Chapter 7.1

Comparison of hair follicle histology between horses with pituitary pars intermedia dysfunction and excessive hair growth and normal aged horses

Marie Innerå*,¹, Annette D. Petersen*, Danielle R. Desjardins†^,2, Barbara A. Steficek†, Edmund J. Rosser Jr*, Harold C. Schott II‡

*Department of Small Animal Clinical Sciences, †Diagnostic Center for Population and Animal Health and ‡Department of Large Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing, MI 48824, USA

Correspondence: Annette D. Petersen, Department of Small Animal Clinical Sciences, Veterinary Medical Center, 736 Wilson Road, Room D208, Michigan State University, East Lansing, MI 48824, USA. E-mail: petersen@cvm.msu.edu

Background – Pituitary pars intermedia dysfunction (PPID) in older equids is commonly recognized by a long hair coat that fails to shed.

Objective – The aim of this study was to compare hair follicle stages in PPID-affected horses with excessively long hair coats with the stages of normal aged horses (controls) and to compare hair follicle stages in PPID-affected horses after 6 months of treatment with pergolide mesylate with those of control horses.

Animals – Eight PPID-affected horses and four normal, age-matched, control horses.

Methods – Skin biopsies were collected from the neck and rump of PPID-affected and control horses. A diagnosis of PPID was established based on hair coat changes and supportive overnight dexamethasone suppression test results. Skin biopsies were repeated after 6 months of treatment with pergolide. The number of hair follicles in anagen (A) or telogen (T) was counted for each skin biopsy using transverse sections.

Results – Pretreatment biopsies had a greater percentage of A follicles (neck 96%, rump 95%) and a lower percentage of T follicles (neck 4%, rump 5%) in PPID-affected horses than in control horses (A, neck 15%, rump 25%; and T, neck 85%, rump 75%). After treatment with pergolide, all PPID-affected horses had improved shedding, and the percentages of A follicles (neck 69%, rump 70%) and T follicles (neck 31%, rump 30%) were not different from untreated control horses (A, neck 68%, rump 82%; and T, neck 32%, rump 18%).

Conclusions – These findings document that excessive hair growth (hypertrichosis) in PPID-affected horses is due to persistence of hair follicles in A. Furthermore, treatment with pergolide improved shedding and reduced the percentage of A follicles in PPID-affected horses.

Introduction

Pituitary pars intermedia dysfunction (PPID), also known as equine Cushing’s disease, is the most common endocrinopathy of equids.^1,2 This disease increases in prevalence with age and may affect 15–20% of equids over 15 years of age.³ Furthermore, a recent report documented clinical findings consistent with PPID in nearly 40% of equids over 30 years of age.⁴

Several clinical signs are observed in PPID-affected equids; a pathognomonic sign in over 80% of cases is a long and often curly hair coat that fails to shed.¹ Hair coat changes with PPID typically progress over several years. Initial changes include delayed shedding and persistence of long hairs under the jaw, along the jugular grooves and ventral neck, and on the palmar and plantar aspects of the limbs. End-stage PPID-affected equids have a generalized, long and shaggy hair coat that fails to shed regardless of season or climate. Other dermatological problems may include excessive or decreased sweating and recurrent pyoderma, notably dermatophilosis.^1,2 Nondermatological clinical signs can include lethargy, muscle wasting, regional fat deposits, polyuria and polydipsia, recurrent infections and chronic laminitis.^1,2

The excessively long hair coat of PPID-affected horses has been described to be caused by hair follicle arrest in anagen (A) or telogen (T);^5,6 however, no data have been presented to support these opposing claims. An equine dermatology textbook stated: ‘To the authors’ knowledge, no studies have been published concerning the hair follicle stages from horses with PPID’.⁷ In our experience, trichograms from horses with PPID reveal hairs in A; furthermore, these hairs are difficult to epilate. Consequently, we hypothesize that excessive hair growth in PPID-affected horses is due to a persistence of hair follicles in the A stage.

The treatment of choice for PPID-affected equids is pergolide mesylate.^8–11 Pergolide is a dopamine agonist that activates dopamine type 2 receptors on pars intermedia melanotrophs, leading to decreased production of pro-opiomelanocortin and pro-opiomelanocortin-derived peptides responsible for the varied clinical signs of PPID. A recent open field trial of pergolide (Prascend™ 1 mg tablets; Boehringer-Ingelheim Vetmedica, Inc., St Joseph, MO, USA) reported improvement in hair coat in 99 of 111 (89%) PPID-affected equids after 6 months of treatment.¹² We hypothesize that the percentages of A and T stages of hair follicles will be similar in PPID-affected horses post-treatment and normal aged horses.

Materials and methods

All procedures performed were approved by the Animal Care and Use Committee of Michigan State University and included owner consent.

Animals

The affected horses were eight horses with PPID, five mares and three geldings, ranging from 20 to 29 years of age (mean 24 years). The group included two Morgan horses, two quarter horses, one Arabian and three mixed breed horses. The eight PPID-affected horses were either client owned (n = 5) or had been donated (n = 3) to Michigan State University and were enrolled in an open field trial to assess the efficacy of pergolide mesylate (Prascend™ 1 mg tablets; Boehringer-Ingelheim Vetmedica, Inc.) for the control of clinical signs of PPID.

The diagnosis of PPID was confirmed by clinical signs (characteristic excessively long hair coats) and overnight dexamethasone suppression test (ODST) results (failure of endogenous cortisol to suppress below 1 μg/dL 19 h after intramuscular administration of 40 μg/kg dexamethasone).¹³ Normal controls were four horses, three mares and one gelding, ranging in age from 21 to 29 years (mean 25 years). The group included two standardbred horses, one quarter horse and one thoroughbred. The aged control horses were reported to have had normal hair coat sheds the previous spring, normal winter hair coats at the study start, and normal ODST results (nonsupportive of PPID). These horses were all donated animals with similar husbandry to the donated PPID-affected horses. During the 6 month treatment period, all 12 horses were housed at pasture, with supplemental hay feeding until pasture grass was available (either at Michigan State University or at the home pasture). All horses had spent their entire lives in the northern hemisphere.

Sample collection

The hair coat was clipped from a 5 cm × 5 cm area of skin on the left side of the neck and a site overlying the left gluteal region. Skin biopsy sites were infiltrated subcutaneously with 1 mL of lidocaine HCl (APP Pharmaceuticals LCC, Schaumburg, IL, USA), and two 8 mm skin punch biopsy samples (Acu-Punch^® Acuderm Inc., Fort Lauderdale, FL, USA) were obtained, one from each site. Samples were fixed in 10% neutral buffered formalin and stored at room temperature until all samples were collected. Specimens were embedded in paraffin and sectioned in a routine longitudinal (vertical) fashion. In addition, biopsies were also sectioned in a transverse (horizontal) plane parallel to the surface of the epidermis, at the level of, or slightly below, entry of the sebaceous duct in the mid-dermis (‘Swiss cheese’ cut).¹⁴ The correct level was approximated to reflect the normal sebaceous gland dermal anatomical placement within the upper one-third of the dermis. Five-micrometre-thick sections were mounted on slides and stained with haematoxylin and eosin. Additional deeper or more superficial sections of the paraffin blocks were made as needed to achieve cross-sectioning at the level of the follicular infundibulum–isthmus junction. Slides were randomly numbered to blind the investigators during histological examination.

Clinical evaluation

For the open field trial, hair coat changes were scored on a 0–3 scoring system, as follows: 0 = normal, no unusual hair growth; 1 = regional hair coat changes, long hair growth restricted to discrete areas (lower jaw, jugular area, and palmar or plantar aspects of limbs); 2 = generalized hair coat changes (slightly to moderately long hair coat that fails to shed out as in previous years); and 3 = severe hair coat changes (severely long and/or curly hair coat over the entire body that fails to shed). All eight PPID-affected horses were enrolled between 1 November and 31 January and had a hair coat score of ‘3’ when pretreatment skin biopsy samples were collected. The four control horses had a hair coat score of ‘0’, and skin biopsy samples were collected during the same time period.

Pergolide treatment

The PPID-affected horses were treated with pergolide (2 μg/kg, orally, once daily). They were re-evaluated by clinical examination and an ODST after 3 and 6 months of treatment. If ODST results remained supportive of PPID after 3 months of treatment, the dose of pergolide was increased to 4 μg/kg, orally, once daily. At the 6 month evaluation (between 1 May and 31 July), a second set of skin biopsy samples from the neck and rump were collected from sites adjacent to the original skin biopsy sites. These biopsy samples were processed as previously described.

Histological examination

Longitudinal (vertical) sections were initially evaluated at low- (×4) and high-power magnification (×20 and ×40) to assess general histological features, including inflammation. Transverse sections were subsequently evaluated under low-power magnification (×4) independently by two investigators (M.I. and A.D.P.). Using a standard light microscope equipped with a 1 mm² optical grid reticule, the number of hair follicles within the same grid area was counted by each investigator, and the growth stage (A or T) was classified for each hair follicle. Criteria previously described for morphological evaluation of human scalp¹⁴ and canine hair follicles¹⁵ were used to classify equine hair follicle growth stages. Anagen follicles were characterized by a hair shaft surrounded by a well-defined inner root sheath, presence of trichohyaline granules in the inner root sheath, and absence of tricholemmal keratinization (Figure 1). Telogen follicles were characterized by absence of a well-defined inner root sheath, tricholemmal keratinization, and volumetric reduction of the outer root sheath (Figure 2). As catagen follicles are difficult to identify on transverse sections and are typically few in number, when suspected they were counted as T, because catagen represents a transition to T.^14,15

Figure 1. (a) Low-power photomicrograph of a haematoxylin and eosin stained longitudinal section of a skin biopsy collected from a horse with pituitary pars intermedia dysfunction showing hair follicles primarily in anagen. (b) A high-power photomicrograph of the same biopsy in transverse section showing an anagen hair follicle with a well-defined inner root sheath, presence of trichohyaline granules in the inner root sheath, and absence of tricholemmal keratinization. The arrows point to the trichohyaline granules in the inner root sheath.

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