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International Journal of Universal Pharmacy and Bio Sciences 3(6): November-December 2014

INTERNATIONAL JOURNAL OF UNIVERSAL

PHARMACY AND BIO SCIENCES IMPACT FACTOR 2.093***

ICV 5.13*** Pharmaceutical Sciences REVIEW ARTICLE……!!!

A REVIEW ON VARIOUS ANIMAL MODELS: INDUCED

OSTEOPOROSIS

Pavani ade1, Dr.Vijay R.Chidrawar

2, Uma Maheshwara Rao V

3

M. Pharmacy Research Scholar, Associate Professor & HOD

2, Dept of Pharmacology, Associate

Professor & Principal3, Dept of Pharmacognosy, CMR College of Pharmacy, Kandlakoya,

Medchal, R R Dist, Hyderabad, India. 501401.

KEYWORDS:

Ovariectomy,

Orchidectomy,

immobilization, and

dietary manipulations.

For Correspondence:

Pavani ade *

Address: M. Pharmacy

Research Scholar, Dept

of Pharmacology, CMR

College of Pharmacy,

Kandlakoya, Medchal, R

R Dist, Hyderabad,

India.501401

Email:

pavani1036@gmail.com

ABSTRACT

osteoporosis is a multifactorial skeletal disease, characterized by

increased porosity of the skeletal resulting from reduced bone mass,

the associated structural changes predispose the bone to fracture. A

large variety of animal species, including rodents, rabbits, dogs, and

primates, have been used as animal models osteoporosis research.

Among these, the laboratory rat is the preferred animal for most

researchers. Its skeleton has been studied extensively and although

there are several limitations to it are the human condition, these can

be overcome through detailed knowledge of its specific traits or with

certain techniques. The rat has been used in many experimental

protocols leading to bone loss, including Ovariectomy,

Orchidectomy, immobilization, and dietary manipulations.

According to this review Ovariectomy animal shown good effects

compared to all animal models.

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INTRODUCTION:

Bone is a most important dynamic tissue; it has multifunctions like it gives shape to body, helps in

movement, take care of blood cell production and weight bearing. By this reason, some of the bone

related problems like osteoporosis, rheumatoid arthritis and osteomalasia are the area of concern in

drug development (Kasabi, Handral and Prabhu., (2012). Osteoporosis mostly effects in older

peoples and especially in post menopausal women because of deficiency of estrogen hormone after

cessation of menopause (Kasabi, Handral and Prabhu., (2012). Osteoporosis is a disease

characterized by low bone mass and microarchitectual deterioration of bone tissue leading to

skeletal fragility and fracture. Reinwald and Burr., (2008). Osteoporosis is a multifactorial disease

and it can be localized or involve the entire skeleton. (Lelovas et al., 2008). Osteoporosis classified

into two types Type -I and Type-II. Type-I again classified into post menopausal and senile, Type II

osteoporosis called secondary osteoporosis (Lelovas et al., 2008). This loss and deterioration of the

structure of bone tissue is caused by imbalance in bone remodeling, due either to an increase

activity or number of osteoclasts and reduced number or activity of osteoblasts (Omara et al., 2009).

Osteoporosis effects mainly sites of fracture include vertebral bodies, distal radius, and the

proximal femur, but some osteoporotic individuals have generalized skeletal fragility and fracture at

other sites, such as ribs and long bones, also most common. The main reason for the osteoporosis is

deficiency of the estrogen hormone. In women the prevalence of vertebral fractures starts to

increase at the time of menopause, in men prevalence of vertebral fracture increased at older ages,

with the ratio 2:1 that of women. The incidence of hip fracture accelerates nearly 10 years after

menopause in women and after age 70 in men. Women have twice as many fractures as men. Post

menopausal osteoporosis is bone resorption (Osteoclasts) relative to bone formation, in conjunction

with an increased rate of bone turnover. The disability, mortality and cost of hip and vertebral

fractures are substantial in the rapidly growing, aging population, this is the reason the prevention

of osteoporosis is a major health problem in world wide. The progressive decreased in bone mass

leads to an increased to fractures, which results in morbidity and mortality (Kasabi, Handral and

Prabhu., (2012). The decrease in ovarian estrogen production is the main cause of rapid hormone

hormone - related bone loss during the first decade after menopause (Menopause, aging, hereditary

factors, inadequate calcium intake and absorption, excessive alcohol intake and cigarette smoking

(Kasabi, Handral and Prabhu., (2012).

Preclinical studies in animal models that are similar to characteristics of human disease processes

are essential for research purposes. Guidelines established by food and drug administration (FDA)

indicate that therapeutic treatments designed to reduce or prevent post menopausal osteoporosis,

should, in the early stage be tested in an ovariectomized rodent model such as the rat because it is

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comparatively characterized in terms of bone loss (Reinwald and Burr, (2008). Even there is no

single animal model that replicates all the characteristics of human osteoporosis. Old work

monkey’s ranks are highly as an appropriate animal model of osteopenia because of an evolutionary

ancestry that has numerous reproductive and physiologic similarities to humans (Reinwald and

Burr., (2008). According to previous studies the practical efficacy of using select large, animal

species specifically dogs, sheep, goats and swine to meet the requisite necessary for an animal

model of bone loss, on that is predominantly associated with deficiency of estrogen hormone and

has some practical relevance to human post menopausal osteoporosis (Reinwald and Burr., (2008).

Animal models requirements

Severely decreased circulating estradiol concentrations in post menopausal women are a major

factor contributing to the accelerated rate of bone loss (Reinwald and Burr., (2008).

The FDA has recommended ovariectomized animals as the preferred animal model for bone loss

research. The engineered commonality in mode of onset of bone loss (i.e., ovarian estrogen

depletion) provides a reasonable basis on which to gauge the potential clinical outcome of a drug or

treatment for osteoporosis.

Animal’s efficacy as a model for postmenopausal osteoporosis in experiments depends on criteria

that include the following.

1. Appropriateness as a model of estrogen deficiency (i.e., significant bone loss and a similar,

if not identical, tissue level mechanism for bone loss induced by estrogen depletion).

2. Specific biological and physiological characteristics (e.g., osteonal bone remodeling).

3. Cost and availability.

4. Housing or spatial requirements.

5. Manageability during an experiment.

6. Reproducible results.

7. Minimal ethical or societal implications.

8. Predictive of skeletal effects of potential osteoporosis therapies in adult’s humans (e.g.,

BMD).

Various animals’ models are present for inducing osteoporosis, large OVX animal models like

dogs, sheep, goats, pigs etc. but compare to rats OVX induced osteoporosis it is have disadvantages

like cost expensive, time taken, labour, availability and difficult to manage

Animal models to induced osteoporosis in rats

1. Ovariectomy induced osteoporosis.

2. Immobilization induced osteoporosis

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3. Glucocorticoid induced osteoporosis

4. Low calcium diet induced osteoporosis

5. Orchidectomy induced osteoporosis

6. Alcohol abuse induced osteoporosis

Ovariectomy induced osteoporosis

Ovariectomy was made by two dorso-lateral incisions, approximately 1 cm long above the ovaries.

With the use of a sharp dissecting scissors, the skin was cut almost together with the dorsal muscles

and the peritoneal cavity was thus accessed. After peritoneal cavity was accessed, the ovary was

found, surrounded by a variable amount of fat. The surgery was done under the cocktail anesthesia

i.e. Ketamine 80 mg/kg, Xylazine 5 mg/kg i.p. The connection between the fallopian tube and the

uterine horn was cut and the ovary moved out. The suturing was performed by using absorbable

catguts. Three single non- absorbable catgut stitches were placed on the skin. In the sham operation

control group, the ovaries were exposed as above and manipulated gently but not excised. The

animals were given antibiotic for four days and Povidine-iodine solution applied locally. Then rats

were allowed for twenty one days for the development of osteoporosis (Kasabi, Handral and

Prabhu., (2012).

Advantages of OVX induced osteoporosis in Female Wistar rats compare to other animal models

It is FDA approval model

Excellent preclinical model

Rapid bone loss of and strength

Reproducible biologic response

Low cost of acquisition

Required little maintenance

Easy and safe to handled

Estrogen

Sex steroids are having an important impact on bone physiology. Estrogen (E) appears to be the

most important sex steroid in preventing osteoporosis in women. Despite the overwhelming

evidence that estrogens modulate bone growth and turnover in vivo, estrogen receptors (ER) were

detected only recently. Two isoforms of ER are ERα and ERβ. Both are present on osteoblasts and

osteoclasts cells. A number of growth factors and cytokines appear to modulate bone resorption in

vitro and in vivo (Krassas and Papadopoulou, 2001).

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The mechanism of action of the ER

The ER is a member of the steroid receptor (SR) superfamily of ligand-dependent transcription

factors. Within this superfamily, the sex steroid receptors are the most conserved both in primary

sequence and mechanism of action. Consequently, a composite model of the mechanism of action

of this class of proteins has emerged. Specifically, ER agonists mediate their effect on gene

transcription via specific intracellular receptor proteins located within target cell nuclei. Upon

interaction with each cognate ligand the latent receptor becomes activated. This event permits the

displacement of heat-shock proteins (HSPs), facilitates receptor dimerization and promotes the

interaction of the receptor with specific steroid response elements (SRE) located within the

regulatory regions of target promoters. At this location depending on the cellular and promoter

context, the ligand-activated receptor can interact with the general transcription apparatus (GTA)

directly or indirectly through adaptor proteins. Ultimately, these interactions stabilize the

transcription pre initiation complex and enhance RNA polymerase activity. Although several

rounds of phosphorylation of the receptor have been shown to occur, it’s in ER signaling has yet to

be determined. In general, E (estrogen) is conditional inhibitors of bone resorption, in contrast to

other inhibitors of bone resorption, such as bisphosphonates and calcitonin, which have far more

predictable and universal effects. Thus, E is potent inhibitors of bone resorption in the setting of

estrogen deficiency but is far less effective in the estrogen -replete organism. In vitro, their action

appears to be influenced by species, age and probably by the presence of other cell types. Taking

these factors together, it would appear likely that E requires the presence of co-factors, second

messengers, or both and that the potency of their action depends on other stimuli to which the target

cell is subjected.

Importance of estrogen

In the quest for animal models that mimic keys aspects of significant postmenopausal bone loss, it

is of interest to consider the extent to which ablation of the ovaries more or less stimulates what

takes place in women that have transitioned to menopause, particularly in terms of reductions in

circulating estradiol concentration estradiol concentrations. Circulating estrogen concentrations of

most healthy women follow n established regular cyclical pattern approximately every 28 days

throughout the reproductive years. The substantial diminution in circulating estrogen concentration

in women in the early years after the transition to menopause accelerates bone turnover rate and is a

predictable impetus for bone loss. Estrous cycles vary in length and frequency among different

species and involve different basal and peak endogenous estradiol exposures. Generally, small short

lived species undergo estrous cycles more frequently; where as larger long lived animals have less

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recurrent regular estrous cycles. Estrous cycling also tends to be more frequent in species that

produce larger litters, raising yet another interspecies incongruity that may deserve some

consideration when selecting animal models. Rodents are adapted to mobilize comparatively larger

amounts of calcium from bone for weaning requirements in a shorter period of time than humans

(Reinwald and Burr., 2008). and possess a profound anabolic capacity that facilitates the

replacement of bone mass that may have been resorbed for lactation purposes relatively rapidly

(Bowman and Miller., (2001). Estrogen is primarily produced in the ovary prior to menopause.

After menopause estrogen production occurs in peripheral tissues (skin, muscle, fat and benign and

malignant breast tissue) through the conversion of androgens to estrogens by the P450 cytochrome

enzyme aromatse (CYP19) (Gaillard and Stearns., (2011).

The role of estrogen on bone tissue in men

The importance of E for bone maturation and development of peak bone mass in men also

suggested. Estradiol is detectable in the serum of healthy men at levels comparable to those in

postmenopausal women. This is a result of peripheral conversion of testosterone by the enzyme

aromatase, a member of the microsomal cytochrome P450 group. Because these levels are rather

low, they were not regarded as physiologically important until epidemiological research into heart

disease risk suggested a protective effect of endogenous E in men. A role for E in skeletal

maintenance in the human male is supported by evidence at the cellular level by animal experiments

and clinical findings. Osteopenia was reported in an aromatase-deficient young man whose

estradiol levels were below 26 pmol/l, but whose testosterone levels were high. It was also reported

in another case with non-functioning ER Serum testosterone and androgen receptors were normal

or increased. In both cases, bone mineral density (BMD) values were similar to those seen in the

converse syndrome of genetic males with androgen insensitivity (androgen receptor defect but

normal testosterone and estradiol levels (Krassas and Papadopoulou, 2001).

Mechanism of bone loss:

Bone metabolism is balance between osteoblastic and osteoclastic activity. Estrogen deficiency is

one of the main factors in mediating age- related bone loss. Clear association is present between

post menopausal estrogen deficiency and the development of osteoporosis. Aromatase and ERs are

both present on bone and estrogen hormone used to regulate bone remodeling by stimulating the

expression of an anti-resorptive factors such as osteoprotegerin (OPG). This results in the

attenuation of receptor activation of RANK and RANK ligand (RANKL) signaling through NF-kβ

pathway leading to inhibition of osteoclastogenesis and an attenuated bone turnover. Deficiency of

estrogen (post- menopausal) is associated with increased expression of measurable markers of bone

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resorption and bone formation (Gaillard and Stearns., (2011). The anti resorptive activity of

estrogen is result of multiple genomic and non genomic effects on bone marrow and bone cells,

which leads to decreased OC formation, increased OC apoptosis and decreased in capacity of

mature OCs to reabsorb bone (D'Amelio et al., 2008)

Glucocorticoid-induced osteoporosis (GIOP)

Various animal models have been proposed for the study of pathophysiology of Glucocorticoid-

induced osteoporosis (GIOP). Contradictory finding have been reported after experimental

administration of GC to rats that may result variations from the background factors such as age of

the animals or the dose of GC. Murine models are most frequently employed and results appear

more consistent. Mice used are usually 5 or 6 month old, which are more respond to peak bone

mass. The continuous administration of GC with slow release pellets for 27 days (a period

equivalent in the mouse to 3-4 years in humans). Here decreases the number of osteoblasts and

osteoclasts progenitors, decrease osteoblasts and osteoclast surface, and increase osteoblasts and

osteocytes apoptosis (Bouvard et al., 2010). After ten days of GCs administration increases

osteoclast number (by reducing osteoclast apoptosis) and decrease in osteoblasts production.

(Weinstein et al., 2002).

Disadvantages

Yielded inconsistent results

Unable to detect bone loss in mature rats

Rat is capable of accurately replicating the glucocorticoid- induced bone loss noted in

adult humans is unavailable.

Long time taken for inducing osteoporosis

Less accurate

Glucocorticoids (GCs):

Glucocorticoids (GCs) were introduced in clinical medicine and the researchers initially involved,

kendall, Hench and Reichstein, won the nobel prize for physiology or medicine in 1950 for research

on the structure and biological effects of adrenal cortex hormones.

Synthetic Glucorticoid (GCs) are used in various disorders including autoimmune, pulmonary,

gastrointestinal disease, rheumatologic and malignancies as well as in organ transplantation

(Bouvard et al., 2010). Glucocorticoid induced osteoporosis is the main cause of secondary

osteoporosis. According to previous studies GCs can cause bone loss and fractures, many patients

receiving or initiating a long term GCs therapy are still now not evaluated for skeletal health. GCs

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have other effects on various systems like sodium metabolism, lipids, skin, muscle and immune

response which can interact with bone metabolism (McDonough et al., 2008).

Direct effects of glucocorticoids on bone cells

Osteoblasts

Glucocorticoids (GCs) decrease the number and the functionality of osteoblasts. These effects cause

to a suppression of bone formation, a central feature in the pathogenesis of GIO. Glucocorticoids

decrease the replication of cells of the osteoblastic lineage, reducing the pool of the cells that may

differentiate into mature osteoblasts (Canalis et al., 2007). In addition, osteoblastic differentiation

and maturation impairs by Glucocorticoids. (Canalis et al., 2005). Under certain experimental

conditions, on the other hand, glucocorticoids have been reported to favor osteoblastic

differentiation (Ejiken et al., 2006). However, the effects of Glucocorticoids to favor osteoblasts

differentiation seem to be highly dependent on practical conditions, and do not reflect the loss of

cells of the osteoblastic lineage regularly seen in glucocorticoid exposure (Canalis et al., 2005). In

the presence of Glucocorticoids bone marrow stromal cells, the precursors of osteoblasts, do not

differentiate or are directed, instead, toward cells of the adipocytic lineage (Ito et al., 2006; Pereira

et al., 2004) An additional mechanism of glucocorticoids inhibit osteoblast cell differentiation is by

opposing Wnt/ β-catenin signaling (Canalis et al 2005; Ohnaka et al., 2006; Smith, 2005). Wnt

signaling has emerged as a key regulator of osteoblastogenesis. Wnt uses four known signaling

pathways, but in skeletal cells the canonical Wnt/β-catenin signaling pathway is operates

(Westendorf et al., 2004). In this pathway, when Wnt is absent, β-catenin is phosphorylated by

glycogen-synthase kinase-3β (GSK-3β), and then ruined by ubiquitination. When Wnt is present, it

binds to specific receptors, called frizzled, and to coreceptors, low density lipoprotein receptor

related proteins (LRP)-5 and -6, leading to inhibition of GSK-3β activity. When GSK-3β is not

active, stabilized β-catenin translocates to the nucleus, where it associates with transcription factors

to regulate gene expression (Glass et al., 2005). Deletions of either Wnt or β-catenin result in the

absence of osteoblastogenesis, and increase of osteoclastogenesis (Glass et al., 2005; Holmen et al.,

2005). The Wnt pathway can be inactivated by Dickkopf, an antagonist that prevents Wnt binding

to its receptor complex. Glucocorticoids enhance Dickkopf expression and maintain GSK 3-β in an

active state, leading ultimately to the inactivation of β-catenin (Ohnaka et al., 2006; Smith, 2005).

Kawano and Kypta, 2003). In addition to inhibiting the differentiation of osteoblasts,

glucocorticoids inhibit the function of the differentiated mature cells. Glucocorticoids inhibit

osteoblast-driven synthesis of type I collagen, the major component of the bone extracellular

matrix, with a resulting decrease in bone matrix available for mineralization (Canalis et al., 2005).

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The decrease in type I collagen synthesis caused by the transcriptional and post-transcriptional

mechanisms. Pro-apoptotic effects of Glucocorticoids on osteoblasts and osteocytes due to

activation of caspase 3, a common downstream effector of several apoptotic signaling pathways

(Liu et al., 2004: O’Brien et al., 2004). Caspases are proenzymes and these are activated through

autocatalysis or a Caspase cascade. Apoptosis by cleaving target cellular proteins by Active

caspases. A key mediator of apoptosis is Caspase 3 and is a common downstream effector of

multiple apoptotic signaling pathways (O’Brien et al., 2004). The inhibitory effects of

Glucocorticoids on osteoblastic cell replication and differentiation and increased in apoptosis of

mature osteoblasts, all contribute to the reduction of the osteoblastic cellular pool and decrease the

bone formation.

Osteocytes

Osteocyte serves as mechanosensors and plays the role in the repair of bone micro damage.

Osteocyte-canalicular network is disrupting by loss of osteocytes and resulting in a failure to detect

signals that normally stimulate processes associated to the replacement of damaged bone.

Disruption of the osteocyte-canalicular network can disrupt fluid flow within the network

unfavorably affecting the material properties of the surrounding bone, independent of changes in

bone architecture or remodeling. Glucocorticoids have an effect on the function of osteocytes by

modifying the elastic modulus surrounding osteocytes lacunae (Lane et al., 2006) Glucocorticoids

induce the apoptosis of osteocytes (Liu et al., 2004). As a result, the normal maintenance of bone

through this mechanism is impaired and the biomechanical properties of bone are impaired. (Lane

et al., 2006).

Osteoclasts

In human subjects, Glucocorticoid-induced osteoporosis (GIO) occurs in two phases, 1) rapid

phase- it is a early phase in which bone mineral density (BMD) is reduced and it is due to excessive

bone resorption, 2) slower phase- in which BMD decreases due to impaired bone

resorption(Canalis et al., 2004) under the influence of two cytokines like macrophage colony

stimulating factor (M-CSF) and receptor activator of NF-kB ligand (RANKL) that differentiate

osteoclasts are members of the monocyte or macrophage family of cells.

Increased the expression of M-CSF and RANKL and decrease the expression of osteoprotegerin

(OPG) by glucocorticoids in stromal and osteoblastic cells. Glucocorticoid suppress the expression

of interferon-β, an inhibitor of osteoclastogenesis (Dovio et al., 2006; Takuma et al., 2003) and also

increase the expression of interleukin-6, an osteoclastogenic cytokine. Glucocorticoid declines the

apoptosis of mature osteoclasts (Jia et al., 2006).

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Consequently, there is enhanced formation of osteoclasts with a prolonged life span explaining, at

the cellular level, the increased and prolonged bone resorption observed in glucocorticoid-induced

osteoporosis (GIO). The direct effects of gucocorticoids on osteoclasts (Bone resorption cells) also

may add to an operational decrease in osteoblasts function during exposure of glucocorticoids. The

net effect of glucocorticoid is to increase osteoclasts number, osteoclasts function, as results

increased bone resorption (Kim et al., 2006). Glucocorticoid increases the expression of selected

matrix metalloproteinase (MMP). Matrix metalloproteinase (MMP1) or collagenase 1 and MMP13

or collagenase 3 are secreted by osteoblasts and both cleave type I collagen fibrils at neutral pH.

Cortisol increases collagenase 3 syntheses by post-transcriptional mechanisms, their binding to

specific RNA sequences and by regulating specific cytosolic RNA binding proteins.

Glucocorticoids may also have effect on bone remodeling at the basic multicellular unit (BMU)

level, mainly manifested as a decreases in wall width ( reduced amount of bone formed per BMU).

In addition, there is some evidence that increased resorption depth (increased amount of bone

resorbed per BMU) may occur particularly at high doses of glucocorticoid and in the early stages of

therapy (Canalis et al., 2007).

Molecular Effects of GCs

The GC Receptor

In bone cells, cellular effects of GCs are initially mediated by the GC receptor which is a member

of the nuclear receptor super family. The receptor is retained in the cytosol (cGCR) in the absence

of ligand, as part of a chaperone containing multiprotein complex (Grad and Picard, 2007). The

location of human cGCR gene is at chromosome 5q31.3 and it is widely expressed in a number of

bone cells including osteoblasts (OB), osteocytes and chondrocytes (Bouvard et al., 2009). The

GCR transcripts are detected in macrophage like cells (Putative osteoclast (OC) precursor),

multinucleated osteoclast like cells and stromal like tumour cells.

Various studies has been suggested that the polymorphism of the cGCR gene is correlated with

bone mineral density (BMD) variation and explain the heterogeneity to GC- associated bone loss

and fractures. Upon hormone binding, translocates the cGCR in to the nucleus, where it acts as a

transcription factor. The subunits of cGCR homodimerize and bind DNA at GC responce element

(GREs) in the locality of target genes. The process which is mediated through positive GREs is

called as transactivaction (Stahn et al., 2007; Lowenberg et al., 2008). On the other way,

transcription of genes can be inhibited by GCs by direct interaction between the GC/GCR complex

and negative GREs or by an interaction of GC/GCR complex monomers with transcription factors.

In this final mechanism called as transrepression, GCs inhibit nuclear translocation and the

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function of several pro-inflammatory transcription factor such as nuclear factor κB or activator

protein 1 and synthesis of inflammatory mediators such as tumor necrosis factor-a (TNF-a), INF-c,

IL-1, and IL-2 is suppressed.

Cellular Effects of GCs

Bone development and homeostasis is a complex process in which a balance between bone

formation (Osteoblasts) and bone resorption (Osteoclasts). In GIOP, GCs stimulate bone resorption

and suppress the bone formation, resulting in bone loss.

In vitro, GCs induces cells of the osteoblast lineage to differentiate into mature osteoblasts (OBs) at

low concentration where as GCs at high concentrations dramatically decline OB number and bone

formation rate (Lane et al., 2006).

Effects of GCs on OB Function

GCs decrease the expression of insulin-like growth factors (IGF) I and II well-known to increase

OB differentiation type I collagen synthesis, bone formation and increase bone collagen

degradation by decreasing the synthesis of collagenases 1 and 3 (Canalis et al., 2007).

Glucocorticoids (GCs) also decrease the expression of non-collagenous proteins such as

osteocalcin, osteopontin, bone alkaline phosphatase and metalloproteinase 1 tissue inhibitor. GCs

also suppress the synthesis of IGFBP-3,4 and 5 (which are binding proteins that can stimulate bone

cell growth) and enhance the expression of IGFBP-6 (a binding protein that selectively blocks the

effects of IGF-II on OBs). By combining these effects together induce a marked decrease in the

OBs number and in their capacity to synthesize bone matrix. GCs also suppress differentiation of

OB by opposing Wnt/b- catenin signaling, is a key regulator of osteoblastogenesis. Osteoblast

differentiation inhibited by GCs through the repression of growth hormone (GH) and bone

morphogenic protein 2 (BMP-2) which increases OB transcription factors (Giustina et al., 2008).

Immobilization induced osteoporosis

It is another method for inducing osteoporosis in rats. There are several methods of immobilization,

which can be either surgical, such as nerve, tendon, and spinal cord resection or conservative such

as casting, suspension, and limb bandaging.

Disadvantages

Rate of bone loss is very slow

More time taken to inducing osteoporosis compare to OVX induced osteoporosis

Alcohol abuse induced osteoporosis

Here mainly osteopenia was studied after administration of low-calcium diet to immature rats. The

rats are used to understand the pathogenesis and severity loss of bone mass after alcohol abuse.

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Orchidectomized (Orx) induced osteoporosis in male rats

These Orx model also widely used for studying bone mass in male rats( Lerouxel et al., 2004;

Audran et al., 2001; Libouban et al., 2001). Androgens withdrawal induced by Orx it cause in

decreased bone mass in experimental animals. Deficiencies of androgens are associated with

accelerated bone turnover and imbalance between bone resorption and bone formation, which cause

in bone loss. Orchidectomized (Orx) results in declined BMC, BMD, bone strength, whole body

weight and lean body mass. The Orx model used for studies of androgen replacement in hypogonal

men. These declines in BMC and BMD were prevented by testosterone administration. The mainly

in Orx rats decreases biochemical serum parameters like serum testosterone and estradiol levels.

These finding suggested that Orx adult male rat model used to examine the effects of both

testosterone and estrogen deficiency on bone structure and bone remodeling.

Androgens play an important role in building the skeleton in young adults and help to prevent bone

loss and osteoporosis in aging men. In addition in hypogonadism or elderly men, bone mass is

related to estrogen levels rather than to testosterone. Therefore, Estrogen replacement therapy has

been proposed to prevent bone loss in males as well as in females.

Animal model – including parameters

Bone volume- decreasing bone volume may be attributed to both increased bone resorption

(Osteoclasts) reduced bone formation (Osteoblasts).

Body mass or body weight (g): When excessive fat mass occurs it is cause the increasing body

weight it may not protect against osteoporosis or osteoporotic fracture.

Postmenopausal women often show increased body weight due to a decrease in basal metabolism,

hormonal level alterations and reduced physical activity (Migliaccio, 2011).

Bone density: Early detection of bone loss by measurement of bone mineral density (BMD) and it

helps to confirm the diagnosis of osteoporosis and assess the future risk of osteoporotic fractures.

Determination of BMD by Dual X-ray Absorptiometry (DEXA) is the very standard method. Bone

mineral density (BMD) changes are late and relatively irreversible, so it is important to have a

means of identifying high risk individuals and to monitor their treatment before fractures occur.

(Civitelli, Villareal and Napoli, 2009).

Biochemical parameters

Bone resorption markers

Amino terminal cross linking telopeptides of type I collagen (NTX)

NTX is a type I collagen is measured by immunoassay based on antibodies against the α2 cross

linked fragment of type I collagen

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Amino terminal cross linking telopeptides of type I collagen (NTX) is a bone resorption marker.

The result of bone resorption (osteoclasts) NTX breakdown product that can be measured in either

serum or urine. After anti resorptive therapy serum NTX decline significantly, but it is less

sensitive than urinary NTX (Abe, Ishikawa and Fukao, 2008) The reason for this unknown, and it is

still unclear whether dietary intake of collagen can interfere with serum NTX levels. Determination

of NTX in 24 hrs urine has a better advantage because prevent the variability due to circadian

changes in bone turnover (Civitelli, Villareal and Napoli, 2009).

Carboxyl- terminal cross linking telopeptides of type I collagen (CTX)

CTX also measured in either urine or serum by enzyme- linked immunosorbent assay (ELISA),

radioimmunoassay (RIA) and electrochemiluminescence assay (Civitelli, Villareal and Napoli,

2009). The C-terminal telopeptides α1 chain of type I collagen undergous β- isomerization and

racemization. Here we has been measured ratio of αCTX/βCTX, this ratio was increased in patients

and decreased in after bisphosphate therapy. In postmenopausal women have higher ratio of

αCTX/βCTX, may results increased risk of fracture compared to women with lower ratios (Garnero

et al., 2002).

PYD and DPD

Pyridinoline (PYD) and deoxypyridinoline (DPD) are covalent pyridinium cross links, during bone

resorption (Osteoclasts), they are produced from breakdown of collagen. They are released into

blood and pass into the urine. Both can be measured by RIA and ELISA. Thus, PYD and DPD offer

reliable assessment of bone resorption (Civitelli, Villareal and Napoli, 2009).

Tartrate-resistant acid phosphatase (TRACP5b)

It is a lysosomal enzyme. TRACP5b is expressed in the osteoclast is the 5b isoform, it is used as

bone resorption marker. It is the only marker of osteoclast activity. In high bone turnover

conditions such as Paget’s disease, bone metastase, multiple myeloma and after ovariectomy

TRACP5b is typically increased (Halleen et al., 2001).

Bone formation markers

Serum osteocalcin

Osteocalcin also known as bone gla protein, it constitutes 15% of the noncollagenous bone matrix

proteins. It is a bone matrix protein synthesized by mature osteoblasts and incorporated into bone

matrix, small portion goes into circulation. Serum osteocalcin measured by using RIA, ELISA, or

chemiluminescence immunoassay methods (Cremers and Garnero, 2006). Osteocalcin is good bone

formation marker.

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Serum alkaline phosphatase and bone-specific alkaline phosphatase

Alkaline phosphatase (ALP) is a glycosyl-phosphatidyl-inosital anchored ectoenzyme present on

the osteoblastic cell membrane. Its exact function is completely unclear; the presence of ALP on the

osteoblastic cell membrane is required for bone mineralization. Immunoassays are used for the

better assessment of bone specific ALP (bone ALP). Bone ALP affords greater specificity for

osteobalst function (Civitelli, Villareal and Napoli, 2009). Exact metabolic function of ALP is

unknown. Alkaline phosphatase is an enzyme that catalyses the alkaline hydrolysis of

monophosphate ester group and is present in high concentration in the bone, liver, placenta,

intestinal epithelium and kidney tubules. Total ALP found in the circulation, 95% of the enzyme in

blood originates from either liver or bone. In health condition the ratio of bone to liver isoforms is

nearly 1:1. The bone isoforms of ALP is produced by bone formation cells (Osteoblasts) as a

tetramer. Bone ALP (BAP) catalyses the hydrolysis of phosphate esters at the osteoblast cell

surface and provide a high concentration of phosphate for the bone mineralization process as part of

the osteobalst cell role in bone remodeling. As a result, bone ALP levels are increased in the

circulation during periods of active bone formation and bone growth.

Serum PICP and serum PINP

Procollagen type I C-terminal propeptide (PICP) and Procollagen type I N-terminal propeptide

(PINP) are peptides derived from post translation cleavage of type I these cleavage products pass

into circulation and these markers are used for bone formation. Serum PICP and PINP can be

determined by either RIA or ELISA and electrochemiluminescence immunoassay (Luftner et al.,

2005). PINP is preferred to PICP as a bone formation marker (Civitelli, Villareal and Napoli, 2009).

Serum total calcium

In bone approximately 99% of body’s calcium was present (Murry et al., 2006). In old age women,

bone resorption (Osteoclasts) is markedly increased in part because of calcium intake is reduced

and inhibited bone formation, in part to preserve serum calcium (Storm et al., 1998). Based upon

these demonstrations that biochemical parameters can give an idea as to the rates of formation and

bone resorption.

Conclusion

The high prevalence, very long term implication, financial burden, high mortality and dramatically

decreased quality of life indicate the severity of osteoporosis. The need to better understand the

multifactorial skeletal disease nature and to develop new preventive and therapeutic methods makes

the use of animal modes necessary for osteoporosis. The observing all animal models and

similarities in pathophysiology responces between human and rat skeleton occur. Based on some

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important points like financial, safety of handle, less time, the rat model is more suitable model in

osteoporosis research.

REFERENCES:

1. Kasabi S, Handral M, Prabhu R. (2012). Evaluation of anti osteoporotic activity of Root

extract of Rubia Cordifolia Ovariectomized rats. International journal of drug development

and research, 4, 163-172.

2. Reinwald S, Burr D. (2008). Review of nonprimate, large Animal Models for osteoporosis

research, J Bone Miner Res, 23, 1353-68.

3. Lelovas PP, Xanthos TT, Thoma SE, Lyritis GP, Dontas IA. (2008). The laboratory rat as an

animal model for osteoporosis research. Comp Med, 58, 424-30.

4. Omara AE, Shaffie MN, Sayed A, Et-Toumy, Aal AW. (2009). Histomorphometric

evaluation of bone tissue exposed to experimental osteoporosis and treated with Retama

Raetam Extract. Journal of applied sciences research, 5, 706-716.

5. Krassas GE, Papadopoulou P. (2001). Oestrogen action on bone cells. J musculoskelet

Neuronal interact, 2, 143-51.

6. Bowman BM, Miller SC. (2001). Skeletal adaptations during mammalian reproduction. J

musculoskelet Neuronal interact, 1, 347-55.

7. Gaillard S, Stearns V. (2011). Aromatase inhibitor-associated bone and musculoskeletal

effects: new evidence defining etiology and strategies for management. Breast Cancer Res,

13, 205.

8. D'Amelio P, Grimaldi A, Di Bella S, Brianza SZ, Cristofaro MA, Tamone C, Giribaldi

G, Ulliers D, Pescarmona GP, Isaia G. (2008). Estrogen deficiency increases

osteoclastogenesis up-regulating T cells activity: a key mechanism in osteoporosis. Bone,

43, 92-100.

9. Bouvard B, Legrand E, Audran M, Chappard D. (2010). Glucocorticoid- induced

Osteoporosis: A review. Clinic Rev Bone Miner Metab, 8, 15-26.

10. Weinstein RS, Chen JR, Powers CC, Stewart SA, Landes RD, Bellido T, Jilka RL, Parfitt

AM, Manolagas SC. (2002). Promotion of osteoclast survival and antagonism of

bisphosphonates-induced osteoclast apoptosis by glucocorticoids. J Clin Invest, 109, 1041-

8.

230 | P a g e International Standard Serial Number (ISSN): 2319-8141

Full Text Available On www.ijupbs.com

11. McDonough AK, Curtis JR, Saag KG. (2008). The epidemiology of glucocorticoid-

associated adverse events. Curr Opin Rheumatol, 20,131-7.

12. Canalis E, Mazziotti G, Giustina A, Bilezikian JP. (2007). Glucocorticoid-induced

osteoporosis: pathophysiology and therapy. Osteoporos Int, 18, 1319-28.

13. Canalis E. (2005). Mechanisms of glucocorticoid action in bone. Curr Osteoporos Rep, 3,

98-102.

14. Eijken M, Koedam M, van Driel M, Buurman CJ, Pols HA, van Leeuwen JP.(2006).

The essential role of glucocorticoids for proper human osteoblast differentiation and matrix

mineralization. Mol Cell Endocrinol. 248, 87-93.

15. Ito S, Suzuki N, Kato S, Takahashi T, Takagi M. (2007). Glucocorticoids induce the

differentiation of a mesenchymal progenitor cell line, ROB-C26 into adipocytes and

osteoblasts, but fail to induce terminal osteoblast differentiation. Bone, 40, 84-92.

16. Pereira RC, Delany AM, Canalis E, (2004). CCAAT/enhancer binding protein homologous

protein (DDIT3) induces osteoblastic cell differentiation. Endocrinology, 145, 1952-60.

17. Ohnaka K, Tanabe M, Kawate H, Nawata H, Takayanagi R.(2005). Glucocorticoid

suppresses the canonical Wnt signal in cultured human osteoblasts. Biochem Biophys Res

Commun, 329, 177-81.

18. Smith E, Frenkel B (2005) Glucocorticoids inhibit the transcriptional activity of LEF/TCF

in differentiating osteoblasts in a glycogen synthase kinase-3beta-dependent and –

independent manner. J Biol Chem, 280, 2388-94.

19. Westendorf JJ, Kahler RA, Schroeder TM. (2004). Wnt signaling in osteoblasts and bone

diseases. Gene, 341, 19-39.

20. Glass DA 2nd, Bialek P, Ahn JD, Starbuck M, Patel MS, Clevers H, Taketo MM, Long

F, McMahon AP, Lang RA, Karsenty G. (2005). Canonical Wnt signaling in differentiated

osteoblasts controls osteoclast differentiation. Dev Cell, 8, 751-64.

21. Holmen SL, Zylstra CR, Mukherjee A, Sigler RE, Faugere MC, Bouxsein ML, Deng

L, Clemens TL, Williams BO. (2005). Essential role of beta-catenin in postnatal bone

acquisition. J Biol Chem, 280, 21162-8.

22. Kawano Y, Kypta R (2003). Secreted antagonists of the Wnt signalling pathway. J Cell Sci,

116, 2627-34.

23. Liu Y, Porta A, Peng X, Gengaro K, Cunningham EB, Li H, Dominguez LA, Bellido

T, Christakos S. (2004). Prevention of glucocorticoid-induced apoptosis in osteocytes and

osteoblasts by calbindin-D28k. J Bone Miner Res, 19, 479-90.

231 | P a g e International Standard Serial Number (ISSN): 2319-8141

Full Text Available On www.ijupbs.com

24. O'Brien CA, Jia D, Plotkin LI, Bellido T, Powers CC, Stewart SA, Manolagas

SC, Weinstein,RS.(2004).Glucocorticoids act directly on osteoblasts and osteocytes to induc

e their apoptosisand reduce bone formationand strength. Endocrinology. 145, 1835-41.

25. Lane NE, Yao W, Balooch M, Nalla RK, Balooch G, Habelitz S, Kinney JH, Bonewald LF.

(2006). Glucocorticoid treated mice have localized changes in trabecular bone material

properties and osteocytes lacunar size that are not observed in placebo-treated or estrogen-

deficient mice. J Bone Miner Res, 21, 466-76.

26. Canalis E, Bilezikian JP, Angeli A, Giustina A. (2004). Perspectives on glucocorticoid-

induced osteoporosis. Bone, 34, 593-8.

27. Dovio A, Perazzolo L, Saba L, Termine A, Capobianco M, Bertolotto A, Angeli A. (2006).

High-dose glucocorticoids increase serum levels of soluble IL-6 receptor alpha and its rato

to soluble gp130: and addition mechanism for early increased bone resorption. Eur J

Endocrinol, 154, 745-51.

28. Takuma A, Kaneda T, Sato T, Ninomiya S, Kumegawa M, Hakeda Y. (2003).

Dexamethasone enhances osteoclast formation synergistically with transforming growth

factor- beta bystimaulating the priming of osteoclast progenitors for differentiation into

osteoclasts. J Biol Chem, 278, 44667-74.

29. Jia D, O'Brien CA, Stewart SA, Manolagas SC, Weinstein RS. (2006). Glucocorticoids act

directly on osteoclasts to increase their life span and reduce bone density. Endocrinology,

147, 5592-9.

30. Kim HJ, Zhao H, Kitaura H, Bhattacharyya S, Brewer JA, Muglia LJ, Ross FP, Teitelbaum

SL. (2006). Glucocorticoids suppress bone formation via the osteoclast. J Clin Invest, 116,

2152-60.

31. Grad I, Picard D. (2007). The glucocorticoid responses are shaped by molecular chaperones.

Mol Cell Endocrinol, 275, 2-12.

32. Stahn C, Lowenberg M, Hommes DW, Buttgereit F. (2007). Molecular mechanisms of

glucocorticoid action and selective glucocorticoid receptor agonists. Mol Cell Endocrinol.

275, 71-8.

33. Lowenberg M, Stahn C, Hommes DW, Buttgereit F. (2008). Novel insights into

mechanisms of glucocorticoid action and the development of new glucocorticoid receptor

ligands. Steroids. 73, 1025-9.

34. Giustina A, Mazziotti G, Canalis E. (2008). Growth hormone, insulin-like growth factors,

and the skeleton. Endocr Rev, 29, 535-59.

232 | P a g e International Standard Serial Number (ISSN): 2319-8141

Full Text Available On www.ijupbs.com

35. Lerouxel E, Libouban H, Moreau MF, Basle MF, Audran M, Chappard D. (2004).

Mandibular bone loss in an animal model of male osteoporosis (orchidectomized rat): a

radiographic and densitometric study. Osteoporos Int. 15, 814-9.

36. Audran M, Chappard D, Legrand E, Libouban H, Basle MF. (2001). Bone microarchitecture

and bone fragility in men: DXA and histomorphometry in humans and in the

orchidectomized rat model. Calcif Tissue Int. 69, 214-7.

37. Libouban H, Moreau MF, Legrand E, Basle MF, Audran M, Chappard D (2001).

Comparison

38. insight dual X-ray absorptiometry (DXA), histomorphometry, ash weight, and

morphometric indices for bone evaluation in an animal model (the orchidectomized rat) of

male osteoporosis. Calcif Tissue Int, 68, 31-7.

39. Civitelli R, Armamento-Villareal R, Napoli N. (2009). Bone turnover markers:

understanding their value in clinical trials and clinical practice. Osteoporos Int, 20, 843-51.

40. Garnero P, Cloos P, Sornay-Rendu E, Qvist P, Delmas PD. (2002). Type I collagen

recemization and isomerization and the risk of fracture in post menopausal women:

the OFELY prospective study. J Bone Miner Res, 17, 826-33.

41. Halleen JM, Alatalo SL, Janckila AJ, Woitge HW, Seibel MJ, Väänänen HK. (2001).

Serum tartrate-resistant acid phosphatase 5b is a specific and sensitive marker of bone

resorption. Clin Chem, 47, 597-600.

42. Cremers S, Garnero P. (2006). Biochemical markers of bone turnover in the clinical

development of drugs for osteoporosis and metastatic bone disease: potential uses and

pitfalls. Drugs 66, 2031-2058.

43. Lüftner D, Jozereau D, Schildhauer S, Geppert R, Müller C, Fiolka G, Wernecke

KD, Possinger K. (2005). PINP as serum marker of metastatic spread to the bone in breast

cancer patients. Anticancer Res, 25, 149-9.

44. Migliaccio S, Greco EA, Fornari R, Donini LM, Lenzi A. (2011). Is obesity in women

protective against osteoporosis? Diabetes Metab Syndr Obes, 4, 273-82.

45. Murray RK, Granner DK and Rodwell VW (2006). The extracellular matrix. In: Harper’s

Illustrated Biochemistry, McGraw Hill Companies.

46. Storm D, Eslin R, Porter ES, Musgrave K, Vereault D, Patton C, Kessenich C, Mohan

S, Chen T, Holick MF, Rosen CJ. (1998). Calcium supplementation prevents seasonal bone

233 | P a g e International Standard Serial Number (ISSN): 2319-8141

Full Text Available On www.ijupbs.com

loss and changes in biochemical markers of bone turnover inelderly New England women: a

randomized placebo-controlled trial. J Clin Endocrinol Metab, 83, 3817-25.

47. Abe Y, Ishikawa H, Fukao A. (2008). Higher efficacy of urinary bone resorption marker

measurements in assessing response to treatment for osteoporosis in postmenopausal

women. Tohoku J Exp Med, 214, 51-59.