Professional Documents
Culture Documents
Abstract
The role of oestrogen receptors in dogs with hair cycle
arrest (alopecia X) was investigated by immunohistochemistry. The purpose of this study was to determine
if hair regrowth in dogs with hair cycle arrest treated
with melatonin was associated with a decrease in
follicular oestrogen receptors. Fifteen Pomeranians
(excluding intact females) with hair cycle arrest were
enrolled. Two biopsies were obtained from alopecic
areas of the trunk before and after 3 months on melatonin.
Haematoxylin and eosin-stained tissues were examined
was demonstrated immunoand oestrogen receptor-
histochemically. Common histopathological findings
included hyperkeratosis, follicular keratosis, excessive
tricholemmal keratinization (flame follicles), thin epidermis, few small anagen bulbs, epidermal pigmentation
and melanin aggregates within follicular keratin. Melanin
aggregates within basal cells and hair were an occasional
finding. After 3 months, 40% (six) dogs had mild to
moderate hair regrowth. Biopsies from six dogs showed
histological evidence of an increase in anagen hairs and
eight dogs had a decrease in epidermal pigmentation.
Moderate to marked staining intensity of oestrogen
was noted in all sebaceous gland basal cells,
receptor-
all small hair bulbs and follicular epithelium of telogen
staining of
hairs. There was no oestrogen receptor-
nuclei within the epidermis, apocrine glands or dermal
fibroblasts. Large anagen hair bulbs had minimal to no
oestrogen receptor staining. Hair regrowth was not asso staining.
ciated with a change in oestrogen receptor-
Accepted 21 March 2006
Introduction
Hair cycle arrest (also known as alopecia X, adrenal
hyperplasia-like syndrome, growth hormone responsive
252
2006 The Authors. Journal compilation 2006 European Society of Veterinary Dermatology. 17; 252258
cream coat colour and two had a blue coat colour. Dogs weighed
between 1.8 and 3 kg. The median melatonin dosage was 1.3 mg kg1
(range 11.7 mg kg1) every 12 h.
Dogs were evaluated by their regular veterinarian (n = 10) or at the
University Veterinary Teaching Hospital (n = 5). Prior to enrolment,
dogs were diagnosed with hair cycle arrest based on clinical signs
(alopecia was bilaterally symmetrical, sparing the head and forelimbs
and was not colour restricted or cyclical in nature), and hypothyroidism and hyperadrenocorticism have been ruled out. Hypothyroidism was excluded based on normal concentrations of thyroxine
and thyrotropin and/or lack of hair regrowth on appropriate thyroid
supplementation. Hypercortisolemia was excluded based on lack of
other clinicopathological abnormalities and cortisol concentrations
within the normal range following either low-dose dexamethasone
screening or adrenocorticotropic hormone stimulation.
Two biopsies were taken from alopecic areas of the trunk using a
6-mm biopsy punch and placed in 10% formalin. The biopsy samples
were mailed overnight, and were transferred into 70% EtOH within
24 h. Biopsies were embedded in paraffin and processed for routine
evaluation using haematoxylin and eosin (H&E). The tissues were
positioned in the paraffin blocks so that both longitudinal and horizontal sections of follicles could be obtained. The sections were microscopically evaluated to confirm the diagnosis of hair cycle arrest
(noninflammatory alopecia), and eliminate diseases that could mimic
this condition (sebaceous adenitis, colour dilution alopecia). A second
set of two biopsies were obtained from similar locations (but not previous biopsy sites) after 3 months on oral melatonin (Natures Bounty,
Bohemia, NY, USA) dosed at 3 mg dog1 every 12 h. This product has
been shown to contain at least 3 mg melatonin per tablet with good
oral bioavailability in dogs.15
Owners and veterinarians evaluated the dogs after 3 months of
oral melatonin for degree of hair regrowth and quality of new hair, if
any. A questionnaire was provided in which clinical description, percentage of body affected (< 25%, 2550%, 5075%, or > 75%) and
quality of hair (totally bald in affected areas, fine undercoat only in
affected areas, moderate undercoat with decreased guard hairs in
affected areas) were recorded. Mild hair regrowth was defined as no
change or only minimal change in percentage of body affected and
quality of new hair was only fine undercoat or sparse guard hairs.
Moderate hair regrowth was defined as a decrease in percentage of
body affected with moderate undercoat and guard hairs. No dog was
categorized as having complete hair regrowth because of the short
duration of the study. At that time a second set of biopsies samples
were obtained from each patient. If there was evidence of hair
regrowth, the biopsies were obtained from that region. The exact
locations of the biopsies were not recorded; however, all biopsies
were obtained from the trunk and biopsies were not obtained from
previous biopsy sites. Handling of the tissue was as described for the
initial biopsies.
Each set of biopsies were described together. The H&E-stained tissues were evaluated for presence of anagen and telogen hair follicles,
thickness of follicular and surface epithelium, presence of epidermal
and/or follicular hyperkeratosis, malformation of the follicles and presence of sebaceous and apocrine sweat glands.
Oestrogen receptors were detected immunohistochemically by
standard methods.16 Briefly, after dewaxing the slides were subjected
to antigen retrieval by steaming at 95 C for 25 min in a pH 6.1
retrieval solution (Dakocytomation Inc., Carpenteria, CA, USA), followed by rinsing in TBS. The slides were then processed on a DAKO
autostainer (Dakocytomation, Inc.) with 60-min incubation with the
mouse monoclonal NCL-ER-H2 antibody (Novocastra Laboratories
Ltd. Newcastle upon Tyne, UK) and completion of the process
employing HRP-labelled DakoEnvision+System reagents (Dakocytomation, Inc.) with the DAB + chromagen (Dakocytomation, Inc.) for
visualization of antibody-antigen binding.
Positively stained nuclei were counted (none = 0; < 25% = 1; 25
50% = 2; > 50% = 3) and the intensity of the reaction was assessed
(+ = mild; ++ = moderate; +++ = marked) within the nuclei of the follicular epithelium, hair bulb, epidermis, dermal fibroblasts and sweat
glands. Uterine tissue obtained from a female dog that was in heat
was used as the positive control. Samples from subjects with hair
regrowth, either from owner/veterinarian observation or from histopathological evidence (many anagen follicles) were compared to
those in which no hair growth was noted and to the initial biopsies for
each animal.
Results
Six (40%) of the dogs had mild to moderate hair regrowth
following 3 months of oral melatonin. Moderate hair
regrowth (defined as a decrease in percentage of body
affected with moderate undercoat and guard hairs) was
reported in three dogs (one orange castrated male, one
orange intact male, and one black spayed female). Mild
hair regrowth (defined as no change or only minimal
change in percentage of body affected and quality of new
hair was only fine undercoat or sparse guard hairs) was
also reported in three dogs (one orange intact male, one
black intact male, and one cream castrated male). No
dog was categorized as having complete hair regrowth
because of the short duration of the study. Owners of
three dogs with no hair regrowth reported a lightening of
the skin while on melatonin.
Histopathological evaluation
All initial biopsies were classified as having a noninflammatory alopecia. The histopathological findings are summarized in Table 1. All tissues showed hyperkeratosis and
follicular keratosis. Telogen hairs with prominent trichilemmal keratinization (flame follicles) were evident in all but
two dogs. The epidermis was 12 cell layers thick in all,
but one dog in which the epidermis was 34 cell layers
thick, and occasional follicles appeared malformed in all
but two dogs (Fig. 1). A median of two anagen bulbs was
noted in biopsies from each dog (range 08). Biopsies
from 11 dogs contained only small anagen bulbs (Fig. 2),
one dog had a large anagen bulb and two dogs had minimal numbers of both large and small anagen bulbs. Sebaceous and apocrine glands were present in all tissues. All
but two had pigmentation of the epidermis. Prominent
aggregates of melanin were present in follicular keratin
from tissue samples from 10 dogs and within basal cells
of the epidermis and hair bulbs in three dogs (two black
and one orange). Three dogs had pigment aggregates in
the sebaceous gland ducts.
After 3 months on melatonin, all skin samples had
hyperkeratosis, follicular keratosis and an epidermis of
12 cell layers thick (Table 1). Flame follicles were noted
in biopsies from 10 of 15 dogs and malformed follicles were
No. (before)
No. (after)
Hyperkeratosis
Follicular keratosis
Prominent tricholemmal keratin
Thin epidermis
Malformed follicles
Epidermal pigmentation
Melanin aggregates
Follicular keratin
Basal cells
Sebaceous gland ducts
15
15
13
14
13
13
15
15
10
15
12
7
10
3
3
8
4
3
2006 The Authors. Journal compilation 2006 European Society of Veterinary Dermatology.
253
Frank et al.
Site
Small hair bulb
Follicular epithelium
Staining intensity
Baseline
Staining intensity
After 3 months
melatonin
++
+++
++
+++
2
2
3
2
2
2
2
1
3
2
(Fig. 3) and one dog had both small and large anagen
bulbs. The three dogs with the large anagen bulbs had an
obvious increase (from 3 to 15, 0 to 6, and 2 to 11) in the
number of anagen follicles present. Owners of two of
these dogs had reported mild to moderate hair regrowth.
Biopsies from six dogs, including these two, showed
increased thickness of the follicular epithelium, suggestive of an increase in anagen hairs, although an increase in
number of anagen bulbs was not observed in four of these
dogs. Of these four dogs, only one owner reported moderate
hair regrowth. Eight of the 13 dogs with prominent epidermal pigmentation prior to melatonin treatment showed only
mild pigmentation within the epidermis after 3 months on
melatonin. Four dogs had aggregates of melanin within
the basal cells of the epidermis and hair bulbs; three dogs
had pigment aggregates in the sebaceous gland ducts.
Immunohistochemical evaluation
The sebaceous glands in all samples had moderate to
marked staining of greater than 50% of the basal cell
nuclei in all tissue samples (Fig. 4). Therefore, this helped
serve as an internal control. There was no staining of
nuclei within the epidermis, apocrine glands or dermal
fibroblasts in any tissue samples.
All of the small hair bulbs had mild to marked staining
intensity of greater than 50% of the nuclei before or after
melatonin treatment (Table 2) (Figs 5 and 6). Most of the
2006 The Authors. Journal compilation 2006 European Society of Veterinary Dermatology.
Discussion
Hair cycle arrest (alopecia X) is described as occurring in
young adult dogs between 1 and 3 years of age.1,2 The
median age of onset of hair loss in the present study was
1 year. As in previous reports, both males and females are
affected with this condition.1,2 There were more males
2006 The Authors. Journal compilation 2006 European Society of Veterinary Dermatology.
255
Frank et al.
2006 The Authors. Journal compilation 2006 European Society of Veterinary Dermatology.
References
1. Scott DW, Miller WH, Griffin CE (eds). Endocrine and metabolic diseases. In: Muller and Kirks Small Animal Dermatology,
6th edn. Philadelphia, PA: W.B. Saunders, 2001: 780886.
2. Schmeitzel LP. Sex hormone-related and growth-hormone
related alopecias. Veterinary Clinics of North America. Small
Animal Practice 1990; 20: 1579601.
3. Schmeitzel LP, Lothrop CD. Hormonal abnormalities in Pomeranians with normal coat and in Pomeranians with growth hormoneresponsive dermatosis. Journal of the American Veterinary
Medical Association 1990; 197: 133341.
4. Frank LA, Hnilica KA, Rohrbach BW et al. Retrospective evaluation of sex hormones and steroid hormone intermediates in dogs
with alopecia. Veterinary Dermatology 2003; 14: 917.
5. Frank LA, Hnilica KA, Oliver JW. Adrenal steroid hormone concentrations in dogs with hair cycle arrest (alopecia X) before and
during treatment with melatonin and mitotane. Veterinary
Dermatology 2004; 15: 27884.
6. Ashley PF, Frank LA, Schmeitzel LP et al. Effect of oral melatonin
administration on sex hormone, prolactin, and thyroid hormone
concentrations in adult dogs. Journal of the American Veterinary
Medical Association 1999; 215: 11115.
7. Oh HS, Smart RC. An estrogen recptor pathway regulates the
telogen-anagen hair follicle transition and influences epidermal
cell proliferation. Proceedings of the National Academy of Sciences
of the USA 1996; 93: 1252530.
8. Smart RC, Oh HS, Chanda S et al. Effects of 17--estradiol and ICI
182 780 on hair growth in various strains of mice. Journal of
Investigative Dermatology Symposium Proceedings 1999; 4:
2859.
9. Chanda S, Robinette CL, Couse JF et al. 17-estradiol and ICI182780 regulate the hair follicle cycle in mice through an estrogen
receptor- pathway. American Journal of Physiology. Endocrinology and Metabolism 2000; 278: E202E210.
10. Ohnemus U, Uenalan M, Conrad F et al. Hair cycle control by
estrogens: Catagen induction via estrogen receptor (ER)- is
checked by ER signaling. Endocrinology 2005; 146: 121425.
11. Schweikert HU, Milewich L, Wilson JD. Aromatization of androstenedione by isolated human hairs. Journal of Clinical Endocrinology and Metabolism 1975; 40: 4137.
12. Bratka-Robia CB, Egerbacher M, Helmreich M et al. Immunohistochemical localization of androgen and oestrogen receptors in
canine hair follicles. Veterinary Dermatology 2002; 13: 1138.
13. Rato AG, Pedrero JG, Martinez MA et al. Melatonin blocks the
activation of estrogen receptor for DNA binding. FASEB Journal
1999; 13: 85768.
14. Kiefer T, Ram PT, Yuan L, Hill SM. Melatonin inhibits estrogen
receptor transactivation and cAMP levels in breast cancer cells.
Breast Cancer Research and Treatment 2002; 71: 3745.
15. Gunter MJ, MacDonald JM, Sartin J et al. The effect of oral melatonin on thyroid and adrenocortical activity in normal dogs. Residents Review and Forum. American Academy of Veterinary
Dermatology and. American College of Veterinary Dermatology,
San Francisco, California, USA, August 2000; 10.
16. Vermeirsch H, Simoens Lauwers H. Immunohistochemical detection of the estrogen receptor- and progesterone receptor in the
canine pregnant uterus and placental labyrinth. The Anatomical
Record 2000; 260: 4250.
17. Animal Forum Web Site. Toy breeds: Pomeranians. Available at
www.animalforum.com/dbreed/topomeranian.htm. Accessed on
24 Oct 2005.
18. Scott DW, Walton DK. Hyposomatotropism in the mature dog: A
discussion of 22 cases. Journal of the American Animal Hospital
Association 1986; 22: 46773.
19. Yager JA, Wilcock BP. (eds) Atrophic dermatoses. In: Color Atlas
and Text of Surgical Pathology of the Dog and Cat Dermatopathology and Skin Tumors. London: Mosby Year Book Europe Ltd,
1994: 217237.
20. Mecklenburg L. Diseases of the hair follicles in domestic animals.
19th Proceedings of the American Academy of Veterinary Dermatology and the American College of Veterinary Dermatology,
Kansas City, Missouri, USA, April 2125, 2004: 5160.
21. Dunstan RW, Credille KM, Mansell J et al. A common sense
approach to the morphology of alopecia: addressing 10 points of
follicular confusion. 16th Proceedings of the American Academy
of Veterinary Dermatology and the American College of Veterinary Dermatology Norfolk, Virginia, USA, April 48, 2001: 55 9.
22. Scott DW, Miller WH, Griffin CE (eds). Acquired alopecias. In:
Muller and Kirks Small Animal Dermatology, 6th edn. Philadelphia,
PA: W.B. Saunders Co, 2001: 887912.
23. Gross TL, Ihrke PJ, Walder EJ et al. (eds). Dysplastic diseases of
the adnexa. In: Skin Diseases of the Dog and Cat, 2nd edn.
Oxford: Blackwell Science Ltd, 2005: 51836.
24. Bagladi M, Scott DW, Miller WH. Sebaceous gland melanosis in
dogs with endocrine skin disease or follicular dysplasia: a retrospective study. Veterinary Dermatology 1996; 7: 8590.
25. Thorton MJ. The biological actions of estrogens on skin. Experimental Dermatology 2002; 11: 487502.
26. Gross TL, Ihrke PJ, Walder EJ et al. (eds). Atrophic diseases of
the adnexa. In: Skin Diseases of the Dog and Cat, 2nd edn.
Oxford: Blackwell Science Ltd, 2005: 480517.
27. Thorton MJ, Taylor AH, Mulligan K et al. Oestrogen receptor beta
is the predominant oestrogen receptor in human scalp skin.
Experimental Dermatology 2003; 12: 18190.
28. Moverare S, Lindberg MK, Faergemann J et al. Estrogen receptor
, but not estrogen receptor , is involved in the regulation of the
hair follicle cycling as well as the thickness of epidermis in male
mice. Journal of Investigative Dermatology 2002; 119: 10538.
29. Ram PT, Kiefer T, Silverman M et al. Estrogen receptor transactivation in MCF-7 breast cancer cells by melatonin and growth factors. Molecular and Cellular Endocrinology 1998; 141: 5364.
Rsum Cette tude rapporte ltude immunohistochimique des rcepteurs aux oestrognes chez les
chiens prsentant un arrt du cycle folliculaire (Alopcie X). Le but tait de dterminer si la croissance des
poils de chiens traits avec la mlatonine tait associe une diminution des rcepteurs folliculaires aux
oestrognes. Quinze Pomeranians (aucune femelle non strilise) prsentant un arrt du cycle folliculaire
ont t enrls. Deux biopsies ont t obtenues sur des zones alopciques du tronc avant et aprs trois
mois de traitement avec la mlatonine. Les sections ont t examines et une tude immunohistochimique
a t ralise pour dmontrer le rcepteur alpha aux oestrognes. Les modifications histopathologiques
regroupaient une hyperkratose, une kratose folliculaire, une kratinisation trichilemmale augmente
(follicules en flammes), un piderme fin, des bulbes anagnes rares une pigmentation pidermique et des
aggrgats de mlanine dans la kratine folliculaire. Des aggrgats de mlanine dans les cellules basales et
les poils taient occasionnellement retrouvs. Aprs trois mois, quarante pour cent (6 chiens) prsentaient
2006 The Authors. Journal compilation 2006 European Society of Veterinary Dermatology.
257
Frank et al.
une repousse modre des poils. Les biopsies des 6 chiens prsentaient une augmentation du nombre de
follicules en phase anagne, et huit chiens avaient une diminution de la pigmentation pidermique. Un
marquage modr marqu du rcepteur aux oestrognes a t not dans toutes les cellules basales des
glandes sbaces, tous les petits bulbes folliculaires, et lpithlium folliculaire des poils en phase tlogne.
Aucun marquage na t not dans lpidermien les glandes apocrines ou les fibroblastes dermiques. Les
follicules anagnes de grande taille ne prsentaient pas de marquage. La repousse pilaire ntait pas associe une modification du marquage du rcepteur aux oestrognes.
Resumen El papel de los receptores de estrgeno en perros con arresto del ciclo de crecimiento folicular
(alopecia X) se investig mediante tcnicas de inmunohistoqumica. El objetivo de este estudio fue determinar si el crecimiento del pelo en perros con arresto del ciclo folicular y tratados con melatonina estaba
asociado a una disminucin en la cantidad de receptores de estrgeno. Quince perros de raza Pomeranian
(excluyendo hembras no esterilizadas) con arresto del ciclo folicular fueron includos en el estudio. Se obtuvieron dos biopsias de las reas con alopecia del tronco antes y despus de tres meses de tratamiento con
melatonina. Se examinaron los tejidos tras tincin con hematoxilina y eosina, mientras que los receptores
de estrgeno se demostraron con una tcnica immunohistoqumica. Los hallazgos histopatolgicos ms frecuentes incluyeron hiperqueratosis, queratosis folicular, excesiva queratinizacin tricolemal (foliculos en
llama), delgadez de la epidermis, reducido nmero de bulbos de pequeo tamao en fase anagnica, pigmentacin de la epidermis y agregados de melanina en la queratina folicular. Los agregados de melanina
en clulas basales y en los pelos fueron un hallazgo ocasional. Despus de tres meses, un 40% (seis perros)
presentaron crecimiento del pelo de modesto a moderado. Las biopsias de seis perros presentaron evidencia histolgica de un aumento en los pelos en fase anagnica y ocho perros presentaron disminucin de la
pigmentacin epidrmica. Una intensidad de tincin de moderada a marcada se observ para el receptor
de estrgeno alfa en todas las clulas basales de las glndulas sebceas, en todos los bulbos pilosos de
pequeo tamao y en el epitelio folicular de los pelos en fase telognica. No se observ tincin nuclear para
el receptor de estrgeno alfa en las clulas epidrmicas, glndulas apocrinas o fibroblastos de la dermis.
Los bulbos pilosos en fase anagnica de tamao grande presentaron tincin mnima o nula.para el receptor
de estrgeno. El crecimiento del pelo tras el tratamiento no estuvo asociado con un cambio en la tincin
para el receptor de estrgeno.
Zusammenfassung Die Rolle von strogenrezeptoren bei Hunden, deren Haarzyklus sich in einer Ruhephase befand (Alopezia X) wurde mittels Immunhistochemie untersucht. Das Ziel dieser Studie war es festzustellen, ob das Nachwachsen der Haare bei Hunden mit Haaren in einer follikulren Ruhephase, die mit
Melatonin behandelt wurden, mit einer Abnahme der follikulren strogenrezeptoren zusammenhngt.
Fnfzehn Pomaranier (mit Ausnahme von unkastrierten Hndinnen), deren Haarzyklus sich in einer Ruhephase befand, wurden verwendet. Zwei Biopsien wurden von den haarlosen Stellen am Rumpf vor und nach
einer 3 monatelangen Behandlung mit Melatonin entnommen. Mit H&E gefrbtes Gewebe wurde untersucht und strogenrezeptor alpha immunhistochemisch dargestellt. Typische histopathologische Befunde
waren Hyperkeratose, follikulre Keratose, exzessive trichilemmale Keratinisierung (Flammenfollikel),
dnne Epidermis, wenige kleine anagene Haarblge, epidermale Pigmentation, und Melaninaggregate im
follikulren Keratin. In den Basalzellen und in Haaren wurden Melaninaggregate gelegentlich gefunden.
Nach 3 Monaten zeigten vierzig Prozent der Hunde (n = 6) geringen bis moderaten Nachwuchs der Haare.
Die Biopsien von 6 Hunden zeigten histologisch eine Zunahme an anagenen Haaren und acht Hunde hatten
eine Abnahme an epidermaler Pigmentation. Mige bis deutliche Frbungsintensitt des strogenrezeptor
alpha wurde in allen Basalzellen der Talgdrsen, in allen kleinen Haarblgen und im follikulren Epithel der
telogenen Haare festgestellt. Die Zellkerne in der Epidermis, den apokrinen Drsen oder den dermalen
Fibroblasten zeigten keine Frbung des strogenrezeptor alpha. Groe anagene Haarblge zeigten minimale oder keine Frbung des strogenrezeptor alpha. Das Nachwachsen der Haare stand nicht im Zusammenhang mit einer Vernderung der Frbung des strogenrezeptor alpha.
258
2006 The Authors. Journal compilation 2006 European Society of Veterinary Dermatology.