Equine Veterinary Journal ISSN 0425-1644 DOI: 10.1111/evj.12450
Lamellar pathology in horses with pituitary pars intermedia dysfunction N. P. KARIKOSKI*, J. C. PATTERSON-KANE†, E. R. SINGER‡, D. McFARLANE§ and C. M. McGOWAN‡ Department of Equine and Small Animal Medicine, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland † Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK ‡ Institute of Ageing and Chronic Disease, Faculty of Health and Life Sciences, University of Liverpool, Liverpool, UK § Department of Physiological Sciences, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, Oklahoma, USA. *Correspondence email: [email protected] Dr. J.C. Patterson-Kane’s present address is: IDEXX Laboratories, Inc., West Sacramento, California, USA. Received: 13.08.14; Accepted: 07.04.15
Summary Reasons for performing study: Hoof lamellar pathology in horses with pituitary pars intermedia dysfunction (PPID) has not been described previously. Objectives: To describe the histomorphometry and pathological lesions in hoof lamellar tissue of animals that had PPID with or without concurrent laminitis, with reference to age-matched controls. We hypothesised that lamellar lesions consistent with laminitis would be associated with PPID, even in animals without current or historical laminitis. Study design: Prospective case–control study. Methods: Mid-dorsal hoof histological sections were obtained post mortem from the forelimbs of 16 PPID-affected animals either with (n = 6) or without laminitis (n = 10) and 10 age- and breed-matched controls. Sections were examined by a blinded veterinary pathologist. The length and width of 10 primary epidermal lamellae were measured using image analysis software. The morphology and pathology of primary and secondary epidermal lamellae were then typed or graded in axial, middle and abaxial regions. Fasting serum insulin, plasma adrenocorticotropin and blood glucose concentration were measured from blood samples taken prior to euthanasia. Results: All animals with PPID and laminitis had fasting hyperinsulinaemia (median 74.1 miu/l, interquartile range 49.9–349.5 miu/l) whereas PPID animals without laminitis had serum insulin concentrations below the upper limit of the reference range (29 ng/l from November to July and >47 ng/l from August to October) [24] and clinical signs. In either situation, clinical signs always included hypertrichosis plus one or more of muscle wastage, hyperhidrosis, polyuria/polydipsia or abnormal fat distribution. The PPID animals were further divided into those with or without current or historical laminitis. Laminitis was diagnosed by an equine veterinarian if there was: lameness involving both fore or all 4 hooves associated with weight shifting, a ‘saw horse’ stance, and/or reluctance to move; increased digital arterial pulses; and sensitivity to hoof-tester pressure applied at the toe(s). The onset of laminitis was recorded as the date the clinical signs were first detected by the owner or the responsible veterinarian. Divergent rings and flat or convex soles were also recorded. When indicated, radiographs confirmed evidence of lamellar disruption including distal phalanx (DP) sinking or rotation. Following euthanasia, the hoof of the right forefoot was bisected sagittally and examined further, including inspection of the outer wall for divergent rings and lack of parallel orientation of the dorsal surface of the DP with the overlying hoof wall [25]. Body condition was scored for all the animals using a 0–5 body condition score (BCS) [26].
Laboratory assays Where possible, the following laboratory assays were performed on the horses and ponies included in the study: plasma ACTH concentration, fasting serum insulin concentration and fasting blood glucose concentration. Blood samples for ACTH measurement were not taken during periods of stress or pain, or from abattoir animals, to reduce the possibility of stress-related increases in plasma ACTH concentrations. Blood samples for ACTH measurement were collected in 10 ml plastic EDTA tubes (Vacuette, EDTA K3)a. The plasma was separated and frozen to 20°C within 1 h. Plasma was sent frozen to the testing laboratory, where ACTH analysis was performed using a chemiluminescent Equine Veterinary Journal 48 (2016) 472–478 © 2015 EVJ Ltd
Lamellar pathology in horses with PPID
immunoassay (Immulite)b,c. Blood samples for insulin testing were collected in 6 ml serum sample tubes following at least 4 h of fasting (Vacuette, 2 serum clot activator)a. The samples were centrifuged and the serum separated within 3 h and frozen to 20°C. Samples were sent frozen to the laboratory where the insulin analysis was performed using a chemiluminescent immunoassay (Immulite)b,c. Blood samples for glucose measurement were collected in 2 ml plastic lithium heparin tubes following at least 4 h of fasting (Vacuette)a. Glucose concentration was measured immediately after blood collection with a glucometer (Ascensia Contour or AlphaTRAK)d,e.
Lamellar sample collection The lamellar samples were collected as in a previous study [20]. Briefly, for each horse/pony, the distal aspect of the right forelimb was disarticulated at the metacarpophalangeal joint within 1 h of death and a sagittal slice (1 cm thick) taken of the entire hoof using a band saw. A square segment was taken from the mid-point of the dorsal hoof wall, and 5 9 5 9 5 mm blocks were cut from it, with a transverse orientation. These blocks were fixed in 4% paraformaldehyde for 24–72 h followed by excision of the hoof wall (to approximately 1 mm from the abaxial margin of the lamellar tissue) and phalangeal bone. The tissue specimens were processed by routine methods for histology and embedded in paraffin wax.
Histomorphometry and categorisation of lamellae Sections were stained using haematoxylin and eosin for examination by a blinded pathologist (J.C.P.K.) or periodic acid Schiff for morphometry (N.P.K.). For primary epidermal lamellar (PEL) typing and measurement, digital images were taken at 409 magnification using image capture software (Cell^D version 2.8)f and a light microscope-attached digital camera (Olympus DP71)g and merged (to image the complete PEL for each specimen; Adobe Photoshop CS5 version 12.0 9 64)h. Ten PEL were randomly selected (http://www.randomizer.org/form.html) and marked in the images for each animal. For secondary epidermal lamellar (SEL) typing, images were taken at 2009 magnification from axial, middle and abaxial regions of each of the 10 randomly selected PEL (10%, 50% and 90% of the length of the PEL, respectively). Measurements of PEL length (PELL) were made as described previously [19]. A line was drawn from the axial to abaxial extent of each of the selected PEL using a drawing tool (Wacom Bamboo)i and image analysis computer software (ImageJ version 1.4.3)j. The width of each PEL (PELW) was measured halfway along the PELL measurement line; this width measurement extended from the bases of SEL on one side to the bases of the SEL on the other. For PEL and SEL typing, previously documented systems were used [20,27]. The PEL type was defined as standard (straight) or curved (with 2 or more bends), and bifurcations were reported when present. The SEL types were defined as standard, tapered, club-shaped, suprabasal layer hyperplasia, bifurcated, fused, separated or keratinised. Morphological types and pathology of the axial and abaxial regions of the PEL were also classified according to a previously defined system [20,27]: the axial region (tip) type as standard, tapered, sharp or bifurcated; and the abaxial region (base) as standard, sharp, bifurcated, ‘proliferative’ (epidermal cells bridging dermal tissue), separated (i.e. epidermal and dermal lamellae had torn apart) or keratinised (dyskeratosis of the bridging epidermal cells). The degree of abaxial dyskeratosis (increased keratinisation), i.e. in the ‘cap horn’ region, was graded as normal, slightly (1–10 extra layers), moderately (10–30 layers) or markedly increased (>30 layers). The degree of epidermal tissue bridging a primary dermal lamella at its abaxial aspect (i.e. joining the adjacent PEL) was defined as none, mild (1–2 epithelial bridges), moderate (>2 epithelial bridges) or extensive (abaxial region completely filled with epidermal cells). Lakes within the cap horn or bridging abaxial epidermal tissue were defined as small to large cavities that were either empty (probably due to processing artefact) or filled with homogeneous eosinophilic (proteinaceous) material. Epithelial islands were noted in some sections (as previously described) [20], and these were defined as isolated round or ovoid structures adjacent to the PEL; their presence in the different regions was noted (abaxial,
473
Lamellar pathology in horses with PPID
N. P. Karikoski et al.
TABLE 1: Sex distribution and median (interquartile range) age, body condition score (BCS), fasting serum insulin concentration, basal plasma adrenocorticotropin (ACTH) concentration and fasting blood glucose concentration in control animals (n = 10), animals with pituitary pars intermedia dysfunction (PPID) without laminitis (n = 10) and animals with PPID and laminitis (n = 6) Sex: mares/ geldings/ stallions
Age (years)
BCS (1–5)
Serum insulin (miu/l)
Plasma ACTH (pg/ml)
Glucose (mmol/l)
Control animals (n = 10)
3/7/0
16.5 (12.0–20.0)
3.0 (2.5–3.6)
2.1 (2.0–3.7)
23.0 (16.8–27.6; n = 5)
4.9 (4.3–5.5; n = 5)
PPID without laminitis (n = 10)
6/4/0
22.0 (20.0–24.8)†
2.5 (2.0–3.0)
2.0 (2.0–3.4)
106.1 (67.5–139.8; n = 10)†
4.7 (4.3–5.3; n = 10)
PPID and laminitis (n = 6)
5/1/0
19.5 (17.8–21.5)
4.0 (4.0–4.5)*
74.1 (49.9–349.5)*
70.5 (33.6–250.0; n = 5)
5.4 (4.3–6.0; n = 4)
Breeds Mixed-breed pony n = 5; Finnhorse n = 1; Standardbred n = 2; Warmbloods n = 2 Warmblood n = 2; Standardbred n = 2; Quarter Horse n = 2; Appaloosa n = 1; mixedbreed pony n = 1; Thoroughbred n = 1; Finnhorse n = 1 Mixed-breed pony n = 2; Russ pony n = 1; Welsh section A n = 1; Finnhorse n = 1; cob n = 1
*P
Lamellar pathology in horses with pituitary pars intermedia dysfunction N. P. KARIKOSKI*, J. C. PATTERSON-KANE†, E. R. SINGER‡, D. McFARLANE§ and C. M. McGOWAN‡ Department of Equine and Small Animal Medicine, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland † Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK ‡ Institute of Ageing and Chronic Disease, Faculty of Health and Life Sciences, University of Liverpool, Liverpool, UK § Department of Physiological Sciences, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, Oklahoma, USA. *Correspondence email: [email protected] Dr. J.C. Patterson-Kane’s present address is: IDEXX Laboratories, Inc., West Sacramento, California, USA. Received: 13.08.14; Accepted: 07.04.15
Summary Reasons for performing study: Hoof lamellar pathology in horses with pituitary pars intermedia dysfunction (PPID) has not been described previously. Objectives: To describe the histomorphometry and pathological lesions in hoof lamellar tissue of animals that had PPID with or without concurrent laminitis, with reference to age-matched controls. We hypothesised that lamellar lesions consistent with laminitis would be associated with PPID, even in animals without current or historical laminitis. Study design: Prospective case–control study. Methods: Mid-dorsal hoof histological sections were obtained post mortem from the forelimbs of 16 PPID-affected animals either with (n = 6) or without laminitis (n = 10) and 10 age- and breed-matched controls. Sections were examined by a blinded veterinary pathologist. The length and width of 10 primary epidermal lamellae were measured using image analysis software. The morphology and pathology of primary and secondary epidermal lamellae were then typed or graded in axial, middle and abaxial regions. Fasting serum insulin, plasma adrenocorticotropin and blood glucose concentration were measured from blood samples taken prior to euthanasia. Results: All animals with PPID and laminitis had fasting hyperinsulinaemia (median 74.1 miu/l, interquartile range 49.9–349.5 miu/l) whereas PPID animals without laminitis had serum insulin concentrations below the upper limit of the reference range (29 ng/l from November to July and >47 ng/l from August to October) [24] and clinical signs. In either situation, clinical signs always included hypertrichosis plus one or more of muscle wastage, hyperhidrosis, polyuria/polydipsia or abnormal fat distribution. The PPID animals were further divided into those with or without current or historical laminitis. Laminitis was diagnosed by an equine veterinarian if there was: lameness involving both fore or all 4 hooves associated with weight shifting, a ‘saw horse’ stance, and/or reluctance to move; increased digital arterial pulses; and sensitivity to hoof-tester pressure applied at the toe(s). The onset of laminitis was recorded as the date the clinical signs were first detected by the owner or the responsible veterinarian. Divergent rings and flat or convex soles were also recorded. When indicated, radiographs confirmed evidence of lamellar disruption including distal phalanx (DP) sinking or rotation. Following euthanasia, the hoof of the right forefoot was bisected sagittally and examined further, including inspection of the outer wall for divergent rings and lack of parallel orientation of the dorsal surface of the DP with the overlying hoof wall [25]. Body condition was scored for all the animals using a 0–5 body condition score (BCS) [26].
Laboratory assays Where possible, the following laboratory assays were performed on the horses and ponies included in the study: plasma ACTH concentration, fasting serum insulin concentration and fasting blood glucose concentration. Blood samples for ACTH measurement were not taken during periods of stress or pain, or from abattoir animals, to reduce the possibility of stress-related increases in plasma ACTH concentrations. Blood samples for ACTH measurement were collected in 10 ml plastic EDTA tubes (Vacuette, EDTA K3)a. The plasma was separated and frozen to 20°C within 1 h. Plasma was sent frozen to the testing laboratory, where ACTH analysis was performed using a chemiluminescent Equine Veterinary Journal 48 (2016) 472–478 © 2015 EVJ Ltd
Lamellar pathology in horses with PPID
immunoassay (Immulite)b,c. Blood samples for insulin testing were collected in 6 ml serum sample tubes following at least 4 h of fasting (Vacuette, 2 serum clot activator)a. The samples were centrifuged and the serum separated within 3 h and frozen to 20°C. Samples were sent frozen to the laboratory where the insulin analysis was performed using a chemiluminescent immunoassay (Immulite)b,c. Blood samples for glucose measurement were collected in 2 ml plastic lithium heparin tubes following at least 4 h of fasting (Vacuette)a. Glucose concentration was measured immediately after blood collection with a glucometer (Ascensia Contour or AlphaTRAK)d,e.
Lamellar sample collection The lamellar samples were collected as in a previous study [20]. Briefly, for each horse/pony, the distal aspect of the right forelimb was disarticulated at the metacarpophalangeal joint within 1 h of death and a sagittal slice (1 cm thick) taken of the entire hoof using a band saw. A square segment was taken from the mid-point of the dorsal hoof wall, and 5 9 5 9 5 mm blocks were cut from it, with a transverse orientation. These blocks were fixed in 4% paraformaldehyde for 24–72 h followed by excision of the hoof wall (to approximately 1 mm from the abaxial margin of the lamellar tissue) and phalangeal bone. The tissue specimens were processed by routine methods for histology and embedded in paraffin wax.
Histomorphometry and categorisation of lamellae Sections were stained using haematoxylin and eosin for examination by a blinded pathologist (J.C.P.K.) or periodic acid Schiff for morphometry (N.P.K.). For primary epidermal lamellar (PEL) typing and measurement, digital images were taken at 409 magnification using image capture software (Cell^D version 2.8)f and a light microscope-attached digital camera (Olympus DP71)g and merged (to image the complete PEL for each specimen; Adobe Photoshop CS5 version 12.0 9 64)h. Ten PEL were randomly selected (http://www.randomizer.org/form.html) and marked in the images for each animal. For secondary epidermal lamellar (SEL) typing, images were taken at 2009 magnification from axial, middle and abaxial regions of each of the 10 randomly selected PEL (10%, 50% and 90% of the length of the PEL, respectively). Measurements of PEL length (PELL) were made as described previously [19]. A line was drawn from the axial to abaxial extent of each of the selected PEL using a drawing tool (Wacom Bamboo)i and image analysis computer software (ImageJ version 1.4.3)j. The width of each PEL (PELW) was measured halfway along the PELL measurement line; this width measurement extended from the bases of SEL on one side to the bases of the SEL on the other. For PEL and SEL typing, previously documented systems were used [20,27]. The PEL type was defined as standard (straight) or curved (with 2 or more bends), and bifurcations were reported when present. The SEL types were defined as standard, tapered, club-shaped, suprabasal layer hyperplasia, bifurcated, fused, separated or keratinised. Morphological types and pathology of the axial and abaxial regions of the PEL were also classified according to a previously defined system [20,27]: the axial region (tip) type as standard, tapered, sharp or bifurcated; and the abaxial region (base) as standard, sharp, bifurcated, ‘proliferative’ (epidermal cells bridging dermal tissue), separated (i.e. epidermal and dermal lamellae had torn apart) or keratinised (dyskeratosis of the bridging epidermal cells). The degree of abaxial dyskeratosis (increased keratinisation), i.e. in the ‘cap horn’ region, was graded as normal, slightly (1–10 extra layers), moderately (10–30 layers) or markedly increased (>30 layers). The degree of epidermal tissue bridging a primary dermal lamella at its abaxial aspect (i.e. joining the adjacent PEL) was defined as none, mild (1–2 epithelial bridges), moderate (>2 epithelial bridges) or extensive (abaxial region completely filled with epidermal cells). Lakes within the cap horn or bridging abaxial epidermal tissue were defined as small to large cavities that were either empty (probably due to processing artefact) or filled with homogeneous eosinophilic (proteinaceous) material. Epithelial islands were noted in some sections (as previously described) [20], and these were defined as isolated round or ovoid structures adjacent to the PEL; their presence in the different regions was noted (abaxial,
473
Lamellar pathology in horses with PPID
N. P. Karikoski et al.
TABLE 1: Sex distribution and median (interquartile range) age, body condition score (BCS), fasting serum insulin concentration, basal plasma adrenocorticotropin (ACTH) concentration and fasting blood glucose concentration in control animals (n = 10), animals with pituitary pars intermedia dysfunction (PPID) without laminitis (n = 10) and animals with PPID and laminitis (n = 6) Sex: mares/ geldings/ stallions
Age (years)
BCS (1–5)
Serum insulin (miu/l)
Plasma ACTH (pg/ml)
Glucose (mmol/l)
Control animals (n = 10)
3/7/0
16.5 (12.0–20.0)
3.0 (2.5–3.6)
2.1 (2.0–3.7)
23.0 (16.8–27.6; n = 5)
4.9 (4.3–5.5; n = 5)
PPID without laminitis (n = 10)
6/4/0
22.0 (20.0–24.8)†
2.5 (2.0–3.0)
2.0 (2.0–3.4)
106.1 (67.5–139.8; n = 10)†
4.7 (4.3–5.3; n = 10)
PPID and laminitis (n = 6)
5/1/0
19.5 (17.8–21.5)
4.0 (4.0–4.5)*
74.1 (49.9–349.5)*
70.5 (33.6–250.0; n = 5)
5.4 (4.3–6.0; n = 4)
Breeds Mixed-breed pony n = 5; Finnhorse n = 1; Standardbred n = 2; Warmbloods n = 2 Warmblood n = 2; Standardbred n = 2; Quarter Horse n = 2; Appaloosa n = 1; mixedbreed pony n = 1; Thoroughbred n = 1; Finnhorse n = 1 Mixed-breed pony n = 2; Russ pony n = 1; Welsh section A n = 1; Finnhorse n = 1; cob n = 1
*P
