EI~SEVIER

Bone Vol. 17, No. 4, Supplement October 1995:191S-196S

THE FERRET AS A SMALL ANIMAL MODEL WITH BMU-BASED REMODELING FOR SKELETAL RESEARCH

M.S. Mackey, M.L. Stevens, D.C. Ebert, D.L. Tressler, K.S. Combs, C.K. Lowry, P.N. Smith, and J.E. McOsker

Procter and Gamble Pharmaceuticals, Norwich, NY

ABSTRACT

The need exists for a small animal model with bone metabolic unit (BMU)-based remodeling to mimic the bone loss found in postmenopausal women. The purpose of this investigation was to evaluate the ferret as a potential model for skeletal research. Specifically, we determined whether the ferret: 1) exhibits evidence of BMU-remodeling, 2) has a skeletal response to parathyroid hormone (PTH) similar to other remodeling species, and 3) loses bone in response to reduced estrogen levels. Methods: Using three sets of experiments, we determined the response of female ferrets to ovariectomy/ light cycle manipulation or to administration of PTH. Scanning electron microscopy, light micros- copy, determination of estrogen levels and/or single-photon absorptiometry (SPA) were used for evaluation. Results: The ferret was found to exhibit BMU-based remodeling, and may therefore provide a small animal remodeling species for skeletal research. Ferrets reach skeletal maturity between four and seven months of age as evidenced by closure of the growth plate and maturation of trabecluae from thin rods to thick rods and plates. PTH treatment resulted in a marked increase in bone mass accompanied by the PTH-induced tunneling phenomenon known to occur in dogs and humans but not rats. The response to PTH supports the use of the ferret in studies of bone anabolic agents. Bone mass in the proximal tibia was significantly reduced when estrogen depletion was induced by either bilateral ovariectomy or short light/dark cycles (8 hour light, 16 hour dark). Main- tenance of intact ferrets under short-light conditions mimiced ovariectomy in terms of serum estro- gen levels, uterine weights, and tibial BMD. The bone loss seen following ovariectomy or short light/dark cycles suggests the ferret as a model for evaluating the effects of estrogen-depletion in a remodeling skeleton. Alteration of light-cycles may offer a reversible alternative to surgical induc- tion of estrogen depletion. Conclusions: Based on these observations, we conclude that the ferret may offer an exciting new model for skeletal research.

INTRODUCTION

Osteoporosis is a disease characterized by loss of bone mass leading to increased bone fragility and subsequent fracture. The most common form of osteoporosis is that resulting from estrogen depletion in women at menopause. The lack of an appropriate, practical, and reproducible animal model of the disease has been a significant impediment to osteoporosis research (4, 6). A small animal model with bone metabolic unit (BMU)-based remodeling which could mimic the estrogen depletion-induced bone loss in women would be of tremendous value in the study of the etiology and treatment of osteoporosis.

Address for correspondence and reprints: Dr. J. McOsker, Procter & Gamble Pharmaceuticals, 1262 Cornel! Park Drive, Cincinnati, OH 45242.

© 1995 by Elsevier Science Inc. 191S 8756-3282/95/$9.50 SSDI 8756-3282(95)00330-G

192S M.S . Mackey et al. Bone Vol. 17, No. 4, Supplement Ferret as a model for skeletal research October 1995:191S-196S

The ferret is a small laboratory animal that can be easily housed and handled, is inexpensive, and is also readily available. However, the skeletal metabolism of ferrets is largely uncharacterized. Our goals in this study were to determine whether the ferret: l) shows evidence of BMU-based skeletal remodeling, 2) has a skeletal response to parathyroid hormone (PTtt) similar to other remodeling species, and 3) loses bone in response to decreased circulating estrogen levels.

MATERIALS AND METHODS

Fifty-eight (58) female ferrets (Mustela putorius furo) were obtained from Marshall Farms (North Rose, NY) at either 4 or 7 months of age. Animals were ovariectomized (bilateral) by the supplier or left intact (no ovariectomy) and were either sacrificed for an initial bone morphology study, or placed in one of two experimental groups outlined below. The animals were pair-housed in suspended stainless steel cages and were provided with environment enrichment devices (e.g., plastic tubes and stainless steel bowls). Ferrets were maintained in a 12-hour light/l 2-hour dark light-cycle unless otherwise stated. High Density Purina Ferret Chow, (1.67% calcium, 1.01% phosphorus, 3.4 IU/ gm vitamin D) and water were given ad libitum. At termination of each experiment, ferrets were euthanized by overdose of pentobarbital. All animals were maintained in accordance with Federal regulations and studies were conducted with approval of the Institutional Animal Care and Use Committee.

As induced ovulators, ferrets remain in estrus unless bred (2). In order to avoid the development of aplastic anemia due to prolonged estrus in the unbred ferret, gonadotropin-releasing hormone (Cystorelin,, Sanofi Animal Health Inc. Overland Park, KS; 20 g/animal) was administered by intramuscular injection approximately 15 days after the detection of the first signs of cstrus (e.g., enlarged vulva) (2).

Bone Morphology Study:

Cross sections of femur from two 7-month-old female ferrets were processed undecalcificd in meth- acrylate to demonstrate the presence or absence of ostconal remodeling. One hundred micron sec- tions were cut using an Exact, saw (Exact Medical Instruments Inc., Oklahoma City, OK). Sections were left unstained for viewing by polarized light or by routine light microscopy. Ten micron yon Kossa stained sections of undecalcified ferret tibia were prepared from 4- and 7-month-old ferrets (n=8 per group) to evaluate the effect of age on growth plate.

Distal femurs from 16 female ferrets (8 each at 4 and 7 months of age) were opened for evaluation of trabecular structure by making a sagittal cut through the distal one-third of the femur with the Exact, saw. Marrow was removed using a high-pressure stream of water (Water Pic,, Teledyne Water Pik, Ft. Collins, CO). Femurs were mounted on stubs using silver paste and sputter coated with gold palladium (3.5 minutes at a current of 30 mAMPs) for later viewing by scanning electron micro- scope (JEOL 840, JEOL Ltd., Tokyo, Japan).

Parathyroid hormone (PTH) treatment:

Sixteen 4-month-old ovariectomized ferrets (two groups of animals, n=8 per group) received subcu- taneous injections along the back of the neck of either 0.02 mg/kg (1-34)hPTH (Bachem Bioscicnce, Inc. Philadelphia, PA) or vehicle [acidified saline (0.001 M HCI) with lmg/ml rat serum albumin] once daily for 12 weeks.

Following euthanasia, tibiae were evaluated for changes in bone mineral density by single-photon absorptiometry (Lunar SP2 Rectilinear Scanner, Lunar, Inc., Madison, WI) by the method of McOsker and Li (3), followed by histological evaluation for the appearance of trabecular tunneling.

Bone Vol. 17, No. 4, Supplement M.S. Mackey et al. 193S October 1995:191 S-196S Ferret as a model for skeletal research

Estrogen depletion:

Twenty-four virgin female ferrets, 7-months of age, were obtained for this study. Estrogen depletion was induced surgically or through housing of the ferrets in an 8-hour light, l 6-hour dark (short) light cycle. Twenty-four virgin 7-month-old female ferrets were either ovariectomized (ovx, n=6) or kept intact (n=l 8). The intact ferrets were randomly assigned to one of three groups (n=6 per group): a baseline control (BC) sacrificed on Day 2; an intact control group maintained on a 16-hour dark schedule (intact control/long light cycle or IC-long); and an intact anestrus group maintained on an 8-hour light, 16-hour dark schedule (intact control/short light cycle or IC-short). The ovx animals (Ovx) were maintained on a 16-hour light, 8-hour dark schedule. Study groups are summarized in the table below.

Study Design

Group Surgical Status Light Cycles Number

BC Intact 16 hr light/8 hr dark n = 6

IC-long Intact 16 hr light/8 hr dark n = 5*

IC-short Intact 8 hr lighl/16 hr dark n -- 6

Ovx Ovariectomy 16 hr light/8 hr dark n = 6

* One animal eliminated due to cystic ovary

After 12 weeks of study (Day 2 for BC), all animals were anesthetized (pentobarbital) and blood was collected by aortic punture for determination of serum estradiol levels. Following the blood sam- pling procedure, the animals were sacrificed by an overdose of pentobarbital. Uteri were collected, trimmed of all fatty attachments, cut at the cervix and weighed. Tibia were also collected and placed in 70% ethanol for determination of BMD as described above.

Unless otherwise noted, data are presented as mean + standard deviation. For statistical analysis a Dunnett 's test was performed to compare each treatment group back to a control. If the parametric assumptions were not satisfied, then a Dunn's test was implemented.

R E S U L T S

Bone Morphology.

Numerous osteons were evident throughout the cross section of cortical bone (Fig. 1) demonstrating BMU-based remodeling in the ferret. Skeletal maturity is reached in the ferret by 7 months of age, as indicated by the closure of the growth plate and maturation of trabeculae from thin rods to thick rods and plates (Figs. 2 and 3). This maturation process has not been reported for the rat.

P T I I Treatment. Twelve weeks of PTH treatment in the ferret resulted in a significant increase in bone mineral den- sity in the proximal tibia compared to vehicle-treated controls (280+18 mg/cm vs. 155+10 mg/cm). PTII treatment also induced tunneling resorption in cancellous bone (Fig. 4).

194S M.S. Mackey et al. Bone Vol. 17, No. 4, Supplement Ferret as a model for skeletal research October 1995:191S-196S

FIG. 1. Evidence for BMU-based remodeling in cross sections of ferret femur. Sections were unstained (left) or viewed by polarized light (right). (Original mag. 4X)

FIG. 2. Scanning electron micrograph of the distal femur from 4-month-old (left) and 7-month- old (right) female ferrets. Note closure of the growth plate and change in trabecular structure from thin rods to thick rods or plates in the 7-month old animal. The bones shown here are representative specimens of the 8 animals evaluated from each age group. (Original mag. 12X)

FIG. 3. Ten micron von Kossa stained sections of undecalcified ferret tibia from 4- (left) and 7-month-old (right) female ferrets. Note closure of growth plate (arrow) (Original mag. 1.25X)

FIG. 4. PTH treatment in the ferret induces tunneling resorption in cancellous bone (right). Tunneling resorption was not observed in the vehicle treated controls (left). (Original mag. 4X)

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Estrogen Depletion.

To determine whether the ferret could be used as a model for postmenopausal osteoporosis a study was conducted to evaluate the skeletal effects of estrogen depletion in adult ferrets. Estro- gen depletion was induced surgically (bilateral ovariectomy) or through alteration of light/dark cycles as outlined in Methods.

All animals in the IC-long group came into estrus during the study and were treated with gona- dotropin-releasing hormone as described in Methods. There was considerable variability in the IC-long serum estradiol levels due to the varying estrous status of the animals. Serum estradiol levels were significantly reduced in the Ovx group relative to the IC-long (Fig. 5). The IC-short and Ovx groups were not different.

Serum Estradiol Uterine Weight 50 , 12

40- E

- - 3 0 - o_

w 20- E

10-

_L_

1 BC IC-Long IC-Short Ovx

FIG. 5. Serum estradiol at termination. *Different from IC-long, p<0.05 (Dunn's).

10-

8 x z

6

4-

0 ~ ~ " BC IC-Long IC-Short Ovx

FIG. 6. Uterine weight at termination. *Different from IC-long, p<0.05(Dunn's).

Uterine weights were determined at termina- tion to further demonstrate estrogen status. Uterine weights were significantly reduced for both IC-short and Ovx groups (Fig 6). There was wide variability in uterine weight for the BC and IC-long groups due to the dif- ference in estrous status of the animals at the time of study termination.

Bone mineral density of the proximal tibia was significantly reduced in the IC-short and Ovx groups compared to BC but not the 1C- long (Fig. 7). The 1C-short and Ovx groups were not different from each other.

220

200-

180- "e, E

~ 160- r n

140-

BMD of the Proximal Tibia

120 BC IC-Long IC-Short Ovx

FIG. 7. Bone mineral density (BMD) of the proximal tibia. * Different from BC, p<0.05.

196S M.S . Mackey et al. Bone Vol. 17, No. 4, Supplement Ferret as a model for skeletal research October 1995:191S-196S

CONCLUSIONS

The ferret provides a convenient, inexpensive, readily available animal model for skeletal studies. Skeletal maturity is reached by 7 months of age allowing investigative studies with an adult skeleton. These studies demonstrate that the ferret is a BMU-based remodeling species, responds to PTH treat- ment in a a manner similar to other remodeling species (dog, human), and loses bone in response to estrogen depletion. The ferret offers a new model with potential utility for skeletal research, includ- ing investigations into the mechanism of estrogen depletion-induced bone loss, and mechanism-of- action of bone therapeutic agents. Altered light cycles may provide a reproducible alternative to surgical induction of estrogen depletion.

A C K N O W L E D G M E N T S

The authors gratefully acknowledge the assistance of Ms. S.K. Berman and Dr. K.E. Gropp with the preparation of this manuscript.

REFERENCES

1. Boyce RW, Paddock CL, Franks AE Jankowsky ML, Eriksen EF. Effects of intermittent hPTH(1- 34) alone and in combination with 1,25(OH)2D3 or risedronate on endosteal bone turnover and the resorptive site of the remodeling unit in canine cancellous bone. Manuscript in preparation. 2. Fox JG. Biology and diseases of the ferret. Philadelphia, PA: Lea and Febiger: 1988. 3. McOsker JE, Li XJ. Use of rectilinear single-photon absorptiometry to evaluate bone-mass changes in rats. Cells and Mater Suppl 1:93-104; 1991. 4. Newman E, Turner AS, Wark JD. The potential of the sheep for the study of osteopenia: current

status and comparison with other animal models. Bone 16(4):277-284S. 5. Parisien M, Silverberg S J, Shane E, de la Cruz L, Lindsay R, Bilezikian JE Dempster, DW The

histomorphometry of bone in primary hyperparathyroidism: preservation of cancellous bone struc- ture. J Clin Endocrinol Metab 70(4):930-938; 1990. 6. Rodgers JB, Monier-Faugere MC, Malluche H. Animal models for the study of bone loss after

cessation of ovarian function. Bone 14:369-377; 1993.