LYMPHATIC RESEARCH AND BIOLOGY Volume 1, Number 3, 2003 © Mary Ann Liebert, Inc.
Progressive Swelling, Hyperkeratosis, and Fibrosis of Distal Limbs in Clydesdales, Shires, and Belgian Draft Horses, Suggestive of Primary Lymphedema HILDE E.V. DE COCK, D.V.M., Ph.D.,1 VERENA K. AFFOLTER, D.V.M., Ph.D.,1 ERIK R. WISNER, D.V.M.,2 GREGORY L. FERRARO, D.V.M.,3 and NIGEL J. MACLACHLAN, D.V.M., Ph.D.1
ABSTRACT Background: A condition characterized by progressive swelling, hyperkeratosis, and fibrosis of the distal limbs has been recognized in Shire, Clydesdale, and Belgian draft horses. This chronic progressive disease starts at an early age, progresses throughout the life of the horse, and often ends in disfigurement and disability of the limbs that inevitably leads to the horse’s premature death. This study was undertaken to better characterize this disease. Methods and Results: Six affected horses were donated for diagnostic workup. A detailed clinical, radiologic, gross, and histologic description is given in this report. The lesions in the limb consisted of progressive development of thick-walled lymphatics, associated with chronic dermal edema, inflammation, fibrosis, neovascularization, and elastin degeneration. In the end stages, arteriosclerosis and fibrosed veins were also present. The clinical signs and pathologic changes in this disease closely resemble the human condition of elephantiasis nostras verrucosa, a state in which chronic lymphedema plays a pivotal pathogenic role.
INTRODUCTION
A
progressive swelling, hyperkeratosis, and fibrosis of distal limbs has been recognized in Shire, Clydesdale, and Belgian draft horses. This chronic progressive disease starts at an early age, progresses throughout the life of the horse, and often ends in disfigurement and disability of the limbs that inevitably leads to the horse’s premature death. The clinical signs and pathologic changes closely resemble a condition known in humans as elephantiasis nostras verrucosa.1–11 CONDITIO N CHARACTERIZED BY
In humans, chronic lymphedema encompasses a spectrum of clinical disorders resulting from regional impairments of lymphatic transport and drainage.4,12,13 Based on preliminary research (Department of Pathology, Microbiology and Immunology and Department of Surgical & Radiological Sciences, University of California Davis, Davis, CA) a similar pathogenic mechanism seems to be involved in the disease that affects these specific draft horse breeds. Since the disease affects only three types of related draft horse breeds, with certain lines more affected than others, a genetic etiology, similar to human heritable primary
Departments of 1Pathology, Microbiology and Immunology and 2Surgical & Radiological Sciences and 3Center for Equine Health, University of California Davis, Davis, California.
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lymphedema, is suspected.4 In this paper we describe the clinical, radiologic, gross, and histologic morphologic findings in six affected animals. MATERIALS AND METHODS Animals Six draft horses (three Shire horses, two Clydesdales, and one Belgian draft horse) were donated to the Center for Equine Health (University of California Davis) for study of a severe skin condition affecting their distal limbs. The mean age of the animals was 15 years (ranging from 9 to 22 years). All animals had no clinical signs other than the skin lesions. Because of perceived severe discomfort associated with the skin lesions, the animals were euthanized after diagnostic workup. Necropsies were performed at the Department of Pathology, Immunology and Microbiology, University of California Davis. In those horses in which imaging studies were to be performed postmortem, 30 mL of heparin (1,000 units/ mL) was administered intravenously immediately prior to euthanasia. Antemortem indirect lymphangiography Following induction of general anesthesia, three affected horses were placed in lateral recumbency with affected limbs on the nondependent side. In one of the horses, iodinated contrast medium (Conray 400, 400 mg of iodine/mL, Mallinckrodt, Hazelwood, MO) was injected subcutaneously at the level of the lateral aspect of the metacarpophalangeal joint of a severely affected forelimb, and at an identical location in a minimally affected, ipsilateral rear limb. Approximately 10–20 mL of contrast medium was administered depending on the size of the limb and the ease of injection. Dorsopalmar/plantar and lateral radiographs of the distal extremities were recorded immediately following contrast administration. Direct lymphangiography was attempted in an additional two horses, but superficial peripheral lymphatics of the distal extremities could not be identified despite subcutaneous administration of Evan’s Blue dye. These studies were therefore aborted.
Postmortem angiography Limbs were removed at a level immediately proximal to the carpus or tarsus. Major arteries of the distal limbs were catheterized and infused with heparinized saline to remove all blood in the arterial and venous segments. Iodinated contrast (Conray 400, 400 mg of iodine/mL) was then injected through the catheters until the isolated system was fully infused with contrast media. Dorsopalmar/ plantar and lateral radiographs of the distal extremities were taken using a standard technique immediately following contrast media administration. Postmortem computed tomography (CT) Two isolated limbs from one horse were prepared according to the methods used for the angiographic studies, as described above. Limbs were positioned on a CT scanner table (FX/I Helical CT scanner, General Electric Co., Milwaukee, WI). Cross-sectional images of the limbs were acquired in helical mode from the mid-metacarpus or metatarsus to the distalmost extent of the limb, using 5-mm-slice collimation, a small field-of-view, and an edgeenhancing reconstruction algorithm. Tissue sampling At necropsy, tissue samples were collected and immediately fixed in 10% neutral buffered formalin. Additional samples collected after angiography from all four distal limbs included: (i) superficial skin and deep tissues at the lateral and medial side of the deep flexor tendon in the pastern region, (ii) deep tissues from the vascular plexus crossing between the deep flexor tendon and the interosseus tendon, and (iii) superficial skin and deep tissue from the fetlock region. Formalin-fixed samples were processed by routine methods to provide paraffin wax sections (4 mm) that were stained with hematoxylin and eosin and acid orcein-Giemsa. RESULTS Lymphangiography A lymphangiogram of a severely affected limb revealed marked dilatation and tortuosity
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of lymphatics of the distal extremity, particularly in the palmar aspect of the metacarpophalangeal joint (Fig. 1). Angiography Angiography revealed excellent anatomical detail of both the arterial and venous systems, depending on the timing of image acquisition following contrast injection. A moderate increase in vascular density was found in the palmar/plantar soft tissues of affected metacarpophalangeal (metatarsophalangeal) joints (Fig. 2). CT CT images provided information similar to that obtained from conventional radiographic angiography studies. The severely affected limb had a markedly increased diameter as compared with the mildly affected limb. The
FIG. 2. Postmortem angiogram of a horse with a mildly affected distal limb. Both the arterial and venous systems are contrast-enhanced in this image. The venous system is markedly distended, and a plethora of small vessels are present in the palmar aspect of the limb at the level of the metacarpophalangeal joint.
distribution of small vessels in the mildly affected limb is evident on cross-sectional image (Fig. 3). In the more severely affected limb, enhanced vessels were not as prominent as in the mildly affected limb, possibly due to increased interstitial pressure and resultant vascular compression. Gross pathology FIG. 1. Lymphangiogram of a severely affected forelimb. Notice the extremely dilated and tortuous lymph vessels of the distal extremity, particularly in the distal aspect of the fetlock.
At necropsy, no significant lesions were found in any organ, other than the skin and subcutis of affected limbs. An overview of the
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FIG. 3. Three-millimeter contrast CT image of a mildly affected limb. The image was acquired at the level of the basilar aspect of the proximal sesamoid bones. A fine vascular network is evident superficially on the palmar aspect of the limb (bottom of image).
horses involved in this study is given in Table 1. Skin lesions were typically milder on the forelimbs and marked-to-severe on the hindlimbs. The milder lesions, found mostly in the
TABLE 1.
CLINICAL , GROSS,
AND
forelimbs, were restricted to the caudal pastern region and consisted of diffuse hyperkeratosis and mild diffuse swelling. More advanced lesions consisted of one or two palpably thickened skin folds predominantly in the rear of the pastern region (Fig. 4). At times, multiple small, well-demarcated ulcerations had developed that occasionally coalesced into larger and more intractable areas of skin ulceration (Fig. 4). On necropsy the more advanced lesions were characterized by mild diffuse accumulation of clear fluid (edema) in the deep dermis and subcutis. In the region above the interosseus and in the deep soft tissues of the fetlock region a small number of dilated, thickwalled, tortuous lymphatics, filled with clear fluid, were present. The most advanced lesions, typically found in the hindlimbs, consisted of a generalized, firm swelling that spread up the limb, often affecting the skin as high as the hock. There was marked hyperkeratosis with numerous crusts. Skin scrapings revealed an infection with mites (Chorioptes spp.) in three animals (horses 1, 3, and 4), and two had numerous maggots invading the skin of the deep folds (horses 1 and 5). Numerous thick skin folds surrounding the entire pastern region and large, poorly defined,
HISTOLOGIC FINDINGS
OF
SIX AFFECTED DRAFT H ORSES
Number (breed)
Sex Age (years) Skin mites (Chorioptes spp.) Secondary bacterial infection Miasis Diameter carpus/tarsus (cm)
Diameter pastern region (cm)
#1 (Shire horse)
#2 (Shire horse)
#3 (Shire horse)
#4 (Clydesdale)
#5 (Clydesdale)
#6 (Belgian draft horse)
Female 22 LF, RF, LH, RH LH, RH*
Female 9 —
Female 22 LH, RH
Female 15 —
Gelding 10 —
LH †
Female 20 LF, RF, LH, RH —
—
—
—
LF LF: 27 RF: 26 LH: 30.5 RH: 35 LF: 51 RF: 42 LH: 44 RH: 47
— LF: 28 RF: 24 LH: 30 RH: 30 LF: 35 RF: 30 LH: 35 RH: 35
— LF: 27 RF: 27 LH: 30 RH: 30 LF: 30 RF: ND LH: 40 RH: ND
— LF: 27 RF: 27 LH: 33 RH: 35 LF: ND RF: 30 LH: 45 RH: 45
LH LF: 29 RF: 27 LH: 76 RH: 34 LF: ND RF: ND LH: ND RH: ND
— LF: 26 RF: 29 LH: 29 RH: 60 LF: 31 RF: 31 LH: 30 RH: 51
LF, left forelimb; RF, right forelimb; LH, left hindlimb; RH, right hindlimb; ND, not done; —, not present. *No significant organisms cultured. † S. aureus cultured.
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FIG. 4. Moderately affected hindlimb. Notice the thick skin folds at the plantar aspect of the fetlock, the fibrous nodules at the lateral aspect of the metatarsophalangeal joint, and small skin ulcerations.
variably sized firm dermal nodules were present (Fig. 5). On necropsy, large amounts of edema fluid escaped from the deep dermal tissues upon incision. In addition, a thick layer of dense fibrous tissue (up to 4 cm) expanded the deep dermis and subcutis (Fig. 6). Large numbers of tortuous, thick-walled, dilated lymphatics expanded the deep soft tissues over the entire length of the distal limbs (Fig. 7), but were most prominent at the fetlock and in the region above the interosseus. The dermal nodules consisted of localized accumulations of dense connective tissue. In most of these animals, both forelimbs or both hindlimbs were equally affected. The mean diameter, measured just distal to the carpus, for the left forelimb was 27.3 6 1.0 cm and for the right forelimb was 26.7 6 1.6 cm (the normal diameter of the forelimb 25–30 cm). The mean diameter at the pastern region for the left forelimb was 36.8 6 9.7 cm and for the right forelimb was 33.3 6 5.9 cm (normal diameter of forelimbs 30–35 cm). The mean diameter just distal to the tarsus in the left hindlimb was 38.1 6 18.6 cm and in the right hindlimb was
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FIG. 5. Severely affected hindlimb. Severe diffuse swelling of the distal hindlimb extending from the hock until the toe associated with thick skin folds, hyperkeratosis, and numerous skin ulcers.
37.3 6 11.3 cm (normal diameter of hindlimbs of 33–38 cm). At the pastern region the mean diameters in the left and right hindlimbs were 38.8 6 6.3 and 44.5 6 6.8 cm, respectively (normal diameter of hindlimbs 35–40 cm). In horses 5 and 6, one hindlimb was extremely swollen and significantly thicker than the other hindlimb (76 cm vs. 34 cm and 60 cm vs. 29 cm,
FIG. 6. This cross section of the skin from the midmetatarsal region of a severely affected hindlimb shows the severe cutaneous edema and fibrosis.
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FIG. 7. This formalin-fixed sample of the deep soft tissues from the fetlock region shows numerous cross sections through a thick-walled tortuous lymph vessel embedded in a dense collagen matrix.
respectively), just distal to the tarsus. In two other animals (horses 1 and 3) secondary bacterial infections of the soft tissues occasioned large pockets of pus scattered throughout the dermis from the toe to the hock. Bacterial culture of horse 1 produced no growth, but bacterial culture of horse 3 revealed Staphylococcus aureus. Histologic changes In the clinically mildly affected extremities, the deep subcutaneous lymph vessels exhibited the most prominent histologic changes. Mildly to moderately dilated lymphatics were, at times, associated with mild, diffuse, subendothelial accumulation of a basophilic matrix. Surrounding some of the lymph vessels were thin, circumferential bands of fibrosis. The medium-sized arteries had focal subendothelial accumulations of a pale basophilic fibrous matrix associated with disruption of the lamina elastica interna. The walls of medium-sized and large veins were mildly but diffusely expanded by deposition of collagen, rather than mucinous stroma. Degeneration of endothelial cells was rarely observed. The overlying epidermis was hyperplastic, with marked compact hyperkeratosis. Follicular hyperkeratosis resulted in follicular casts. Occasional superficial folliculitis was present.
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In clinically advanced cases with marked limb swelling, changes in lymphatics, arteries, and veins became more prominent, with additional changes observed, particularly in the deep dermis and subcutaneous tissues. The severity of the changes varied among extremities and also within a given extremity. The lymphatics, whose changes were most prominent in the deeper soft tissues, were always more severely affected than the corresponding arterial or venous structures. The lymph vessels were markedly dilated and tortuous, with severe subendothelial accumulation of a basophilic mucinous matrix and perilymphatic fibrosis (Fig. 8). The appearance of the latter varied from thin, indistinct layers to thick, prominent layers of broad, dark eosinophilic collagen bundles arranged circumferentially around the lymphatics. There was variable proliferation of the middle and deep vascular plexi of the skin. The deep soft tissues were variably expanded by an edematous connective tissue that contained myriad small, newly formed arterial structures. In the most severe cases, marked smooth muscular hyperplasia was seen in both newly formed and pre-existing arterioles. Arteriosclerotic lesions, characterized by prominent subendothelial plaques, involved the entire subendothelial layer. The vasa vasorum were very prominent and increased in number. Moderate subendothelial fibrosis and accumulation of a basophilic matrix were present in the large veins as well. Variable numbers of lymphocytes were distributed diffusely throughout the deep dermis and, occasionally, formed small lymphoid aggregates. Superficial skin changes in the more advanced cases consisted of prominent proliferation of the superficial vascular plexus and perivascular dermatitis predominated by lymphocytes and eosinophils. Erosions and ulcerations with serocellular crusting were often severe. In the two horses (1 and 3) with secondary bacterial infections, multiple pyogranulomata were scattered throughout the dermis and subcutis. In the case with negative culture (horse 1), numerous intralesional gram-positive cocci surrounded by splendore hoepli material were found (botryomycosis). Acid orcein-Giemsa stains of the lesional tissues revealed a clear change in elastic fibers
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Clydesdale, and Belgian draft horses of this study bear striking similarities with those seen in chronic lymphedema in humans, a condition predisposing to the cutaneous changes described in elephantiasis nostras verrucosa, mossy foot and limb, lymphostatic papillomatosis cutis, and pachyderma vegetans.5–7,9 In humans, chronic lymphedema is associated with diffuse enlargement of affected limbs, non-pitting edema, formation of thick skin folds and diffuse lichenification, hyperkeratosis, and formation of nodules and/or verrucous plaques of the skin.1–11 This spectrum of dermal lesions was also found in the affected draft breeds. Clinically, the lesions in these draft breeds begin at a young age and slowly progress to complete disfigurement and nonfunctionality of the limbs. In Belgian draft horses, the first skin folds are often noticed at 2 years of age. This condition significantly reduces the life-
FIG. 8. Histologic picture of the lymph vessels of the deep soft tissues in a normal (top) and an affected (bottom) draft horse. Notice the marked perilymphatic fibrosis and neovascularization in the tissue of the affected horse. Hematoxylin and eosin. Bars 5 50 mm.
when compared with non-affected skin of another draft horse breed, such as the Suffolk. In the initial stages of the disease, only minimal differences, characterized by occasional breaks in the elastic layer in the subepidermal dermis, were seen. In severely affected extremities, the elastic layer of the subepidermal dermis and elastic fibers surrounding the lymphatics of the deep dermis appeared markedly distorted, and separated by fibrous tissue (Fig. 9). The staining intensity of the elastic fibers changed from black to dark brown. The latter observation may indicate a change in biochemical components of the elastic fiber. DISCUSSION The clinical presentation and morphologic features of the lesions observed in the Shire,
FIG. 9. Histologic picture of the lymph vessels of the deep soft tissues in a normal (top) and an affected (bottom) draft horse. Notice the dense band of elastic fibers (black) supporting the normal lymphatics (top) compared with the thin disorganized elastic fibers surrounding the affected lymphatic (bottom). Acid orcein-Giemsa. Bars 5 50 mm.
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span of Belgian draft horse stallions (W. Beeckman, personal communication). Belgian draft horse mares are generally less affected, as are both Shire and Clydesdale stallions and mares. In these populations lesions usually begin to interfere with normal movement at an age of 15 years. In analogy to the description of human chronic lymphedema,5,7,10 light microscopic features in the horses is characterized by marked dermal edema, progressive fibrosis of the dermis and subcutaneous tissues, and fibrotic thickening of the lymphatic vessel walls, associated with elastic fiber degeneration in the subcutaneous and dermal tissues. These changes, in addition to the fibrosis of veins and arteries that may be found in the deep tissues, are all features of chronic lymphedema, and are not observed with tissue edema secondary to hypoproteinemia or renal or cardiac failure.10 Moreover, complete necropsy of these animals revealed no cardiac or renal lesions, which supports the hypothesis of a primary lymphatic disorder. Venous or arterial insufficiency can also result in diffuse limb swelling 4 that may superficially resemble lymphedema. Although performed on a limited number of animals, all diagnostic approaches (lymphangiography, angiography, and morphologic evaluation) suggested primary pathology of the lymphatic system. Lymphangiography revealed marked dilatation and tortuosity of lymphatics of the distal extremity, particularly in the region of the palmar aspect of the fetlock. Angiography demonstrated a moderate increase of vascular density, a feature of chronic lymphostasis known as lymphostatic angiopathy,12 in the fetlock region. Additional studies on several of these animals, using ultrasonography (M.B. Whitcomb, personal communication), have previously shown clear evidence of lymphostasis, predominantly in the palmar/plantar region. Human hereditary lymphedema is estimated to occur in about one in 6,000 live births. In this and other forms of chronic lymphedema, the degeneration of cutaneous elastic fibers is thought to play an important mechanistic role.12 In normal skin, a dense elastin meshwork connects the epidermis with the dermal lymphatics and surrounds the dermal lymphatics.12 Lymphatic vessels are thus believed
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to spring back to their original shape following compression and manipulation of the skin or muscles. Within the current investigation, fragmentation and disorganization of elastic fibers supporting the lymphatics within the dermis were striking features in the draft horses examined. It is unclear at this point whether these changes play any role in the development and progression of the disease. Future study, intended to discriminate inherited from acquired mechanisms and to further describe the pathogenetic changes in the elastic elements of the skin, will be invaluable to elucidate this condition of draft horses. In conclusion we herein report the spontaneous occurrence, in three draft horse breeds (Shire, Clydesdale, and Belgian), of a possible congenital condition analogous to the primary lymphedema observed in humans. Further characterization of this disease, particularly at the molecular level, may prove valuable not only because it might ultimately lead to therapeutic modalities for this condition in animals, but also because the equine disease may represent an excellent animal model for the further study of issues related to human chronic lymphedema.
ACKNOWLEDGMENTS We are grateful to the Center for Equine Health for providing us with the animals and financial support. We thank Jerry Fields, Monica Kratochvil, and Esi Djan of the Veterinary Medical Teaching Hospital, Pathology Department, University of California Davis for their excellent technical assistance.
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5. Price EW. Nodular subepidermal fibrosis in non-filarial endemic elephantiasis of the legs. Br J Dermatol 1973;89:451–456. 6. Richards RN. Verrucous and elaphantoid lymphedema: morphologic spectrum and terminology. Int J Dermatol 1981;20:177–187. 7. Rowley MJ, Rapini RP. Elephantiasis notras. Cutis 1995;49:91–96. 8. Saccoman S, Rifleman GT. Elephantiasis nostras verrucosa. J Am Podiatric Med Assoc 1995;85:265–267. 9. Sanders LJ, Slomsky JM, Burger-Caplan C. Elephantiasis nostras: an eight-year observation of progressive nonfilarial elephantiasis of the lower extremity. Cutis 1988;42:406–411. 10. Schiff BL, Kern AB. Elephantiasis nostras. Cutis 1980;25:88–89. 11. Vaccaro M, Borgia F, Guarneri F, Cannavo SP. Ele-
phantiasis nostras verrucosa. Int J Dermatol 2000; 39:760–773. 12. Daroczy J. Pathology of lymphedema. Clin Dermatol 1995;13:433–444. 13. Szuba A, Rockson SG. Lymphedema: anatomy, physiology and pathogenesis. Vasc Med 1997;2:321–326.
Address reprint requests to: Hilde De Cock, D.V.M., Ph.D. Department of Pathology, Microbiology and Immunology University of California Davis One Shields Avenue Davis, CA 95616-8739 E-mail: hedecock@ucdavis.edu
This article has been cited by: 1. Dr. Hilde E.V. De Cock, Verena K. Affolter, Thomas B. Farver, Leen Van Brantegem, Brad Scheuch, Gregory L. Ferraro. 2006. Measurement of Skin Desmosine as an Indicator of Altered Cutaneous Elastin in Draft Horses With Chronic Progressive Lymphedema. Lymphatic Research and Biology 4:2, 67-72. [Abstract] [PDF] [PDF Plus]