Calcif Tissue Int (1999) 64:318–324
© 1999 Springer-Verlag New York Inc.
Histomorphometric Evaluation of the Effects of Ovariectomy on Bone
Turnover in Rat Caudal Vertebrae
N. Miyakoshi, K. Sato, T. Abe, T. Tsuchida, Y. Tamura, T. Kudo
Department of Orthopedic Surgery, Akita University School of Medicine, 1-1-1 Hondo, Akita 010-8543, Japan
Received: 12 August 1997 / Accepted: 11 May 1998
Abstract. The purpose of this study was to learn whether
caudal vertebrae can be used to evaluate the effects of ovari-
ectomy (OVX) in rats. Seven-month-old female Wistar rats
were divided into two groups: the OVX group and the un-
treated control group. All rats were killed at 8 weeks and
their 4th lumbar (L4), 1st caudal (C1), 3rd caudal (C3), and
5th caudal (C5) vertebrae were processed undecalcified and
sectioned with Villanueva bone stain for quantitative bone
histomorphometry. Both length of vertebral bodies and the
cancellous tissue area in C1 were similar in size to L4 but
significantly bigger than C3 and C5. Within the groups,
cancellous bone volume (BV/TV) and trabecular thickness
in both groups gradually increased in caudal vertebrae in
relation to the distal direction. Between the groups, OVX
rats exhibited a significantly lower BV/TV relative to con-
trol rats at L4 and C1, however, no significant difference
were seen at C3 and C5. Bone formation-related parameters
such as osteoid and mineralizing surface, and eroded surface
were higher in the OVX group than in the control group in
caudal as well as in lumbar vertebrae. By quantitative analy-
sis of bone marrow composition, yellow marrow volume in
C3 and C5 was significantly higher than that in L4 and C1,
in both groups. Our results suggest that C1 is similar to L4
in size, bone turnover, and bone marrow composition. How-
ever, further experiments are needed to evaluate the possi-
bility that C1 vertebra could be used as an alternative site
for histomorphometric evaluation of bone changes in OVX
rats.
Key words: Bone histomorphometry — Caudal vertebrae
— Ovariectomy — Rat.
The rat is an appropriate small animal model for skeletal
biological studies especially in experimental osteopenia. In
most analyses using spinal bone in rats, lumbar vertebrae
were usually used. The caudal vertebrae of rats bears a
morphological resemblance to that of the human iliac crest
because of their relative slow rate in longitudinal bone
growth and the presence of some remodeling activity which
is similar to that of adult human bone [1]. Therefore, caudal
vertebrae can be an ideal site for monitoring the experimen-
tal bone diseases. Some researchers used caudal vertebrae
Correspondence to: N. Miyakoshi
[1–13] because the rat long tail has enough bone for evalu-
ating changes in bone volume. However, these studies did
not evaluate the lumbar vertebrae simultaneously, and pre-
sumed that the caudal vertebrae have the same bone turn-
over as the lumbar vertebrae.
Estrogen deficiency is a well-known cause of osteopo-
rosis. Since Saville [14] showed that castration of young rats
resulted in bone loss, osteopenia has been consistently ob-
served in ovariectomized rats [10, 15–20], and to date,
ovariectomized rats have been most commonly used as an
experimental postmenopausal osteopenic model. Although
bone morphometrical studies of cancellous bone in rat lum-
bar vertebrae in normal and ovariectomized rats have been
performed [18, 21], the relationship between lumbar and
caudal vertebrae, especially in bone turnover, is still un-
clear.
Caudal vertebrae are more ideal sites than other spinal
bones in rats for monitoring bone mineral content in vivo
because of the lower repositioning error [5]. However, bone
mineral changes in caudal vertebrae do not always coincide
with other sites such as appendicular bones [3, 13]. In vivo,
observations with dual energy X-ray absorptiometry (DXA)
in caudal vertebrae in OVX rats showed increased bone
mineral density [13]. Because DXA cannot distinguish be-
tween cancellous and cortical bones, we must evaluate why,
in vivo, bone mineral increases in caudal vertebrae in OVX
rats, as opposed to OVX being the most representative ex-
perimental osteopenic model.
Li et al. [22] found in ovariectomized rats that cancellous
bone in the 1st lumbar vertebra (red marrow site) was de-
creased after ovariectomy, whereas the 5th caudal vertebra
(yellow marrow site) was not decreased; they concluded
that unlike skeletal sites with red marrow, estrogen deple-
tion does not induce cancellous osteopenia at skeletal sites
with yellow marrow. However, they did not quantitate the
amount of red and yellow marrow in both sites.
The caudal vertebrae are shallow under the skin and can
be exposed easier than the lumbar vertebrae that are deep
into paravertebral muscles. If we could use the caudal ver-
tebrae as an alternative to the lumbar vertebrae, surgical
experiments on the spinal bone in rats could be done easily;
furthermore, we might use the caudal vertebrae for longi-
tudinal histological studies as iliac biopsies in humans. The
purpose of this study was therefore to determine whether the
caudal vertebrae indicate the same bone turnover and bone
changes as the lumbar vertebrae in normal and in OVX rats;
this was done by bone histomorphometric analysis, includ-
N. Miyakoshi et al.: Bone Histomorphometry in Rat Caudal Vertebrae
Table 1. Length of vertebral bodies and cancellous tissue area
Length (mm)
Control OVX All
Vertebrae (n ⳱ 6) (n ⳱ 6) (n ⳱ 12)
L4 7.02 ± 0.45 7.15 ± 0.28 7.08 ± 0.36
C1 6.48 ± 0.59 6.47 ± 0.76 6.48 ± 0.65
C3 5.33 ± 0.45a**b* 5.60 ± 0.71a** 5.47 ± 0.58a***b**
C5 5.57 ± 1.56a* 5.55 ± 1.12a** 5.56 ± 1.29a***b**
All values are expressed as means ± SD. Lower case letters indicate significant differences between aL4 and bCl of the same group.
* P < 0.05, **P < 0.01, ***P < 0.001
ing the evaluation of the marrow composition, in lumbar
and caudal vertebrae simultaneously.
Materials and Methods
Experimental Design
Seven-month-old adult female Wistar rats with a mean body
weight of 241.8 g (224.0–265.0 g), were allocated to two groups:
the untreated control group (n ⳱ 6) and the OVX group (n ⳱ 6).
Rats in the OVX group were anesthetized with an intraperitoneal
injection of Nembutal (Abott Laboratories, Illinois, USA), and
underwent bilateral ovariectomies, from a dorsal approach, at the
beginning of the experiment. The animals were kept in conven-
tional metal cages under specific pathogen-free conditions. They
received a standard diet (CE-2: CLEA Japan Inc., Tokyo, Japan)
containing 1.14% calcium, 1.06% phosphorus, and 250 IU of vi-
tamin D per 100 g. Food and water were given ad libitum. The
duration of the experiment was 8 weeks.
For the assessment of bone formation by bone histomorphom-
etry, double labeling was performed by giving intraperitoneal in-
jections of 30 mg/kg of tetracycline 12 days before and 10 mg/kg
of calcein 4 days before the animals were killed under Nembutal
anesthesia. Their L4, C1, C3, and C5 vertebrae were removed and
freed of soft tissue.
Sample Preparation
All four vertebrae from each rat were fixed with 70% ethanol and
stained with Villanueva bone stain for 14 days. They were then
dehydrated in a graded series of ethanol and acetone, and embed-
ded in methylmethacrylate. Then 10-␮m-thick sections of the ver-
tebral body were obtained in the midsagittal plane using a micro-
tome (Polycut S, R. Jung, Heidelberg, Germany).
Evaluation of Vertebral Sizes
We measured the length of the vertebral bodies and cancellous
tissue area for evaluation of vertebral sizes. The length of vertebral
bodies was measured with calipers soon after the autopsy. Can-
cellous tissue area was defined as the same area as tissue volume
area in bone histomorphometry. These two parameters in each
vertebra in the control and OVX groups and the sum of both
groups were compared.
Histomorphometric Analysis
Bone histomorphometry in the cancellous area of vertebral bodies
was performed with a semiautomatic tracing system consisting of
a digitizer (KD 3030 L, Graphtec Corp. Tokyo, Japan) connected
319
Cancellous tissue area (mm2)
Control OVX All
(n ⳱ 6) (n ⳱ 6) (n ⳱ 12)
4.04 ± 0.26 4.02 ± 0.40 4.03 ± 0.32
3.54 ± 0.57 3.94 ± 0.51 3.74 ± 0.56
3.00 ± 0.78a** 2.84 ± 0.91a**b** 2.92 ± 0.81a***b**
3.18 ± 0.59a* 3.18 ± 0.48a*b* 3.18 ± 0.51a***b*
to a personal computer (PC 9801 RX, NEC, Tokyo) with software
(Cosmozone 1S, Nikon, Tokyo) using an integrating eye piece
(Integrationsplatte II, Carl Zeiss Co., Ltd. Tokyo). The measure-
ment magnification was ×312.5. The volume of the cancellous
tissue area (tissue volume) was measured in areas 0.4 mm from
both cranial and caudal growth plate-metaphyseal junctions to ex-
clude the primary spongiosa, as well as 0.4 mm from endocortical
surfaces.
Nomenclature and symbols used in conventional bone histo-
morphometry are the same as those described by Parfitt et al. [23].
The validity of their method is confirmed only in the human iliac
trabecular bone, and the isotropism of the structure has not been
fully confirmed in the trabecular bone in rats. We assumed the
isotropism in the trabecular structure in this animal model and used
the method of Parfitt et al. The following histomorphometrical
parameters were measured: percent bone volume (BV/TV, %),
defined as the percentage of trabecular bone volume (BV) to tissue
volume (TV); trabecular thickness (Tb.Th, ␮m), calculated as the
BV/TV divided by half the bone surface (BS); trabecular number
(Tb.N, mm−1); trabecular separation (Tb.Sp, ␮m); percent osteoid
surface (OS/BS, %), defined as the percentage osteoid surface to
BS; percent mineralizing surface (MS/BS, %), defined as the per-
centage of mineralizing surface to BS; mineral apposition rate
(MAR, ␮m/day); surface referent bone formation rate (BFR/BS,
␮m3/␮m2/year); and percent eroded surface (ES/BS, %), defined
as the percentage eroded surface to BS. Tb.N and Tb.Sp were
calculated from the following expressions: Tb.N ⳱ (BV/TV)/
Tb.Th, Tb.Sp ⳱ 1/Tb.N − Tb.Th. MS/BS was obtained from the
equation MS/BS ⳱ 1/2 × sLS/BS + dLS/BS (sLS: single-labeled
surface, dLS: double-labeled surface). MAR was obtained by di-
viding the distance between the two labels on the trabeculae by the
label period. BFR/BS was calculated by the formula MAR × MS/
BS.
Bone Marrow Composition
Bone marrow composition was analyzed by percentages of red and
yellow marrow to total marrow tissue: percent red marrow (%),
defined as the percentage of red marrow to total marrow tissue;
percent yellow marrow (%), defined as the percentage yellow mar-
row to total marrow tissue.
Data Analysis
Statistical differences among each numbered vertebra within the
groups were compared using Fisher’s protected least significant
difference method (post-hoc test) for multiple comparisons in a
one-way analysis of variance. Differences between control and
OVX groups at each vertebra were evaluated with Student’s t test
or Welch’s t test, as appropriate. Statistical computation of the data
was performed using a statistical package (Statview 4.0, Macin-
tosh, Abacus Concepts, Inc., California, USA). Statistical signifi-
cance was set at the 5% level.
320 N. Miyakoshi et al.: Bone Histomorphometry in Rat Caudal Vertebrae

Significantly different from the control group

9.8 ± 3.4a***b*@*
Results

34.7 ± 3.9a***b*

9.0 ± 4.6a*@**
111.9 ± 12.5a***

1.30 ± 0.80@*
At the end of the experiment, OVX rats weighed signifi-

8.9 ± 5.2@*

1.43 ± 0.18
3.22 ± 0.33
cantly more than control rats by the influence of estrogen

202.1 ± 19.9
deficiency [24]. The mean body weight of OVX rats was
270 ± 12.3 g, whereas the control weighed an average of
243.5 ± 13.1 g. This difference is highly significant at the
level of P ⳱ 0.004. General conditions were the same in the
C5

two groups.

8.4 ± 2.8a***b**
a***b*

10.7 ± 3.4a*@**

1.48 ± 0.58@**
@**
33.0 ± 5.4 **

Vertebral Size: Length and Cancellous Tissue Area

1.46 ± 0.11
2.96 ± 0.31
a

225.6 ± 63.2
114.1 ± 8.7

10.2 ± 4.2

The length of the vertebral bodies and cancellous tissue area

among the vertebrae were significantly different in both
control and OVX rats. The post-hoc analysis showed that
C3

values in the L4 and C1 were higher than in the C3 and C5

@
18.1 ± 2.4@*** 13.8 ± 2.0a*@**

(Table 1). No significant differences were seen in each lum-

78.0 ± 9.4@** 101.6 ± 10.4a***
28.9 ± 1.9 **

16.5 ± 5.3@*** 14.3 ± 5.0@**

9.8 ± 3.3@**

1.76 ± 0.44@*** 1.33 ± 0.43@*

All values are expressed as means ± SD. Lower case letters indicate significant differences between aL4 and bCl of the same group.

bar and caudal vertebra between control and OVX groups.

@

1.38 ± 0.08
2.96 ± 0.33
250.7 ± 35.2

Bone Histomorphometry
C1

Table 2 summarizes the results of bone histomorphometry.

BV/TV, Tb.Th, Tb.N, and Tb.Sp were compared within the
12.9 ± 2.5@***

groups of each numbered vertebra to evaluate the volumet-

@
25.4 ± 4.3 *

ric and structural difference between the different numbered

1.37 ± 0.18
3.38 ± 0.22
OVX (n ⳱ 6)

233.5 ± 41.0

vertebrae. Within the groups, BV/TV and Tb.Th in control

and OVX groups gradually increased in proportion to the
distal direction, and significant differences were seen be-
tween L4 and C3/C5 in BV/TV in OVX group, and were
L4

also seen between L4 and three other caudal vertebrae in

97.1 ± 10.9 112.6 ± 10.2a* 124.6 ± 14.0a*** 125.1 ± 12.2a***

Tb.Th in both groups; Tb.N and Tb.Sp showed no signifi-

cant difference among L4 and three other caudal vertebrae.
1.41 ± 0.11
3.05 ± 0.33

0.39 ± 0.20
37.5 ± 3.9

211.2 ± 41.6

5.7 ± 1.3
2.3 ± 1.4
2.8 ± 1.5

In the control group there were no significant difference

among the four vertebral sites in all dynamic parameters. In
the OVX group, however, OS/BS in C3 and C5 were sig-
nificantly less than that in L4, and ES/BS gradually and
C5

significantly decreased in proportion to the distal direction.

Comparing the groups, OVX rats exhibited a signifi-
cantly lower BV/TV in control rats at L4 and C1, however,
1.34 ± 0.22
2.97 ± 0.49

0.46 ± 0.18
34.5 ± 4.6

213.0 ± 32.1 232.0 ± 33.0 223.2 ± 28.4

7.0 ± 2.9
4.2 ± 2.6
3.4 ± 0.9

there were no significant differences at C3 and C5. Bone

formation-related parameters, except MAR: OS/BS, MS/
BS, and BFR/BS were significantly higher in the OVX
group than in the control group at all vertebral levels. ES/BS
C3

was significantly higher in OVX rats than in control rats at

L4, C1, and C5.
1.18 ± 0.13 1.32 ± 0.27
3.12 ± 0.39 2.86 ± 0.38

(␮m3/␮m2/y) × 10-1 0.40 ± 0.12 0.50 ± 0.20

8.4 ± 1.4
31.6 ± 4.9 32.8 ± 1.9

4.5 ± 2.7
3.7 ± 0.8

Bone Marrow Composition

C1

Histologically, L4 and C1 contained mainly red marrow

Control (n ⳱ 6)

* P < 0.05, **P < 0.01, ***P < 0.001

whereas C3 and C5 contained mostly yellow marrow (Fig.

8.8 ± 2.2
2.8 ± 1.3
3.4 ± 0.9

1). Quantitative analysis showed that percent red marrow

Table 2. Bone histomorphometry

at the same numbered vertebrae.

was gradually decreased and yellow marrow was gradually

increased in the distal direction (Table 3). In both control
and OVX rats, percent red marrow in the C3 and C5 was
L4

significantly lower than that in L4 and C1, and yellow mar-

row in the C3 and C5 was significantly higher than that in
L4 and C1. No significant differences were seen between
control and OVX rats in the four vertebral sites.
Tb.N(/mm2)
Tb.Th (␮m)

Tb.Sp (␮m)

(␮m/day)
BV/TV (%)

MS/BS (%)
OS/BS (%)

ES/BS (%)
Parameters

BFR/BS

Discussion
MAR

Some researchers [4, 7, 10–12] attempted bone histomor-

N. Miyakoshi et al.: Bone Histomorphometry in Rat Caudal Vertebrae 321

90.39 ± 5.41a***b***c*
phometry on rat caudal vertebrae, however, they did not

80.18 ± 18.44a***b***
compare caudal vertebrae with lumbar vertebrae concur-
rently. In the present study, we evaluated bone histomor-

37.08 ± 10.89a**
phometric analysis on both lumbar (L4) and caudal verte-
brae (C1, C3, and C5) simultaneously and observed all cau-
20.34 ± 7.05
dal vertebrae to show similar bone turnover to lumbar
(n ⳱ 12)

vertebrae, regardless of the fact that cancellous bone loss

did not occur in C3 and C5 which contained mainly yellow
All

marrow.
We selected the proximal five caudal vertebrae because
further distal dissection would have been difficult due to
79.22 ± 23.63a***b***
90.62 ± 4.93a***b***

strong ligamentous connections within each vertebrae as the

size of the body progressively decreased. We suspected that
the more distal vertebrae had the fewer cancellous bone
areas for bone histomorphometry.
18.61 ± 6.98
34.06 ± 6.31

There is little in the literature that describes the size and

All values are expressed as means ± SD. Lower case letters indicate significant differences between aL4, bCl, and cC3 of the same group.
(n ⳱ 6)

bone histomorphometric results in the rat caudal vertebrae.

OVX

Nakamura et al. [6, 7] measured the length and cancellous

bone volume of the first caudal vertebral bodies in normal
male Wistar rats aged 2 years and over. Their length was 7.2
± 0.52 mm, and cancellous bone volume was about 25%. In
81.15 ± 13.69a***b***
90.17 ± 6.31a***b***

the present study, in both control and OVX rats, cancellous

Yellow marrow (%)

bone volume and trabecular thickness were gradually in-

40.10 ± 14.12a**

creased in the distal direction, with no significant changes

observed in trabecular number and separation. These obser-
22.07 ± 7.30

vations indicate that trabecular thickness in the caudal ver-

tebrae became gradually thicker with increment in bone
(n ⳱ 6)
Control

mass distally without structural change.

Osteopenia has been a consistent finding in ovariecto-
mized rats [10, 14–20]. Histomorphometric studies have
shown that this phenomenon is associated with increased
9.61 ± 5.41a***b***c*

bone turnover, with resorption exceeding formation [16–

19.82 ± 18.44a***b***

20]. In our study, fluorochrome-based indices of bone for-

mation, such as mineralizing surface and bone formation
62.92 ± 10.89a**

rate, in OVX rats were more increased in control rats in each

caudal vertebra, and the eroded surface as well. These ob-
79.66 ± 7.05

servations indicate that the same high turn-over state in the

(n ⳱ 12)

lumbar vertebrae also occurs in the caudal vertebrae.

Nagashima [13] measured bone mineral density (BMD)
All

of OVX rats aged 14 weeks in vivo longitudinally (4–24

weeks postovariectomy) in different sites: the skull, distal
femur, proximal tibia, and 4th–6th caudal vertebrae. Distal
20.79 ± 23.63a***b***
9.38 ± 4.93a***b***

femoral and proximal tibial BMD decreased, however,

BMD of the skull and caudal vertebrae increased. Lindsay et
al. [3] reported similar results. They used unknown aged
Sprague-Dawley rats and measured femoral and 7th caudal
81.39 ± 6.98
65.94 ± 6.31

vertebral density by photon absorptiometry after a 10-month

experimental period, and ovariectomy resulted in osteope-
(n ⳱ 6)

nia of the femur but not of the caudal vertebra. We sus-

OVX

pected several reasons why no significance was seen in C3

and C5. C3 and C5 were smaller in their size and cancellous
bone area than C1, and therefore significance was hardly
* P < 0.05, **P < 0.01, ***P < 0.001

revealed. another reason was a limited experimental dura-

18.85 ± 13.69a***b***
9.84 ± 6.31a***b***
Table 3. Bone marrow composition

tion. It is also important to note that 8 weeks postovariec-

tomy may not be of significant duration to achieve a steady
59.90 ± 14.12a**
Red marrow (%)

state in the bone-mass response to estrogen deficiency in

77.93 ± 7.30

caudal vertebrae. However, considering the result of Na-

gashima [13] and Lindsay et al. [3], the different time
(n ⳱ 6)
Control

course of bone mineral change at various sites may be due

to different turnover rate and the proportion of trabecular
bone.
Our quantitative measurement of red and yellow marrow
for the four sites confirmed the Li et al. [22] finding. These
Vertebrae

authors found that ovariectomy does not induce cancellous

osteopenia at skeletal sites with yellow marrow in rats. In
C3
C5
C1

our present study, yellow marrow-enriched C3 and C5 did

L4
322
Fig. 1. Bone changes in control and OVX rats at L4, C1, C3, and
C5 vertebrae. (A) L4 in the control, (B) L4 in the OVX, (C) C1 in
the control, (D) C1 in the OVX, (E) C3 in the control, (F) C3 in
the OVX, (G) C5 in the control, and (H) C5 in the OVX group. L4
and C1 in the OVX group (B,D) show reduced cancellous bone
N. Miyakoshi et al.: Bone Histomorphometry in Rat Caudal Vertebrae
volume (BV) compared with those in the control group (A,C). In
contrast, BV is preserved in the C3 and C5 (F,H) of OVX rats.
Concerning bone marrow composition, red marrow is dominant in
L4 and C1 (A,B,C,D), and yellow marrow is dominant in C3 and
C5 (E,F,G,H) in both groups. (Villanueva bone stain, ×40).
N. Miyakoshi et al.: Bone Histomorphometry in Rat Caudal Vertebrae 323

Fig. 1. Continued
324
not show osteopenia, but red marrow-enriched C1 and L4
did show osteopenia. Therefore, different marrow compo-
sition between C1 and two other caudal vertebrae could
possibly be the reason why C3 and C5 did not lose cancel-
lous bone in ovariectomized rats.
In this study, we have not measured the relative impact
of weight bearing and muscular strain at lumbar and caudal
vertebrae. Muscular strain may be likely greater at the cau-
dal vertebrae than at the lumbar vertebrae. C1 connects to
sacrum and has lesser movement than C3 or C5. Therefore
the mechanical feature of C1 may be similar to lumbar
vertebrae than other caudal vertebrae. Further studies in-
volving time course, impact of weight bearing and repro-
ducibility are needed before we could conclude that C1
could be used as an alternative site for histomorphometric
evaluation of bone changes in OVX rats. The advantage
with C1 is that it could be used for longitudinal studies
because of its easy accessibility.
In summary, this study is the first study by bone histo-
morphometric analysis to determine the relationship be-
tween lumbar and caudal vertebrae in normal and OVX rats
in that it involves less work. The results showed that C1 has
the more similar size and bone marrow composition to L4
than C3 or C5. Bone histomorphometric analysis showed
that all three caudal vertebrae (C1, C3, and C5) have similar
bone turnover to lumbar vertebrae in normal and OVX rats.
However, significant cancellous bone-loss occurred only in
C1 at 8 weeks postovariectomy. Further experiments are
needed to evaluate if C1 vertebra could be used as an al-
ternative site for histomorphometric evaluation of bone
change in OVX rats.
Acknowledgment. We wish to thank Subburaman Mohan, Ph.D.
for helpful discussion during the manuscript preparation.
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