Abstract
OBJECTIVE To identify signs of tissue-specific cortisol activity in samples of suspensory ligament (SL) and neck skin tissue from horses with and without pituitary pars intermedia dysfunction (PPID).
SAMPLE Suspensory ligament and neck skin tissue samples obtained from 26 euthanized horses with and without PPID.
PROCEDURES Tissue samples were collected from 12 horses with and 14 horses without PPID (controls). Two control horses had received treatment with dexamethasone; data from those horses were not used in statistical analyses. The other 12 control horses were classified as old horses (≥ 14 years old) and young horses (≤ 9 years old). Standard histologic staining, staining for proteoglycan accumulation, and immunostaining of SL and neck skin tissue sections for glucocorticoid receptors, insulin, 11β hydroxysteroid dehydrogenase type 1, and 11β hydroxysteroid dehydrogenase type 2 were performed. Findings for horses with PPID were compared with findings for young and old horses without PPID.
RESULTS Compared with findings for old and young control horses, there were significantly more cells stained for glucocorticoid receptors in SL samples and for 11 β hydroxysteroid dehydrogenase type 1 in SL and skin tissue samples from horses with PPID. Insulin could not be detected in any of the SL or skin tissue samples. Horses with PPID had evidence of SL degeneration with significantly increased proteoglycan accumulation. Neck skin tissue was found to be significantly thinner in PPID-affected horses than in young control horses.
CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that tissue-specific dysregulation of cortisol metabolism may contribute to the SL degeneration associated with PPID in horses.
Idiopathic chronic degeneration of the SL is common in aged horses,1 and it is strongly associated with PPID.2 Similar to horses with PPID, humans with CD are at risk of tendon and ligament degeneration or rupture.3,4 This CD-associated tendon and ligament degeneration is proposed to be the result of excessive circulating concentrations of cortisol.3 Cushing disease in humans is characterized phenotypically by signs of cortisol excess, and there is evidence of tissue dysregulation of cortisol.5 In horses with PPID, some clinical findings, such as muscle wasting, are consistent with signs of glucocorticoid excess in other species. Polyuria and polydipsia can be caused by cortisol excess, hyperglycemia, and low serum antidiuretic hormone activity; furthermore, alterations in cortisol metabolism may have a role in the development of laminitis.6 Unlike humans with CD, elevated plasma total cortisol concentration is commonly not observed in horses and ponies with PPID.7 However, free, unbound cortisol fractions, which are considered biologically active and available to bind cytoplasmic steroid receptors to mediate most of the systemic effects of cortisol, are significantly higher in horses with PPID, compared with healthy control horses.7
Glucocorticoid activity and action depend on not only circulating concentration but also tissue-specific intracellular metabolism by 11β hydroxysteroid dehydrogenases. The enzyme 11βHSD1 converts inactive cortisone to active cortisol in key metabolic tissues including the liver, adipose tissue, and skeletal muscle. The enzyme 11βHSD2 is predominantly expressed in the kidneys, colon, and sweat glands, and it catalyzes the inactivation of cortisol to cortisone, providing substrate for 11βHSD1 in peripheral tissues.5
Laminitis is common in horses with PPID,8 and high concentrations of 11βHSD1 have been detected in lamellar and subcutaneous tissues of horses with acute or chronic laminitis, suggesting a possible involvement of cortisol dysregulation in laminitic horses.8 Short- or long-term administration of synthetic glucocorticoids can lead to effects similar to those associated with endocrine disorders.7 Glucocorticoids have negative effects on tendon cell viability in human tendon cell explants,9 and medication with dexamethasone induces tenocyte apoptosis and reduction of proliferation in canine calcaneal tendons, leading to degeneration and a loss of tendon strength.10
Approximately a third of PPID-affected horses also have insulin dysregulation,11 with insulin dysregulation being a key component of equine metabolic syndrome.12 Equine metabolic syndrome and PPID are different endocrine disorders, but they can coexist in a given horse.7 Insulin dysregulation has been detected in Peruvian Paso horses with breed-specific SL degeneration13; however, in those horses, tissue insulin concentration was not evaluated. In tendon tissues of nondiabetic humans and rats, a population of insulin-producing, beta-cell–like cells has been identified; such cells are absent in rats with experimentally induced diabetes and the tendon strength is reduced by approximately 40%.14 An evaluation of insulin concentration in horses with signs of tendon degeneration might therefore be of interest.
The purpose of the present study was to evaluate SL samples obtained from horses with and without PPID for the presence and distribution of GCRs, insulin, PA, 11βHSD1, and 11βHSD2. Additionally, skin samples were similarly evaluated to investigate the presence of a systemic disorder and to test whether such skin samples may be useful for identification of such systemic disease. The hypothesis investigated in this study was that insulin, GCRs, and 11βHSD1 would be present in SL and skin tissue samples of horses and that their expressions in PPID-affected horses would differ from those in horses without PPID. Moreover, we proposed that low and similar expressions of 11βHSD2 in both tissue types and groups of horses would be present. Identification of a possible association between PPID and SL degeneration would increase the prospects of future studies into causality probably necessitating appropriate animal experiments.
Materials and Methods
Samples
Tissue samples were collected over the course of a year from horses euthanized for clinical reasons at the Equine Clinic, University of Veterinary Medicine Vienna, Austria. The horses were euthanized by IV injection of thiopentala and euthanasia solution (composed of embutramide, mebezonium iodide, and tetracaine).b Owner's written consent for postmortem use of tissue samples was obtained on admission of each horse.
Group assignment of tissue samples was based on the horses' clinical signs, results of postmortem assessments, or review of clinical records. Horses were included in the PPID-affected group if they had 1 or more clinical signs attributable to PPID before euthanasia (hypertrichosis, typical PPID-associated fat distribution, laminitis, or chronic infection), increased seasonally adjusted plasma ACTH concentration, postmortem histologic evidence of PPID, or all of the aforementioned characteristics. Horses with evidence of PPID during postmortem examination and histologic evaluation of the pituitary gland were also included in the PPID-affected group in the absence of clinical record data. One horse included in the PPID-affected group was treated with pergolide for unknown duration; its plasma ACTH concentration in March exceeded the upper reference limit (62 pg/mL), and clinical diagnosis was confirmed by histopathologic findings. Horses were classed as controls for the purpose of the study if they had no clinical record of PPID or postmortem or histopathologic findings indicative of or questionable for the presence of PPID. Control horses were allocated into 2 age groups: old horses (≥ 14 years old) and young horses (≤ 9 years old). Tissue samples from non-PPID–affected horses with a history of dexamethasone treatment in the 14-day period preceding euthanasia were also evaluated, but data for those horses were not included in the statistical analyses.
Among the 12 PPID-affected horses, reasons for euthanasia were acute colic (n = 6), hyperextension of the metatarsophalangeal joint (2), recurring laminitis (1), progressive cachexia (1), multiple melanomas (1), and sinusitis attributable to squamous cell carcinoma (1). Among the 12 non-PPID–affected control horses for which data were included in the statistical analyses, reasons for euthanasia were acute colic (n = 7), chronic laminitis (1), acute postparturient laminitis (1), fracture (1), cachexia (1), and osteosarcoma (1). The 2 dexamethasone-treated control horses were euthanized because of muscle atrophy or laminitis.
Tissue sample collection and preparation
All tissue samples were collected from the horses immediately following euthanasia. Skin samples were taken from the left side of the neck; this body site was selected to provide a similar source location in all horses and as a location that could also be easily used for biopsies in vivo. Samples of the SL were taken from the midbody of the right forelimb and the right hind limb approximately 1 to 2 cm proximal to the division into branches. Each pituitary gland was fixed in neutral-buffered 10% formalin, and SL and neck skin samples were fixed in neutral-buffered 4% formalin. All tissue samples were then embedded in paraffin.
Histologic examination of pituitary gland, SL, and neck skin samples
Median sections of pituitary glands were stained with H&E stain. One cross-sectional and 3 serial longitudinal (5-μm) sections of each SL sample and 3 serial sections of each skin sample were prepared on slides and stained with H&E, Alcian blue (pH, 2.5), or safranin-O stain by use of standard protocols.
Two longitudinal sections and 2 cross-sectional sections of SL from each horse (2 sections of the right forelimb and 2 sections of the right hindlimb) stained with H&E stain were used for evaluation of longitudinal arrangement of collagen fibers, matrix changes (eg, presence of chondrocytes or hemorrhage), and width of interstitial connective tissue septa as well as proliferation of vascular structures within the interstitial connective tissue septa. Findings for each of the aforementioned characteristics were graded for each section by means of a grading score used in a previous study,2 wherein 0 = no pathological changes, 1 = mild signs of degeneration, 2 = moderate signs of degeneration, and 3 = marked signs of degeneration. Muscle components were screened for size, number of muscle islands, and signs of degeneration.
Alcian blue and safranin-O stains were used for grading of PA in 2 longitudinal sections of SL (1 section from a forelimb and 1 section from a hindlimb). A scale of 0 to 3 was used for assessment of both stain distributions. In Alcian blue–stained sections, no blue staining between SL fibers was designated as 0, single areas of stain accumulation were designated as 1, low numbers of areas of stain accumulation were designated as 2, and high numbers of areas of stain accumulation or diffuse blue staining was designated as 3. In safranin-O–stained sections, green staining was designated as 0 and single areas of red stain accumulation were designated as 1, low numbers of areas of red stain accumulation were designated as 2, and high numbers of areas of red stain accumulation or diffuse red staining were designated as 3.
Skin samples were screened on the H&E-stained slides and scored for PA, similar to the SL samples. Skin thickness was measured at 3 different sites in each H&E-stained slide with imaging software.c
All stained slides were identified numerically for blinded evaluation (by 1 investigator [SCH]) by means of light microscopy.d Digital images were captured with a digital microscope camera and associated software.e
A total histologic score (0 to 18) was calculated based on the 6 characteristics (scored as 0 to 3, as described) indicating SL degeneration: longitudinal arrangement of collagen fibers, matrix changes (eg, presence of chondrocytes or hemorrhage), width of interstitial connective tissue septa, proliferation of vascular structures within the interstitial connective tissue septa, PAs stained with safranin-O, and PAs stained with Alcian blue stain.
Immunohistochemical analyses of neck skin and SL samples
For immunohistochemical anaylsis, 4 serial skin sections and 4 longitudinal SL sections from both the right forelimb and the right hindlimb were used from each horse. These were dewaxed by incubating them in xylol (for 8 minutes, twice) and then rehydrated through a series of graded ethanol solutions (100%, 96%, and 70%; 3-minute exposure to each). After peroxidase blocking with 3% H2O2 for 15 minutes, sections were rinsed with tap water. For antigen retrieval, all samples were incubated for 2.5 hours in a 65°C water bath with target citrate solution (pH, 6; then rinsed with PBS solution) and blocked for nonspecific binding with 1.5% goat serum (10% for 11βHSD1- and 11βHSD2-specific antibodies) to decrease background staining. Neck skin and SL samples were incubated overnight (approx 16 hours) at 4°C with the specific antibody against GCRs,f insulin,g 11βHSD1,h or 11βHSD2.i Prior to the study, a dilution series was performed and dilutions of PBS solution were used as follows: 1:100 for GCR, 11βHSD1, and 11βHSD2 staining and 1:200 for insulin staining. As positive controls, sections of horse ovaries were used for GCR staining,j sections of horse and rat pancreas were used for insulin staining, and sections of horse liver and horse kidneys were used for 11βHSD1 and 11βHSD staining15 (Supplementary Figure S1, available at http://avmajournals.avma.org/doi/suppl/10.2460/ajvr.79.2.199). As the negative control for each immunohistochemical stain, 1 sample of skin and SL, respectively, was prepared as described but without the addition of the specific antibody.
The next day, after a 5-minute rinse with PBS solution, all sections were incubated for 30 minutes with a secondary polyclonal horseradish peroxidase-conjugated anti-mouse antibody used in the dilution provided, and diaminobenzidinek was used for development. After another rinse with PBS solution for 5 minutes, nuclei were stained with hematoxylin, followed by rinses with tap water for 10 minutes, 96% alcohol for 2 minutes, 100% alcohol for 2 minutes twice, and xylol for 2 minutes twice. Slides were covered with a resinous mounting mediuml and a cover glass.
The distributions of the immunohistochemical stains in SL and neck skin sections were evaluated with light microscopy. Photographs of 2 randomly selected visual fields (100X magnification) in each section stained for GCRs, insulin, 11βHSD1, and 11βHSD2 were converted to grayscale images with software.c The range of grayscale values indicative of a positive response to the immunohistochemical stain was identified from the positive control slides, and the percentage of these values in randomly selected fields of view was determined. Furthermore, for comparison of the distribution of the immunohistochemical staining, similar percentages were calculated for areas of MFC and areas of SL fibers.
Statistical analysis
The 2 control horses that had been treated with dexamethasone were excluded from the statistical analysis. For each of the other horses, the mean of the sum of all scores of each of the 2 SLs (right forelimb and right hindlimb) was determined; as such, the maximum value was 18. Also, for each horse, the mean value of the 3 measured skin thickness was calculated. All numerical data (skin thickness values [in millimeters] and percentage of the immunohistochemical staining within each slide or MFC and SL fiber area) and ordinal data (histologic scores) were evaluated for normal distribution via the Kolmogorov-Smirnov testm and shown to be nonparametric. Therefore, differences between groups were tested with a Kruskal-Wallis test and post hoc testing according to Dunn-Bonferroni. Percentage data of MFC and SL fibers within the same SL sample were compared with a Wilcoxon matched pair test. Values of P < 0.05 were considered significant.
Results
For all of the results, data for the laminitic horses were within the range of the data for the other horses in their respective groups.
Case and control horses
Twelve horses with postmortem changes indicative of PPID were selected for the study. The horses with PPID were 16 to 25 years old; there were 5 mares and 7 geldings. Breeds included warmbloods (n = 9), Quarter Horse (1), Thoroughbred (1), and Lusitano (1). Nine of the horses also had clinical signs typical of PPID; in 8 of the 9 horses, signs were accompanied by high plasma ACTH concentration (range, 62 to 1,250 pg/mL) as determined by use of seasonally adjusted reference ranges (reference limits, 29 pg/mL in November through July and 47 pg/mL in August through October). The 3 remaining horses had no record of clinical signs of PPID or plasma ACTH concentration.
The control horses (for which data were used in statistical analyses) included 6 aged horses (age range, 14 to 23 years); there were 2 mares, 3 geldings, and 1 stallion. Breeds included warmbloods (n = 2), Standardbreds (2), Haflinger (1), and Shire Horse (1). There were 6 young control horses (age range, 2 to 9 years) comprising 2 mares and 4 geldings. Breeds included Standardbreds (n = 2), Quarter Horses (2), warmblood (1), and Thoroughbred (1). These horses had no clinical record or postmortem signs of PPID. An additional 2 horses without PPID (for which data were not used in statistical analyses) had been treated with dexamethasone. Both horses were 12-year-old mares. One horse was a warmblood (euthanized because of laminitis and treated with dexamethasone 1 day prior to euthanasia) and the other horse was an Icelandic horse (euthanized because of muscle atrophy and treated with dexamethasone 13 days prior to euthanasia).
Histologic findings
Pituitary glands of horses with PPID were macroscopically enlarged and roughly spherical (up to 3.6 cm in diameter; Table 1). These glands were graded histologically16 on a scale of 1 to 5 and assigned grades of 4 (pars intermedia adenomatous hyperplasia with microadenomas [1 to 5 mm in diameter]) or 5 (adenoma [> 5 mm in diameter] in the pars intermedia or pars anterior; Figure 1). The pituitary glands of all other horses had a normal macroscopic appearance and a histologic grade of 1 (ie, within normal limits).
Dimensions of the pituitary glands (measured at necropsy) of horses with and without PPID.
| Pituitary gland dimension | |||
|---|---|---|---|
| Group | Length (cm) | Height (cm) | Width (cm) |
| Horses with PPID (n = 11) | 2.3 (1.8–3.6) | 1.5 (0.9–2.5) | 1.9 (1.6–2.4) |
| Old horses without PPID (n = 6) | 2.1 (1.8–2.4) | 0.9 (0.7–1.2) | 1.9 (1.8–2.2) |
| Young horses without PPID (n = 6) | 1.8 (1.7–1.9) | 0.9 (0.7–1.2) | 1.8 (1.6–1.9) |
| Dexamethasone-treated non-PPID–affected horses (n = 2) | 1.9 and 2.2 | 0.8 and 0.9 | 1.8 and 2.5 |
Values are reported as median (range) or the actual measurement. Non-PPID–affected control horses (excluding the dexamethasone-treated non-PPID–affected horses) were grouped as old horses (≥ 14 years old) and young horses (≤ 9 years old). The 2 horses that had received treatment with dexamethasone were both 12-year-old mares; data from these horses were not used in any statistical analyses.
Representative photographs (A, C, and E) and photomicrographs (B, D, and F) of corresponding sections of pituitary glands removed from euthanized horses without (A and B) or with (C through F) PPID. A—Macroscopically normal appearance of the pituitary gland of a 16-year-old horse without PPID. Bar = 1 cm. B—Section from the gland in panel A. Notice the clear separation of the neurohypophysis and adenohypophysis. H&E stain; bar = 2 mm. C—Macroscopic appearance of an enlarged pituitary gland of a 21-year-old horse with PPID. Colloid-filled cysts and areas of bleeding are evident. Bar = 1 cm. D—Section from the gland in panel C. Microadenoma activity, bleeding, and colloid-filled cysts can be distinguished. H&E stain; bar = 2 mm. E—Macroscopic appearance of the pituitary gland of a 17-year-old horse with PPID. A macroadenoma and multifocal bleeding as well as compression of neurohypophysis and pars anterior are visible. Bar = 1 cm. F—Section from the gland in panel E. Multifocal bleeding is present, and the neurohypophysis is visible only as a narrow band displaced dorsally (upper left) with degenerative lesions and pigment accumulation. H&E stain; bar = 2 mm.
Citation: American Journal of Veterinary Research 79, 2; 10.2460/ajvr.79.2.199
Histologic examination of H&E-stained sections of SLs revealed significantly more degeneration in SLs of horses with PPID (Figure 2), compared with findings for young and old control horses (P = 0.000). Similar to the findings in our previous study,2 SLs from horses with PPID had greater thickness of interstitial septa and increased vascularization, whereas longitudinal arrangement of collagen fibers was reduced and there were inclusions of cartilage, extracellular matrix, and hemorrhage. In SL tissue, PA was detected with Alcian blue stain as well as safranin-O stain; staining intensity for PA did not differ significantly between these 2 staining methods. In horses with PPID, PA in SL tissue was significantly (P = 0.005) greater than that in any of the control groups. The PA was present between collagen fibers, within the interstitial septa, and surrounding blood vessels. There was no difference in total SL histologic score between young and old control horses, and the 2 control horses that had been treated with dexamethasone had SL conformation comparable to that of the young and old control horses (Figure 3). The H&E-stained neck skin sections in all groups appeared normal. Proteoglycan accumulation was detected in all neck skin samples with either Alcian blue or safranin-O stain. The PA in the neck skin samples was located in a physiological pattern (eg, in the hair follicles), similar to that detected following H&E staining. There were no significant differences in PA grade among groups. However, neck skin was significantly (P = 0.024) thinner in horses with PPID, compared with that in young control horses (Figure 4).
Representative photomicrographs of longitudinal midbody sections of the right forelimb SL with degenerative appearance from a euthanized PPID-affected horse. A—Notice the complete diffuse collagen arrangement (longitudinal collagen fiber arrangement, grade 3) with few tenocytes in parallel alignment and a high number of tenocytes without their normal fiber spindle shape and with more rounded nuclei. H&E stain; bar = 800 μm. B—With Alcian blue stain, PA is moderate and visible as diffuse blue staining between SL fibers (PA, grade 2). Bar = 800 μm. C—With safranin-O stain, PA is evident as diffuse red staining between all fibers (PA, grade 3).
Citation: American Journal of Veterinary Research 79, 2; 10.2460/ajvr.79.2.199
Scatterplot of the total histologic scores determined in SL samples of forelimbs (circles) and hind lmbs (triangles) obtained from each of 12 horses with PPID, 6 old (≥ 14 years old) horses without PPID, and 6 young (≤ 9 years old) horses without PPID. Individual horses are identified with the same colored circles and triangles on the same vertical line, and the mean of these 2 values was used for statistical analysis. Of each SL examined, 1 transverse and 1 longitudinal section stained with H&E stain were used for scoring of longitudinal arrangement of collagen fibers, matrix changes (eg, presence of chondrocytes or hemorrhage), and width of interstitial connective tissue septa as well as proliferation of vascular structures within the interstitial connective tissue septa. Two other longitudinal sections of each SL were stained with safranin-O stain and Alcian blue stain for evaluation of PAs. Findings for each of the aforementioned characteristics were graded by means of a grading score used in a previous study,2 wherein 0 = no pathological change, 1 = mild signs of degeneration, 2 = moderate signs of degeneration, and 3 = marked signs of degeneration. Overall histologic scores were calculated as the sum of these grading scores, with a possible maximum of 18 (ie, a score of 3 for 6 characterisitics [longitudinal arrangement of collagen fibers, matrix changes {eg, presence of chondrocytes or hemorrhage}, width of interstitial connective tissue septa, proliferation of vascular structures within the interstitial connective tissue septa, PAs stained with safranin-O stain, and PAs stained with Alcian blue stain]).
Citation: American Journal of Veterinary Research 79, 2; 10.2460/ajvr.79.2.199
Box-and-whisker plots of the thickness (mm) of neck skin samples collected from 12 horses with PPID, 6 old (≥ 14 years old) horses without PPID, and 6 young (≤ 9 years old) horses without PPID. Three serial sections of each skin sample were prepared on slides and stained with H&E stain, skin thickness was measured with imaging software at 3 different sites in each H&E-stained slide, and a mean value for each horse was calculated. The boundaries of each box represent the 25th and 75th percentiles, and the line within each box represents the median value. The asterisk and the circle represent outliers.
Citation: American Journal of Veterinary Research 79, 2; 10.2460/ajvr.79.2.199
Results of immunohistochemical analyses
The percentages of grayscale values equivalent to staining for GCRs in SL fibers and MFC were significantly (P = 0.000) greater in horses with PPID, compared with findings for young and old control horses (Figure 5). Although not significant, the percentages of grayscale values equivalent to staining for GCRs in neck skin were greater in horses with PPID, compared with findings for young and old control horses (Figure 6). Staining for GCRs was located in the nuclei and cytosol of SL fibers, muscle and fat cells within the SL, and epidermal cells, dermal fibroblasts, cells of hair outer root sheaths, and sweat glands in skin.
Box-and-whisker plots of GCR-specific staining (as a percentage of the overall surface determined by means of grayscale analysis) in the fibers (dark gray bars) and MFC (light gray bars) of SL samples collected from the 12 horses with PPID, 6 old (≥ 14 years old) horses without PPID, and 6 young (≤ 9 years old) horses without PPID. For immunohistochemical analysis, 4 longitudinal SL sections from both the right forelimb and the right hind limb were used from each horse. Sections were incubated overnight with the specific antibody against GCRs. The distribution of the immunohistochemical stain in SL sections was evaluated with light microscopy. Photographs of 2 randomly selected visual fields (100X magnification) in each section stained for GCRs were converted to grayscale images with software. The range of grayscale values indicative of a positive response to the immunohistochemical stain was identified from the positive control slides (sections of horse ovaries), and the percentage of these values in randomly selected fields of view was determined. See Figure 4 for key.
Citation: American Journal of Veterinary Research 79, 2; 10.2460/ajvr.79.2.199
Box-and-whisker plots of GCR-specific staining (as a percentage of the overall surface determined by means of grayscale analysis) in the neck skin samples collected from the 12 horses with PPID, 6 old (≥ 14 years old) horses without PPID, and 6 young (≤ 9 years old) horses without PPID. For immunohistochemical analysis, 4 serial neck sections were used from each horse. Sections were incubated overnight with specific antibody against GCRs. The distribution of the immunohistochemical stain in neck skin sections was evaluated with light microscopy. See Figures 4 and 5 for key.
Citation: American Journal of Veterinary Research 79, 2; 10.2460/ajvr.79.2.199
The results for the control horses that had been treated with dexamethasone were within the range of findings for the horses with PPID. Within the SLs of PPID-affected horses, GCRs were found in higher quantities in MFC than in SL fibers, but this difference was not significant (Figure 5). Among the SL fibers, nuclear staining for GCRs was variable in that a fiber with a GCR-stained nucleus was often adjacent to another fiber with an unstained nucleus (Figure 7). In skin sections, GCR-specific staining was preferentially seen in epidermis, dermal fibroblasts, and hair outer root sheaths (Figure 8). No positive staining for insulin was seen in any SL or neck skin sections from horses in any group. Perinuclear staining for 11pHSD1 was seen in SL fibers as well as in MFC of SLs. There was significantly more staining for 11pHSD1 in horses with PPID, compared with findings for young (P = 0.014) and old (P = 0.000) control horses (Figure 9). Staining percentages for 11pHSD1 in MFC and SL fibers were similar. Skin sections from horses with PPID had significantly higher amounts of 11pHSD1, compared with findings for young (P = 0.043) and old (P = 0.004) control horses, with staining for 11pHSD1 in epidermal cells, dermal fibroblasts, hair outer root sheaths, and glandular cells. Results of 11pHSD1 staining of SL and neck skin samples from the control horses that had been treated with dexamethasone were within the range of findings for horses with PPID. With regard to staining for 11PHSD2, there were no significant differences in skin sections or in SL sections among the groups of horses (Figure 10), and the staining percentages for 11PHSD2 in MFC and SL fibers were similar.
Representative photomicrographs of longitudinal midbody sections of the right hind limb SL from a euthanized PPID-affected horse following immunohistochemical staining for GCRs, 11βHSD1, or 11βHSD2. A—Section with GCR-specific staining. Brown staining indicates that GCRs are located in tenocyte nuclei. Variable receptor staining within SL fibers is also evident; stained tenocytes are visible adjacent to unstained tenocytes. High levels of staining were also seen in fibrocytes within the interstitial connective tissue septa, in endothelial cells in vascular structures of the SL, and in MFC. GCR-specific immunohistochemical stain; bar = 800 μm. B—Section with 11βHSD1-specific staining. Brown-red staining indicative of 11βHSD1 activity is present surrounding the nuclei of all SL components. Highest intensity is evident within the interstitial connective tissue septa, endothelial cells, and fibrocytes. 11βHSD1-specific immunohistochemical stain; bar = 800 μm. C—Section with 11 βHSD2-specific staining. The localization of staining indicative of 11βHSD2 activity is the same as that of the staining indicative of 11βHSD1 activity. 11βHSD2-specific immunohistochemical stain; bar = 800 μm.
Citation: American Journal of Veterinary Research 79, 2; 10.2460/ajvr.79.2.199
Representative photomicrographs of skin tissue samples from the left lateral aspect of the neck of a euthanized PPID-affected horse following immunohistochemical staining for GCRs, 11βHSD1, or 11βHSD2. A—Section with GCR-specific staining. Receptor staining can be seen in nuclei of epidermal keratinocytes, dermal fibroblasts, hair outer root sheaths, and glandular cells. GCR-specific immunohistochemical stain; bar = 2,000 μm. B—Section with 11βHSD1-specific staining. Brown-red staining indicative of 11βHSD1 activity is present surrounding the nuclei of cells, as perinuclear staining. 11βHSD1-specific immunohistochemical stain; bar = 2,000 μm. C—Section with 11βHSD2-specific staining. The localization of the brown-red staining indicative of 11βHSD2 activity is the same as that of the staining indicative of 11βHSD1 activity. 11βHSD2-specific immunohistochemical stain; bar = 2,000 μm.
Citation: American Journal of Veterinary Research 79, 2; 10.2460/ajvr.79.2.199
Box-and-whisker plots of 11βHSD1-specific staining (as a percentage of the overall surface determined by means of grayscale analysis) in the SL (A) and neck skin (B) samples collected from the 12 horses with PPID, 6 old (≥ 14 years old) horses without PPID, and 6 young (≤ 9 years old) horses without PPID. For immunohistochemical anaylsis, 4 serial skin sections and 4 longitudinal SL sections from both the right forelimb and the right hind limb were used from each horse. Sections were incubated overnight with the specific antibody against 11pHSD1. The distribution of the immunohistochemical stain in SL and neck skin sections were evaluated with light microscopy. Photographs of 2 randomly selected visual fields (100X magnification) in each section stained for 11βHSD1 were converted to grayscale images with software. The range of grayscale values indicative of a positive response to the immunohistochemical stain was identified from the positive control slides (sections of horse liver and kidneys), and the percentage of these values in randomly selected fields of view was determined. See Figure 4 for key.
Citation: American Journal of Veterinary Research 79, 2; 10.2460/ajvr.79.2.199
Box-and-whisker plots of 11βHSD2-specific staining (as a percentage of the overall surface determined by means of grayscale analysis) in the SL (A) and neck skin (B) samples collected from the 12 horses with PPID, 6 old (≥ 14 years old) horses without PPID, and 6 young (≤ 9 years old) horses without PPID. For immunohistochemical anaylsis, 4 serial skin sections and 4 longitudinal SL sections from both the right forelimb and the right hind limb were used from each horse. Sections were incubated overnight with the specific antibody against 11pHSD1. The distribution of the immunohistochemical stain in SL and neck skin sections were evaluated with light microscopy. Photographs of 2 randomly selected visual fields (100X magnification) in each section stained for 11βHSD2 were converted to grayscale images with software. The range of grayscale values indicative of a positive response to the immunohistochemical stain was identified from the positive control slides (sections of horse liver and kidneys), and the percentage of these values in randomly selected fields of view was determined. See Figure 4 for key.
Citation: American Journal of Veterinary Research 79, 2; 10.2460/ajvr.79.2.199
Discussion
The results of the present study indicated that horses with PPID had evidence of higher amounts of GCRs in SL samples and greater activity of 11pHSD1 in SL and neck skin samples, suggesting altered tissue cortisol distribution in those horses, compared with non-PPID–affected control horses. In SL tissue, the amount of GCRs and activity of 11pHSD1 were found to be high in horses with PPID, and findings for non-PPID-affected control horses treated with dexamethasone were similar. In horses with PPID, chronic inflammation and oxidative damage may develop in multiple organs,n and inflammation-associated upregulation of 11pHSD1 activity has been detected in chronically inflamed tissues, including skin, synovium, and bone marrow fibroblasts, in humans with rheumatoid arthritis.17 Contrary to this finding, no inflammatory cells were found in SL or neck skin tissue samples obtained from horses with PPID in the present study.
Proteoglycan accumulation is a pathological feature both in humans with Achilles tendinopathy and in horses with degenerative SL desmitis.18,19 Such PA was documented in horses with PPID in the present study by staining of SL tissue sections with safranin-O stain (which preferentially stains aggrecan-related proteoglycans and degradation products of aggrecan20) and Alcian blue stain (which binds to proteoglycans with glycosaminoglycan chains, such as decorin21). Structural integrity and normal mechanical function of tendons depend on the precise alignment of type I collagen, the most important structural component; this alignment is regulated by proteoglycans.21 In cases of tendinopathy, there is a 5-fold increase in the content of water-binding proteoglycans, such as aggrecan; this increase likely contributes to tissue swelling, a feature common to human tendinopathy22 and equine SL degeneration.1,13,18,23 The extracellular matrix, including collagen and proteoglycan molecules, has an important role in tendon remodeling as a response to changes in loading and mobilization. Mechanical tension (tensional load) increases the synthesis of decorin, whereas the production of the large proteoglycan aggrecan is stimulated in tendons by compression.21 In the present study, there were increases in both types of proteoglycans in SL tissue of horses with PPID, as indicated by the greater staining intensity in SL tissue sections from affected horses, compared with findings for sections from control horses, with both Alcian blue and safranin-O staining methods.
Skin thinning was evident in horses with PPID in the present study. Skin thinning is a clinical sign also associated with CD in humans24; other signs include central obesity, proximal muscle atrophy, insulin dysregulation, and tendon degeneration,3,5 similar to the signs of PPID in horses.2,n These clinical features are associated with cortisol excess; however, it is a paradox that horses with PPID have serum cortisol concentrations that are within reference interval most of the time.25 Recent evidence in people suggests that measurement of free cortisol concentration rather than total cortisol concentration in serum samples might more accurately reflect systemic cortisol activity,26 and veterinary researchers found free, unbound cortisol concentration to be significantly higher in PPID-affected horses, compared with results for healthy horses, independent of sex and age.7 Although 90% of plasma cortisol is bound to cortisolbinding globulin in healthy horses, the amount of free, unbound, biologically active cortisol seems to be elevated in horses with PPID,7 a possible reason for more availability of cortisol in peripheral tissues.
The tissue effect of glucocorticoids depends on the tissue-based conversion of inactive cortisone to active cortisol by 11βHSD1 and 11βHSD2,5 with the 11βHSD2 isoform predominantly expressed in mineralocorticoid-receptive tissues (eg, the kidneys, colon, and sweat glands) where it protects the mineralocorticoid receptor by inactivating cortisol that would otherwise activate it inappropriately.8 In contrast, the 11βHSD1 isoform is expressed in glucocorticoid target tissues, where it ensures adequate concentrations of cortisol to activate the GCRs. Dysfunction of these local controls on cortisol activity have been proposed to play a role in the pathophysiology of cardiovascular disease in humans.27 Key metabolic target tissues include adipose tissue and skeletal muscle,5,8 both of which are present in equine SLs.28 In the present study, the amount of GCRs and activity of 11βHSD1 were significantly increased in PPID-affected horses, compared with young and old non-PPID–affected control horses. Furthermore, within the SLs of PPID-affected horses, GCRs were found in higher quantities in MFC than in SL fibers (although this difference was not significant). Because glucocorticoids are known to reduce tensile strength,9,10,29 this may be a possible explanation for SL degeneration in PPID-affected horses. In humans with CD, systemic cortisol concentration is usually high30; downregulation of GCRs may therefore be induced by high concentration of circulating cortisol to maintain normal glucocorticoid homeostasis in cells.31 In skin samples of humans with CD and mice with experimentally induced CD, 11βHSD1 activity was found to be elevated; this was thought to be the underlying cause for the development of the clinical signs of CD.5 In horses aged 12 to 15 years affected by naturally occurring chronic laminitis of unknown origin for at least 6 months' duration and not screened for PPID, a similar elevation in 11βHSD1 activity was detected in lamellar and skin samples; this was also the case in a pony aged 20 years with pars intermedia adenoma of 2 to 3 years' duration.8 Unbound serum cortisol concentration is higher in PPID-affected horses than in control horses,7 whereas total serum cortisol concentration in PPID-affected horses and controls is similar.25 If the tissue-specific expression patterns of the different isoforms of GCRs detected in rodents and humans30 as well as the tissue-specific variability of isozymes of 11β-hydroxysteroid dehydrogenase documented in mice and humans15 are also present in horses, then cortisol dysregulation in a specific tissue may be a nonlinear effect of overall increased serum concentration of total or free cortisol, or both.
In the present study, 4 horses that had laminitis were included. Of those 4 horses, 1 had PPID (with recurring laminitis) and 2 were classified as old controls (1 with chronic laminitis and 1 with acute, postparturient laminitis); 1 was a non-PPID–affected horse with acute laminitis after a single dexamethasone treatment. In the present study, data for laminitic horses were within the range of the findings for the other horses in their groups. This was in contrast to 11βHSD1 activities in skin and lamellar tissue samples from horses with chronic and acute laminitis in another study,8 where 11βHSD1 activities were significantly elevated in acute laminitis cases and chronic cases had slightly higher 11βHSD1 activities than did healthy controls. In both that study8 and the study of the present report, the insulin dysregulation status of the horses was not assessed; therefore, it is possible that underlying insulin dysregulation may have caused laminitis in some of the horses of either study, and may be an explanation for the difference between the study results.
In the present study, 11βHSD1 activity in neck skin and SL samples from the old horses without PPID that had acute or chronic laminitis was comparable to findings for other old control horses. However, the PPID-affected horse with acute laminitis had 11βHSD1 activities in neck skin and SL samples that were within the range of findings for other horses with PPID, which were significantly higher than those for old control horses. The acutely laminitic horse that had received dexamethasone had 11βHSD1 activity in neck skin and SL samples similar to findings for horses with PPID. It is possible that naturally occurring laminitis in the horses of the present study was not associated with changes similar to those of laminitic horses in the aforementioned study,8 wherein only 1 horse with chronic laminitis had PPID and acute cases were young horses with experimentally induced laminitis8; furthermore, the tissues selected for investigation in that study8 differed from those used in the present study.
In immunohistochemical studies of mouse and human skin,5,15 11βHSD1 activity was detected in epidermal keratinocytes, dermal fibroblasts, and hair outer root sheaths. In the present study, sections of equine neck skin tissue had staining patterns for GCRs, 11βHSD1, and 11βHSD2 that were similar to those in sections of human skin.15 Also, there were high expressions of GCR and 11βHSD1 associated with a reduced skin thickness in neck skin tissue sections from horses with PPID, compared with findings for control horses.
Tendons of rats and humans without diabetes mellitus harbor a population of insulin-producing, pancreatic beta-cell–like cells.14 In rats with induced diabetes mellitus, these insulin-producing, pancreatic beta-cell–like cells were not present, and 5 days after induction of diabetes, tendon tensile strength was 39% lower than that in healthy individuals, suggesting an involvement of insulin-producing cells in tendinopathy. However, no insulin was detected in SLs of the horses used in the present study, despite the fact that sections of horse pancreas reacted positively with the insulin-specific mouse antibody at all dilutions tested (Supplementary Figure 1). Compared with pancreatic cells, a lower expression of insulin in tendon cells might be a possible reason for this lack of staining. Other antibodies used for immunohistochemical analyses in the present study were also not specific horse antibodies. The GCR-specific antibody had already been tested in sections of horse ovaries,j and ovarian tissue samples were used as a positive control in the present study. The 11βHSD1-specific antibody had been successfully used in reproductive tissue of male horses32 and in positive control sections of horse liver stained for 11βHSD1. The 11βHSD2-specific antibody has not been used in horse studies before, to our knowledge, but positive control sections of horse kidney tissue stained for 11βHSD2 in the present study. Because 11βHSD2 preferentially acts with mineralocorticoid receptors, additional staining for this type of receptor in the tissues investigated in the present study could have provided confirmation of this immunostaining. However, 11βHSD2 mRNA has been detected at low concentration in samples of ovine uterus, whereas no expression of mineralocorticoid receptor was found.33
The present study had several limitations. No samples other than SL, skin, and pituitary gland tissues were collected for further examination; thus, the ovarian cycle of the mares included in this study was not considered. Although this is a limitation of the study, a recent study7 of cortisol concentrations in horses of either sex and different ages with and without endocrine disease did not find any differences related to sex, even with consideration of the effect of seasons. Also, mares undergoing ovariectomy did not have differences in serum cortisol concentrations before and after surgery, which suggested no influence of the ovarian cycle on serum cortisol measurements.34 Furthermore, in the present study, there was a majority of geldings and stallions in all groups (7/12 horses with PPID, 4/6 old control horses, and 4/6 young control horses), which probably limited the influence of the ovarian cycle on the results. Another limitation of the study was that the control horse groups may have included very early cases of PPID, which were not detected because no in vivo testing was carried out. A more stringent testing prior to euthanasia would have been possible in a study involving live animals; it is conceivable that elimination of any horses with early PPID from the control groups might have resulted in even larger differences between PPID-affected and non-PPID–affected horses.
A negative effect of glucocorticoids on tendon fibroblasts has already been shown in human and canine tendon explants and cell cultures.9,10 Dexamethasone, as was administered to 2 non-PPID–affected horses in the present study, affects tenocyte viability, proliferation, collagen type I content, proteoglycan synthesis, and migration in cell culture.10 Tenocytes are responsible for the structural integrity of tendons including synthesis and maintenance of collagen and other components of extracellular matrix. Suppression of tenocyte activity by glucocorticoids and the subsequent altered matrix synthesis and modulation, with disturbance of proteoglycan production in particular, may lead to altered integrity and strength of tendons.9 Because the SL is in many ways similar to a tendon and also contains mainly tenocytes, tissue cortisol dysregulation could explain the degenerative histologic appearance of SL tissue in horses with PPID in the present study. Most degenerative SL lesions and most 11βHSD1-specific staining were in the same location (in close proximity to vessels in the interstitial septa), which supports a connection. Even though no inflammatory cells were found in SL or neck skin samples of the PPID-affected horses in the present study, we cannot rule out that the higher expression of 11βHSD1 might have been a consequence of the detected SL lesions rather than of the inciting cause, given that higher 11βHSD1 activity was found in human fibroblasts in response to inflammatory stimuli.17
With only 2 dexamethasone-treated control horses investigated, no conclusions regarding the effects of dexamethasone on equine neck skin and SL can be drawn. However, similar to findings for horses with PPID, high activity of 11βHSD1 and a high amount of GCRs were found in samples from those 2 horses; this was consistent with elevated 11βHSD1 staining in human skin and in outer hair follicle root sheath cells in murine skin after glucocorticoid treatment.15 These findings justify the future investigation of SL tissues in a larger number of horses treated with glucocorticoids to evaluate potential detrimental effects on tendon tissue.
Results of the present study provided evidence of an association of changes in protein expression in the SLs of horses with PPID, and this may suggest peripheral tissue cortisol dysregulation in SLs of such horses. Further studies of the causality of these associations are needed. However, it is recommended that horses with SL degeneration should be screened for PPID and that horses with PPID should be screened for SL degeneration to allow timely treatment with corrective trimming or shoeing or appropriate medication.
Acknowledgments
This manuscript represents a portion of the doctoral thesis submitted by Sina Hofberger to the University of Veterinary Medicine, Vienna as partial fulfillment of the requirements for a Doctorate in Veterinary Medicine degree.
Supported by the Austrian Science Fund (FWF): P22598.
None of the authors of this paper had a financial or personal relationship with other people or organizations that could have inappropriately influenced or biased the content of the paper.
The authors thank Professor Peter Böck for instruction and evaluation of histological samples; Claudia Höchsmann, Brigitte Machac, and Florian Schneckenleitner for technical assistance; and Lyndsey Boswell for sample staining.
ABBREVIATIONS
| CD | Cushing disease |
| GCR | Glucocorticoid receptor |
| MFC | Muscle and fat components |
| PA | Proteoglycan accumulation |
| PPID | Pituitary pars intermedia dysfunction |
| SL | Suspensory ligament |
| 11βHSD1 | 11β hydroxysteroid dehydrogenase type 1 |
| 11βHSD2 | 11β hydroxysteroid dehydrogenase type 2 |
Footnotes
Thiopental, Inresa, Freiburg im Breisgau, Germany.
T61, Intervet, Vienna, Austria.
ImageJ, version 1.47, National Institute of Health, Bethesda, Md.
Reichert-Jung, Wien, Austria.
Nikon Ds-Fi1 camera and NIS-Element software, Nikon Instruments Inc, Amsterdam, The Netherlands.
Monoclonal mouse antibody, AB2768, Abcam, Milton, Cambridge, England.
Monoclonal mouse antibody, AB6995, Abcam, Milton, Cambridge, England.
Polyclonal rabbit antibody, AB39364, Abcam, Milton, Cambridge, England.
Polyclonal rabbit antibody, AB115696, Abcam, Milton, Cambridge, England.
Scarlet D, Walter I, Aurich C. Glucocorticoid receptors are expressed in ovaries of newborn and adult female horses (abstr). Reprod Fertil Dev 2014;27:142.
Sigma-Aldrich, St Louis, Mo.
DPX, Fluka, Sigma-Aldrich, St Louis, Mo.
SPSS statistics, version 19, SPSS/IBM, Armonk, NY.
Morgan R, Hadoke P, Walker B, et al. Abnormal glucocorticoid metabolism in horses with metabolic syndrome (abstr). Endocrine Abstracts 2014:34.
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