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Keywords:

  • horse;
  • hoof;
  • anatomy;
  • Thoroughbred;
  • laminar junction;
  • morphology;
  • epidermal laminae

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Authors' declaration of interests
  8. Sources of funding
  9. Acknowledgements
  10. Manufacturers' addresses
  11. References

Reasons for performing the study: Hoof health is a major concern of horse owners as well as the equine industry. However, many questions remain concerning regional variations of laminar junction and its potential to remodel.

Hypothesis: To examine regional variations in the morphology of the laminar junction and thickness of the hoof wall in Thoroughbred horses.

Methods: The forefeet of 25 Thoroughbred cadavers were examined. Each hoof was divided into 20 blocks through 4 proximodistal slices (below the coronary band, each 1 cm apart) and 5 circumferential positions (toe, medial and lateral quarters and heels). In each block, 25 central primary epidermal laminae (PEL) were considered. Orientation of each lamina in relation to the hoof wall (LO), degree of bending (IA) and the spaces between the adjacent laminae (LS) were measured. Thickness of the hoof wall and number of branched PEL were also measured. Data were analysed using a split-block design in ANOVA.

Results: There were significant differences between the 2 proximal and 2 distal slices in LO and IA data, but not in LS data. Circumferentially, toe blocks were different from heel and quarters blocks. Lateral and medial heels as well as the quarters were mostly different. The hoof wall was slightly thicker laterally than medially. There were more branched PEL on the lateral side of the left hooves and on the medial side of the right hooves.

Conclusions: These data add to the circumstantial evidence supporting the hypothesis of adaptive remodelling in the laminar junction. Results of this study signify the capability of PEL to remodel in response to applied stress to the regions of the hoof.

Potential relevance: A deeper understanding of the gross and cellular processes of laminar remodelling may well prove to be complementary to an understanding of their failure in laminitis.


Abbreviations:
PEL

Primary epidermal laminae

SEL

Secondary epidermal laminae

SM

Stratum medium

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Authors' declaration of interests
  8. Sources of funding
  9. Acknowledgements
  10. Manufacturers' addresses
  11. References

One of the many adaptations of the equine hoof to an unguligrade posture is seen in the convolution of the dermoepidermal junction deep to the hoof wall into 2 generations of laminae or lamellae. Approximately 600 primary epidermal laminae (PEL), each bearing 100–150 nonkeratinised secondary epidermal laminae (SEL), with their dermal counterparts, magnify the area of the epidermal basement membrane to more than 2 m2 (Pollitt 1998). The logical interpretation is that this modification reduces magnitudes of physical stress and strain in the living cells to either side of the membrane. Cellular remodelling allows the PEL to slide distally past the SEL and dermal laminae during normal hoof growth (Pollitt 1992). Remodelling also changes the number of PEL and SEL from neonate to adult, by an active process of bifurcation of existing laminae (Bowker 2003a; Sarratt and Hood 2005). Presumably, the lamina splits longitudinally, bifurcates and eventually divides into 2 laminae. A third effect of remodelling is to produce differences in the density, orientation and curvature of PEL. It has been shown that the density of PEL is uniform in the neonate, but in the adult it is highest at the toe and reduces towards the quarters and heels (Douglas and Thomason 2000; Bowker 2003a). Orientation of the PEL, seen on horizontal sections, is radial at the toe (i.e. close to perpendicular to the wall); however, it diverges from this radial orientation at the quarters and heels. Such differences in laminar morphology have been interpreted as regional responses to variation in loading (Faramarzi 2003). At the toe the distal phalanx pulls caudally and distally, whereas it also tends to shear the junction at the quarters and heels (Thomason et al. 2001). In asymmetrical hooves, particularly in those with a lateral flare, there is an abrupt change in the number and density of the laminae in the lateral quarter (Bowker 2003b; Lancaster et al. 2007). This accumulated circumstantial evidence supports the intuitive suggestion that observed remodelling of epidermal laminae is in part due to variation in stress levels.

We have previously shown correlations between the shape of the hoof capsule and morphology of the laminar junction in Standardbred and Thoroughbred horses (Thomason et al. 2008). In this study, we examined regional variations (both proximodistally and circumferentially) in the number and density of PEL, aspects of their shape and size, as well as their bifurcation in matureThoroughbred forefeet. The hypothesis tested was that the laminar junction is capable of change and remodelling. Our purpose was to add another piece to the jigsaw puzzle of understanding the structure-function relationship of the laminar junction in a healthy hoof. Completing the puzzle will achieve 2 goals: 1) to determine whether the remodelling response is truly adaptive, i.e. appropriate for the stimulus of changes in stress level and 2) aid in understanding the role of physical stress in the breakdown of the laminar junction or in ameliorating such stress during treatment (and recovery) of laminitis. The contribution of the present work is to quantify morphological pattern variations in PEL and in healthy Thoroughbred feet in order to set a baseline for comparison of similar data from other breeds and identify anomalous patterns in Thoroughbreds.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Authors' declaration of interests
  8. Sources of funding
  9. Acknowledgements
  10. Manufacturers' addresses
  11. References

Forty-seven forefeet (24 left and 23 right) were obtained on the day of the death from 25 mature Thoroughbred horses of mixed gender and age (2–17 years). The animals were destroyed for reasons other than musculoskeletal pathology. The hooves were frozen immediately after euthanasia.

Identifying sample blocks of PEL

Each hoof was sliced on a band saw parallel to the coronary band to yield 4 slices, approximately 1 cm thick beginning one third of the toe length distal to coronary band; selecting 5 circumferential positions provided 20 sampling blocks on each hoof (Fig 1). At each sampling site 25 central PEL were examined.

image

Figure 1. Using 4 proximodistal slices and 5 circumferential positions, 20 blocks were allocated on each hoof. Medial heel (MH), medial quarter (MQ), toe, lateral quarter (LQ) and lateral heel (LH).

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Measurements on the PEL

High resolution, low magnification digital photographs (Olympus Camedin E-10)1 were taken of all blocks of PEL and the adjacent wall. The XY coordinates of 3 points were digitised for all PEL in each block (Optimas 6.5 image analysis software)2. One point was at the inner end of each lamina, one at the midpoint of straight laminae or at the centre of the curvature if it was bent and the third at the outer end of the lamina (Fig 2). Coordinates of the grooves were taken and used to construct a cubic spline curve to represent the outer boundary of the wall. From the laminar coordinates, 3 descriptors of PEL shape were calculated. 1) Laminar spacing (LS): the spacing between pairs of laminae, 2) laminar orientation (LO): the angle between the axis of the laminae projected to the hoof wall and a tangent to the cubic spline representing the wall at the point of intersection and 3) internal angle (IA): the angle, subtended between inner and outer segments of each lamina.

image

Figure 2. Measurements on primary epidermal laminae. Laminar spacing (LS) is the distance between 2 points at the outer end of adjacent laminae. Laminar orientation (LO) is the angle between the line connecting first and third points on one lamina and the tangent line to the hoof wall at this point. Internal angle (IA) is the angle subtended by the 2 segments of each lamina. SM: stratum medium.

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Thickness of the hoof wall and number of branched PEL

The thickness of the stratum medium (SM), which is equivalent to the hoof wall and the thickness of the stratum lamellatum (SL), which is equivalent to the depth (radial length) of the primary laminae, were measured on slice 3 (approximately midway down the length of the hoof wall). The number of branched (bifurcated) PEL at the central 25 laminae at each of 20 blocks was also counted.

Statistical analysis

A randomised complete block, split plot design of ANOVA was used to examine the statistical effects of individual, bilateral and positional variation on PEL morphology (using Proc MIXED in SAS)3. The same statistical procedure was used to analyse the thickness of the SM and SL; data were normally distributed and no transformation was used. A logistic regression test was used to analyse the number of branched PEL by position. For all statistical analysis significance was set at a value of P<0.05.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Authors' declaration of interests
  8. Sources of funding
  9. Acknowledgements
  10. Manufacturers' addresses
  11. References

Laminar spacing (LS)

There was no significant difference between left and right feet (P = 0.819). All the circumferential blocks were significantly different from each other (P<0.0001). Laminar spacing ranged from 0.36–0.53 mm; it was smaller (laminae were closely spaced) at the toe and increased towards the quarters and heels. In all slices the adjacent heel and quarter blocks were significantly different. The medial and lateral quarters were significantly different. Likewise, medial and lateral heels significantly differed from each other. The laminae were slightly wider spaced (greater LS) at the lateral heel (0.53 mm) compared to the medial heel (0.50 mm). Proximodistally, there were no significant differences between any of the slices.

Laminar orientation (LO)

Laminar orientation was significantly different between left and right hooves (P = 0.003). Laminar orientation ranged from 86.47–104.85°; the angles greater than 90° indicated the orientation to the opposite side, i.e. outer part of the laminae were oriented toward the toe (cranially). Mean LO was slightly greater on right hooves (100.1°) compared to the left ones (95.1°).

All the toe blocks were significantly different (P<0.0001) from the adjacent quarter blocks. The adjacent heel and quarter blocks were not significantly different at the 2 proximal slices but they were different at the 2 distal slices; there were 2 exceptions with this pattern on slice 1 (P = 0.09). The outer parts of the laminae at the quarters and heels (with exception of slice 1 of the left hooves) were oriented cranially, with a larger angle at the quarters compared to the adjacent heels. Medial (MQ/MH) and lateral (LQ/LH) parts of the hooves were not significantly different (Fig 3a).

image

Figure 3. Positional differences (mean ± s.d.) between circumferential and proximodistal blocks for a) laminar orientation (LO) and b) internal angle plotted by circumferential (MH: medial heel, MQ: medial quarter, Toe, LH: lateral heel and LQ: lateral quarter) and proximodistal (slices A–D) positions. LO: black diamonds indicate right and hollow diamonds indicate left hooves. Diff: Different. Horizontal ellipses demonstrate comparisons between circumferential blocks and vertical ellipses show comparisons between proximodistal blocks.

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At the toe, none of the proximodistal slices differed significantly; all the laminae were almost perpendicular to the outer hoof wall, the mean LO was 88.4° (± 1.6) on the left and 89.2° (± 1.7) on the right hooves. The outer parts of most laminae were oriented cranially at the quarters. At the heels, LO decreased from the most proximal slice to the most distal slice.

Internal angle (IA)

Internal angle was significantly different between the slices (P<0.0001) and blocks (P<0.0001). At the toe the laminae were mostly straight or slightly curved (90–3.72°/90°+ 2.1°); the curvature of the laminae increased circumferentially. At the quarters and heels IA ranged from -14.56 to 13.10°; the negative angle indicates curvature to the opposite side i.e. outer part of the laminae were curved toward the toe (cranially). Adjacent quarter and heel blocks showed this pattern: they were significantly different at the 2 distal slices but not at the 2 proximal slices; there were 2 exceptions for this pattern (Fig 3b). Medial and lateral heels were not significantly different in most cases.

None of the proximodistal blocks at the toe were significantly different from each other; the quarters followed a similar pattern. At the quarters, the outer parts of the laminae were curved caudally in all slices. At the heels, the outer parts of the laminae located at the 2 proximal slices were curved caudally and the laminae located at the 2 distal slices were curved cranially. Although the differences between those slices were not always significant, the pattern was quite obvious.

Thickness of the stratum medium (SM) and lamellatum (SL)

The left and right hooves were not significantly different. The SM and SL were both thicker at the toe and their thicknesses decreased towards the heels at both sides (Fig 4). The thicknesses of SM and SL were significantly different between the adjacent quarter and heel, but not between the medial and lateral quarters. At the heels, SM was significantly thicker laterally (7.3 compared to 6.7 mm). However, for SL, this difference (2.42 compared to 2.21 mm) was not statistically significant (P = 0.0952).

image

Figure 4. Mean thickness of the stratum medium and lamellatum.

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Laminar branching

The number of branching varied from 0–7; there were also some variations among horses. The left and right hooves were not significantly different. The highest number of the branched PEL was observed at the toe and at the quarters (Fig 5). Medial and lateral quarters were significantly different only on the left hooves. On the left hooves, there was more branching at the lateral side of the hoof compared to the medial side. This pattern was reversed on the right hooves but this difference was not statistically significant.

image

Figure 5. Average number of branched primary epidermal laminar (PEL) on the left and right hooves.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Authors' declaration of interests
  8. Sources of funding
  9. Acknowledgements
  10. Manufacturers' addresses
  11. References

With each footfall the forehoof capsule experiences 2 types of forces: weight of the animal and ground reaction forces (GRF). The dorsal part of the wall rotates distally and palmarly and the laminar junction experiences large amounts of tensile forces (Douglas et al. 1996). High density of the laminae at the dorsum is required to dissipate the high tensile forces at this region (Douglas et al. 1998). The straight (IA close to zero) and perpendicular (LO close to 90°) shape of the laminae at the toe is presumably related to the high radial tension at this region. The palmar movement of the toe is accompanied with an abaxial movement of the quarters and heels, in particular at the distal border; which in turn, generates transverse shear forces between the hoof capsule and the distal phalanx. Consequently, lesser tensile and greater shear forces are applied to the quarters and heels. The direction of the applied forces may explain the diversity of the laminar morphology at those regions. There is greater variation at the quarters and heels which is consistent with the variations in loading pattern in the hoof capsule. We showed (Thomason et al. 2008) differences in laminar morphology between Thoroughbred and Standardbred horses which is presumably related to different loading patterns. It appears that this is not a one way relationship; not only does loading affect laminar morphology but laminar morphology may reciprocally influence the distribution of forces but to a lesser extent.

Likewise, laminar morphology is correlated with some variables of the hoof shape (Faramarzi 2003). Bowker (2003b) reported greater laminar spacing (fewer laminae) at the steeper side of the asymmetrical hooves and vice versa. Lancaster et al. (2007) reported abrupt changes in laminar morphology and argued that those changes could occur in response to trauma to the hoof wall or as preclinical forms of laminitis. It is likely that laminae respond to applied stresses and undergo a self-healing process; pathological changes are seen when the stress exceeds the adaptive capacity of the laminae. Understanding laminar remodelling provides fundamental knowledge that facilitates early recognition of laminar pathology such as that seen in laminitis.

Laminar spacing (LS)

The convoluted architecture of the laminae provides further contact surface between the distal phalanx and inner hoof wall and adds more strength to the laminar junction. It is likely that increased contact surface at the toe reduces the stress on the laminar junction; the higher number of the laminae at the toe (smaller LS) could be related to higher stress at this region. It has been suggested that dermal laminae provide more flexibility (Sarratt and Hood 2005; Lancaster et al. 2007); greater LS at the quarters and heels is associated with thicker dermal laminae; this may provide further flexibility for the abaxial movement of the hoof wall at these regions. Higher amounts of dermal tissues at the periphery is also associated with more vasculature and, therefore, more blood perfusion (Lancaster et al. 2007); the higher blood supply may be correlated with the abaxial movement of the hoof wall outskirts, in particular at the heels.

Laminar orientation (LO)

The proximodistal similarity between the toe and quarter slices (with a few exceptions) indicates a similar response (presumably to a similar stimulus or stimuli) at these regions. Considering the adjacent quarters and heels, the opposite trend between the 2 proximal and 2 distal slices, implies existence of different stresses and/or different responses at those regions.

Internal angle (IA)

The similarity between the proximodistal blocks at the toe and quarters indicates that curvature of the laminae does not significantly change as the laminae slide distally. Examining the laminae just distal to the coronary band may reveal if the laminae are curved as they are produced at the coronary band or if they curve later on (e.g. just distal to the coronary band) as they move distally. The difference between the 2 proximal and the 2 distal slices at the heels could be related to experiencing different stresses and/or different responses by those slices. One reason for such a difference could be the position of the distal phalanx compared to the proximodistal slices. Although the exact position of the distal border of the distal phalanx was not addressed in this study, it was located approximately at the level of the 2nd and 3rd slices at the quarters and heels. The opposite pattern in the adjacent heel and quarter blocks between the 2 proximal and distal slices could have occurred due to a similar reason. Lower density of the laminae at the heels may allow for more diversity in the curvature of the laminae at the heels.

Thickness of the stratum medium and lamellatum

The hoof wall (SM) was thicker at the lateral side compared to the medial side; the differences were statistically significant for the heels and nonsignificant for the quarters. The reason for such a difference is not quite known. It could be genetics, biomechanical adaptation or related to the hoof shape i.e. lateral wall flare. Sarratt and Hood (2005) reported a proximodistal increase in the primary laminar length (depth) in the dorsal laminae, while our study focused on circumferential variation in slice 3 only and showed longer laminae (greater SL) at the toe compared to the quarters and the heels.

Laminar branching

The proximodistal similarity in the number of the branched PEL implies that branching does not initiate or change at the middle or distal slices; it is likely that this happens at the more proximal parts of the hoof i.e. proximal to slice 4 in this study. It appears that the branching process gives rise to new laminae. Bidwell and Bowker (2006) reported early signs of laminar bifurcation in 2 month old foals; the bifurcation process progresses and gives rise to new laminae as the foals grow to yearlings. Creation of the new laminae is likely to be a response to increased stress in areas of the hoof as the animal grows and becomes more active. However, the role of keratinocyte mitosis and associated growth factors is not well known. A better understanding of the bifurcation process provides insight into laminar recovery after laminitic insult.

The observed changes in laminar architecture, particularly the differences between the 2 proximal and distal slices, indicate the potential of the laminae to change and remodel i.e. in response to variation in regional stresses. Our results also provide evidence that although the bulk of the primary laminae are produced at the coronary band, the laminae may potentially change or even proliferate in some extents as they move distally. This work used a relatively large sample size and provides a reference for laminar morphology in Thoroughbreds and for the first time compared left and right hooves. It is likely that the side differences (e.g. more branched laminae on the lateral side of the left hooves and on the medial side of the right hooves) are related to the biomechanics of the asymmetrical loading seen in the gallop of Thoroughbreds (counter clockwise racing tracks in North America). The samples were collected over a period of time and the exercise history of all horses was not known. However, a number of the samples were collected from racehorses with recent history of training. This work adds to the body of knowledge on the potential of healthy laminae to remodel.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Authors' declaration of interests
  8. Sources of funding
  9. Acknowledgements
  10. Manufacturers' addresses
  11. References

A small section of this study was presented at the American Association of Veterinary Anatomists Convention (July 2007; University of Prince Edwards Island, PEI, Canada). An abstract (<250 words) was presented (Circumferential and proximodistal morphology of the laminar junction in the forehoof of Thoroughbred horses).

The author would like to sincerely thank Dr. Jeffrey Thomason and also Mr. William Sears for statistical analysis. This study was performed at the Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada N1G 2W1.

Manufacturers' addresses

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Authors' declaration of interests
  8. Sources of funding
  9. Acknowledgements
  10. Manufacturers' addresses
  11. References

1 Olympus UK Ltd, Essex, UK.

2 Optimas 6.5 image analysis software; BioScan, Inc. Edmonds, Washington, USA.

3 SAS, Statistical Analysis Services Institute, Cary, North Carolina, USA.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Authors' declaration of interests
  8. Sources of funding
  9. Acknowledgements
  10. Manufacturers' addresses
  11. References
  • Bidwell, L.A. and Bowker, R.M. (2006) Evaluation of changes in architecture of the stratum internum of the hoof wall from fetal, new born, and yearling horses. Am. J. vet. Res. 67, 1947-1955.
  • Bowker, R.M. (2003a) The growth and adaptive capabilities of the hoof wall and sole: functional changes in response to stress. Proc. Am. Ass. equine Practnrs. 49, 146-168.
  • Bowker, R.M. (2003b) Contrasting structural morphologies of ‘good’ and ‘bad’ footed horses. Proc. Am. Ass. equine Practnrs. 49, 186-209.
  • Douglas, J.E., Biddick, T.L., Thomason, J.J. and Jofriet, J.C. (1998) Stress-strain behaviour of the equine laminar junction. J. expt. Biol. 201, 2287-2297.
  • Douglas, J.E., Mittal, C., Thomason, J.J. and Jofriet, J.C. (1996) The modulus of elasticity of equine hoof wall: implications for the mechanical function of the hoof. J. expt. Biol. 199, 1829-1836.
  • Douglas, J.E. and Thomason, J.J. (2000) Shape, orientation and spacing of the primary epidermal laminae in the hooves of neonatal and adult horses (Equus caballus). Cells Tissues Organs 166, 304-318.
  • Faramarzi, B. (2003) Morphology of the Equine Laminar Junction Correlated with External Anatomy of the Hoof. MSc Thesis, University of Guelph, Guelph, Ontario, Canada. pp 53-60.
  • Lancaster, L.S., Bowker, R.M. and Mauer, W.A. (2007) Density and morphological features of primary epidermal laminae in the feet of three-year-old racing Quarter Horses. Am. J. vet. Res. 68, 11-19.
  • Leach, D.H. and Oliphant, L.W. (1983) Ultrastructure of the equine hoof wall secondary epidermal lamellae. Am. J. vet. Res. 44, 1561-1570.
  • Pollitt, C.C. (1992) Clinical anatomy and physiology of the normal equine foot. Equine vet. Educ. 4, 219-224.
  • Pollitt, C.C. (1998) The anatomy and physiology of the hoof wall. Equine vet. Educ. Manual 4 , 3-10.
  • Sarratt, S.M. and Hood, D.M. (2005) Evaluations of architectural changes along the proximal to distal regions of the dorsal laminar interface in the equine hoof. Am. J. vet. Res. 66, 277-283.
  • Thomason, J.J., Douglas, J.E. and Sears, W. (2001) Morphology of the laminar junction in relation to the shape of the hoof capsule and distal phalanx in adult horses (Equus caballus). Cells Tissues Organs 168, 295-311.
  • Thomason, J.J., Faramarzi, B., Revill, A. and Sears, W. (2008) Quantitative morphology of the equine laminar junction in relation to capsule shape in the forehoof of Standardbreds and Thoroughbreds. Equine vet. J. 40, 473-480.