epidermis using monoclonal antibodies. J Cell BioI 98:580, 1982
Address reprint requests to Deborah A. Grosenbaugh, DVM, PhD Abbott Laboratories, Department 32p Building Al-2 1401 Sheridan Road North Chicago, IL 60048