Morphological and Histochemical Analysis of a Case of Superficial Digital Flexor Tendon Injury in the Horse

J. Comp. Path. 1999 Vol. 120, 403–414

Morphological and Histochemical Analysis of a Case of Superficial Digital Flexor Tendon Injury in the Horse A. Kobayashi, M. Sugisaka, K. Takehana, M. Yamaguchi∗, Eerdunchaolu, K. Iwasa and M. Abe Department of Veterinary Anatomy, School of Veterinary Medicine, Rakuno Gakuen University, 582 Bunkyodai-Midorimachi, Ebetsu, Hokkaido 069–0851, Japan and ∗Department of Veterinary Biosciences, School of Veterinary Medicine, Ohio State University, 1900 Coffey Road, Columbus, OH 43210, USA Summary This report compares the morphology and the concentrations of glycosaminoglycans (GAGs) in an injured superficial digital flexor tendon (SDFT) of a horse with those of a normal tendon. An injured 6-year-old male Thoroughbred exhibited heat and swelling around the SDFT of the right forelimb. On histopathological examination, exuberant granulation was observed in the affected tendon, with activated tenocytes, angiogenesis, haemorrhage, and infiltration of small numbers of leucocytes. The collagen fibres were loosely packed and irregularly arranged. The diameter of control collagen fibrils was 20–360 nm and that of affected collagen fibrils 20–240 nm. In the analysis of GAGs in the matrix, hyaluronic acid (HA), dermatan sulphate (DS), and chondroitin sulphate (CS) were found to be major components in both control and affected tendons. Increases in DS in the affected tendon were striking. Our observations suggest that fibrillogenesis was activated by increases in DS and decreases in HA and CS. It is also assumed that absence of collagen fibrils of normal thickness and in a parallel arrangement reflected the morphological and biochemical characteristics of fibrillogenesis in the injured tendon. If the inflammatory features of an injured tendon could be altered, it might return eventually to its normal structure.  1999 W.B. Saunders Company Limited

Introduction Superficial digital flexor tendon (SDFT) injury affects up to 7% of racing horses (Rooney and Genovese, 1981; Rantanen et al., 1983; Genovese et al., 1990; Goodship, 1993). An injured tendon heals slowly, usually over 8–12 months, by scar formation rather than by tissue regeneration (Goodship, 1993). Heat, swelling (apparent on palpation of the SDFT) and pain occur. A variable degree of lameness is seen but usually resolves rapidly. The goals of therapy for acute tendon injury are to decrease inflammation, to minimize formation of scar tissue, and to promote restoration of normal tendon structure and function (Henninger, 1994). Although the importance of injured tendons has long been recognized, little is known about their anatomical and biochemical properties. A tendon is composed of fine filaments 0021-9975/99/040403+12 $12.00

 1999 W.B. Saunders Company Limited

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which can be teased apart easily at a cut surface (Davidson, 1982). These colinear filaments are referred to as fascicles and constitute the principle unit of tendon structure. The fascicles are coherent bundles of collagen fibres lying between tenocytes (Evans and Barbenel, 1975; Davidson, 1982). Collagen fibres and tenocytes are oriented in parallel to the longitudinal axis of the tendon. The cross-sectional area (CSA) of SDFTs varies between proximal and distal regions, and injury occurs most commonly in the mid-metacarpal region, where CSA is smallest and the tendon is not enclosed within a synovial sheath (Webbon, 1973, 1977, 1978). Webbon also suggested that the lateral aspect of the SDFT of horses is more cellular (with more fibroblasts) than the medial aspect, and that the tendons of horses from birth to 2 years of age are more cellular than those of older horses. In the damaged part of the tendon, collagen produced by fibroblasts initially forms fibrils of small diameter which are randomly oriented (Ketchum, 1979; Davidson, 1982). Most authors agree that the physical properties of tendon collagen are largely dependent on intra- and inter-molecular cross-linking (Parry et al., 1978; Goodship, 1993; Henninger, 1994). According to Parry et al. (1978), large collagen fibrils may contribute to the high tensile strength attributable to lateral cross-linking, small collagen fibrils may inhibit deformation, and the composition of the fibre and matrix of the tendon may inhibit the propagation of cracks. Haut et al. (1992) found that the total collagen content of the canine patellar tendon decreased with age, while the insoluble collagen content increased, possibly affecting the tendon’s elasticity. The equine SDFT undergoes an increase in structural organization and an increase in non-reducible cross-links with maturation and aging; these changes are associated with an increase in elasticity (Gillis et al., 1997). Jones and Bee (1990) reported that tendon proteoglycans exhibit both quantitative and qualitative variations in response to the functional demands imposed. Proteoglycans interact with and influence collagen fibrillogenesis (Parry et al., 1982; Vogel et al., 1984; Scott and Hughes, 1986). However, no comparisons of glycosaminoglycans (GAGs) in injured and normal SDFTs have been reported to our knowledge. The purpose of this study was to make such a comparison and also to compare the morphology of injured and normal SDFTs in the horse. Materials and Methods Case History and Samples A 6-year-old male Thoroughbred (weight 480 kg), with 3 years of racing experience and no history of tendon injury, was injured during daily training. The horse was lame and showed signs of pain upon palpation of its right forelimb. Acute heat production and swelling of the SDFT of the right forelimb were noted. Ultrasonography confirmed injury to the SDFT. The horse was donated to the Department of Veterinary Pathology and Veterinary Anatomy, Rakuno Gakuen University for teaching and research purposes 2 days after lameness became apparent. The animal was killed with an intravenous injection of an overdose of sodium pentobarbital. Typical inflammation of the SDFT was observed 20 mm distal to the middle of the metacarpal bone after removal of the overlying skin, the inflamed region

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Fig. 1.

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Dorsoventral and lateral thickness of the affected SDFT (right) and same site in the control (left). The affected area is located centrally in the cross-section of the injured tendon, with oedematous swelling and dark red discoloration (inside circle).

being approximately 20 mm in length. A 15-mm length of the affected region was removed with a razor blade, leaving 2·5 mm of inflamed tendon at each end. The cross-section of the removed portion was then immediately photographed. The sample of injured tendon was divided into three portions for (1) light microscopy, (2) electron microscopy, and (3) analysis of GAGs. The sample for GAG analysis was immediately frozen in liquid nitrogen and stored at −80°C until used. No evidence of any other abnormality was noted at post-mortem examination. A control sample was taken from the central region of the SDFT of the left forelimb of the same horse, and divided into three portions as described above. Light Microscopy (LM) The samples were fixed in 10% neutral buffered formalin (0·1  phosphate buffer, pH 7·4) for 48 h, followed by dehydration and embedding in paraffin wax by conventional methods. Sections were cut at 5 lm, dewaxed and stained with haematoxylin and eosin (HE) for LM. Electron Microscopy Samples were carefully cut into blocks (1×1×1 mm for transmission electron microscopy [TEM] and 1×1×5 mm for scanning electron microscopy [SEM]). Each sample was immersed in glutaraldehyde 3% in 0·1  phosphate buffer at pH 7·4 for 5 min at 4°C; it was then subjected to continuous fixation in the glutaraldehyde solution for 3 h at room temperature and before preparation of samples for TEM and SEM. For TEM, glutaraldehyde-fixed samples were further treated with 1% OsO4 for 2 h at room temperature. They were then dehydrated in a graded ethanol series (70%, 80%, 90%, 95% and 100%; three times successively) and embedded in Quetol 812 resin (Nissin EM, Tokyo, Japan). Samples were sectioned (60 nm) with a Reichert Supernova system (Leica, Vienna, Austria) with diamond knives. The sections were stained with uranyl acetate 1% in distilled water and then with a solution of lead citrate in distilled water. They were then examined by TEM ( JEM-1220; JEOL, Tokyo, Japan) at an accelerating voltage of 80 kV. For SEM, glutaraldehyde-treated samples were washed three times with 0·1  phosphate buffer (pH 7·4) and post-fixed with 1% OsO4 for 2 h at room temperature. Conductive staining was performed by the 1% thiocarbohydrazide (T) and 1% OsO4 (O) method (Familiari et al., 1992) at room temperature, with staining in T for 20 min,

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Fig. 2.

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Cross section of the control (a) and the affected SDFT (b). In the affected area, increased numbers of activated tenocytes and haemorrhage can be observed. HE. ×180.

in O for 60 min, in T for 20 min, and in O for 60 min. Samples were dehydrated through a graded ethanol series and processed for freeze-drying by t-butyl alcohol substitution (Inoue and Osatake, 1987). The specimens were examined by field emission SEM ( JSM-6000F; JEOL, Tokyo, Japan), without metal coating, at an accelerating voltage of 3 or 5 kV. GAG Analysis The sample (approx. 5 g) was homogenized for 1 min at 2000 rpm in a super-cooled metal homogenizer in a walk-in freezer. The sample powder (−80°C) was dissolved in 0·5  NaOH for removal of GAGs from core proteins of proteoglycans at 4°C for 15 h. The mixture was then neutralized with 1  HCl at 4°C. The protein in the mixture was denatured by heating at 100°C for 30 min. The mixture was brought to pH 8·0 with 1  Tris-HCl buffer, and digestion with 1% pronase (actinase E; Seikagakukougyo, Tokyo) was performed twice at 50°C for 24 h. Trichloroacetic acid (30%) was then added at 4°C and after 1 h the mixture was centrifuged at 15 000 rpm (4°C) to remove the precipitated proteins. The supernate was dialysed against distilled water at 4°C for 4 days. The dialysed sample was freeze-dried and subjected to twodimensional (2D) electrophoretic analysis of GAGs on a cellulose acetate membrane (Hata, 1973). GAGs were “visualized” with a solution containing alcian blue 8GX (Merck, Darmstadt, Germany) 0·1% and acetic acid 0·1%. Quantitation of GAG content by assay for hexosamine was carried out by the method of Hata (1973). Hyaluronic acid (HA), dermatan sulphate (DS), heparin (HP), and chondroitin 4sulphate (C4S) were used as standard GAGs (Nacalai Tesque, Kyoto, Japan, and Sigma, Tokyo, Japan).

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Fig. 3.

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Control (a) and affected tissue (b). In the affected area, the tenocytes had extensively developed, rough endoplasmic reticulum in their cytoplasm. Uranyl acetate and lead citrate. TEM. ×10 000.

Results The affected site of injury to the tendon was located 2 cm distal to the midmetacarpal bone. The dorsoventral thickness (about 15 mm) of the affected site was approximately 1·5 times that of the corresponding site of the control SDFT, and the lateral thickness (about 24 mm) of the affected site was approximately 1·3 times that of the control. The affected area was located centrally in a cross-section of the injured tendon, with oedematous swelling and dark red discoloration (Fig. 1). Light microscopy revealed that the control area was composed of tenocytes, with collagen fibres filling the gaps between them. In the affected area, however, exuberant granulation (with activated tenocytes, angiogenesis, haemorrhage, and infiltration of small numbers of leucocytes) was observed, and collagen fibres were loosely packed and irregularly organized (Fig. 2). The proportion of the affected area filled by the ground substance was larger than in the control. The respective relative amounts of collagen fibrils, matrix and

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Control (a) and affected tissue (b). In the control sample, note the high density of collagen fibrils. In the affected area, a low density of collagen fibrils and decreased number of fibrils with large diameters can be seen. Uranyl acetate and lead citrate. TEM. ×75 000.

cell components were 86:12:2 in the control tissue and 33:34:33 in the affected tissue. TEM revealed that many tenocytes in the affected area had extensively developed rough endoplasmic reticulum in their cytoplasm (Fig. 3). The average diameter of the fibrils in the affected sample (61·7±36·5 nm) was significantly different (P<0·002) from that in the control (69·0±54·4 nm) (Fig. 4). The diameters of the collagen fibrils ranged from 20–240 nm (n=182) in the affected area and from 20–360 nm (n=360) in the control (Fig. 5). SEM revealed that both affected and control collagen fibrils were composed of many subfibrils of 10–20 nm diameter, which were wound helically around the axes of collagen fibrils at a constant angle of about 17°, all forming righthanded helical structures. There were more particles on the surface of each collagen fibril in the affected tissue than in the control (Fig. 6). In the analysis of GAGs in the matrix, HA, DS and chondroitin sulphate

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Frequency (%)

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Fig. 5.

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140 200 260 Diameter of collagen fibrils (nm)

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Graph showing the frequency distribution of collagen fibril diameters in the affected and control areas. – –Α– –, Control tendon (n=360); —Φ— affected tendon (n=182).

(CS) were found to be major components in both areas (Fig. 7). Their respective relative concentrations were approximately 14:45:41 in the control area but 8:67:25 in the affected area, the increase of DS in the affected area being considerable (Fig. 8). Discussion Equine SDFT injury occurs as a result of ischaemic degeneration of tendon fibres due to small ruptures or to overfatigue caused by excessive tension, impacts or external injury (Shiraishi et al., 1985; Thomson, 1978). The horse in this study had no prior history of SDFT injury. Detection of clinical signs often lags behind development of tendon lesions, and subtle clinical signs may be the harbinger of a tendon injury severe enough to preclude further athletic performance (Stromberg, 1973). It is unlikely that we witnessed the initial phase of SDFT injury. However, the injury was probably recent because exuberant granulation was observed. Haemorrhage, in particular, is typical of an inflammatory reaction in its infancy. Most SDFT lesions develop in the central region (Pool and Meagher, 1990; Haut et al., 1992; Marr et al., 1993) as in the present case. Light microscopy revealed that, in the affected area, collagen fibres were loosely packed and irregularly organized. Williams et al. (1984) reported that, one week after experimental injury, new small blood vessels colonized the areas of haemorrhage in which fibrin and damaged collagen fibres were still present, while invading connective-tissue cells were producing a new matrix in adjacent areas. These results support our belief that the injury occurred 1–7 days before diagnosis. In the affected SDFT, the mean diameter of collagen fibrils was significantly decreased, probably as a reflection of the synthesis of new collagen fibrils and of enzymatic effects. Thus, the affected tendon probably lacked high tensile strength.

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Fig. 6.

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Control (a) and affected tissue (b). Collagen fibrils are composed of many subfibrils. The subfibrils are helical in relation to the collagen fibril main axis (see superimposed lines). Many particles can be seen on the surface of each collagen fibril in the affected tissue. SEM. ×75 000.

Subfibrillar architecture is consistently related to the functional properties of collagen (Raspanti et al., 1990). In the present study, SEM revealed that subfibrils of collagen fibrils did not differ between affected and control tissue; consequently, they may not have played an important role in regulation of collagen synthesis in the injured SDFT. Particles on the surface of collagen fibrils in the affected area may have been related to increased matrix. There was an increase in the relative amounts of matrix and cellular components in the affected area. This may have been related to increases in numbers of tenocytes and in components of the matrix as a result of inflammation and haemorrhage. The many activated tenocytes may have produced matrix and collagen molecules. In typical acute inflammation, the rates of production of collagenase and hyaluronic acid are increased (Mori, 1991). The increase in matrix (possibly containing collagenase and other enzymes)

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Fig. 7.

Analysis of GAGs in the matrix by two-dimensional electrophoresis on cellulose acetate membrane. Hyaluronic acid (HA), dermatan sulphate (DS) and chondroitin sulphate (CS) were found in both the control (a) and the affected area (b).

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Fig. 8.

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DS Components of GAG

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Histogram showing relative concentrations of hyaluronic acid (HA), dermatan sulphate (DS) and chondroitin sulphate (CS). The increase in concentration of DS in the affected area was considerable. Ε, Control; Φ, affected.

may have been associated with the increase in the number of collagen fibrils of small diameter. Gaughan et al. (1991) found that instillation of HA into the sheath surrounding a collagenase-injured tendon improved the histological appearance and maturation of the repair tissue at 8 weeks after injury. It has been suggested that HA decreases the formation of adhesions and improves gliding function in human flexor tendon injuries (Gaughan et al., 1991). However, the importance of gliding function in the unsheathed portion of the SDFT in horses is unknown, and adhesions can serve as a potential source of additional

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blood supply to the relatively avascular central core of the tendon. It is difficult to explain how the peritendinous injection of HA can affect the intratendinous repair process. Commercially available GAGs have been used in the treatment of tendon injury. The composition of GAG in the extracellular matrix changes during tendon repair and may be important in the regulation of collagen synthesis (Dorner, 1968; Toole and Gross, 1971; Alexander and Donoff, 1980; Garg et. al., 1989; Mast et al., 1991). The presence of large tenocytes with well developed rough endoplasmic reticulum, possibly related to recovery, and the increases in volume of the matrix and in the relative concentration of DS, are characteristics of tendon injuries (Scott and Orford, 1981). In general, increases in concentrations of collagen molecules and DS are linked during the wound-healing process (Scott and Orford, 1981). In typical acute inflammation, production of collagenase and HA are increased; and increased matrix formation at the start of granulation, in particular the synthesis of HA and CS, restrains fibrosis. In chronic inflammation, increasing concentrations of DS and decreasing concentrations of heparan sulphate (HS) play a role in fibrosis (Mori, 1991). In this study, biochemical analysis indicated chronic inflammation, characterized by increased concentrations of DS in the affected area. However, morphological study indicated acute inflammation, characterized by haemorrhage. DS exists as DS proteoglycan (decorin) in vivo, and decorin functions to regulate the organization of collagen fibres (Shinkai, 1991). It seems possible that tendon repair is characterized by morphological signs of acute inflammation and biochemical signs of chronic inflammation. Our observations may indicate that fibrillogenesis was activated by increasing concentrations of DS and decreasing concentrations of HA and CS. Since no collagen fibrils of normal thickness and with parallel arrangement were seen in the injured tendon, the morphological and biochemical characteristics of the injured tendon may have reflected fibrillogenesis. We found no evidence to indicate that the injured tendon would have healed and returned to normal. However, if the inflammatory characteristics of such injured tendons could be changed, complete recovery might occur. This study, although of only a single case, may prove useful in relation to future work on treatment and prognosis. Acknowledgments The authors thank all the staff of the Department of Veterinary Pathology, Rakuno Gakuen University, and of Shadai Farm, for the collection of specimens. We are also grateful to Dr Ryu-ichiro Hata for instruction on microanalysis of glycosaminoglycans and Dr Masa-aki Oikawa for advice. This work was partly supported by Gakujutsufrontier Cooperative Research in Rakuno Gakuen University. References Alexander, S. A. and Donoff, R. B. (1980). The glycosaminoglycans of open wounds. Journal of Surgical Research, 29, 422–429. Davidson, P. F. (1982). Tendon. In: Collagen in Health and Disease, 1st Edit, M. I. V. Jayson and J. B. Weiss, Eds, Churchill Livingstone, Edinburgh, pp. 498–505.

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Received, June 18th, 1998 Accepted, December 7th, 1998