Fig 1.
Transection details and ultrasound.
A) Schematic diagram of the equine forelimb showing the metacarpal region of the superficial digital flexor tendon (SDFT; width not to scale) used in this study. The tendon was divided into 12 regions; longitudinally lateral and medial (cyan line) and the 1–4cm, 4–7cm and 7–10cm proximal and distal to the midmetacarpal (yellow lines; transection/lesion site indicated by the triangle). Each region was then further longitudinally divided into three portions for different analyses; gene expression (black), biomechanics (grey; not presented here) and histology (white). The histology sections are viewed from the face of the tendon indicated by the white arrows. B) Gross morphology of a representative transected tendon at harvest, six weeks after surgery presented at the same vertical scale (ruler shows cm) as in A) Inset is a control tendon at the lesion area for comparison. C) Ultrasonographs of the operated SDFT of one horse pre-surgery and two, four and six weeks post-transection at three indicated locations along the tendon. The SDFT is outlined with green dashes with areas of hypoechogenicity indicated with arrows.
Table 1.
Primer sequences for real-time PCR of equine tendon cDNA.
Fig 2.
Representative light microscopic images of haematoxylin and eosin-stained sections (A) from each location from a control and transected SDFT mapped to a diagram of the SDFT as depicted in Fig 1A. (B) Topographically-mapped box plot of overall histopathology scores ((n = 6 per group and region) of partially transected tendons (dark bars) compared with control SDFT (light bars). The lateral lesion site in the transected tendons is indicated by a triangle. As the horizontal scale indicates, expression on lateral side increases from right to left for display symmetry. Tendon regions in the central diagram are shaded if the score difference between control and transected tendons (indicated P values) is significant at the 5% level by Mann-Whitney U. Overall histopathology scores were higher in regions near the site of transection compared to regions further away in the partially transected tendons (P < 0.009, Kruskal-Wallis analyses). There were no significant differences in histological parameters between medial and lateral halves of the tendons. Lines within the boxes represent the median, the boxes represent the 25th and 75th percentiles, and the lines outside the boxes correspond to the minimum and maximum values. C) Topographical maps of significant changes in the indicated histopathology scores. Full box plots for these scores are in S2 Fig.
Fig 3.
Collagen alignment and proteoglycan scores.
Representative microscopic images of (A) picrosirius red-stained sections (polarised light) and (C) toluidine blue-stained sections (normal light) from each location from a control and transected SDFT mapped to a diagram of the SDFT. Topographically-mapped box plots of (B) collagen fiber alignment scores and (D) proteoglycan scores of partially transected tendons (dark bars) compared with control SDFT (light bars). The lateral lesion site in the transected tendons is indicated by a triangle. As indicated on the horizontal logarithmic scale, expression on lateral side increases from right to left for display symmetry. Tendon regions in the central diagram are shaded if the score difference between control and transected tendons (indicated P values) is significant at the 5% level by Mann-Whitney U.
Fig 4.
Topographically-mapped box plots (n = 6 per group and region) of (A) aggrecan, (B) versican, (C) biglycan and (D) lumican gene expression by partially transected tendons (dark bars) compared with control SDFT (light bars). The lateral lesion site in the transected tendons is indicated by the black triangle. As the horizontal logarithmic scale indicates, expression on lateral side increases from right to left for display symmetry. Tendon regions in the central diagram are shaded if the score difference between control and transected tendons (indicated P values) is significant at the 5% level by Mann-Whitney U. RFU = relative fluorescent units. Differences in expression between different locations as assessed by mixed model regression are summarized in Table 2.
Fig 5.
Topographically-mapped box plots of (A) ADAMTS4, (B) ADAMTS5, (C) MMP3 and (D) MMP14 gene expression (n = 6 per group and region) by partially transected tendons (dark bars) compared with control SDFT (light bars). The lateral lesion site in the transected tendons is indicated by the black triangle. As the horizontal logarithmic scale indicates, expression on lateral side increases from right to left for display symmetry. Tendon regions in the central diagram are shaded if the score difference between control and transected tendons (indicated P values) is significant at the 5% level by Mann-Whitney U. RFU = relative fluorescent units. Differences in expression between different locations as assessed by mixed model regression are summarized in Table 2.
Fig 6.
Topographically-mapped box plots of the alpha-1 chains of collagen types I (COL1A1; A); II (COL2A1; B) and III (COL3A1; C) gene expression (n = 6 per group and region) by partially transected tendons (dark bars) compared with control SDFT (light bars). The lateral lesion site in the transected tendons is indicated by a triangle. As the horizontal logarithmic scale indicates, expression on lateral side increases from right to left for display symmetry. Tendon regions in the central diagram are shaded if the score difference between control and transected tendons (indicated P values) is significant at the 5% level by Mann-Whitney U. D) RFU = relative fluorescent units. Differences in expression between different locations as assessed by mixed model regression are summarized in Table 2.
Table 2.
Mixed regression modelling of gene expression data.
Table 3.
Partial correlations of histology score parameters and gene expression.
Fig 7.
Representative sections of a non-operated control (A,C,E,G,I,K) and transected stress-derived tendon (B,D,F,H,J,L) from the lateral side proximal to the lesion are shown. Positive immunostaining is seen as red/brown colour after incubating with antibodies to the aggrecan G1 domain (A,B); the versican GAG-alpha binding region (C,D); the versican GAG-beta binding region (E,F); the C-terminus of biglycan (G,H) and fibromodulin (I,J); and rabbit IgG (K,L; negative control). Scale bar indicates 0.1mm.