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Skeletal alterations in hypophysectomized ratsI. A histomorphometric study on tibial cancellous bone

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THE ANATOMICAL RECORD 241505-512 (1995)
Skeletal Alterations in Hypophysectomized Rats: 1. A
Histomorphometric Study on Tibia1 Cancellous Bone
JAMES K. YEH, MENG-MENG CHEN, AND JOHN F. ALOIA
Department of Medicine, Winthrop-Uniuersity Hospital, Mineola (J.K.Y.,M.-M.C.,
J.FA.), and Health Sciences Center, State University of New York, Stony Brook (J.K.Y.,
J.F.A.),New York
ABSTRACT
Background: Hypophysectomy (HX) results in a cessation
of bone growth and a decrease in bone metabolism. The purpose of this
study is to examine the effect of HX on the static and dynamic histomorphometry of cancellous bone in the secondary spongiosa of the proximal
tibial metaphysis in rats.
Methods: Female rats, at 2 or 3 months of age, were HX and sacrificed at
0,5 days, 2 and 5 weeks after the surgery. Age-matched intact rats served
as controls. Cancellous bone histomorphometry was performed on doublefluorescent labeled, 30-um-thick sections of the proximal tibia. Tartrateresistant acid phosphatase histomorphometry was performed at 5 days on
HX and control rats to evaluate the resorption in the metaphyseal bone.
Results: Although the intact rats gained in body weight, tibial length,
tibial weight, and density after 5 weeks, these changes did not occur following HX. As compared to the basal group, HX resulted in a decrease in
the density and dry weight of the metaphysis. The histomorphometric data
showed that the cancellous bone volume and trabecular number of the
secondary spongiosa were decreased and the separation was increased in
the HX rats. The dynamic results showed that HX significantly decreased
longitudinal growth rate and tissue-based bone formation and resorption.
However, the bone surface-based eroded surface, labeled surface, the mineral apposition rate, and the bone formation rate did not differ between the
intact and the HX rats at either the 2 or 5 weeks study. Five days after HX,
the bone surface and tissue-based osteoclast surfaces were significantly
lower in the HX than in the intact rats.
Conclusions: Pituitary hormone deficiency results in cancellous bone
loss. The bone loss is due primarily to the suppression of longitudinal
growth-dependentbone gain and the inhibition of tissue-based bone turnover with a lower bone formation relative to bone resorption. The surfacebased bone turnover is not affected. o 1995 WiIey-Liss, Inc.
Key words: Bone formation, Bone resorption, Bone remodeling, Hypophysectomy, Cancellous bone
In the growing rat, skeletal growth, modeling by
drifts, and remodeling by basic multicellular units
(BMU) are active simultaneously, but their rates may
vary between the different bones of the skeleton (Jee et
al., 1983a; Kimmel and Jee, 1982; Sontag, 1986). Bone
modeling changes the size and shape of cortical bone by
resorption and formation drifts, whereas bone remodeling replaces bone by bone formation coupled to antecedent resorption (Frost, 1973a,b; Jee et al., 1983a). In
cancellous bone, a balanced bone formation and resorption with reversal cement lines occurs in the secondary
spongiosa of the trabecular bone envelope, indicating
that bone remodeling is predominant in this area
(Baron et al., 1984; Li et al., 1990). Longitudinal bone
growth is the result of a strictly regulated endochondral ossification process in the long bones (Loveridge
0 1995 WILEY-LISS, INC.
and Farquharson, 1993). The chondrocytes in this tissue area are found in distinct morphologic zones, such
as resting, proliferative, hypertrophic, and calcifying
zones. The zones and the process are regulated by a
number of growth mediators, including growth factors
and hormones (Ohlsson et al., 1993).
Pituitary hormones are essential for normal growth
and hypophysectomy (HX) can result in a reduction in
lean mass and body weight gain and decreased skeletal
Received May 16, 1994; accepted November 14, 1994.
Address reprint requests to James K. Yeh, Metabolism Laboratory,
Department of Medicine, Winthrop-University Hospital, Mineola, NY
11501.
506
J.K. YEH ET AL.
growth (Isaksson et al., 1987; Kember, 1971; Martinez
et al., 1991; Schlechter et al., 1986; Thorngren et al.,
1973). The HX model has been extensively used to
study the regulation of skeletal growth by pituitary
hormones. The most striking effect of HX on bone is a
cessation of longitudinal growth, reflected by a cessation of proliferation and maturation of chondrocytes in
the growth plate cartilage (Isaksson et al., 1987; Kember, 1971; Thorngren et al., 1973). Using a matrix-induced endochondral bone differentiation system, Reddi
and Sullivan (1980) demonstrated that HX results in
delayed and reduced osteogenesis. A reduction of metaphyseal bone mass and tartrate resistant acid phosphatase (TRAP)-positive multinuclear cells and mononuclear cells in the metaphyseal bone of HX rats has
also been reported (Hock and Fonseca, 1990; Lewinson
et al., 1993). The evidence indicates that not only is the
longitudinal bone growth decreased after HX, but
metaphyseal bone formation and resorption activity in
the whole metaphyseal tissue are also decreased after
HX. However, i t was not determined whether this decrease is due to a loss in the quantity of cancellous bone
or a loss in the turnover rate per bone surface.
The present study examined the static and dynamic
histomorphometry of metaphyseal cancellous bone in
the secondary spongiosa of the tibia1 metaphysis of HX
rats. Evidence is provided that HX induced cancellous
bone loss by suppressing longitudinal growth-dependent bone gain and by decreasing both tissue-based
bone formation and resorption without a significant
effect on the bone surface-based formation rate.
MATERIALS AND METHODS
Animal Care
Age-matched HX and intact control female SpragueDawley rats were purchased from Taconic Farms (Germantown, NY). Hypophysectomy was done a t the
Taconic Farm when the rats were 3 months of age.
Upon arrival, 3 days postoperatively, until the end of
the experiment, HX rats were supplemented daily with
hydrocortisone in the form of sodium succinate (100
pg/lOOg) and thyroxine at 2 pg/lOOg, subcutaneously.
The purpose of physiological replacement doses of hydrocortisone and thyroxine are to maintain O2 consumption and basic metabolism (Barnes and Eltherington, 1973). They were also given 3% sucrose water
ad libitum and allowed free access to a standard pelleted chow diet (Rodent Laboratory Chow 5001, Ralston Purina). All animals were placed into individual
cages (25 x 20 x 18 cm3) and housed at a temperature
of 2 ~ 8 ° CAnimals
.
were maintained in accordance with
the NIH Guide for the Care and Use of Laboratory
Animals, and animal protocols were approved by the
Laboratory Animal Care Committee of the WinthropUniversity Hospital. Gain in the body weight of the
rats was monitored weekly and their serum level of
insulin-like growth factor-I was measured upon sacrifice to assess the completeness of HX.
In experiment one, 30 rats with a n average of body
weight 233 f 14.1 g were divided into three groups: 10
basal controls, 9 age-matched intact controls, and 11
HX. Animals in the basal group were sacrificed a t the
age of 3 months and the other two groups were sacrificed 5 weeks later. In experiment two, 15 rats (3
months of age) with a n average body weight of 250
*
11.3 g were divided into two groups; 8 age-matched
intact controls and 7 HX, and sacrificed a t 3 months
plus 2 weeks of age. In experiment three, 12 rats (2
months of age) with a n average of body weight 188 2
8.7 g were divided into two groups: 6 age-matched controls and 6 HX, and sacrificed a t 2 months plus 5 days
of age.
Preparation of Specimens
In experiments one and two, all rats were labeled
with 15 mg/kg of demeclocycline intraperitoneally
(Sigma, St. Louis, MO) and 8 m g k g of calcein subcutaneously (Sigma) at 10 and 3 days, respectively, before
sacrifice. On the final day of the study, the rats were
bled from the choroidal aorta and jugular vein under
anesthesia with carbon dioxide. The gastrocnemius
muscle from the right limb was dissected out and
weighed immediately. The right tibia was removed and
fixed immediately in a 70% ethanol solution for 2 days.
The carcass was kept at -30°C until the left tibia was
dissected for dry weight. The proximal section of each
right tibia was then cut using a n Isomet saw (Buehler,
Lake Bluff, IL) and stained by Villanueva Osteochrome
Bone Stain (Polysciences, Warrington, PA) for 5 days.
The specimens were destained and dehydrated with sequential changes (70,95, and 100%) of ethanol solution
and xylene and then embedded in methyl methacrylate
(Eastman Organic Chemicals, Rochester, NY). Proximal tibiae were frontier cut longitudinally to 200 pm
thickness with a n Isomet saw, ground to 30 pm thickness, and then coverslipped with Eukitt (Calibrated
Instruments, Hawthorne, NY).
In experiment three, the left tibia was dissected and
the proximal tibia was fixed immediately in 70% ethanol solution when the animal was sacrificed. The
bones were then demineralized for 28 days in 10%
EDTA solution (pH 7.4) containing 7.5% sucrose. The
specimen were washed with phosphate-buffered saline
solution and dehydrated with sequential changes (70,
95, and 100%) of ethanol solution and embedded in
glycol methacrylate (JB-4 embedding kit; Polysciences,
Warrington, PA) (Liu et al., 1987). Proximal tibiae
were frontier cut longitudinally to 4 pm thickness with
a microtome (Reichert-Jung 2040, Cambridge Instruments, Buffalo, NY), then the sections were mounted
on glass slides for staining. Tartrate-resistant acid
phosphatase was localized using the hexasolitized
pararosaniline method as described by Liu et al. (1987).
Alpha-naphthyl-phosphate disodium salt (Sigma)
served a s substrate. The enzyme was allowed to react
for 20 minutes at 37°C in the presence of 50 mM L( )tartaric acid in 0.1 M acetate buffer (pH 5.0). Weigert’s
iron Hematoxylin solutions (Poly Scientific, Bay Shore,
NY) were used to stain the nuclears of cells and Fast
Green solutions were used for general evaluation of
tissue.
+
Bone Histomorphometry
A digitizing morphometry system was used to measure bone histomorphometric parameters. The system
consists of a n epifluorescence microscope (Olympus,
BH-2), a digitizing pad (Numonics 2206) coupled to a n
IBM computer, and the morphometry program OsteoMetrics (OsteoMetrics, Atlanta, GA). We measured the
secondary spongiosa of the metaphysis in the region
HYPOPHYSECTOMY AND CANCELLOUS BONE IN RATS
1-4 mm proximal to the growth plate-metaphyseal
junction in the intact rats, as described previously
(Chen et al., 1992; Li et al., 1990; Wronski et al., 1987).
Since the longitudinal growth of HX rats was profoundly decreased, the region of bone measured in the
HX was age-equivalent in comparison to the region of
age-matched intact rats a t 2 and 5 weeks time points.
The bone age was calculated based on the longitudinal
growth rate (LGR) (Jee et al., 1983a; Kimmel and Jee
1980). Bone age = distance from growth platelaverage
LGR. The LGR of the proximal tibia, as measured a t 0,
2, and 5 weeks interval, was 77.9, 44.4 and 29.4 pml
day, respectively, in intact rats, and 77.9,4.97 and zero
pmlday, respectively, in the HX rats. In the 2-week
study, the bone age of the intact control in the distance
of 1 mm is calculated as 1,000 pml(Basa1 LGR +
2-week LGR)/2 = 1,000 pml(77.9 pmlday + 44.4 pml
day)/2 = 16.3 days. Thus the age-equivalent distance
estimated in HX is bone age x (Basal LGR + 2-week
LGR)/2 = 16.3 x (77.9 pmlday + 4.97 pmlday)/2 =
676 pm. Similarly, the bone age of the intact control in
the distance of 1 mm is 19.8 days in the 5-week study,
whereas the age-equivalent distance in HX rat is estimated as 547 pm. Thus the region of metaphysis measured in the 2-wk HX rats was 0.68 mm to 3.68 mm
from the growth plate in comparison with the region of
1-4 mm in the age-matched intact rats. The region
measured in the 5-wk HX rats was from 0.55 to 3.55
mm from the growth plate.
Histomorphometric parameters measured and calculated were percentage bone volume, trabecular width,
trabecular number, trabecular separation, and longitudinal growth rate, labeled surface, mineral apposition rate, bone surface- and tissue-based formation
rates, and bone surface- and tissue-based eroded surfaces in experiments one and two, according to the
nomenclature standardized by Parfitt et al. (1987). In
experiment three, the resorption surface having TRAPpositive cells contacted were calculated as bone surface- and tissue-based in the region measured.
Tibial Bone Weigh1
The left tibia was dissected free of soft tissue and
placed in a volumetric flask filled with deionized water. The flask was placed in a desiccator connected to a
vacuum for 2 h. After the trapped air diffused out of the
bone, the wet weight of the bone was taken using a
Sartorius balance provided with a thin wire to which
the blotted bone was attached. The bone was reweighed
after submersion in deionized water. The difference between the weight of the bone in air and that in water is
the volume of the bone, and the density was calculated
as mg wet weight per ul bone volume. The bone was
then extracted twice with 70% ethanol for 48 h, absolute ethanol for 24 h, and absolute ethanol and chloroform (1:l) solution twice for another 48 h. The dehydrated and defatted bones were then dried in a 80°C
oven under vacuum for 48 h to measure the dry weight.
The left tibia was separated perpendicular to the longitudinal axis by an Isomet saw. The metaphysis (the
proximal tibia) were collected 7 mm length from the
top. The dry weight of the segment and the volume
after rehydration and submersion in deionized water
were measured as described above.
507
Serum IGF-I
Serum levels of IGF-I were measured with a commercial RIA kit after acid-ethanol precipitation (Nichols
Institute, San Juan Capistrano, CA) (Yeh et al., 1994).
Rabbit polyclonal antihuman IGF-I antiserum (cross
reacts with rat IGF-I) and recombinant human IGF-I
as standard and tracer were used to measure the relative quantity of rat IGF-I.
Statistical Analysis
All data are presented as mean k S.D. in tables and
mean -t S.E. in figures. Comparison of the two groups
was done by Student’s t-test or Fisher’ Protected Least
Significant Difference using the mean square of the
error from the ANOVA as indicated in tables and figures.
RESULTS
Body Weight and Tibial Bone Mass
Hypophysectomized rats failed to gain body weight,
tibial length, tibial wet and dry weight, whereas the
age-matched intact control rats had a significant increase in these variables (P<O.Ol) after 5 weeks of the
experiment (Table 1). None of the serum levels of IGF-I
were above 100 ng/ml in the 2 and 5 weeks HX rats
(mean2S.D. of 76.9211 ng/ml), whereas the levels in
the basal and age-matched intact control groups were
12872153 ng/ml and 1235+152 nglml, respectively.
As compared to the basal levels with the intact control rats, the bone density and bone dry weight of the
metaphysis increased with age (Table 1).Conversely,
HX resulted in a significant decrease in the density
and a nonsignificant decrease in the dry weight of the
metaphysis. The dry weight and the density of the
metaphysis were significantly higher in the control
than in the HX rats.
Cancellous Bone Volume and the Architecture
Table 2 and Figure 1C,D show that the intact rats
gained in cancellous bone (BV/TV) of the proximal
tibia with age, from 26.1729% to 38.0127.56%, and
the HX rats resulted in a significant decrease in the
bone volume from 26.1729% to 13.4+5.32% at the
5-week study. The HX-induced bone loss was accompanied by a decrease in the trabecular number (P<O.Ol)
and an increase in trabecular separation (P<O.Ol)
without a significant effect on trabecular width. Trabecular width increased and the trabecular separation
decreased (nonsignificantly) in the intact control as
compared to the basal group animals.
Dynamic Histomorphometry of the Cancellous Bone
Figure 2 shows that, in the intact animals, the longitudinal growth of tibia decreased with age. In HX
rats, longitudinal growth of the tibia was only 10% of
the age-matched controls in the 2-week study and it
was undetectable in the 5-week study (Fig. lA,B).
When both the cancellous bone formation and resorption parameters were calculated based on the unit per
tissue area (Fig. 2), these parameters were significantly lower in the HX than in the age-matched intact
rats by 2 and 5 weeks of surgery. However, when the
bone formation and resorption parameters such as total
labeled surface, mineral apposition rate, and the bone
508
J.K. YEH ET AL.
TABLE 1. Effect of 5 weeks of hypophysectomy on the body weight, gastrocnemius muscle weight, and the
weight, volume, and the density of total tibia and tibial metaphysis'
Group
BASAL (10)
CON (9)
HX (11)
Body weight
Initial
Final
(€9
(g)
233 t 13.8
235 t 11.4
230 t 15.5
280 + 15.2**
221 t 18.7+
Gastrocnemius
Muscle weight2
(g)
1.51 t 0.05
+
1.95 -t 0.15*
1.44 t 0.05+
Tibia1 metaphysis
Volume
Dry weight
(mg)
139 t 11.8
167 t 17.2**
126 t 7.15+
Density
(id)
+
108 + 11.8
123 t 12.2**
112 t 13.5
1.281 t .077
1.358 t .09
1.13 t .118**+
+
Total tibia
Wet Weight
(mg)
BASAL (10)
CON (9)
HX (11)
582 t 39.5
666 t 43.5**
560 t 25.2 + +
Volume
(Id)
384 + 27.6
421 t 28.6**
364 t 17.3
++
Density
1.516 t 0.023
1.583 t 0.016**
1.536 -t 0.02*
++
Dry Weight
(mg)
Length
(mm)
391 ? 24.4
464 t 35.0**
384 t 17.8 + +
37.1 t ,336
38.4 t .549**
37.1 t ,463 + +
'Values are meanLf3.D. Number of rats in each group is presented in the parenthesis.
2Muscle weight is the gatrocnemius muscle of right limb.
*;**Significantly different from the BASAL group by Fisher's Least Significant Difference test (P<0.05;P<O.Ol).
+ ;+ +Significantly different from the CON group by Fisher's Least Significant Difference test (P<0.05; P<O.Ol).
TABLE 2. Static histomorphometric changes in the secondary spongiosa of proximal tibia of the
hypophysectomized rat'
Cancellous bone area
(%I
2-weeks experiment
CONTROL (8)
HX (7)
5-weeks experiment
BASAL (10)
CONTROL (9)
HX (11)
Trabecular thickness
(pm)
Trabecular number
(#/mm)
Trabecular separation
(pn)
24.3 t 5.58
18.9 t 6.53
56.8 2 2.40
52.4 t 3.71
4.27 t 0.85
3.56 t 1.02
268 t 74
357 t 129
26.2 t 9.2
38.0 t 7.6*
13.4 & 5.32*,+
54.3 t 4.31
60.0 -t 8.94
53.1 t 6.21
4.75 t 1.29
6.55 t 0.68*
2.50 2 0.86*,
248 -t 103
139 t 30
582 t 269*,+
+
++
+
'Values are mean*S.D. Number of rat in each group is in the parenthes.
*: Significantly different from the BASAL group by the Fisher's Least Significant Difference test (FYO.05).
+ + : Significantly different from the CONTROL group by the Fisher's Least Significant Difference test (P<O.Ol).
formation rate, and eroded surface were measured and
calculated based on the unit per bone surface, none of
the parameters were different between the two groups
at either the 2-week or 5-week study periods (Fig. 3).
In order to investigate cancellous bone loss after HX,
we examined the osteoclast population in the secondary spongiosa of the tibial metaphysis, as identified by
a TRAP-positive staining, at the fifth day after HX.
Figure 4 shows that the osteoclast surfaces (based on
the unit per bone surface and tissue area) of the HX
were <40% of the age-matched intact rats (P<O.OOl).
DISCUSSION
Hypophysectomy in the growing rats resulted in depressed weight gain, decreased longitudinal growth of
the tibia, and decreased serum level of IGF-I after HX,
which demonstrate that the surgery was complete.
However, the decrease in longitudinal bone growth was
not complete at 2 weeks. Since the half-life of rat
growth hormone is only a few hours (Badger et al.,
1991), the remaining longitudinal growth activity
could be due to the residual chondrocytes at 2 weeks
after the surgery. Kember (1971) observed that when
the rat was HX at 6 weeks of age, the cell division rate
in the cartilage plate and the tibial growth rate were
about one-fifth of that found in normal rats 2 wks later.
The reduction was observed largely in the hypertrophic
cell number of the growth cartilage with little change
in the size of the proliferation zone. Their findings and
our results indicate that the suppression of longitudinal growth occurs rapidly within 2 weeks after the HX.
A cessation of growth plate growth could be complete
before 5 weeks. The effect of HX on the LGR vs. time
needs additional study.
Although several hormones are needed for normal
longitudinal bone growth, growth hormone (GH) has a
direct effect on longitudinal growth (Loveridge and
Farquharson, 1993; Schlechter et al., 1986). Receptors
to GH are found predominantly on the stem cells in the
resting zone of the growth plate (Barnard et al., 1988),
whereas proliferating chondrocytes have the highest
concentration of IGF-I receptors (Trippel et al., 1986).
It has been suggested that GH is essential to prime a
prechondrocyte to initiate a subsequent clonal expansion, whereas the IGF-I effect rather reflects a stimulatory effect on already differentiated proliferative
chondrocytes (Bentham et al., 1993; Nilsson et al.,
1989; Ohlsson et al., 1992).
In the present study, we observed that there was a
significant loss in the dry weight and the density of the
metaphysis a s compared to the basal levels. Bone loss
in the metaphysis was due primarily to the loss in cancellous bone volume as revealed by the static histomorphometry.
Since HX results in a decrease in longitudinal bone
growth, the width of the cartilaginous plate and pri-
HYPOPHYSECTOMY AND CANCELLOUS BONE IN RATS
509
Fig. 1. Photomicrograph of tibia1 metaphysis to show the fluorescent labeling of the growth plate and
the primary spongiosa in intact (A) and HX (B) rats, and the cancellous bone of secondary spongiosa in
intact (C) and HX (D) rats from the 5-wk study. Original magnification, x 15.
mary spongiosa are diminished. Thus the region of
bone in the secondary spongiosa to be compared between intact and HX rats has to be age equivalent (Jee
et al., 1983a; Kimmel and Jee, 1980). Accordingly, the
cancellous bone volume was measured in the region of
1-4 mm from the growth plate in the intact rats and
0.55-3.55 mm from the growth plate in the HX rats by
the method of Kimmel and Jee (1980). The cancellous
bone volume is significantly lower in the HX rats
(13.4+5.32%) than either the basal control rats
(26.2+9.2%) or age-matched control rats (38.0+7.56%)
a t 5 weeks. However, this method may not be perfect
since the depression of longitudinal growth rate after
HX is not linear as discussed above. Nevertheless, the
cancellous bone volume measured in either the region
of 1-4 mm or 0-3 mm from the growth plate in the HX
rats (12926.4% and 14.014.42%, respectively) was
still significantly lower than the region of 1-4 mm in
the basal control rats (26229.2%) or age-matched control rats (38.0+7.56%). These findings indicate that
HX indeed results in a cancellous bone loss in the secondary spongiosa.
In normal growing animals, skeletal longitudinal
growth forms primary spongiosa. The primary spongiosa is soon replaced by lamellar bone through remodeling activity and become secondary spongiosa. In the
510
J.K. YEH ET AL.
A.
I:
pO.001:"sage-maiched control (I-tesl)
a: pO.001;vs basal cmuoi (FLSD-lesl).
b pO.001:vs agematched control @L.$D-le~l)
10
.-E
'El
.OD
30
5
E
0
0
2
5
WEEKS
B.
70
"2
1
1
I-
0
2
2
5
0
2
5
0
2
5
B.
1
0.3
I
I
0.0
C.
5
WEEKS
Fig. 2. Effects of hypophysectomy on the longitudinal growth rate
(A) and the bone formation rate (B) of the secondary spongiosa of
proximal tibia. All values shown are meaniSE. *, Significantly different from the age-matched intact control (P<O.OOl) by Student
t-test from the 2-wk study. (a) Significantly different from the basal
group (P<O.OOl) and (b) significantly different from the age-matched
intact control group by Fisher's Least Significant Difference test from
the 5-wk study.
intact rats of the current study, there is a n increase in
the cancellous bone volume of the secondary spongiosa,
trabecular number, and thickness, but a decrease in
trabecular separation by 5 weeks. This indicates the
cancellous bone mass is increased and architecture of
the bone is improved in the young rats during the ages
of 3-4 months. An increase in the cancellous bone volume suggests t h a t some degree of modeling-dependent
bone gain may occur during this growing period, even
though bone remodeling is predominant in the cancellous bone of secondary spongiosa. Conversely, HX results in a suppression in the longitudinal growth-dependent bone gain by a decrease in longitudinal growth
rate. This leads to a decrease in the formation of new
bone in primary spongiosa, which normally consists of
a high percentage of bone (Kimmel and Jee, 1980). Besides, HX decreased both tissue-based bone formation
and resorption, which suggested that HX inhibited tissue-based bone turnover. Therefore, another factor of
bone loss induced by HX should be due to a lower bone
formation activity relative to resorption activity. However, HX does not affect both surface-based bone formation and resorption.
In the HX rats, we observed a significant reduction
WEEKS
Fig. 3.The effects of hypophysectomy on the labeled surface 1%)(A),
mineral apposition rate (um/d) (B), and bone formation rate (bone
surface based, um3/um2/d)(C) of the cancellous bone in the secondary
spongiosa of proximal tibia. All values shown are meant-SE. (a) Significantly different from the basal group (P<O.OOI) by Fisher's Least
Significant Difference test from the 5-wk study.
in bone surface- and tissue-based osteoclast surfaces at
5 days after the surgery. This finding is consistent with
a report of significant reduction in the number of osteoclasts per metaphyseal area at 10 days after HX
(Lewinson et al., 1991). Although the tissue-based
eroded surface a t 5 weeks after the surgery was decreased, the bone surface-based eroded surface did not
differ between the HX and intact rats. These findings
suggest that HX may result in a transient decrease in
resorption activity.
A decrease in the cancellous bone mass of the HX
rats is accompanied with a poor trabecular architecture. We detected a significant decrease in the trabecular number and a n increase in the trabecular separation with a nonsignificant decrease in the trabecular
511
HYPOPHYSECTOMY AND CANCELLOUS BONE IN RATS
A.
a
L -
4
0
5
2
WEEKS
C.
lo
-
E
1
T
E
E
28
8
we
2 6
-"
-
2" 021
I.
e
B
01
2s
5 .s
-::
4
w
2
0
0.5
d -
W
8
0.8
E4:
-01
W
0.2
6
Control
Hypophysectomy
0.0
Control
Hypophysectomy
Fig. 4. The effect of hypophysectomy on the surface- and tissue-based eroded surfaces (A and B) and/or
osteoclast surfaces (Cand D) of the cancellous bone in the secondary spongiosa of proximal tibia at 5
days, 2 and 5 weeks. All values shown are meaniSE. *, Significantly different from the control
(P<O.OOl) by Student t-test. (a) Significantly different from the basal group (P<O.OOl) and (b) significantly different from the age-matched intact control group by Fisher's Least Significant Difference test
from the 5-wk study.
thickness. The alteration in the bone architecture is
similar to the changes observed after immobilization
(Chen et al., 1992; Li et al., 1990), ovariectomy in rats
(Mori et al., 1992), or age-related loss of cancellous
bone in women (Li, 1988, 1989): decreased trabecular
bone number and increased trabecular separation prior
to the decrease in trabecular thickness.
Using low doses of cortisol in intact rats (0.5 to 5.0
mgkgld) and in intact rabbits (0.05-1 mglkgld), Jee et
al. (1966, 1970) observed an increase in the number of
osteoclast and bone resorption activity in bone. In the
current study, administration of hydrocortisone at a
dose of 1mglkgld to the HX rats resulted in a decrease
in the osteoclast number a t 5 days and an inhibition in
tissue-based bone formation and resorption activity at
2 and 5 weeks. This discrepancy suggests that the decrease in osteoclast number and tissue-based turnover
of the HX animal could be due primarily to pituitary
hormone deficiency instead of hydrocortisone replacement. A group of HX rats without hydrocortisone treatment is required to confirm this suggestion.
The mechanism is unknown whereby HX rats experience a decrease in cancellous bone mass, and both in
tissue-based formation and resorption activity without
an effect on surface-based bone formation and resorption on the trabecular surface. Certainly, pituitary hormone deficiency after HX could be one of the major
factors causing the skeletal alteration. Other factors,
as a consequence of HX, could also attribute to the
alteration. Skeletal bone is capable of adapt to mechanical usage (Frost, 1990a,b).It has been suggested (pers.
comm. with Dr. Harold Frost) that during growth the
secondary metaphyseal spongiosa is normally underloaded and exists in a state of partial disuse. In the
current study we observed that the HX rats were physically inactive and ceased gain in weight and muscle
mass. These responses resemble the condition of partial
unloading and are similar to reduced gravity in spaceflight. In fact, bone alteration after HX has many similarities to the result of spaceflight; decreased longitudinal bone growth (Jee et al., 1983b, Montufar-Solis et
al., 1992, Wronski et al., 1987) decreased cancellous
bone mass and the bone formation with or without decreased the bone resorption (Morey and Baylink, 1978;
Cann and Adachi, 1983; Jee et al., 1983b; Wronski et
al., 1987).
In conclusion, the current study demonstrates that
HX results in cancellous bone loss in the secondary
spongiosa of the proximal tibia. Dynamic analysis
shows that HX results in a transient decrease in osteoclast number in the early time period and decreased
both tissue-based bone formation and resorption without significantly affecting the surface-based bone formation. The bone loss could be due primarily to suppressed longitudinal growth-dependent bone gain and
suppressed tissue-based bone turnover with a lower
bone formation relative to bone resorption. Hypophysectomized rat may be a good model for the study of
512
J.K. YEH ET AL.
pituitary hormone deficiency-induced bone loss in humans.
LITERATURE CITED
Badger, T.M., W.J. Millard, S.M. Owens, J. LaRovere, and D. OSullivan 1991 Effects of gonadal steroids on clearance of growth hormone at steady state in the rat. Endocrinology, 128r1065-1072.
Barnard R., K.M. Haynes, G.A. Werther, and M.J. Waters 1988 The
ontogeny of growth hormone receptors in the rabbit tibia. Endocrinology, 122t2562-2569.
Barnes, C.D., and L.G. Eltherington 1973 Drug Dosage in Laboratory
Animals, rev. ed. Univ. of California Press, Berkeley.
Baron R., R. TTOSS,
and A Vignery 1984 Evidence of sequential remodeling in rat trabecular bone: Morphology, dynamic histomorphometry, and changes during skeletal maturation. Anat Rec.,
208:137-145.
Bentham J., C. Ohlsson, A. Lindahl, O.G.P. Isaksson, and A. Nilsson
1993 A double staining technique for detection of growth hormone and insulin-like growth factor-I binding to rat tibial epiphyseal chondrocytes. J . Endocrinol., 137:361-367.
Cann, C.E., and R.R. Adachi 1983 Bone resorption and mineral excretion in rats during spaceflight. Am. J. Physiol., 244.R327R331.
Chen, M.M., W.S.S. Jee, H.Z. Ke, B.Y. Lin, Q.N. Li, and X.J. Li 1992
Adaptation of cancellous bone to aging and immobilization in
growing rats. Anat Rec., 234.317-334.
Frost, H.M. 1973a Bone Modeling and Skeletal Modeling Errors.
Charles C. Thomas, Springfield, IL.
Frost, H.M. 1973b Bone Remodeling and Its Relation to Metabolic
Bone Disease. Charles C. Thomas, Springfield, IL.
Frost, H.M. 1990a Skeletal structural adaptations to mechanical usage (SATMU): 1. Redefining Wolffs Law: The bone modeling
problem. Anat Rec., 226:403-413.
Frost, H.M. 1990b Skeletal structural adaptations to mechanical usage (SATMU): 1. Redefining Wolffs Law: The remodeling problem. Anat. Rec., 226.414-422.
Hock, J.M., and J. Fonseca 1990 Anabolic effect of human synthetic
parathyroid hormone-(l-34) depends on growth hormone. Endocrinology, 127.1804-1810.
Isaksson, O.G.P., A. Lindahl, A. Nilsson, and J. Isgaard 1987 Mechanism of the stimulatory effect of growth hormone on longitudinal bone growth. Endocrine Rev., 8:426-438.
Jee, W.S.S., E.L. Blackwood, N.L. Dockum, R.K. Haslam, and F.A.
Kincl 1966 Bio-assay of responses of growing bones to cortisol.
Clin. Ortho. Re. Res., 49:39-63.
Jee, W.S.S., J. Inoue, K.W. Jee and T. Haba 1983a Histomorphometric
assay of the growing long bone. In: Handbook of Bone Morphology. H. Takahashi, ed. Nishimura, Niigata City, Japan, pp. 101122.
Jee, W.S.S., H.Z. Park, W.E. Roberts, and G.H. Kenner 1970 Corticosteroid and Bone Am. J. Anat., 129(41:477-479.
Jee, W.S.S., T.J. Wronski, E.R. Morey, and D.B. Kimmel1983bEffects
of spaceflight on trabecular bone in rats. Am. J . Physiol., 244:
R310-R314.
Kember, N.F. 1971 Growth hormone and cartilage cell division in
hypophysectomized rats. Cell Tissue Kinet., 4r193-199.
Kimmel, D.B., and W.S.S. Jee 1980 A quantitative histologic analysis
of the growing long bone metaphysis. Calcif. Tiss. Int., 32.113122.
Kimmel, D.B., and W.S.S. Jee 1982 A quantitative histologic study of
bone turnover in young adult beagles. Anat. Rec., 203:31-45.
Lewinson, D., P. Shenzer, and Z. Hochberg 1993 Growth hormone
involvement in the rermlation of tartrate-resistant acid Dhosphatase-positive cells &at are active in cartilage and bone resorption. Calcif. Tiss. Int., 52:216-221.
Li, X.J., W.S.S. Jee, H.Z. Ke, S. Mori, T. Akamine 1991 Age-related
changes of cancellous and cortical bone histomorphometry in female Sprague-Dawley rats. Cells Mater, Suppl., 1:25-35.
Li, X.J., W.S.S. Jee, S.Y. Chow, and D.M. Woodbury 1990 Adaptation
of cancellous bone to aging and immobilization in the rat: A single photon absorptiometry and histomorphometry study. Anat.
Rec., 227r12-24.
Liu C.C., R. Sanghvi, J.M. Burnell, and G.A. Howard 1987 Simultaneous demonstration of bone alkaline and acid phosphatase activities in plastic-embedded sections and differential inhibition of
the activities, Histochemistry, 86:559-565.
Loveridge N., and C. Farquharson 1993 Studies on growth plate chondrocytes in situ: cell proliferation and differentiation. Acta Paediatr Suppl., 391:42-48.
Martinez, D.A., A.C. Vailas. and R.E. Grindeland 1991 Cortical bone
maturation in young hypophysectomized rats. Am. J. Physiol.,
260:E690-E694.
Montufar-Solis, D., P.J. Duke, and G. Durnova 1992 Spaceflight and
age affect tibial epiphyseal growth plate histomorphometry. J.
Appl. Physiol., 73, Suppl:19%25S.
Morey, E.R., and D.J. Baylink 1978 Inhibition of bone formation during space flight. Science, 20lt1138-1141.
Mori, S., W.S.S. Jee, and X.J. Li 1992 Production of new trabecular
bone in osteopenic ovariectomized rats by prostaglandin E,. Calcif. Tiss. Int., 50r80-87.
Mosekilde Li 1988 Age-related changes in vertebral trabecular bone
architecture-assessed by a new method. Bone, 9:247-250.
Mosekilde Li 1989 Sex differences in age-related loss of vertebral
trabecular bone mass and structure-biomechanical consequences.
Bone, 10:425-432.
Nilsson A., A. Lindahl, S. Eden, and O.G.P. Isaksson 1989 Demonstration of growth hormone receptors in cultured rat epiphyseal
chondrocytes by specific binding of growth hormone and immunohistochemistry. J . Endocrinol., 122~69-71.
Ohlsson C., A. Nilsson, O.G.P. Isaksson, and A. Lindahl 1992 Effects
of growth hormone and insulin-like growth factor-I on DNA synthesis and matrix production in rat epiphyseal chondrocytes in
monolayer culture. J . Endocrinol., 133:291-300.
Ohlsson, C., J. Isgaard, J . Tornell, A. Nilddon, O.G.P. Isaksson, and A.
Lindahl 1993 Endocrine regulation of longitudinal bone growth.
Acta Paediatr. Suppl., 391t33-40.
Parfitt A.M., M.K. Drezner, F.H. Glorieux, J.A. Kanis, H. Malluche,
P.J. Meunier, S.M. Ott, and R.R. Reeker 1987 Bone histomorphometry: Standardization of nomenclature, symbols and units.
J . Bone Miner. Res., 2:595-610.
Reddi, A.H., and N.E. Sullivan 1980 Matrix-induced endochondral
bone differentiation: Influence of hypophysectomy, growth hormone, and thyroid-stimulating hormone. Endocrinolom,
-. 107:
1291-1299.
Schlechter, N.L., S.M. Russell, S. Greenberg, E.M. Spencer, and C.S.
Nicoll 1986 A direct growth effect of growth hormone in rat hindlimb shown by arterial infusion. Am. J . Physiol., 250:E231-E235.
Sontag W. 1986 Quantitative measurements of periosteal and cortical-endosteal bone formation and resorption in the midshaft of
female rat femur. Bone 7.55-62.
Thorngren, K.G., L.I. Hansson, K. Menander-Sellman, and A. Stenstrom 1973 Effect of hypophysectomy on longitudinal bone
growth in the rat. Calcif. Tiss. Res., IIr281-300.
Trippel S.B., J.J. Van Wyk, and H.J. Mankin 1986 Localization of
somatomedin-C binding to bovine growth-plate chondrocytes in
situ. J . Bone Joint Surg., 68:897-902.
Wronski, T.J., E.R. Morey-Holton, S.B. Doty, A.C. Maese, and C.C.
Walsh 1987 Histomorphometric analysis of rat skeleton following
spaceflight. Am. J. Physiol., 252:R252-R255.
Yeh, J.K., J.F. Aloia, M.M. Chen, N. Ling, H. Koo, and W.J. Millard
1994 Effect of growth hormone administration and treadmill exercise on serum and skeletal IGF-I in rats. Am. J . Physiol., 266:
R129-E1.?5
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