215 | P a g e International Standard Serial Number …. RPA1415135015.pdf · related problems like...
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215 | P a g e International Standard Serial Number (ISSN): 2319-8141
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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:
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.
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