Abstract
The digital cushion (DC) plays a role in absorbing and dampening forces applied to the foot and therefore supports internal structures such as navicular bone; yet, its architecture is not well-known. The goal of this study was to characterize the microanatomical structure of the DC in horses with clinically sound hooves. Both forefeet from the cadavers of 12 adult Quarter horses were cut and sectioned and samples of the following 4 regions of the DC were obtained: axial proximal (AxProx), axial distal (AxDis), abaxial lateral (AbxLat), and abaxial medial (AbxMed). The samples were processed and stained with hematoxylin and eosin, Masson’s trichrome, and Weigert’s elastic stains. On each slide, 2 central 3- × 3-mm areas were microscopically assessed and all measurements were done within the 9-mm2 area. The number of detected collagen bundles, nerve fascicles, vessels, and the diameter of wall thickness and lumen of blood vessels were measured. Elastic fiber profiles were categorized based on relative density of elastic fibers detected in the field. The percentage of samples in which chondrocytes and adipose tissues were either present or absent was calculated. Significant structural differences were identified among the 4 regions of the DC. The AxDis region contained more collagen bundles (P < 0.0001) and less elastic fiber profiles than the AxProx region (P < 0.0001). The AxDis also contained more collagen bundles than the AbxMed and AbxLat (P < 0.0001) regions. Our findings provide insight into the structure of the DC of mature Quarter horses. The structural differences in the various regions of the DC are presumably related to the different functional properties of those regions; yet more research is warranted.
Résumé
Le coussinet plantaire (CP) joue un rôle en absorbant et diminuant les forces appliquées au pied et par conséquent supporte les structures internes telles que l’os naviculaire; pourtant son architecture n’est pas très bien connue. Le but de la présente étude était de caractériser la structure micro-anatomique du CP chez des chevaux avec des sabots cliniquement sains. Les deux pattes avants provenant de 12 chevaux Quarter Horse furent coupées et sectionnées et des échantillons des quatre régions suivantes du CP obtenus : axial proximal (AxProx), axial distal (AxDis), abaxial latéral (AbxLat), et abaxial médial (AbxMed). Les échantillons ont été traités et colorés avec hématoxyline et éosine, trichrome de Masson, et coloration de Weigert pour les fibres élastiques. Sur chaque lame, deux zones centrales de 3 × 3 mm ont été évaluées en microscopie et toutes les mesures effectuées dans cette zone de 9 mm2. Le nombre de paquets de collagène, de faisceaux nerveux, et de vaisseaux sanguins a été déterminé ainsi que les diamètres de l’épaisseur de la paroi et de la lumière des vaisseaux sanguins mesurés. Les profils des fibres élastiques ont été catégorisés sur la base de la densité relative des fibres élastiques détectées dans le champ. Le pourcentage d’échantillons dans lesquels des chondrocytes et du tissu adipeux étaient présents ou absents a été calculé. Des différences structurelles ont été identifiées parmi les quatre régions du CP. La région AxDis contenait plus de paquets de collagène (P < 0,0001) et moins de profils de fibres élastiques que la région AxProx (P < 0,0001). La région AxDis contenait également plus de paquets de collagène que les régions AbxMed et AbxLat (P < 0,0001). Nos résultats donnent un aperçu de la structure du CP de chevaux Quarter Horse matures. Les différences structurales parmi les différentes régions du CP sont probablement liées aux différentes propriétés fonctionnelles de ces régions; mais plus de recherche sont requises.
(Traduit par Docteur Serge Messier)
Introduction
The hoof is subject to 2 major forces with each footstep: loading from the weight of the animal and ground reaction forces. Such forces generate extensive shock waves and stress within the tissues of the hoof as well as within proximal structures. The complicated anatomy of the hoof plays a crucial role in its ability to absorb and dissipate applied forces (1). Integrity and proper function of each of the hoof’s anatomical components and associated structures are vital for the hoof to withstand applied forces and maintain its proper function. Any dysfunction in the components of the hoof may lead to force being transferred to more sensitive structures, either within the hoof itself or to more proximal structures, causing subsequent injury. The different components of the hoof, including the laminar junction, hoof wall, bars, ungual cartilages, frog, and digital cushion (DC), each play a significant role in dissipation and distribution of force.
The exact mechanism of force distribution and anti-concussive properties of the equine foot is not fully understood (2). Clinicians previously assumed that the DC merely acts as a fat pad to dampen concussive forces. More recent studies, however, have shown that the DC plays a more complicated role, also contributing to the hemodynamic pump mechanism within the hoof (3,4). Bowker et al (2) reported differences in the composition of the DC in horses from different geographical areas. The authors speculated that adaptation to the applied forces, as well as external factors, such as substrate, trimming, breed, and age, contribute to observed differences. Another study noted a greater amount of adipose tissue in foals than in adult horses and concluded that mechanical loading affects the composition of the DC (5). It is likely that functional properties of the DC are associated with its composition (5). However, the histological properties and structural variation in different regions of the DC have not been thoroughly characterized (2).
The goal of this study was to examine the regional distribution of connective tissue, nervous tissues, and vascular structures of the DC in Quarter horses with sound hooves. We hypothesized that the composition and arrangement of different fibers and tissues of the axial and abaxial regions of the DC vary.
Materials and methods
Samples
Both forehooves from 12 Quarter horses with a mean age of 23.5 y (± 7.3 y) were collected. All horses were humanely euthanized for reasons unrelated to this study. The Institutional Animal Care and Use Committee (IACUC) of the Western University of Health Sciences approved the study protocol.
Study design
Each forelimb was cut at the level of the metacarpophalangeal joint (fetlock) and sectioned with a band saw by making 1 midsagittal cuts at the midline and 2 parasagittal cuts, each 2 cm lateral and medial to the original sagittal cut. Using a scalpel blade, tissue samples approximately 1 cm3 in size were collected from the following 4 distinct regions of the DC: axial proximal (AxProx); axial distal (AxDis); abaxial lateral (AbxLat); and abaxial medial (AbxMed) (Figure 1, A to C). Tissue samples were immediately placed in 10% neutral buffered formalin for further processing. Tissue samples were processed, sectioned at a thickness of 5 to 7 μm, and mounted on glass slides by a reference laboratory (AML Laboratories, Saint Augustine, Florida, USA). Sections were stained with hematoxylin and eosin (H&E), Masson’s trichrome, and Weigert’s elastic stains. Slides were examined using light microscopy (Nikon Model Eclipse E200; Nikon Instruments, Melville, New York, USA).
Figure 1 (A–C).
Representation of hoof sectioning (dotted lines) and sampled regions: axial proximal (AxProx) (a); axial distal (AxDis) (b); abaxial medial (AbxMed) (c); and abaxial lateral (AbxLat) (d) are shown with black squares (A). Axial samples were obtained from sagittal sections (B); abaxial samples were obtained from each parasagittal section (C); each blue box designates the site where samples of 1 cm3 were collected.
Based on the size of tissue samples on slides and for consistency, 2 random 3- × 3-mm areas were identified on each slide. Both squares were placed approximately at the center of the slide, while avoiding a cut, irregular edge or any other artifacts due to the nature of the tissue under study. For all measurements, the mathematical mean of the 2 squares was calculated and analyzed. Collagen bundles, elastic fiber profile, number of blood vessels detected, average diameter of the wall (tunica media) and lumen of arteries/arterioles/veins, nerve fascicles, and the presence or absence of adipocytes and chondrocytes were evaluated per each 3- × 3-mm area on each slide. Collagen bundles were evaluated by counting isolated collagen bundles oriented as a group. Elastic fiber profiles were rated for relative density using a scale of 0 when no elastic fibers were detected to 3 when elastic fibers were densely arranged as stained black/dark purple patches occupying most of the measured field (Figure 2, A to D). Wall thickness (WT) and lumen diameter (LD) of the blood vessels were directly measured and their ratio (WT/LD) was calculated. The diameter of the vessel (tunica media and lumen) was measured using 2 perpendicular axes (shortest and longest diameters); the averages of long and short axes were calculated to measure the diameter. The diameter of the tunica media and lumen were measured in a similar fashion. The numbers of detected nerve fascicles were also counted. Adipocytes and chondrocytes were evaluated based on their absence or presence, where 0 or 1 were assigned respectively, to calculate the percentage of the samples containing adipose and chondrocytes.
Figure 2 (A–D).
Black/dark patches represent elastic fiber profiles, which were rated for relative density using a scale of 0 to 3: grade 0 — almost no elastic fibers detected (A); grade 1 — only a few elastic fibers detected (B); grade 2 — moderate amount of elastic fibers detected (C); and grade 3 — elastic fibers densely arranged as stained black patches occupying most of the measured field (D). Line scale = 200 μm.
Statistical analysis
Kolmogorov-Smirnov and Shapiro-Wilk tests were used to determine the normal distribution of the data. As none of the variables were normally distributed, Kruskal-Wallis analysis was used to compare the 4 regions. When significant, a Mann-Whitney test followed by Bonferroni correction were conducted to determine statistical differences between each of the measured values at the axial and abaxial regions of the DC. After required adjustments, values of P < 0.05 and P < 0.008 were considered significant for Kruskal-Wallis and Mann-Whitney tests, respectively. For each measured variable and for each region, the statistical difference between the left and right hooves was investigated using a Mann-Whitney test and P < 0.05 was considered significant. All analyses were done using a commercial statistical software (SPSS Statistics for Windows, Version 18.0; IBM, Armonk, New York, USA).
Results
None of the measured variables were statistically different between the left and right hooves. All variables were evaluated within 2 central 3- × 3-mm areas on each slide and an average was calculated. Significant differences in tissue composition were revealed between the axial and abaxial regions of the DC, particularly in the distribution of the collagen and elastic fibers. The AxDis region contained more collagen bundles (P < 0.0001) and less elastic fiber profiles than the AxProx region (P < 0.0001). The AxDis contained significantly more collagen bundles (P < 0.0001) than the AxProx (P < 0.0001) and AbxMed and AbxLat (P < 0.0001) regions. Also, significantly lower elastic fiber profiles were found at the AxDis region than at the AxProx (P < 0.0001), AbxMed, and AbxLat (P < 0.0001) regions (Table IA and IB and Figure 3). More nerve fascicle/bundles were detected in the AxProx and abaxial regions than in the AxDis region. However, the differences in bundle distribution of the nerve fascicles among the regions were not statistically significant (P = 0.092 to 0.128). On average, adipose tissue was detected more frequently in the abaxial regions (AbxMed and AbxLat) than in the axial (AxProx and AxDis) regions. While those differences were not statistically significant (P = 0.013 to 0.074), they clearly confirmed the trend. In general, the tissue composition of the AbxMed and AbxLat regions was similar, except for the cartilaginous tissues where larger numbers of slides containing chondrocytes were detected at the AbxLat than at the AbxMed region (Table II). While more arteriole and venule profiles were found at the AxProx than at the other 3 regions, these values were not statistically significant.
Table I.
Mean and standard deviation of structures quantified per 2 adjacent 3-× 3-mm samples in the axial distal (AxDis), axial proximal (AxProx), abaxial medial (AbxMed), and abaxial lateral (AbxLat) regions of the equine digital cushion. The number of observed collagen bundles, nerve fascicles, arteries, arterioles, veins, and venules on each slide (per 9 mm2) were counted; mathematical mean and standard deviation for each region are shown in A. Elastic fibers were rated for relative density, which was categorized using a scale of 0 to 3 (ranging from no elastic fibers to densely arranged elastic fibers). Mathematical mean (of grading) and standard deviation for each region were calculated (B).
| A | ||||||
|---|---|---|---|---|---|---|
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| ||||||
| Region | Collagen bundles | Nerve fascicles | Arteries | Arterioles | Veins | Venules |
| AxDis | 4.2 ± 3.3 | 0.3 ± 0.6 | 0.6 ± 1.1 | 5.9 ± 8.1 | 0.6 ± 1.1 | 7.9 ± 10.5 |
| AxProx | 1.2 ± 0.7 | 1.1 ± 1.5 | 0.7 ± 0.8 | 8.7 ± 11.1 | 0.9 ± 1.2 | 11.3 ± 12.1 |
| AbxMed | 1.5 ± 0.9 | 0.9 ± 1.5 | 0.8 ± 0.9 | 5.7 ± 6.7 | 0.7 ± 0.8 | 7.1 ± 7.1 |
| AbxLat | 1.6 ± 1.1 | 0.8 ± 1.4 | 0.7 ± 1.1 | 3.3 ± 2.2 | 0.5 ± 1.2 | 4.9 ± 3.8 |
|
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| B | ||||||
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| Region | Elastic fibers | |||||
|
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| AxDis | 1.1 ± 0.7 | |||||
| AxProx | 2.3 ± 0.7 | |||||
| AbxMed | 2.3 ± 0.9 | |||||
| AbxLat | 2.5 ± 0.8 | |||||
Figure 3.
Comparison of the elastic fiber profiles (based on average density rating using a 0–3 scale) and the number of observed collagen bundles (per 3-× 3-mm block) among the regions of the equine digital cushion. X-axis shows different regions of the DC and Y-axis designates the density of elastic fibers (left) and number of detected collagen bundles (right). Axial distal (AxDis) region compared with axial proximal (AxProx) (P < 0.0001), abaxial medial (AbxMed) (P < 0.0001), and abaxial lateral (AbxLat) (P < 0.0001) regions (as shown with a, b, and c on the graph). A higher number of collagen bundles was detected at the AxDist region than at AxProx (P < 0.0001), AbxMed (P < 0.0001), and AbxLat (P < 0.0001) regions (as shown with d, e, and f on the graph).
Table II.
Percentage of image fields (within two 3- × 3-mm samples) containing cartilage and/or adipose tissues in the axial distal (AxDis), axial proximal (AxProx), abaxial medial (AbxMed), and abaxial lateral (AbxLat) regions of the equine digital cushion. Adipocytes and chondrocytes, rated according to their presence or absence per 9-mm2 area on each slide (where 0 and 1 numbers/codes were assigned). A mathematical mean for each region was calculated and is shown here.
| Region | Adipose | Cartilage |
|---|---|---|
| AxDis | 47 | 4 |
| AxProx | 41 | 2 |
| AbxMed | 72 | 8 |
| AbxLat | 79 | 21 |
Measurements of the blood vessel profiles including WT and LD were obtained and WT/LD ratio was calculated (Table III). No statistically significant differences were detected in profile measurements of blood vessels among the 4 different regions. Each of the 4 regions stained with either H&E, Masson’s trichrome, or Weigert’s elastic stains is shown in Figures 4 and 5.
Table III.
Mean and standard deviation of wall thickness (WT), lumen diameter (LD), and their ratio (WT/LD), of the blood vessels within 9-mm2 area on each slide of the equine digital cushion. The calculated WT/LD shows the actual ratio and is independent of the vessel diameter.
| Vessel type | Wall thickness (μm) | Lumen diameter (μm) | WT/LD |
|---|---|---|---|
| Arteries | 47 ± 41 | 74 ± 76 | 0.6 ± 0.5 |
| Arterioles | 18 ± 8 | 14 ± 6 | 1.3 ± 1.3 |
| Veins | 29 ± 20 | 225 ± 285 | 0.1 ± 0.1 |
Figure 4 (A,B).
A microscopic image of the equine digital cushion at the axial proximal (AxProx) (A) and axial distal (AxDis) (B) regions stained with Weigert’s elastic. Notice the differences in the amount of elastic fibers present in the proximal compared with the distal region. Artery (a); vein (v); nerve fascicles (n); collagen fibers/bundles (cf); and elastic fibers in black/dark purple. Line scale = 100 μm.
Figure 5 (A,B).
A microscopic image of the equine digital cushion at the abaxial lateral (AbxLat) region stained with Masson’s trichrome (A) and abaxial medial (AbxMed) region stained with hematoxylin and eosin (B). Notice the presence of arteries (a); arterioles (aa); nerve fascicles (n); veins (v); collagen fibers/bundles (cf); and adipocytes (ad). Line scale = 100 μm.
Discussion
The digital cushion (Pulvinus digitalis) fills the space between the frog, coffin bone, and deep digital flexor tendon/navicular bone as it extends between the paired ungual cartilages (2). Previous reports have divided the equine DC into pars torica (between the heels) and pars cunealis (overlying the frog) based on its anatomical location (5,6). Pars torica comprises most of the DC, while the pars cunealis is a much smaller section. We examined the axial and abaxial regions of the DC that are closest to the pars torica. This study examined the structural differences between these regions and showed microanatomical variations, which are presumably related to their respective functional properties. To the best of our knowledge, the histological and functional properties of different regions of the equine DC, particularly the abaxial regions, have not been fully studied.
There are significant inconsistencies in previous reports describing the composition of the DC. One study stated that the DC is composed of only elastic fibers and adipose tissue (7). Another study examined the histology of the DC and reported that the equine DC consists primarily of tightly packed collagen fibers with only a few interspersed elastic fibers and very little adipose tissue (8). A more recent study indicated that the DC contains connective and adipose tissues, elastic fibers, and tissues with matrix rich in hyaluronic acid (5). Collectively, these studies disagree about the architecture of the DC. Such differences might be due to variability in sectioning techniques and can partially be explained by examining the distinct regions of the DC.
Our results clearly showed variable presence of collagen bundles, adipose tissue, elastic fiber profiles, blood vessels, as well as nerve fascicles throughout the DC, although only the distribution of the collagen bundles and elastic fiber profiles were statistically significant. We observed a larger number of slides containing chondrocytes/chondroblasts and adipose tissue (per 3- × 3-mm area of interest) at both abaxial regions compared with the axial regions of the DC, although the differences were not statistically significant. A previous study showed differences in DC composition between foals and adult horses (5). Further research is warranted to determine whether differences in the DC observed in this study are related to the breed, age, or exercise regimen of the horses. Räber et al (9) examined the DC in cows and concluded that the presence of a variable amount of adipose tissue reflects the fact that such tissue acts as a mechanical insulator, rather than a thermoregulatory pad, and efficiently absorbs forces by simultaneously providing soft and resilient consistency.
Significant differences were noted in connective tissue as the wedge-shaped DC tapered in width distally and abaxially. The AxDist region was composed of tightly interwoven collagen bundles with very little to no elastic fibers. Elastic fibers deform under physiological forces and subsequently release stored energy to drive passive recoil; such capability is vital for normal function of many dynamic tissues (10). The presence of a moderate to large amount of elastic fiber profiles in the DC, with its high resilience may allow the elastic fibers to support the tensile strength of collagen bundles (11). Elastic fiber-rich dynamic tissues are therefore able to deform and store energy under normal physiological loads and use this energy to drive recoil back to a resting state (12). Collagen is characterized by high tensile strength and poor shear strength, whereas its stretch capability is limited to approximately 5% of the initial length. However, elastic fibers can stretch by as much as 2 1/2 times their original length (13).
It is likely that the distribution of the connective tissue of the more proximal region may permit expansion, i.e., more elastic fiber profiles, while the densely interwoven collagen of the distal region may limit movement and possibly provides more strength. The distal region of the DC fixes the axial projections of the ungual cartilages into place (2,5). The proximal region stretches and expands with abaxial rotation of the ungual cartilages, which causes the observed drop in pressure within the cushion during loading (2,14). Additionally, lower vascular density and fewer elastic fiber profiles and nerve fascicles were observed in the distal region than in the proximal region. Lower vascular density in the distal regions can be associated with branching of vessels as they descend.
While different components of the equine hoof, including the laminar junction, digital cushion, and related connective tissues, contribute to absorption and dissipation of applied forces, the functional characteristics of the DC are not well-known (2). It has been suggested that the DC absorbs the energy generated during the impact/stance phase, although the exact mechanism has been subject to speculation (2). Two theories have traditionally been suggested, namely, the pressure theory and the depression theory. The pressure theory states that pressure against the sole and frog during the stance phase causes compression of the DC and lateral expansion of the ungual cartilages and hoof wall (15). According to the depression theory, applied forces are transmitted to the hoof wall by the laminar junction and the lowering of the second phalanx during stance is associated with the compression of the DC, which expands the hoof wall (16). Both theories rely on the DC absorbing the force of the impact as it is compressed.
A more recent study measured the in-vivo internal pressure of the DC during locomotion and reported a negative pressure in the DC during the stance phase, rather than a positive pressure, which would be expected if the DC was being compressed. As this indicates expansion of the volume inside the hoof wall, the authors questioned the accuracy of the pressure theory (14). Another study examined the in-vitro displacement of the DC using cadaver feet and stated that DC displacement is not dependent on the degree of solar support and questioned the accuracy of both pressure and depression theories (17). The distribution of elastic and fibrocartilaginous tissues and extensive vasculature reported in this study provide further evidence of the role of the DC in regulating blood flow and its contribution to the absorption and dissipation of applied forces. Our results showed a large number of vessels within the DC, particularly at the proximal parts, which in addition to providing nutrients/oxygen to the hoof, could contribute to the hemodynamic mechanism of the hoof.
It has been stated that age and the intensity of exercise affect the composition of the DC in cows (18). Wilhelm et al (5) reported the presence of more adipose tissue, particularly in pars torica, in foals than in adult horses. As the equine DC probably responds and adapts to its environment, it is likely that certain factors, such as exercise regimen, trimming protocols, ground reaction forces, and substrate, stimulate changes in the DC, such as the development of fibroelastic tissues, although further investigation is warranted (18,19). One study detected more consistent fibrocartilaginous and elastic tissues in the DC of Arabian, Morgan, and Tennessee walking horses than in Standardbred, Thoroughbred, and Quarter horses (2). The authors concluded that, in addition to the potential genetic predisposition, various external factors, such as weight of the horse, concussive forces, and age, may affect the composition of the DC (2). A recent study showed that daily exercise caused a 37% increase in mean volume and an 18% increase in the surface area of the DC in cows and concluded that those changes have a positive effect on the hoof health and welfare of cows (18). Another study investigated the effect of diet on the composition of the digital cushion of the hind claw of 46 bulls and reported that the total amount of polyunsaturated fatty acid in the DC varied significantly (20). To our knowledge, the effect of diet on the composition of the digital cushion in horses has not been reported.
Although this study provided valuable data on the histologic composition of different regions of the DC in mature Quarter horses, it is not without its limitations. The horses used in this study were relatively old, retired pasture horses from the same breed. Further study is therefore warranted to determine whether the composition of the DC varies in young or athletic horses. More investigation is needed regarding adaptive responses in the DC, particularly the role of age and exercise regimen. Furthermore, comparison of compositional differences between breeds may provide insight into foot health among breeds.
Our results showed significant differences among the composition of the digital regions of the DC. Such differences are presumably associated with the biomechanical function of different regions of the DC; yet, elucidation of the functional properties of different regions of the DC warrants further research.
Acknowledgments
The authors thank Morgan Nowlin, Lena Chitgar, Naomi Chow, Mailie Fanning, and Danielle Demel for assistance with data collection.
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