Introduction concomitant with the BMD-and BMC-reduction in SCI patients [6].

1. Introduction concomitant with the BMD-and BMC-reduction in SCI patients [6].

Spinal cord injury (SCI) is well known to cause Osteoporosis after SCI has been studied in rapid bone loss and thereby increase the risk of

animal models. Jiang et al. [7] reported that the dry fractures. Both bone mineral density (BMD) and

bone weight and ash weight of the femur/tibia and the bone mineral content (BMC) are reduced by bone

BMD of the proximal tibia were significantly demineralization in SCI patients; this occurs in a

reduced 3 weeks after SCI in rats, and these relatively early phase (within 12 months) after the

reductions could be observed even 6 months after the injury [1-3]. The reduction in BMD and BMC is

injury. In addition, bone histomorphometry revealed found in the pelvic and lower limbs of SCI patients

deterioration in the trabecular bone structure of the [4], and the most severely damaged sites are the

tibial metaphysis 3 weeks after SCI in rats [7]. They trabecular metaphyseal-epiphyseal areas of the distal

also reported that deterioration in bone mass, cortical femur and the proximal tibia [5]. In addition,

bone geometric structure and trabecular deterioration of bone microstructure or geometry is

microstructure of the proximal tibial metaphysis is grosser in rats with SCI than in rats with sciatic

Corresponding author: Akira Minematsu, Ph.D., professor, neurectomy (NX) or ovariectomy (OVX) [8, 9]. Thus, research fields: osteoporosis and rehabilitation. E-mail: a.minematsu@kio.ac.jp.

although SCI is reported to damage both bone mass

Time Course of Changes in Trabecular Bone Microstructure in Rats with Spinal Cord Injury

(eg. BMD and BMC) and quality (eg. geometry and

2.2 Sample Collections

microstructure), the initial bone loss caused by SCI Rats underwent the SCI or sham operation were

has not been fully studied in rats yet. Therefore, the anesthetized with intraperitoneal injection of

purpose of this study was to examine changes in trabecular bone microstructure (TBMS) during the

pentobarbital sodium and sacrificed by exsanguination initial post-SCI period in rats. The authors report

from the carotid artery 1, 3, or 5 weeks after the herein that SCI causes reduced bone mass and

surgery. To disregard circadian variation in serum deterioration of TBMS as early as 1 week.

biochemical data, all blood samples were collected in the morning. Following exsanguination, both tibias

2. Materials and Methods

were isolated and stored in 70% ethanol until analyzed.

The study was performed according to the Guide for Animal Experimentation at the Kio

2.3 Trabecular Bone Microstructure (TBMS) Analysis University and the animal experimental protocol was

approved by the Committee for Research Facilities Using an X-ray micro-computed tomography and Laboratory Animal Science of the Kio (micro-CT; ©Hitachi Medical Corporation, Tokyo, University.

Japan), the proximal tibias were scanned at 60 kV, 100 µA, with a voxel size of 26.4 µm in the

2.1 Animals high-definition mode. A BMD phantom was Thirty male Wistar rats aged 8 weeks (Japan SLC,

simultaneously scanned to measure tissue mineral Inc., Hamamatsu, Japan) were housed in an animal

density (TMD) and to calculate BMC and volumetric research facility (temperature, 23 ± 2 °C; humidity,

BMD (vBMD: BMC/tissue volume) under the same

55 ± 5%) with a 12:12-h light-dark cycle. They were scanning condition. Scanned data were transmitted to fed a standard laboratory diet (CE-2; CLEA, Inc.,

a personal computer, in which TBMS of the region of Hamamatsu, Japan) and water ad libitum throughout

interest (ROI) was analyzed by a special software for the experiment. Sixteen rats were assigned to the SCI

bone analysis, TRI BON 3D (Ratoc ystem group and 14 rats to the SHAM group. All rats were

Engineering Co., Ltd., Tokyo, Japan). The ROI was a anesthetized by intraperitoneal injection of portion of 2 mm-length at the tibial metaphysis, and pentobarbital sodium (40 mg/kg). Rats in the SCI

the first slice was scanned 1 mm-distal from the group underwent laminectomy, in which the lower

demarcation of physeal-metaphyseal. tissue volume thoracic spinal cord was surgically transected using

(TV), bone volume (BV), bone volume fraction micro scissors. Rats in the SHAM group underwent a

(BV/TV), trabecular thickness (Tb. Th), trabecular sham operation, in which the lower thoracic spinal

width (Tb. W), trabecular number (Tb. N), trabecular cord was left intact. All treated rats were kept on a

separation (Tb. Sp), connectivity density (Conn. D), warming pad until they awoke from the anesthesia,

and trabecular bone pattern factor (TBPf) were and were given a subcutaneous injection of assessed as parameters of TBMS in the proximal enrofloxacin (10 mg/kg). During the period of the

tibia.

first week after the surgery, small amounts of diet

2.4 Dry Bone and Ash Weight Measurement were placed on the floor of the cage in order to allow

the rats to eat them easily. Manual bladder expression After TBMS analysis, in order to obtain dry bone was performed for SCI rats until reflexive voiding

weight, the tibias were dehydrated in 100% ethanol had recovered.

for 48 h, and then dried at 100 °C for 24 h in a drying

Time Course of Changes in Trabecular Bone Microstructure in Rats with Spinal Cord Injury

machine (ADVANTEC®, Tokyo, Japan). Finally, the significantly greater, in the SCI group than in the tibias were burned to ash at 600 °C for 24 h with an

SHAM group (Table 2).

electric furnace (NITTO KAGAKU Co., Ltd.,

3.3 Blood Biochemical Analysis

Nagoya, Japan), and the ash thus obtained was weighed.

Serum concentrations of IP and ALP were significantly lower in the SCI group than in the

2.5 Serum Biochemical Analysis SHAM group 1 week after SCI (Table 3). However,

Serum was separated from coagulated blood by there was no difference in all the measured serum centrifugation at 2500 rpm for 20 min, and then stored

biochemical parameters between the SCI and the at -80 ℃ until analyzed. Serum concentrations of

SHAM groups 3 and 5 weeks after SCI. calcium (Ca), inorganic phosphate (IP), total protein (TP), creatinine (CRE) and alkaline phosphatase (ALP)

4. Discussion

were determined. This paper is the first to report that SCI causes reduced bone mass and deterioration of

2.6 Statistical Data Analysis TBMS as early as 1 week. The mechanisms of

All values are expressed as mean ± standard SCI-induced bone loss are multifactorial [10], and deviation (SD). Difference between the SCI and

include not only lack of mechanical stress like SHAM groups was assessed using unpaired t-test. A P

the immobilization-induced bone loss but also value of < 0.05 was considered statistically significant.

metabolic, hormonal and neural factors related to All statistical analyses were performed by Excel

bone resorption. Ju et al. [11] reported that BMD, Statistics software (Excel 2010 ver.1.08 for Windows,

BV, BV/TV, Tb. Th, Tb. N and Conn. D decrease 14 Social Survey Research Information Co., Ltd., Tokyo,

days after tail suspension, while Tb. Sp increases. Japan).

Jiang et al. [8] reported that NX resulted in reduction in dry bone weight, ash weight, BV/TV, Tb. Th, Tb.

3. Results

N, and in enhancement in Tb. Sp after 21 days. Thus,

3.1 Body Weight, Dry Bone Weight, Ash Weight and the immobilization leads to bone loss and Bone Mass Parameters

deterioration of TBMS in a few weeks. Similarly, OVX was found to lead to reduction in BMD,

Body weight was significantly lighter in the SCI BV/TV, Tb. Th, Tb. N and Conn. D, and to group than in the SHAM group 1 and 5 weeks after

enhancement in Tb. Sp after 3 weeks [9]. However, SCI (Table 1). Likewise, dry bone weight, ash weight,

the bone loss and deterioration of TBMS caused by BMC and vBMD in the tibia were significantly lighter

SCI were more severe than those observed after NX in the SCI group than in the SHAM group 1, 3 and 5

and OVX [7-9].

weeks after SCI (Table 1). Our results are similar to those of the previous studies [7, 8]; in the present study, the significantly

3.2 Trabecular bone Microstructure (TBMS) lighter dry bone weight and ash weight of the tibia

Parameters were observed as early as 1 week after SCI, and these

All TBMS parameters in the SCI group were reductions became greater as the time advanced. different from those of the SHAM group; TV, BV,

Jiang et al. [7] reported that the water content of the BV/TV, Tb. Th, Tb. W, Tb. N, and Conn. D were

forelimb bones is increased 3 weeks after SCI, but is significantly smaller, but Tb. Sp and TBPf were

decreased 6 weeks and 6 months after SCI; it is

Time Course of Changes in Trabecular Bone Microstructure in Rats with Spinal Cord Injury

Table 1 Body weight, dry bone weight, ash weight and bone mass parameters in the tibia.

SHAM SCI Body weight (g)

SHAM SCI

SHAM SCI

339.8 ± 11.6 289.2 ± 23.3** Dry bone weight (mg)

443.8 ± 9.7 372.6 ± 27.4** Ash weight (mg)

265.6 ± 6.7 219.1 ± 13.7** TMD (mg/cm 3 )

278.9 ± 10.7 280.3 ± 22.8 BMC (µg)

523.1 ± 102.4 277.0 ± 135.2** vBMD (mg/cm 3 )

47.4 ± 8.1 25.5 ± 10.9** ** Significantly different from the SHAM group (P < 0.01).

TMD: tissue mineral density; BMC: bone mineral density; vBMD: volume bone mineral density.

Table 2 Trabecular bone microstructure parameters in the proximal tibia.

SHAM SCI TV (mm 3 )

SHAM SCI

SHAM SCI

10.88 ± 0.61 10.68 ± 0.90 BV (mm 3 )

1.87 ± 3.6 0.97 ± 0.44** BV/TV (%)

17.1 ± 2.7 9.0 ± 3.4** Tb. Th (µm)

101.9 ± 6.2 96.7 ± 8.9 Tb. W (µm)

150.9 ± 12.1 139.5 ± 11.9* Tb. N (1/mm)

1.22 ± 0.20 0.70 ± 0.22** Tb. Sp (µm)

202.5 ± 15.4 280.2 ± 80.7** Conn. D (1/mm)

19.9 ± 4.7 8.2 ± 5.8** TBPf (1/mm)

12.1 ± 1.8 15.5 ± 3.1** * Significantly different from the SHAM group (P<0.05).

** Significantly different from the SHAM group (P<0.01). TV: tissue volume; BV: bone volume; Tb.Th: trabecular thickness; Tb.W: trabecular width; Tb.N: trabecular number; Conn.D: connectivity density; Tb.Sp: trabecular separation; TBPf: trabecular bone pattern factor.

Table 3 Serum biochemical parameters.

SHAM SCI Calcium (mg/dL)

SHAM SCI

SHAM SCI

10.1 ± 0.2 9.9 ± 0.3 Inorganic phosphate (mg/dL) 9.4 ± 0.5

7.8 ± 0.7 7.9 ± 0.5 Total protein (g/dL)

6.2 ± 0.2 6.1 ± 0.3 Creatinine (mg/dL)

0.28 ± 0.03 0.27 ± 0.03 Alkaline phosphatase (IU/L) 1353 ± 130

1045 ± 68 1081 ± 121 * Significantly different from the SHAM group (P<0.05).

Time Course of Changes in Trabecular Bone Microstructure in Rats with Spinal Cord Injury

likely that this decrease is due mainly to bone loss [5, 16]. Levels of serum osteocalcin, bone overloading of the forelimb. They also reported that

formation marker, were higher in the post-injury the water content of femur and tibia is significantly

period from 16 to 24 weeks in SCI patients than in lower 3 weeks after SCI, but is significantly higher 6

able-bodied people, but returned to those of months after SCI, while the ash weight of all bone

able-bodied people 36 weeks after the injury [17]. On tissues remained unchanged in SCI rats. A decrease

the other hand, levels of serum type I collagen in BMC may lead to increased water content,

C-telopeptide, bone resorption maker, were higher although the effects of SCI on water content are not

in the post-injury period from 16 to 48 weeks in clarified yet [7]. These results suggest that bone

SCI patients than in able-bodied people, but returned composition such as water or mineral content change

to those of able-bodied people 71 weeks after the in relation to the degree and acuteness of SCI. Ash

injury [17]. In an animal study, osteoclast activity weight was reported to be highly correlated with

increases after SCI; rats with SCI showed about 3-fold BMC measured in situ by a dual-energy X-ray

increase in the number of mature osteoclast (tartrate absorptiometry (DXA), and ash weight was resistant acid phosphatase-positive multinucleated

regarded as a better predictive index for femoral cells) at the growth plate after 10 days compared with mechanical failure loads, as compared with normal rats [14]. Furthermore, Morse et al. [14] DXA-assessed BMC [12]. Although our BMC was

reported that the bone formation rate defined as bone calculated with not DXA but micro-CT both BMC

formation rate/bone surface (BFR/BS) decreases after and ash weight were significantly lowed 1 week after

SCI. This may be due mainly to the increased SCI, suggesting that loss of bone mineral and bone

osteoclast activity at the growth plate, which may strength occurred during the first week period

prevent new bone formation, resulting in both bone following SCI.

loss and TBMS deterioration. Using the same rat SCI Bone mass accounts for 70% of the variation in

model as herein, however, Jiang et al. [7] showed that bone strength that is decreased by loss of bone mass

bone mass and microarchitectural parameters [13]. However, the remaining 30% of the variation in

decreased 3 weeks after the surgery, in spite of an bone strength is ascribable to bone quality, especially

increase in mineral apposition rate (MAR) and

a major structural factor of bone quality, TBMS [14]. BFR/BS. Such deterioration in bone mass and In our study, deterioration of TBMS and loss of bone

microarchitectural parameters appeared to be mass occurred as early as 1 week after SCI; the bone

dependent of the eroded surface/bone surface (ES/BS) loss was likely preceded by the deterioration in TBMS.

[7]. The ES/BS remained higher in the SCI rats than in Morse et al. [14] reported that BMC, BV, BV/TV, Tb.

the SHAM rats throughout the experiment, although a Th, and Conn. D are decreased 10 days after SCI;

decrease in MAR and BFR/BS occurred between 6 these parameters except Tb. Th are approximately half

weeks and 6 months after the surgery [7]. In addition, the control value in agreement with the present results.

it was suggested that increased osteoclast activity, Voor et al. [15] reported that the cancellous bone

without a concomitant increase in osteoblast activity, volume fraction in the proximal tibia becomes

is one of the reasons underlying the imbalance approximately 75% of baseline value within 3 weeks

between bone formation and resorption after SCI [18]. after SCI.

Bone formation and resorption are increased Bone turnover increases after SCI, and immediately after SCI. This increase in bone

uncoupling of bone formation and resorption leads to resorption is steadily maintained during the post-SCI

Time Course of Changes in Trabecular Bone Microstructure in Rats with Spinal Cord Injury

period, while the increased bone formation rate spinal cord injury: A cross-sectional observational returnes to normal levels. Thus, SCI ultimately leads studyin 100 paraplegic men.” Osteoporosis Int. 15:

180-189.

to decrease in bone parameters, especially BMC and [5] Jiang, S. D., Jiang, L.S., and Dai, L. Y. 2006. TBMS.

“Mechanism of osteoporosis in spinal cord injury.” Clin. The present study is limited by the defects of data

Endocrinol. 65: 555-565.

concerning bone metabolism available by serum [6] Modlesky, C. M., Majumdar, S., Narasimhan, A., and Dudley, G. A. 2004. “Trabecular bone microarchitecture

biomarkers and histomorphometric analyses. is deteriorated in men with spinal cord injury.” J. Bone Nevertheless, it seems very likely that reduction in

Miner Res. 19: 48-55.

bone mass and deterioration of TBMS are caused by [7] Jiang, S. D., Jiang, L. S., and Dai, L. Y. 2007. “Change in an excess of bone resorption over bone formation in bone mass, bone structure, bone biomechanical properties, and bone metabolism after spinal cord injury: A 6-month

the early stages after SCI. longitudinal study in growing rats.” Calcif. Tissue Int. 80:

167-175.

5. Conclusions

[8] Jiang, S. D., Jiang, L. S., and Dai, L. Y. 2006. “Spinal

Bone mass and TBMS were rapidly damaged by cord injury causes more damage to bone mass, bone

structure, biomechanical properties and bone metabolism SCI. Transected rat model of SCI resulted in rapid

than sciatic neurectomy in young rats.” Osteoporosis Int. reduction of dry bone weight, ash weight and

17: 1552-1561.

BMC in the tibia. In addition, SCI also caused [9] Jiang, S. D., Shen, C., Jiang, L. S., and Dai, L. Y. deterioration of TBMS as early as 1 week after the 2007. “Differences of bone mass and bone structure in osteopenic rat models caused by spinal cord

injury, and this deterioration persisted up to at least 5 injury and ovariectomy.” Osteoporosis Int. 18: 743-750.

weeks after SCI. All TBMS parameters were also [10] Maimoun, L., Fattal, C., Micallef, J. P., Peruchon, E., and damaged by SCI, and this microstructural

Rabischong, P., 2006. “Bone loss spinal cord-injured deterioration appeared to lead to a reduction in patients: from physiopathology to therapy.” Spinal Cord

44: 203-210.

trabecular bone volume. Based on our results, it [11] Ju, Y. I., Sone, T., Okamoto, T., and Fukunaga, M. 2008.

should be noted that in SCI patients, interventions for “Jump exercise during remobilization restores integrity of preventing bone loss should start as soon as possible

the trabecular architecture after tail suspension in young after the injury.

rat.” J. Appl. Physiol. 104: 1594-1600. [12] Lochmuller, E. M., Miller, P., Burklein, D., Wehr, U.,

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June 2014, Vol. 8, No. 6, pp. 529-532

Journal of Life Sciences, ISSN 1934-7391, USA

DAVID PUBLISHING

A Primary Hepatobiliary Neoplasia in a Persian Cat

Mohamed Shokry 1 , El-Sayed Berbish 1 and Iman Shaheed 2

1. Departments of Surgery, Faculty of Veterinary Medicine, Cairo University, Giza 12211, Egypt 2. Departments of Pathology, Faculty of Veterinary Medicine, Cairo University, Giza 12211, Egypt

Received: April 27, 2014 / Accepted: May 31, 2014 / Published: June 30, 2014.

Abstract: Biliary carcinoma was diagnosed in a 7-year female Persian cat. The presented clinical signs were not conclusive for hepatic neoplasms and shared many other common health problems in cats i.e. anorexia, vomiting and dehydration. Blood laboratory analysis showed marked elevation of liver enzymes. Ultrasonography revealed a mixed echogenic pattern with multiple hypoechoic cyst-like cavities. At necropsy, the liver showed diffuse multi-lobulated masses affecting the parenchyma of all the lobes of the liver . Histopathology confirmed the neoplastic nature of the hepatic parenchymatous masses.

Key words: Hepatobiliary, neoplasia, alpha-fetoprotein.