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The influence of trimming of the hoof wall on the damage of laminar tissue after loading: An in vitro study
The Veterinary Journal 250 (2019) 63–70
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The Veterinary Journal journal homepage: www.elsevier.com/locate/tvjl
The influence of trimming of the hoof wall on the damage of laminar tissue after loading: An in vitro study S. Moellera,* , B. Patan-Zugajb , T. Däullaryc , A. Tichyd , T.F. Lickaa,e a
University Clinic for Horses, Department of Companion Animals and Horses, University of Veterinary Medicine, Veterinärplatz 1, 1210 Vienna, Austria Department for Pathobiology, University of Veterinary Medicine, Veterinärplatz 1, 1210 Vienna, Austria Institute of Tissue Engineering and Regenerative Medicine, University Clinic Würzburg, Röntgenring 11, 97082 Würzburg, Germany d Department of Biomedical Sciences, University of Veterinary Medicine, Veterinärplatz 1, 1210 Vienna, Austria e Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush Campus, Midlothian EH25 9RG, Scotland, UK b c
A R T I C L E I N F O
A B S T R A C T
Article history: Accepted 2 July 2019
Laminitis is associated with failure of the suspensory apparatus of the distal phalanx (SADP) connecting the distal phalanx to the hoof wall. The specific aim of this study was to examine in vitro whether thinning of the hoof wall leading to increased deformability influences the damage of the laminar tissue created by loading of the hoof. Paired cadaver forelimbs from twelve horses were used. For each pair, the hoof wall from one hoof was thinned by 25%; this was ascertained by radiography. The contralateral hooves were used as controls. In a material testing machine, hooves were loaded in a proximodistal direction at 0.5 mm/s until a cut-off value of 8 kN or 14 mm was reached. Afterwards, samples of the SADP were taken for histology. Image-based evaluation of the destruction of the SADP was performed using quantitative histogram analysis. Additionally, three examiners masked to treatment (trimmed/ untrimmed) qualitatively evaluated SADP destruction. During hoof loading with forces from 0.5 to 1.8 times the body mass of the donor horses, hooves with thinned hoof wall underwent significantly more deformation (P < 0.05). Quantitative histogram analysis detected a shift to higher brightness values and a higher pixel intensity in control hooves, representing disruption in the histologic analysis. Qualitative evaluation of histology sections showed significantly more disruption of the SADP in untrimmed hooves (P = 0.03). These results confirm the hypothesis that reduced hoof wall thickness can decrease disruption of laminar tissue in vitro, thus supporting the evaluation of hoof wall reduction as a prophylactic measure in horses at imminent risk of SADP failure. © 2019 Elsevier Ltd. All rights reserved.
Keywords: Histology Hoof wall Laminitis Suspensory apparatus of the distal phalanx Trimming
Introduction Laminitis is a common and often life-threatening condition in horses (Hunt, 1993; Heymering, 2010; Wylie et al., 2011); it is now recognised as a manifestation of systemic disease, or limb overloading (Patterson-Kane et al., 2018). In laminitis, lameness is caused by loss of function of the suspensory apparatus of the distal phalanx (SADP), leading to distal displacement and/or rotation of the distal phalanx (DP) and eventually causing irreparable damage to the hoof and the bony structures (Pollitt, 2004). The severity of lamellar destruction is related to the degree of rotation of the DP, and this negatively impacts prognosis (Hunt, 1993; Pollitt, 2004). Therefore, prevention of damage to the SADP and protection of its anatomical function is important (van Eps and
* Corresponding author. E-mail address: [email protected] (S. Moeller). https://doi.org/10.1016/j.tvjl.2019.07.002 1090-0233/© 2019 Elsevier Ltd. All rights reserved.
Pollitt, 2009a). Besides systemic anti-inflammatory, analgesic or vasodilatory medication, local prophylactic measures such as cryotherapy, optimizing hoof shape, and frog and/or solar support systems or shoes, are important for management and prevention of equine laminitis (van Eps and Orsini, 2016; O’Grady, 2017; Agass, 20191 ). However, other prophylactic measures have not been thoroughly investigated. It is generally accepted that laminitis is extremely rare in foals (Frame et al., 1988; French and Pollitt, 2004; Schvartz et al., 2012). The cause of this low prevalence is unknown, and may include lower body mass and limb loading, as well as the absence or rarity of some typical risk factors for laminitis, such as endocrinopathic diseases (e.g. pituitary pars intermedia dysfunction, equine
1 See: Agass, R., 2019. Management of acute laminitis. UK-Vet Equine 3, 43-48. https://www.magonlinelibrary.com/doi/abs/10.12968/ukve.2019.3.2.43 (Accessed 2 July 2019).
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metabolic syndrome; Menzies-Gow, 2018; de Laat, 2019), carbohydrate overload, or breed predisposition (Alford et al., 2001; Coleman et al., 2018). However, it is unclear why foals do not develop laminitis in circumstances which commonly result in laminitis in ponies, since ponies can have a similar body mass and limb loading (Dorn et al., 1975). Furthermore, colic, gastrointestinal diseases, and musculoskeletal infections causing severe sepsis are relatively common in foals (Dunkel and Wilkins, 2004; Glass and Watts, 2017), but the development of laminitis in foals after colic surgery has not been described (MacKinnon et al., 2013; Scharner et al., 2015). Foals have a thinner hoof wall than mature horses, and also have a different laminar orientation and conformation of the primary epidermal lamellae (PEL; Douglas and Thomason, 2000; Bidwell and Bowker, 2006). The distinct changes in hoof conformation in growing foals from 4 to 8 months of age, as shown by Faramarzi et al. (2017), may be caused by an increase in growth factors, keratin hardness and overall hardness of the horn material (Wattle, 1998; Bidwell and Bowker, 2006). It is possible that foal hoof capsules deform more readily during loading, and this shape adaptation may be in part responsible for the low incidence of laminitis in foals. This preliminary study was designed to investigate the relationship between SADP damage and the deformability of the hoof capsule in cadaver hooves. During loading of the foot, shear forces between the SADP and the hoof wall depend on the difference in the mechanical properties between these two layers. Our hypothesis was that reduced hoof wall thickness would result in an increased deformability of the entire horn capsule and reduced disruption of the laminar tissue during loading compared to untrimmed hoof walls. Materials and methods Cadaver limbs Paired forelimbs disarticulated at the metacarpophalangeal joint were collected after euthanasia of donor horses at the Equine Clinic at the University of Veterinary Medicine, Vienna, using a convenience sampling method (one gelding, three stallions and eight mares; mean standard deviation [SD] age 15 6.81 years and body mass 477.33 102.13 kg). Horses with abnormal hoof growth (e.g. horses with hoof canker, hoof cracks and/or changes in the white line) were excluded. Reasons for euthanasia included fractures (n = 4), wounds (n = 1), lymphoma (n = 1), and colic (n = 6). If present, horse shoes were removed. Limbs were stored in a waterproof packing in a freezer ( 20 C) for a maximum of 2 weeks and were thawed 2 h prior to preparation by immersion in a warm water bath. Shore d hardness testing In all hooves, shore d hardness was measured three times at 24 locations (See Appendix: Supplementary Fig. S1) using a shore d testing device (Sauter HBD 100-0, Sauter GmbH, ISO Norm 868) using a scale from 1 to 100; this was repeated after hoof wall thinning in the trimmed hooves. Radiography Dorsopalmar and lateromedial radiographs were acquired and measurements of the stratum externum and medium were made using Fiji2 software (2.0.0-rc-65/ 1.51s) using a previously described method (Goulet et al., 2015; Appendix: Supplementary Fig. S2). Superficial hoof wall layer thickness values were obtained at three different levels perpendicular to the dorsal margin of the hoof wall: distal to the level of the extensor process; at the level of the solar margin of the distal phalanx; and midway between these two points. At the dorsopalmar projection, measurements were made halfway between the lateral/medial solar margin of the distal phalanx and the level of the medial/lateral solar foramen. After loading of the limbs, radiographs and hoof wall measurements were repeated. Hoof preparation Paired forelimbs were disarticulated at the distal interphalangeal joint (DIPJ). Using a convenience sampling method, one hoof was selected for thinning of the hoof wall (trimmed group T), with the contralateral hoof used as control hoof
2
See: Fiji Is Just ImageJ, https://fiji.sc/ (accessed 27 February 2019).
Fig. 1. Loading test curves of paired forelimbs (trimmed = black line; control = grey line) of one horse. Two sudden force decreases, defined as disruptive events (DEs) of 0.13 kN (white arrow) and 0.6 kN (black arrow) are seen in the test curve of the control hoof. Force values at each displacement of 1 mm were included in further analysis (arrows). Loads were also expressed as percentage of the body mass of each horse.
(control group C). Based on the radiographic measurements, approximately 25% of the superficial hoof wall layer was removed. According to Lancaster et al. (2013), this zone represented the zone of highest tubular density of the hoof wall starting from the outermost edge of the stratum externum to the dermal edge of the stratum internum. Trimming was performed with an angle grinder starting 5 mm distally to the coronary band and was continued to the solar margin including the heels. Hoof loading Each hoof was placed on a wooden wedge so that the dorsal hoof wall was approximately perpendicular to the ground. The hoof was then loaded through the articular surface of the DP in a material testing machine (Walter and Bai AG). Loading was performed in a proximodistal direction at a speed of 0.5 mm/s until either 8 kN or 14 mm distal movement of the force transducer was reached. The slope of the test curves (kN/mm) and maximum displacement (mm) were noted. Sudden force decreases of >0.1 kN were defined as disruptive events (DE; Fig. 1). The decrease in total force (kN), total displacement (mm) and occurrence of the DEs were recorded. After loading, hooves were visually examined for gross damage. Sample collection for histology and determination of water content Immediately after loading and repeat radiography, a full-thickness sample (10 10 5 mm) of the mid-dorsal hoof wall was taken from each hoof; laminar tissue was removed and fixed in 4% buffered formaldehyde, embedded in paraffin, sectioned (3 mm) and stained with haematoxylin and eosin (H and E). The remaining horn was weighed, dried to constant weight at 110 C and re-weighed, and % water content was calculated. Histological analysis Measurements were obtained on digitised sections of 10 consecutive primary epidermal lamellae (PEL) using Aperio ImageScope software (Version 11.2.0.780; Fig. 2). Total primary epidermal lamellar length (TPELL) and keratinised primary epidermal lamellar length (KPELL) were measured (mm). Length and width of 10 secondary epidermal lamellae (SEL) on each side of the mid-section of each selected PEL were measured (mm). Angle measurements ( ) were carried out using Fiji1 (2.0.0-rc-65/1.51s) between the selected SELs and their specific PEL. A convenience sample of half of the sections were measured a second time to assess repeatability. The disruption of the SADP after loading was evaluated using Fiji-histogram analysis in the same region (Fig. 3). Initially, the background brightness was determined for each section as the mean brightness values of five background pixels multiplied by factor 0.85, as disruption of tissue allows the background to be seen within the slice. Pixels of brightness values exceeding this threshold were taken as white, and the relationship of white and non-white pixels was calculated. Altogether, 20 histograms were made for each hoof and of these, the median number of pixels at each brightness value was calculated. Qualitative evaluation of measurement area was performed by presenting histology sections of the SADP of paired forelimbs to three independent evaluators masked to treatment (trimmed/untrimmed). Sections were assessed for both hooves from each horse, using a grading system with half-points from 0 (no disruption) to 3 (marked disruption). Differences of 0.3 points between the mean evaluations of two paired hooves were defined as different. Furthermore, each hoof was compared to the contralateral hoof based on the histology sections, and ranked for more, similar, or less disruption of the SADP.
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Fig. 2. Photomicrographs of haematoxylin and eosin stained lamellar tissue. (a) Measurements of primary epidermal lamellae (PELs) were obtained using the lineal-tool of ImageScope. A continuous line was drawn starting from the base of each PEL at the level of the tip of the adjacent primary dermal lamella (PDL) to the tip of the consecutive PEL including secondary epidermal lamellae (SELs) for total primary epidermal lamellar length (TPELL) measurements (A) and excluding SELs for keratinised primary epidermal lamellar length (KPELL) measurements (B). (b) The SEL length was measured from the base of each SEL at the margin to the keratinised axis of the related PEL to the tip (A). Width measurements were made in the middle section of each SEL (B). The angle between the keratinised axis of the PEL and the SELs was calculated (C).
Fig. 3. Photomicrograph of haematoxylin and eosin stained lamellar histology (a), illustrating the region of interest (ROI) applied for creating the associated histogram (b). Using the selection-tool (rectangle), the ROI was outlined as small as possible including only lamellar tissue of 10 consecutive secondary epidermal lamellae (SEL) and a correspondent histogram (b) was created. The number of pixels (y-axis) for each brightness value is depicted applying a scale of 256 brightness values (x-axis) ranging from 0 (black) to 255 (white).
Statistical analysis
Results
Statistical analysis was performed using SPSS (SPSS Statistics 25). Normal distribution of data was verified with the Kolmogorov–Smirnov tests and results from horses with and without colic were compared using a linear mixed-effects model. Shore d hardness values, radiographic hoof wall measurements, water content, initial and repeated histological measurements of length, width and angles were further evaluated by a linear mixed-effects model investigating potential differences between groups. A paired t-test was used to compare points in the test curves. Rankings of histology results were evaluated using Friedman tests. Median histology scores were compared over the categories of the DEs using unpaired t-tests. All correlations were calculated as Pearsons coefficient. P-values 0.05 were considered statistically significant.
Results are presented as mean values SD. As there were no significant differences between horses with and without colic (P > 0.05); these data sets were pooled. Shore d hardness, radiographic examination and water content There were no significant differences in the shore d measurements between locations (P > 0.05). Therefore, the mean shore d hardness for each hoof was used for further evaluation (Table 1).
Table 1 Mean value standard deviations (SD) for superficial hoof wall layer thickness, shore d hardness and water content (%) of trimmed (T) and control hooves (C), before and after hoof wall thinning, and before and after loading. Shore d hardness Mean SD Hooves group T (before thinning of superficial hoof wall layer) Hooves group T (after thinning of superficial hoof wall layer) Before loading After loading Hooves group C (before loading) Hooves group C (after loading) a,b,c,d
68.66 2.97 – 68.59 2.48 – 68.11 4.88 –
Superficial hoof wall layer thickness mm Dorsal Mean SD
Superficial hoof wall layer thickness mm Medial Mean SD
Superficial hoof wall layer thickness mm Lateral Mean SD
Water content (%)
10.49 mma 0.31 mm
7.79 mma 1.59 mm
7.9 mma 1.33 mm
–
–
–
–
–
–
– b
7.55 mm 0.34 mm 10.43 mma 1.71 mm –
– b
5.86 mm 1.1 mm 7.83 mma 1.36 mm –
– b
6.06 mm 0.89 mm 8.08 mma 1.79 mm –
For each variable, statistically significant differences are indicated by different superscripted letters within the column.
Mean SD
29.19%c 3.11% – 26.3%d 1.92
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Fig. 4. Comparison of the mean slope (a) and mean maximal displacement at 8 kN (b) for control (C) and trimmed (T) hooves. Both bar graphs indicate a higher deformability for group T hooves, with a significantly lower slope (P = 0.004) and higher mean values for maximal displacement at 8 kN (P = 0.001). Additionally, a moderate positive correlation between shore d hardness and the overall slope was found (r = 0.308). Error bars show the standard deviations from the mean. (** P 0.01, *** P 0.001.).
There was no significant difference in shore d hardness between C and T hooves prior to trimming and in T hooves before and after trimming (P > 0.05). There was no significant correlation between shore d hardness and water content of the hooves (P > 0.05), but water content in horn samples of trimmed hooves was significantly higher than in control hooves (P < 0.05; Table 1). Neither initial or repeat radiographs showed signs of laminitis, distal phalanx changes, or hoof wall defects. As there were no significant differences in the radiographic measurements between the different locations, mean dorsal hoof wall values were used for further analysis. There was no significant difference between medial and lateral hoof wall measurements (P > 0.05). Trimming reduced the hoof wall thickness dorsally by 28 3%, medially by 24 7%, and laterally by 23 7% (Table 1). Hoof loading Each loading ended at the maximum test load of 8 kN, and a displacement of 14 mm was not reached. After loading, the articular cartilage showed the indentation of the force transducer; however, there were no signs of articular cartilage cracks. Additionally, no hoof showed signs of horn damage. Comparing the individual test curves of the donor horses, marked differences were observed; however, the test curves of paired hooves were much more similar (See Appendix: Supplementary Fig. S3). In 11/ 12 horses, the trimmed hooves were more displaced at the endpoint of 8 kN than the control hooves. Most hooves showed a biphasic test curve with an extended linear phase, except for one horse, which showed an overall linear curve trend. This horse was also the youngest (2 years) and lightest (250 kg) (See Appendix: Supplementary Fig. S3). Trimmed hooves showed significantly more deformation than control hooves during testing (Figs. 4a, b; P = 0.004, P = 0.001, respectively), with significant differences at displacements of the force transducer from 4 to 9 mm (Fig. 5a; P4 mm = 0.049, P5 mm = 0.039, P6 mm = 0.011, P7 mm = 0.004, P8 mm = 0.001, P9 mm = 0.028) and applied loads of 0.5 to 1.8 times body mass (50–180%; Fig. 5b). Taking into account all hooves, there were significant correlations between DE incidence and total displacement (r = 0.730; P 0.01), between DE incidence and total force decrease (r = 0.876; P 0.01), and between total displacement and total force decrease (r = 0.716, P 0.01). There were no significant differences in the occurrence of DEs and their total force decrease (T: 0.168 0.091 kN; C: 0.224 0.214 kN) between groups. Histologic evaluation and histogram analysis Histological measurements of length, width and angles are summarised in Table S1 (Appendix: Supplementary material).
There was no significant difference between either repeated measurements or between groups (P > 0.05). There was a significant positive correlation between SEL length (r = 0.827, P 0.01), width (r = 0.546, P 0.01), and angle measurements (r = 0.725, P 0.01) of both sides of the PEL. A significant positive correlation was also found between SEL length and angle measurements on both sides (r = 0.48, P 0.01). Disruption of the connective tissue and a varied lamellar morphology was observed in all hooves (Fig. 6). Histogram analysis showed higher numbers of pixels in C hooves at higher brightness values (Fig. 7). Histological sections of lamellar tissue of group C hooves presented significantly higher grades of damage than those of group T hooves (Fig. 8; P = 0.002) and were ranked significantly more often by three independent evaluators masked to treatment (trimmed/untrimmed) to have greater SADP disruption (P = 0.03; Table 2). All evaluators (A, B, C) evaluated the paired hooves of six horses (2, 7–10, 12) similarly. In five of these pairs, the control hooves had more disruption of the SADP (2,7–10) and only in one pair, the trimmed hoof had more SADP disruption (12). Each evaluator ranked the control hoof from seven hoof pairs as having more SADP disruption, while the trimmed hoof was ranked as having more SADP disruption only in one hoof pair (evaluators B, C) or in three hoof pairs (evaluator A). Of 36 evaluations (12 hoof pairs evaluated by three evaluators each), the control hooves showed more SADP disruption in 21 cases, while no difference was noted in 10 cases, and only in five hoof pairs the trimmed hooves were designated to show more SADP disruption. Histology sections of hooves with three DEs had significantly higher histology scores (1.9 0.79) than histology sections of hooves with no DEs (1.0 0.75; P = 0.004) or with two DEs (0.9 0.39; P = 0.001). Discussion This in vitro study demonstrated that reducing hoof wall thickness increases deformability of the hoof with decreased disruption of the SADP after hoof loading. The number of samples used was based on studies by Maierl et al. (2002), who determined the mechanical properties of the bovine SADP in 13 donor animals, and Dippel et al. (2016), who used 12 equine forelimbs to investigate intra-articular pressure of the DIPJ during loading. For this study, only forelimbs were used, because laminitis more commonly affects the front feet (Wylie et al., 2013). An in vitro approach was chosen as an initial test in order to adhere to the principles of replacement, reduction and refinement.3 Radiographic measurements of hoof wall thickness are commonly
3
See: The 3Rs. https://www.nc3rs.org.uk/the-3rs (Accessed 2 July 2019).
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Fig. 5. (a) Paired t-test evaluation of displacement of the force transducer in mm (x-axis) comparing trimmed (T, black line) and control hooves (C, grey line). Between the displacement of 4–9 mm measured at each mm point, the curve trend of T and C hooves was determined to be statistically significant different (P4 mm = 0.049, P5 mm = 0.039, P6 mm = 0.011, P7 mm = 0.004, P8 mm = 0.001, P9 mm = 0.028). The referring mean force values ranged from 1.83 0.92 kN to 6.93 0.78 kN for group T and from 2.44 0.68 kN after 4 mm to 6.7 0.38 kN after 9 mm for group C hooves. (b) Relationship between loads applied to the hooves in percentage of body mass (y-axis) and displacement of the force transducer in mm (x-axis). Trimmed hooves (T, black line) and control hooves (C, grey line) were compared using a paired t-test. Differences at an interval of 10 percent were statistically significant from 50% to 180% of body mass (asterisks), with displacement ranging from 4.69 0.99 mm to 8.39 0.85 mm in T hooves and from 4.1 0.93 mm to 6.93 1.05 mm in C hooves. Also, differences were statistically significant from 200% to 220% of body mass with displacement ranging from 8.29 0.28 mm to 8.85 0.15 mm in T hooves and from 6.76 0.39 mm to 7.4 0.001 mm in C hooves. (* P 0.05, ** P 0.01.).
Fig. 6. Photomicrographs of haematoxylin and eosin stained sections of lamellar tissues of control (a, c) and trimmed (b, d) hooves showing the variety of histological appearances after loading. (a) Secondary epidermal lamellae (SEL) appeared elongated, narrowed and slightly curved. Disruption of the connective tissue (arrowheads) was clearly visible in whitened areas between the SELs due to the absence of secondary dermal lamellae (SDL). In this case, disruption occurred irregularly and asymmetrically. (b) Characteristic symmetrical orientation of the SELs with an evenly distributed tissue-disruption (arrowheads). (c) Extensively lengthened SELs with a clear increase in the angle between the axes of SEL and primary epidermal lamella (PEL). Margins of SELs were poorly defined and could hardly be separated. (d) Almost perpendicular orientation of the SEL to the PEL axis. SELs were uniform in length and disruptive tissue was hardly identifiable by a slight loss of density within the SDL.
used for the non-invasive assessment of laminitis (Goulet et al., 2015; Mullard et al., 2018), and this was therefore considered appropriate. Previous studies have reported that the effects of a single freeze thaw cycle on laminar tissue and its mechanical properties are minor (Moon et al., 2006; Kochová et al., 2013; Boettcher et al., 2014); therefore, frozen limbs were used. Durometers have previously been used to detect hoof horn hardness (Hinterhofer et al., 2001; Patan and Budras, 2003; Tocci et al., 2015). Our study demonstrated that shore d hardness did not change significantly with trimming, but significantly increased water content in the hoof horn after trimming was found. In this
regard, shore d hardness measurements appeared to be less sensitive than water content measurements. These seemingly contradictory findings may be explained by the fact that shore d hardness reflects the quality of the horn surface tested, even though the tubule density is slightly lower at the layer exposed in the trimmed hooves. However, water content refers to the volume of the horn, thus including the inner layers of the hoof wall with much smaller tubule density (Lancaster et al., 2013). Therefore, shore d hardness seems suitable to monitor slight thinning of the hoof wall, as performed in this study. However, the positive correlation between shore d hardness and the overall slope of the test curves illustrated that
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S. Moeller et al. / The Veterinary Journal 250 (2019) 63–70 Table 2 For each pair of hooves, the hoof evaluated with more histological disruption of the suspensory apparatus of the distal phalanx than its paired hoof is listed, based on the evaluation by three independent, masked evaluators.
Fig. 7. Histogram analysis depicting comparative total brightness distribution of all slides of trimmed (black line) and control (grey line) hooves starting from the threshold for ‘background brightness’ in percentage of total pixels. For better understanding and comparability, a line diagram was used. Brightness values of histological sections of control hooves show a right shifting to higher brightness values (area 240–250 on x-axis, arrowheads) and a higher number of pixels (area 225–235 on x-axis, arrows) compared to sections of trimmed hooves, while control hooves show more pixels in the lower range above the threshold of the brightness values (area 200–225 on x-axis).
thinning of the hoof wall was sufficient to increase deformability without weakening the horn, as this remained intact after loading. In contrast, dorsal hoof wall resection (where approximately 25% of the entire horn capsule is removed) leads to loss of structural integrity and stability of the horn capsule and can cause severe complications (Rucker, 2010). In general, dorsal hoof wall resection is primarily used in chronic laminitis cases to obtain decompression when oedema and lamellar separation, causing severe reduction in SADP functionality (Parks and O’Grady, 2003; Baker, 2012). The method proposed in our study would rather be considered in the early stages or even prophylactically, as it might help to decrease stress associated with the transmission of forces from the outer hoof wall over the dermoepidermal interface to the distal phalanx (Kasapi and Gosline, 1997). The reduction of the hoof wall thickness and increased deformation remain to be critically evaluated in live horses where the sole and frog could be overloaded, although increased loading of sole and frog has been recommended to reduce loading on the affected hoof wall (Parks, 2003; Sleutjens et al., 2018). Nonetheless, application of this method should be considered very carefully, as hoof renewal time is between 9 and 12 months (Kainer, 2002). Additional supportive methods such as soft bedding or noninvasive support systems (foam pads, frog supports, soft boots etc.) should therefore be maintained. Alternatively, a combination of moderate thinning of the hoof wall and soaking could be used, as
Horse
Evaluator A
Evaluator B
Evaluator C
1 2 3 4 5 6 7 8 9 10 11 12
C C T T ND ND C C C C C T
C C ND C ND ND C C C C ND T
ND C C C ND ND C C C C ND T
C, control; T, trimmed; ND, no difference.
the exposed inner coronary hoof horn has higher water uptake capacity than the outer coronary horn (Patan and Budras, 2003), leading to increased deformability of the hoof capsule. This could possibly also minimise lamellar damage during loading. The water content and the hydration gradient of hoof horn from the inner to the outer region have been investigated (Douglas et al., 1996; Hampson et al., 2012). The inverse correlation between water content and stiffness as reported by Bertram and Gosline (1987) could not be confirmed statistically in the present study. However, in laminitic horses, watering and soaking of the hooves in warm or cold water have been used as treatments for centuries (Slater et al., 1995; Wagner and Heymering, 1999; Heymering, 2010). Kochová et al. (2013) reported nonlinear mechanical behaviour of the SADP and an effect of the structure of the SADP on its mechanical properties. Also, a largely biphasic force-displacement curve, with a linear phase followed by a plateau, has been reported in hoof wall samples (Goodman and Haggis, 2009). In the present study, the force-displacement of the hoof horn probably remained within the linear phase, but we consider that the force-displacements represent the composite characteristics of the SADP and the hoof wall. Using similar measurement methods, Patan-Zugaj et al. (2012) reported that higher loads (from 9 to 14 kN) were required to dislocate the DP completely from the horn capsule, supporting the results of our study. In the present study, SADP damage was significantly reduced by thinning of the hoof wall, and the test curves indicated that this significant difference started at loads of 50% body mass. Comparatively, loads of 30% of the body mass are reached in a
Fig. 8. Photomicrographs illustrating the haematoxylin and eosin stained histologic appearance of lamellar tissues of the control (a) and trimmed (b) forelimb of one horse. The loss of tissue integrity and disruption in the dermal-epidermal junction (arrowheads) was more severe in the control forelimb.
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standing horse (Kainer, 2002) and of 60–70% in a normal walking horse (Hodson et al., 2000). In the acute phase of laminitis, Hood et al. (2001) determined loads on a single forelimb during weight shifting of approximately 28% of body mass. Several studies have reported histological evaluation of the SADP in healthy and laminitic hooves (Kawasako et al., 2009; van Eps and Pollitt, 2009b; de Laat et al., 2013), and samples from frozen SADP tissues are commonly used in this field of research (Bidwell and Bowker, 2006; Lancaster et al., 2007). The measurement protocol used in this study has been described previously (van Eps and Pollitt, 2009b; Asplin et al., 2010; de Laat et al., 2011), and the histological appearance of the epidermal lamellae reported here is consistent with the findings of Kawasako et al. (2009), who reported variation of the SADP structure with age and training. Our histological evaluations by trained, masked evaluators clearly showed differences in tissue disruption between C and T hooves. Additionally, the occurrence of DEs could serve as an indicator of the severity of SADP disruption, as the histology scores were significantly higher in hooves with two and three DEs. Laminitis is regarded as a clinical syndrome resulting from several systemic diseases preceded by a subclinical phase with early lamellar lesions (Patterson-Kane et al., 2018). Early therapeutic intervention (prior to the development of the clinical syndrome) are recommended because structural changes of the SADP are initiated at the cellular level and even minor interventions could possibly have a significant effect on tissue preservation and clinical outcome (de Laat et al., 2013; Karikoski et al., 2014). The results of this preliminary study on cadaver specimens cannot be extrapolated to live horses without taking into account several limitations. The potential benefits of weakening the hoof in live horses remain unsubstantiated. Firstly, the water content of the cadaveric hooves is not similar to that in fresh samples from live horses, which was reported to decrease significantly 1 h following removal (Hopegood, 2002). However, water content results in the present study are comparable to other studies of hoof horn samples (Butler and Hintz, 1977; Leach, 1980), some of which also investigated previously frozen feet (Hampson et al., 2012). The loading force, velocity and direction used in our study does not represent the physiological loading in live horses, which depends partly on body and limb conformation, hoof shape and speed of locomotion. The loading force in our study was similar to the load transferred through the horse’s foot when standing (30% of body mass; Kainer, 2002) or during normal walking (60–70% of body mass; Hodson et al., 2000). However, the angle of loading and the speed of displacement did not represent the normal stresses on the hoof wall. The testing conditions described were chosen for the purpose of standardisation. Similarly, non-physiological testing procedures have been used in many other studies on the mechanical qualities of the SADP in ungulates (Douglas et al., 1998; Maierl et al., 2002; Kochová et al., 2013). In the present study, the occurrence of DEs during loading was documented, and interpreted as abrupt disruptions of the SADP itself. However, it is possible that some of these DEs might have been associated with cartilage deformations during loading, as indentations in the articular surface of the distal phalanx were noted in all feet in both groups. Conclusions This study indicates that reduced hoof wall thickness and higher deformability can potentially reduce damage to the SADP and increase its resilience to stresses and strains in vitro. This confirmed the hypothesis that thinning of the superficial hoof wall layer can potentially serve as a prophylactic measure for horses at imminent risk of laminitis before radiological signs of DP displacement and/or rotation are apparent. However, therapeutic/prophylactic hoof
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thinning in live horses is not without risk, and further research is needed before considering this procedure in clinical cases. Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of this paper. Acknowledgements The authors would like to thank Prof. Dr Johann Kofler for the co-supervision of this work and Mag. Stefan Kummer for the technical support. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.tvjl.2019.07.002. References Alford, P., Geller, S., Richardson, B., Slater, M., Honnas, C., Foreman, J., Robinson, J., Messer, M., Roberts, M., Goble, D., et al., 2001. A multicenter, matched casecontrol study of risk factors for equine laminitis. Preventive Veterinary Medicine 49, 209–222. Asplin, K.E., Patterson-Kane, J.C., Sillence, N., Pollitt, C.C., McGowan, C.M., 2010. Histopathology of insulin-induced laminitis in ponies. Equine Veterinary Journal 42, 700–706. van Eps, A.W., Pollitt, C.C., 2009b. Equine laminitis model: lamellar histopathology seven days after induction with oligofructose. Equine Veterinary Journal 41, 735–740. Baker, W.R., 2012. Treating laminitis. Beyond the mechanics of trimming and shoeing. Veterinary Clinics of North America: Equine Practice 28, 441–455. Bertram, J.E.A., Gosline, J.M., 1987. Functional design of horse hoof keratin: the modulation of mechanical properties through hydration effects. Journal of Experimental Biology 130, 121–136. Bidwell, L.A., Bowker, R.M., 2006. Evaluation of changes in architecture of the stratum internum of the hoof wall from fetal, newborn, and yearling horses. American Journal of Veterinary Research 67, 1947–1955. Boettcher, H.S., Knudsen, J.C., Andersen, P.H., Danscher, A.M., 2014. Technical note: effects of frozen storage on the mechanical properties of the suspensory tissue in the bovine claw. Journal of Dairy Science 97, 2969–2973. Butler, K.D., Hintz, H.F., 1977. Effect of level of feed intake and gelatin supplementation on growth and quality of hoofs of ponies. Journal of Animal Science 44, 257–261. Coleman, M.C., Belknap, J.K., Eades, S.C., Galantino-Homer, H.L., Hunt, R.J., Geor, R.J., McCue, M.E., McIlwraith, C.W., Moore, R.M., Peroni, J.F., et al., 2018. Case-control study of risk factors for pasture- and endocrinopathy-associated laminitis in North American horses. Journal of the American Veterinary Medical Association 253, 470–478. de Laat, M.A., 2019. Science in brief: progress in endocrinopathic laminitis research: have we got a foothold? Equine Veterinary Journal 51, 141–142. de Laat, M.A., van Eps, A.W., McGowan, C.M., Sillence, M.N., Pollitt, C.C., 2011. Equine laminitis: comparative histopathology 48 hours after experimental induction with insulin or alimentary oligofructose in standardbred horses. Journal of Comparative Pathology 145, 399–409. de Laat, M.A., Patterson-Kane, J.C., Pollitt, C.C., Sillence, M.N., McGowan, C.M., 2013. Histological and morphometric lesions in the pre-clinical, developmental phase of insulin-induced laminitis in Standardbred horses. The Veterinary Journal 195, 305–312. Dippel, M., Ruczizka, U., Valentin, S., Licka, T.F., 2016. Influence of increased intraarticular pressure on the angular displacement of the isolated equine distal interphalangeal joint. Journal of Equine Veterinary Science 38, 54–63. Dorn, C.R., Gamer, H.E., Coffman, J.R., Hahn, A.W., Tritschler, L., 1975. Castration and other factors affecting the risk of equine laminitis. The Cornell Veterinarian 65, 57–64. Douglas, J.E., 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. Douglas, J.E., Mittal, C., Thomason, J.J., Jofriet, J.C., 1996. The modulus of elasticity of equine hoof wall: implications for the mechanical function of the hoof. Journal of Experimental Biology 199, 1829–1836. Douglas, J.E., Biddick, T.L., Thomason, J.J., Jofriet, J.C., 1998. Stress/strain behaviour of the equine laminar junction. Journal of Experimental Biology 201, 2287–2297. Dunkel, B., Wilkins, P., 2004. Infectious foal diarrhoea: pathophysiology, prevalence and diagnosis. Equine Veterinary Education 16, 94–101. Faramarzi, B., Salinger, A., Kaneps, A., Nout-Lomas, Y., Greene, H., Dong, F., 2017. Quantitative analysis and development of the fore feet of Arabian foals from
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The influence of trimming of the hoof wall on the damage of laminar tissue after loading: An in vitro study S. Moellera,* , B. Patan-Zugajb , T. Däullaryc , A. Tichyd , T.F. Lickaa,e a
University Clinic for Horses, Department of Companion Animals and Horses, University of Veterinary Medicine, Veterinärplatz 1, 1210 Vienna, Austria Department for Pathobiology, University of Veterinary Medicine, Veterinärplatz 1, 1210 Vienna, Austria Institute of Tissue Engineering and Regenerative Medicine, University Clinic Würzburg, Röntgenring 11, 97082 Würzburg, Germany d Department of Biomedical Sciences, University of Veterinary Medicine, Veterinärplatz 1, 1210 Vienna, Austria e Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush Campus, Midlothian EH25 9RG, Scotland, UK b c
A R T I C L E I N F O
A B S T R A C T
Article history: Accepted 2 July 2019
Laminitis is associated with failure of the suspensory apparatus of the distal phalanx (SADP) connecting the distal phalanx to the hoof wall. The specific aim of this study was to examine in vitro whether thinning of the hoof wall leading to increased deformability influences the damage of the laminar tissue created by loading of the hoof. Paired cadaver forelimbs from twelve horses were used. For each pair, the hoof wall from one hoof was thinned by 25%; this was ascertained by radiography. The contralateral hooves were used as controls. In a material testing machine, hooves were loaded in a proximodistal direction at 0.5 mm/s until a cut-off value of 8 kN or 14 mm was reached. Afterwards, samples of the SADP were taken for histology. Image-based evaluation of the destruction of the SADP was performed using quantitative histogram analysis. Additionally, three examiners masked to treatment (trimmed/ untrimmed) qualitatively evaluated SADP destruction. During hoof loading with forces from 0.5 to 1.8 times the body mass of the donor horses, hooves with thinned hoof wall underwent significantly more deformation (P < 0.05). Quantitative histogram analysis detected a shift to higher brightness values and a higher pixel intensity in control hooves, representing disruption in the histologic analysis. Qualitative evaluation of histology sections showed significantly more disruption of the SADP in untrimmed hooves (P = 0.03). These results confirm the hypothesis that reduced hoof wall thickness can decrease disruption of laminar tissue in vitro, thus supporting the evaluation of hoof wall reduction as a prophylactic measure in horses at imminent risk of SADP failure. © 2019 Elsevier Ltd. All rights reserved.
Keywords: Histology Hoof wall Laminitis Suspensory apparatus of the distal phalanx Trimming
Introduction Laminitis is a common and often life-threatening condition in horses (Hunt, 1993; Heymering, 2010; Wylie et al., 2011); it is now recognised as a manifestation of systemic disease, or limb overloading (Patterson-Kane et al., 2018). In laminitis, lameness is caused by loss of function of the suspensory apparatus of the distal phalanx (SADP), leading to distal displacement and/or rotation of the distal phalanx (DP) and eventually causing irreparable damage to the hoof and the bony structures (Pollitt, 2004). The severity of lamellar destruction is related to the degree of rotation of the DP, and this negatively impacts prognosis (Hunt, 1993; Pollitt, 2004). Therefore, prevention of damage to the SADP and protection of its anatomical function is important (van Eps and
* Corresponding author. E-mail address: [email protected] (S. Moeller). https://doi.org/10.1016/j.tvjl.2019.07.002 1090-0233/© 2019 Elsevier Ltd. All rights reserved.
Pollitt, 2009a). Besides systemic anti-inflammatory, analgesic or vasodilatory medication, local prophylactic measures such as cryotherapy, optimizing hoof shape, and frog and/or solar support systems or shoes, are important for management and prevention of equine laminitis (van Eps and Orsini, 2016; O’Grady, 2017; Agass, 20191 ). However, other prophylactic measures have not been thoroughly investigated. It is generally accepted that laminitis is extremely rare in foals (Frame et al., 1988; French and Pollitt, 2004; Schvartz et al., 2012). The cause of this low prevalence is unknown, and may include lower body mass and limb loading, as well as the absence or rarity of some typical risk factors for laminitis, such as endocrinopathic diseases (e.g. pituitary pars intermedia dysfunction, equine
1 See: Agass, R., 2019. Management of acute laminitis. UK-Vet Equine 3, 43-48. https://www.magonlinelibrary.com/doi/abs/10.12968/ukve.2019.3.2.43 (Accessed 2 July 2019).
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metabolic syndrome; Menzies-Gow, 2018; de Laat, 2019), carbohydrate overload, or breed predisposition (Alford et al., 2001; Coleman et al., 2018). However, it is unclear why foals do not develop laminitis in circumstances which commonly result in laminitis in ponies, since ponies can have a similar body mass and limb loading (Dorn et al., 1975). Furthermore, colic, gastrointestinal diseases, and musculoskeletal infections causing severe sepsis are relatively common in foals (Dunkel and Wilkins, 2004; Glass and Watts, 2017), but the development of laminitis in foals after colic surgery has not been described (MacKinnon et al., 2013; Scharner et al., 2015). Foals have a thinner hoof wall than mature horses, and also have a different laminar orientation and conformation of the primary epidermal lamellae (PEL; Douglas and Thomason, 2000; Bidwell and Bowker, 2006). The distinct changes in hoof conformation in growing foals from 4 to 8 months of age, as shown by Faramarzi et al. (2017), may be caused by an increase in growth factors, keratin hardness and overall hardness of the horn material (Wattle, 1998; Bidwell and Bowker, 2006). It is possible that foal hoof capsules deform more readily during loading, and this shape adaptation may be in part responsible for the low incidence of laminitis in foals. This preliminary study was designed to investigate the relationship between SADP damage and the deformability of the hoof capsule in cadaver hooves. During loading of the foot, shear forces between the SADP and the hoof wall depend on the difference in the mechanical properties between these two layers. Our hypothesis was that reduced hoof wall thickness would result in an increased deformability of the entire horn capsule and reduced disruption of the laminar tissue during loading compared to untrimmed hoof walls. Materials and methods Cadaver limbs Paired forelimbs disarticulated at the metacarpophalangeal joint were collected after euthanasia of donor horses at the Equine Clinic at the University of Veterinary Medicine, Vienna, using a convenience sampling method (one gelding, three stallions and eight mares; mean standard deviation [SD] age 15 6.81 years and body mass 477.33 102.13 kg). Horses with abnormal hoof growth (e.g. horses with hoof canker, hoof cracks and/or changes in the white line) were excluded. Reasons for euthanasia included fractures (n = 4), wounds (n = 1), lymphoma (n = 1), and colic (n = 6). If present, horse shoes were removed. Limbs were stored in a waterproof packing in a freezer ( 20 C) for a maximum of 2 weeks and were thawed 2 h prior to preparation by immersion in a warm water bath. Shore d hardness testing In all hooves, shore d hardness was measured three times at 24 locations (See Appendix: Supplementary Fig. S1) using a shore d testing device (Sauter HBD 100-0, Sauter GmbH, ISO Norm 868) using a scale from 1 to 100; this was repeated after hoof wall thinning in the trimmed hooves. Radiography Dorsopalmar and lateromedial radiographs were acquired and measurements of the stratum externum and medium were made using Fiji2 software (2.0.0-rc-65/ 1.51s) using a previously described method (Goulet et al., 2015; Appendix: Supplementary Fig. S2). Superficial hoof wall layer thickness values were obtained at three different levels perpendicular to the dorsal margin of the hoof wall: distal to the level of the extensor process; at the level of the solar margin of the distal phalanx; and midway between these two points. At the dorsopalmar projection, measurements were made halfway between the lateral/medial solar margin of the distal phalanx and the level of the medial/lateral solar foramen. After loading of the limbs, radiographs and hoof wall measurements were repeated. Hoof preparation Paired forelimbs were disarticulated at the distal interphalangeal joint (DIPJ). Using a convenience sampling method, one hoof was selected for thinning of the hoof wall (trimmed group T), with the contralateral hoof used as control hoof
2
See: Fiji Is Just ImageJ, https://fiji.sc/ (accessed 27 February 2019).
Fig. 1. Loading test curves of paired forelimbs (trimmed = black line; control = grey line) of one horse. Two sudden force decreases, defined as disruptive events (DEs) of 0.13 kN (white arrow) and 0.6 kN (black arrow) are seen in the test curve of the control hoof. Force values at each displacement of 1 mm were included in further analysis (arrows). Loads were also expressed as percentage of the body mass of each horse.
(control group C). Based on the radiographic measurements, approximately 25% of the superficial hoof wall layer was removed. According to Lancaster et al. (2013), this zone represented the zone of highest tubular density of the hoof wall starting from the outermost edge of the stratum externum to the dermal edge of the stratum internum. Trimming was performed with an angle grinder starting 5 mm distally to the coronary band and was continued to the solar margin including the heels. Hoof loading Each hoof was placed on a wooden wedge so that the dorsal hoof wall was approximately perpendicular to the ground. The hoof was then loaded through the articular surface of the DP in a material testing machine (Walter and Bai AG). Loading was performed in a proximodistal direction at a speed of 0.5 mm/s until either 8 kN or 14 mm distal movement of the force transducer was reached. The slope of the test curves (kN/mm) and maximum displacement (mm) were noted. Sudden force decreases of >0.1 kN were defined as disruptive events (DE; Fig. 1). The decrease in total force (kN), total displacement (mm) and occurrence of the DEs were recorded. After loading, hooves were visually examined for gross damage. Sample collection for histology and determination of water content Immediately after loading and repeat radiography, a full-thickness sample (10 10 5 mm) of the mid-dorsal hoof wall was taken from each hoof; laminar tissue was removed and fixed in 4% buffered formaldehyde, embedded in paraffin, sectioned (3 mm) and stained with haematoxylin and eosin (H and E). The remaining horn was weighed, dried to constant weight at 110 C and re-weighed, and % water content was calculated. Histological analysis Measurements were obtained on digitised sections of 10 consecutive primary epidermal lamellae (PEL) using Aperio ImageScope software (Version 11.2.0.780; Fig. 2). Total primary epidermal lamellar length (TPELL) and keratinised primary epidermal lamellar length (KPELL) were measured (mm). Length and width of 10 secondary epidermal lamellae (SEL) on each side of the mid-section of each selected PEL were measured (mm). Angle measurements ( ) were carried out using Fiji1 (2.0.0-rc-65/1.51s) between the selected SELs and their specific PEL. A convenience sample of half of the sections were measured a second time to assess repeatability. The disruption of the SADP after loading was evaluated using Fiji-histogram analysis in the same region (Fig. 3). Initially, the background brightness was determined for each section as the mean brightness values of five background pixels multiplied by factor 0.85, as disruption of tissue allows the background to be seen within the slice. Pixels of brightness values exceeding this threshold were taken as white, and the relationship of white and non-white pixels was calculated. Altogether, 20 histograms were made for each hoof and of these, the median number of pixels at each brightness value was calculated. Qualitative evaluation of measurement area was performed by presenting histology sections of the SADP of paired forelimbs to three independent evaluators masked to treatment (trimmed/untrimmed). Sections were assessed for both hooves from each horse, using a grading system with half-points from 0 (no disruption) to 3 (marked disruption). Differences of 0.3 points between the mean evaluations of two paired hooves were defined as different. Furthermore, each hoof was compared to the contralateral hoof based on the histology sections, and ranked for more, similar, or less disruption of the SADP.
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Fig. 2. Photomicrographs of haematoxylin and eosin stained lamellar tissue. (a) Measurements of primary epidermal lamellae (PELs) were obtained using the lineal-tool of ImageScope. A continuous line was drawn starting from the base of each PEL at the level of the tip of the adjacent primary dermal lamella (PDL) to the tip of the consecutive PEL including secondary epidermal lamellae (SELs) for total primary epidermal lamellar length (TPELL) measurements (A) and excluding SELs for keratinised primary epidermal lamellar length (KPELL) measurements (B). (b) The SEL length was measured from the base of each SEL at the margin to the keratinised axis of the related PEL to the tip (A). Width measurements were made in the middle section of each SEL (B). The angle between the keratinised axis of the PEL and the SELs was calculated (C).
Fig. 3. Photomicrograph of haematoxylin and eosin stained lamellar histology (a), illustrating the region of interest (ROI) applied for creating the associated histogram (b). Using the selection-tool (rectangle), the ROI was outlined as small as possible including only lamellar tissue of 10 consecutive secondary epidermal lamellae (SEL) and a correspondent histogram (b) was created. The number of pixels (y-axis) for each brightness value is depicted applying a scale of 256 brightness values (x-axis) ranging from 0 (black) to 255 (white).
Statistical analysis
Results
Statistical analysis was performed using SPSS (SPSS Statistics 25). Normal distribution of data was verified with the Kolmogorov–Smirnov tests and results from horses with and without colic were compared using a linear mixed-effects model. Shore d hardness values, radiographic hoof wall measurements, water content, initial and repeated histological measurements of length, width and angles were further evaluated by a linear mixed-effects model investigating potential differences between groups. A paired t-test was used to compare points in the test curves. Rankings of histology results were evaluated using Friedman tests. Median histology scores were compared over the categories of the DEs using unpaired t-tests. All correlations were calculated as Pearsons coefficient. P-values 0.05 were considered statistically significant.
Results are presented as mean values SD. As there were no significant differences between horses with and without colic (P > 0.05); these data sets were pooled. Shore d hardness, radiographic examination and water content There were no significant differences in the shore d measurements between locations (P > 0.05). Therefore, the mean shore d hardness for each hoof was used for further evaluation (Table 1).
Table 1 Mean value standard deviations (SD) for superficial hoof wall layer thickness, shore d hardness and water content (%) of trimmed (T) and control hooves (C), before and after hoof wall thinning, and before and after loading. Shore d hardness Mean SD Hooves group T (before thinning of superficial hoof wall layer) Hooves group T (after thinning of superficial hoof wall layer) Before loading After loading Hooves group C (before loading) Hooves group C (after loading) a,b,c,d
68.66 2.97 – 68.59 2.48 – 68.11 4.88 –
Superficial hoof wall layer thickness mm Dorsal Mean SD
Superficial hoof wall layer thickness mm Medial Mean SD
Superficial hoof wall layer thickness mm Lateral Mean SD
Water content (%)
10.49 mma 0.31 mm
7.79 mma 1.59 mm
7.9 mma 1.33 mm
–
–
–
–
–
–
– b
7.55 mm 0.34 mm 10.43 mma 1.71 mm –
– b
5.86 mm 1.1 mm 7.83 mma 1.36 mm –
– b
6.06 mm 0.89 mm 8.08 mma 1.79 mm –
For each variable, statistically significant differences are indicated by different superscripted letters within the column.
Mean SD
29.19%c 3.11% – 26.3%d 1.92
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Fig. 4. Comparison of the mean slope (a) and mean maximal displacement at 8 kN (b) for control (C) and trimmed (T) hooves. Both bar graphs indicate a higher deformability for group T hooves, with a significantly lower slope (P = 0.004) and higher mean values for maximal displacement at 8 kN (P = 0.001). Additionally, a moderate positive correlation between shore d hardness and the overall slope was found (r = 0.308). Error bars show the standard deviations from the mean. (** P 0.01, *** P 0.001.).
There was no significant difference in shore d hardness between C and T hooves prior to trimming and in T hooves before and after trimming (P > 0.05). There was no significant correlation between shore d hardness and water content of the hooves (P > 0.05), but water content in horn samples of trimmed hooves was significantly higher than in control hooves (P < 0.05; Table 1). Neither initial or repeat radiographs showed signs of laminitis, distal phalanx changes, or hoof wall defects. As there were no significant differences in the radiographic measurements between the different locations, mean dorsal hoof wall values were used for further analysis. There was no significant difference between medial and lateral hoof wall measurements (P > 0.05). Trimming reduced the hoof wall thickness dorsally by 28 3%, medially by 24 7%, and laterally by 23 7% (Table 1). Hoof loading Each loading ended at the maximum test load of 8 kN, and a displacement of 14 mm was not reached. After loading, the articular cartilage showed the indentation of the force transducer; however, there were no signs of articular cartilage cracks. Additionally, no hoof showed signs of horn damage. Comparing the individual test curves of the donor horses, marked differences were observed; however, the test curves of paired hooves were much more similar (See Appendix: Supplementary Fig. S3). In 11/ 12 horses, the trimmed hooves were more displaced at the endpoint of 8 kN than the control hooves. Most hooves showed a biphasic test curve with an extended linear phase, except for one horse, which showed an overall linear curve trend. This horse was also the youngest (2 years) and lightest (250 kg) (See Appendix: Supplementary Fig. S3). Trimmed hooves showed significantly more deformation than control hooves during testing (Figs. 4a, b; P = 0.004, P = 0.001, respectively), with significant differences at displacements of the force transducer from 4 to 9 mm (Fig. 5a; P4 mm = 0.049, P5 mm = 0.039, P6 mm = 0.011, P7 mm = 0.004, P8 mm = 0.001, P9 mm = 0.028) and applied loads of 0.5 to 1.8 times body mass (50–180%; Fig. 5b). Taking into account all hooves, there were significant correlations between DE incidence and total displacement (r = 0.730; P 0.01), between DE incidence and total force decrease (r = 0.876; P 0.01), and between total displacement and total force decrease (r = 0.716, P 0.01). There were no significant differences in the occurrence of DEs and their total force decrease (T: 0.168 0.091 kN; C: 0.224 0.214 kN) between groups. Histologic evaluation and histogram analysis Histological measurements of length, width and angles are summarised in Table S1 (Appendix: Supplementary material).
There was no significant difference between either repeated measurements or between groups (P > 0.05). There was a significant positive correlation between SEL length (r = 0.827, P 0.01), width (r = 0.546, P 0.01), and angle measurements (r = 0.725, P 0.01) of both sides of the PEL. A significant positive correlation was also found between SEL length and angle measurements on both sides (r = 0.48, P 0.01). Disruption of the connective tissue and a varied lamellar morphology was observed in all hooves (Fig. 6). Histogram analysis showed higher numbers of pixels in C hooves at higher brightness values (Fig. 7). Histological sections of lamellar tissue of group C hooves presented significantly higher grades of damage than those of group T hooves (Fig. 8; P = 0.002) and were ranked significantly more often by three independent evaluators masked to treatment (trimmed/untrimmed) to have greater SADP disruption (P = 0.03; Table 2). All evaluators (A, B, C) evaluated the paired hooves of six horses (2, 7–10, 12) similarly. In five of these pairs, the control hooves had more disruption of the SADP (2,7–10) and only in one pair, the trimmed hoof had more SADP disruption (12). Each evaluator ranked the control hoof from seven hoof pairs as having more SADP disruption, while the trimmed hoof was ranked as having more SADP disruption only in one hoof pair (evaluators B, C) or in three hoof pairs (evaluator A). Of 36 evaluations (12 hoof pairs evaluated by three evaluators each), the control hooves showed more SADP disruption in 21 cases, while no difference was noted in 10 cases, and only in five hoof pairs the trimmed hooves were designated to show more SADP disruption. Histology sections of hooves with three DEs had significantly higher histology scores (1.9 0.79) than histology sections of hooves with no DEs (1.0 0.75; P = 0.004) or with two DEs (0.9 0.39; P = 0.001). Discussion This in vitro study demonstrated that reducing hoof wall thickness increases deformability of the hoof with decreased disruption of the SADP after hoof loading. The number of samples used was based on studies by Maierl et al. (2002), who determined the mechanical properties of the bovine SADP in 13 donor animals, and Dippel et al. (2016), who used 12 equine forelimbs to investigate intra-articular pressure of the DIPJ during loading. For this study, only forelimbs were used, because laminitis more commonly affects the front feet (Wylie et al., 2013). An in vitro approach was chosen as an initial test in order to adhere to the principles of replacement, reduction and refinement.3 Radiographic measurements of hoof wall thickness are commonly
3
See: The 3Rs. https://www.nc3rs.org.uk/the-3rs (Accessed 2 July 2019).
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Fig. 5. (a) Paired t-test evaluation of displacement of the force transducer in mm (x-axis) comparing trimmed (T, black line) and control hooves (C, grey line). Between the displacement of 4–9 mm measured at each mm point, the curve trend of T and C hooves was determined to be statistically significant different (P4 mm = 0.049, P5 mm = 0.039, P6 mm = 0.011, P7 mm = 0.004, P8 mm = 0.001, P9 mm = 0.028). The referring mean force values ranged from 1.83 0.92 kN to 6.93 0.78 kN for group T and from 2.44 0.68 kN after 4 mm to 6.7 0.38 kN after 9 mm for group C hooves. (b) Relationship between loads applied to the hooves in percentage of body mass (y-axis) and displacement of the force transducer in mm (x-axis). Trimmed hooves (T, black line) and control hooves (C, grey line) were compared using a paired t-test. Differences at an interval of 10 percent were statistically significant from 50% to 180% of body mass (asterisks), with displacement ranging from 4.69 0.99 mm to 8.39 0.85 mm in T hooves and from 4.1 0.93 mm to 6.93 1.05 mm in C hooves. Also, differences were statistically significant from 200% to 220% of body mass with displacement ranging from 8.29 0.28 mm to 8.85 0.15 mm in T hooves and from 6.76 0.39 mm to 7.4 0.001 mm in C hooves. (* P 0.05, ** P 0.01.).
Fig. 6. Photomicrographs of haematoxylin and eosin stained sections of lamellar tissues of control (a, c) and trimmed (b, d) hooves showing the variety of histological appearances after loading. (a) Secondary epidermal lamellae (SEL) appeared elongated, narrowed and slightly curved. Disruption of the connective tissue (arrowheads) was clearly visible in whitened areas between the SELs due to the absence of secondary dermal lamellae (SDL). In this case, disruption occurred irregularly and asymmetrically. (b) Characteristic symmetrical orientation of the SELs with an evenly distributed tissue-disruption (arrowheads). (c) Extensively lengthened SELs with a clear increase in the angle between the axes of SEL and primary epidermal lamella (PEL). Margins of SELs were poorly defined and could hardly be separated. (d) Almost perpendicular orientation of the SEL to the PEL axis. SELs were uniform in length and disruptive tissue was hardly identifiable by a slight loss of density within the SDL.
used for the non-invasive assessment of laminitis (Goulet et al., 2015; Mullard et al., 2018), and this was therefore considered appropriate. Previous studies have reported that the effects of a single freeze thaw cycle on laminar tissue and its mechanical properties are minor (Moon et al., 2006; Kochová et al., 2013; Boettcher et al., 2014); therefore, frozen limbs were used. Durometers have previously been used to detect hoof horn hardness (Hinterhofer et al., 2001; Patan and Budras, 2003; Tocci et al., 2015). Our study demonstrated that shore d hardness did not change significantly with trimming, but significantly increased water content in the hoof horn after trimming was found. In this
regard, shore d hardness measurements appeared to be less sensitive than water content measurements. These seemingly contradictory findings may be explained by the fact that shore d hardness reflects the quality of the horn surface tested, even though the tubule density is slightly lower at the layer exposed in the trimmed hooves. However, water content refers to the volume of the horn, thus including the inner layers of the hoof wall with much smaller tubule density (Lancaster et al., 2013). Therefore, shore d hardness seems suitable to monitor slight thinning of the hoof wall, as performed in this study. However, the positive correlation between shore d hardness and the overall slope of the test curves illustrated that
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S. Moeller et al. / The Veterinary Journal 250 (2019) 63–70 Table 2 For each pair of hooves, the hoof evaluated with more histological disruption of the suspensory apparatus of the distal phalanx than its paired hoof is listed, based on the evaluation by three independent, masked evaluators.
Fig. 7. Histogram analysis depicting comparative total brightness distribution of all slides of trimmed (black line) and control (grey line) hooves starting from the threshold for ‘background brightness’ in percentage of total pixels. For better understanding and comparability, a line diagram was used. Brightness values of histological sections of control hooves show a right shifting to higher brightness values (area 240–250 on x-axis, arrowheads) and a higher number of pixels (area 225–235 on x-axis, arrows) compared to sections of trimmed hooves, while control hooves show more pixels in the lower range above the threshold of the brightness values (area 200–225 on x-axis).
thinning of the hoof wall was sufficient to increase deformability without weakening the horn, as this remained intact after loading. In contrast, dorsal hoof wall resection (where approximately 25% of the entire horn capsule is removed) leads to loss of structural integrity and stability of the horn capsule and can cause severe complications (Rucker, 2010). In general, dorsal hoof wall resection is primarily used in chronic laminitis cases to obtain decompression when oedema and lamellar separation, causing severe reduction in SADP functionality (Parks and O’Grady, 2003; Baker, 2012). The method proposed in our study would rather be considered in the early stages or even prophylactically, as it might help to decrease stress associated with the transmission of forces from the outer hoof wall over the dermoepidermal interface to the distal phalanx (Kasapi and Gosline, 1997). The reduction of the hoof wall thickness and increased deformation remain to be critically evaluated in live horses where the sole and frog could be overloaded, although increased loading of sole and frog has been recommended to reduce loading on the affected hoof wall (Parks, 2003; Sleutjens et al., 2018). Nonetheless, application of this method should be considered very carefully, as hoof renewal time is between 9 and 12 months (Kainer, 2002). Additional supportive methods such as soft bedding or noninvasive support systems (foam pads, frog supports, soft boots etc.) should therefore be maintained. Alternatively, a combination of moderate thinning of the hoof wall and soaking could be used, as
Horse
Evaluator A
Evaluator B
Evaluator C
1 2 3 4 5 6 7 8 9 10 11 12
C C T T ND ND C C C C C T
C C ND C ND ND C C C C ND T
ND C C C ND ND C C C C ND T
C, control; T, trimmed; ND, no difference.
the exposed inner coronary hoof horn has higher water uptake capacity than the outer coronary horn (Patan and Budras, 2003), leading to increased deformability of the hoof capsule. This could possibly also minimise lamellar damage during loading. The water content and the hydration gradient of hoof horn from the inner to the outer region have been investigated (Douglas et al., 1996; Hampson et al., 2012). The inverse correlation between water content and stiffness as reported by Bertram and Gosline (1987) could not be confirmed statistically in the present study. However, in laminitic horses, watering and soaking of the hooves in warm or cold water have been used as treatments for centuries (Slater et al., 1995; Wagner and Heymering, 1999; Heymering, 2010). Kochová et al. (2013) reported nonlinear mechanical behaviour of the SADP and an effect of the structure of the SADP on its mechanical properties. Also, a largely biphasic force-displacement curve, with a linear phase followed by a plateau, has been reported in hoof wall samples (Goodman and Haggis, 2009). In the present study, the force-displacement of the hoof horn probably remained within the linear phase, but we consider that the force-displacements represent the composite characteristics of the SADP and the hoof wall. Using similar measurement methods, Patan-Zugaj et al. (2012) reported that higher loads (from 9 to 14 kN) were required to dislocate the DP completely from the horn capsule, supporting the results of our study. In the present study, SADP damage was significantly reduced by thinning of the hoof wall, and the test curves indicated that this significant difference started at loads of 50% body mass. Comparatively, loads of 30% of the body mass are reached in a
Fig. 8. Photomicrographs illustrating the haematoxylin and eosin stained histologic appearance of lamellar tissues of the control (a) and trimmed (b) forelimb of one horse. The loss of tissue integrity and disruption in the dermal-epidermal junction (arrowheads) was more severe in the control forelimb.
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standing horse (Kainer, 2002) and of 60–70% in a normal walking horse (Hodson et al., 2000). In the acute phase of laminitis, Hood et al. (2001) determined loads on a single forelimb during weight shifting of approximately 28% of body mass. Several studies have reported histological evaluation of the SADP in healthy and laminitic hooves (Kawasako et al., 2009; van Eps and Pollitt, 2009b; de Laat et al., 2013), and samples from frozen SADP tissues are commonly used in this field of research (Bidwell and Bowker, 2006; Lancaster et al., 2007). The measurement protocol used in this study has been described previously (van Eps and Pollitt, 2009b; Asplin et al., 2010; de Laat et al., 2011), and the histological appearance of the epidermal lamellae reported here is consistent with the findings of Kawasako et al. (2009), who reported variation of the SADP structure with age and training. Our histological evaluations by trained, masked evaluators clearly showed differences in tissue disruption between C and T hooves. Additionally, the occurrence of DEs could serve as an indicator of the severity of SADP disruption, as the histology scores were significantly higher in hooves with two and three DEs. Laminitis is regarded as a clinical syndrome resulting from several systemic diseases preceded by a subclinical phase with early lamellar lesions (Patterson-Kane et al., 2018). Early therapeutic intervention (prior to the development of the clinical syndrome) are recommended because structural changes of the SADP are initiated at the cellular level and even minor interventions could possibly have a significant effect on tissue preservation and clinical outcome (de Laat et al., 2013; Karikoski et al., 2014). The results of this preliminary study on cadaver specimens cannot be extrapolated to live horses without taking into account several limitations. The potential benefits of weakening the hoof in live horses remain unsubstantiated. Firstly, the water content of the cadaveric hooves is not similar to that in fresh samples from live horses, which was reported to decrease significantly 1 h following removal (Hopegood, 2002). However, water content results in the present study are comparable to other studies of hoof horn samples (Butler and Hintz, 1977; Leach, 1980), some of which also investigated previously frozen feet (Hampson et al., 2012). The loading force, velocity and direction used in our study does not represent the physiological loading in live horses, which depends partly on body and limb conformation, hoof shape and speed of locomotion. The loading force in our study was similar to the load transferred through the horse’s foot when standing (30% of body mass; Kainer, 2002) or during normal walking (60–70% of body mass; Hodson et al., 2000). However, the angle of loading and the speed of displacement did not represent the normal stresses on the hoof wall. The testing conditions described were chosen for the purpose of standardisation. Similarly, non-physiological testing procedures have been used in many other studies on the mechanical qualities of the SADP in ungulates (Douglas et al., 1998; Maierl et al., 2002; Kochová et al., 2013). In the present study, the occurrence of DEs during loading was documented, and interpreted as abrupt disruptions of the SADP itself. However, it is possible that some of these DEs might have been associated with cartilage deformations during loading, as indentations in the articular surface of the distal phalanx were noted in all feet in both groups. Conclusions This study indicates that reduced hoof wall thickness and higher deformability can potentially reduce damage to the SADP and increase its resilience to stresses and strains in vitro. This confirmed the hypothesis that thinning of the superficial hoof wall layer can potentially serve as a prophylactic measure for horses at imminent risk of laminitis before radiological signs of DP displacement and/or rotation are apparent. However, therapeutic/prophylactic hoof
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thinning in live horses is not without risk, and further research is needed before considering this procedure in clinical cases. Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of this paper. Acknowledgements The authors would like to thank Prof. Dr Johann Kofler for the co-supervision of this work and Mag. Stefan Kummer for the technical support. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.tvjl.2019.07.002. References Alford, P., Geller, S., Richardson, B., Slater, M., Honnas, C., Foreman, J., Robinson, J., Messer, M., Roberts, M., Goble, D., et al., 2001. A multicenter, matched casecontrol study of risk factors for equine laminitis. Preventive Veterinary Medicine 49, 209–222. Asplin, K.E., Patterson-Kane, J.C., Sillence, N., Pollitt, C.C., McGowan, C.M., 2010. Histopathology of insulin-induced laminitis in ponies. Equine Veterinary Journal 42, 700–706. van Eps, A.W., Pollitt, C.C., 2009b. Equine laminitis model: lamellar histopathology seven days after induction with oligofructose. Equine Veterinary Journal 41, 735–740. Baker, W.R., 2012. Treating laminitis. Beyond the mechanics of trimming and shoeing. Veterinary Clinics of North America: Equine Practice 28, 441–455. Bertram, J.E.A., Gosline, J.M., 1987. Functional design of horse hoof keratin: the modulation of mechanical properties through hydration effects. Journal of Experimental Biology 130, 121–136. Bidwell, L.A., Bowker, R.M., 2006. Evaluation of changes in architecture of the stratum internum of the hoof wall from fetal, newborn, and yearling horses. American Journal of Veterinary Research 67, 1947–1955. Boettcher, H.S., Knudsen, J.C., Andersen, P.H., Danscher, A.M., 2014. Technical note: effects of frozen storage on the mechanical properties of the suspensory tissue in the bovine claw. Journal of Dairy Science 97, 2969–2973. Butler, K.D., Hintz, H.F., 1977. Effect of level of feed intake and gelatin supplementation on growth and quality of hoofs of ponies. Journal of Animal Science 44, 257–261. Coleman, M.C., Belknap, J.K., Eades, S.C., Galantino-Homer, H.L., Hunt, R.J., Geor, R.J., McCue, M.E., McIlwraith, C.W., Moore, R.M., Peroni, J.F., et al., 2018. Case-control study of risk factors for pasture- and endocrinopathy-associated laminitis in North American horses. Journal of the American Veterinary Medical Association 253, 470–478. de Laat, M.A., 2019. Science in brief: progress in endocrinopathic laminitis research: have we got a foothold? Equine Veterinary Journal 51, 141–142. de Laat, M.A., van Eps, A.W., McGowan, C.M., Sillence, M.N., Pollitt, C.C., 2011. Equine laminitis: comparative histopathology 48 hours after experimental induction with insulin or alimentary oligofructose in standardbred horses. Journal of Comparative Pathology 145, 399–409. de Laat, M.A., Patterson-Kane, J.C., Pollitt, C.C., Sillence, M.N., McGowan, C.M., 2013. Histological and morphometric lesions in the pre-clinical, developmental phase of insulin-induced laminitis in Standardbred horses. The Veterinary Journal 195, 305–312. Dippel, M., Ruczizka, U., Valentin, S., Licka, T.F., 2016. Influence of increased intraarticular pressure on the angular displacement of the isolated equine distal interphalangeal joint. Journal of Equine Veterinary Science 38, 54–63. Dorn, C.R., Gamer, H.E., Coffman, J.R., Hahn, A.W., Tritschler, L., 1975. Castration and other factors affecting the risk of equine laminitis. The Cornell Veterinarian 65, 57–64. Douglas, J.E., 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. Douglas, J.E., Mittal, C., Thomason, J.J., Jofriet, J.C., 1996. The modulus of elasticity of equine hoof wall: implications for the mechanical function of the hoof. Journal of Experimental Biology 199, 1829–1836. Douglas, J.E., Biddick, T.L., Thomason, J.J., Jofriet, J.C., 1998. Stress/strain behaviour of the equine laminar junction. Journal of Experimental Biology 201, 2287–2297. Dunkel, B., Wilkins, P., 2004. Infectious foal diarrhoea: pathophysiology, prevalence and diagnosis. Equine Veterinary Education 16, 94–101. Faramarzi, B., Salinger, A., Kaneps, A., Nout-Lomas, Y., Greene, H., Dong, F., 2017. Quantitative analysis and development of the fore feet of Arabian foals from
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