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the continuous assessment scores should not
contribute to the final examination scores. Another
one is the ruling on balance between medical and
non-medical academic staff which should be 70 :
30 ratio as well as the ratio between full-time and
part-time staff where full-time faculty should bemore than 60%.
Staff-student ratio is also stipulated for all the
various teaching-learning activities such as tutorials-
not exceeding 16 students per group; problem-based
sessions not exceeding 12 students per group;
clinical teachings in skills lab setting not exceeding
10 students per group and bed side clinical teaching-
not exceeding 8 students per group. The overall staff
: student ratio should be 1 : 4. Another new ruling
is on the hospitals used with a ratio of 1 student to 5
beds. The hospitals recognized for this purposemuch have the basic disciplines available ie.
Medicine, Pediatrics, Surgery, O & G, Orthopedics,
Radiology and Pathology.
With the revised Guidelines for Accreditation,
a rating scheme for accreditation was also adopted.
The rating is based on the guidelines which sets out
good practice in nine areas and the rating system
uses a percentage scoring scale that indicates the
degree of institutional and programme compliance
to the standards for each area and criterion.
Compliance is rated according to 5 Levels: Level 5
Excellent, Level 4 Good, Level 3 Satisfactory,
Level 2 - Less than satisfactory and Level 1
Unsatisfactory. The accreditation period given to a
particular medical school is then based on the overall
rating points of the compliance obtained.
As for the process of accreditation, before a
particular medical course is started, a team is sent
to evaluate the curriculum and consider the schools
plans and implementation details of at least the first
two years of the programme. The team may go for
a re-visit if there are areas of concern noted in the
earlier visit to see if these concerns have beenovercome. A pre-accreditation visit is carried out
about 1 year before the formal accreditation visit to
enable the school to know and rectify deficiencies
before the formal accreditation survey, which is
conducted when the first batch of students is in the
final year. Thereafter, the accreditation survey is
done every 1, 3 or 5 years depending on the length
of accreditation duration given.
Despite a structured and comprehensive
accreditation system for the course and the medical
school, it does not necessarily guarantee a very goodmedical graduate as the graduates own personal
traits and behaviour would play a large bearing on
the quality of the graduate. To assess this quality, a
rating of medical graduates has been developed. The
rating system is based on knowledge, basic
procedural skills, interpersonal skills, personality/
attitudes, discipline, continuing professional
development and leadership qualities. From these,an overall score is obtained and rating is given as
either A, B, C or D. This would be useful to assess
the overall quality of medical graduates from any
medical school and would provide important
feedback to the medical schools to overcome
deficiencies, if any.
In conclusion, a quality assurance mechanism
is in place in Malaysia to ensure quality medical
education and medical graduates. This involves the
key stakeholders such as the Malaysian Medical
Council, Malaysian Qualifying Agency, Ministry ofHigher Education, Ministry of Health and the Public
Services Department. The standard set is similar to
the World Federation for Medical Education and
would also change and evolve over time in response
to continuous improvement in quality. The
introduction of ratings for medical schools and
graduates will certainly spur medical schools to
strive for improvement.
Acknowledgements :-
Prof. Dato Dr. Mafauzy Mohamed was the
previous editor of MJMS from 2000 to December
2007. We wish him best wishes for his future
endevour. MJMS grew significantly under his
editorialship.
Corresponding Author :
Prof. Dato Dr. Mafauzy Mohamed FRCP,
Professor of Medicine & Director Health Campus,
Universiti Sains Malaysia, Health Campus,
16150 Kubang Kerian, Kelantan, Malaysia
Tel: + 609 -766 4545
Fax: + 609- 765 2678
Email: [email protected]
References
1. Guidelines For The Accreditation of Basic Medical
Education Programmes In Malaysia. Malaysian
Medical Council. August 2007.
2. Rating For Accreditation of Undergraduate Medical
Programme In Malaysia. Malaysian Medical Council.
August 2007.
Rahmattullah Khan bin Abdul Wahab Khan
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3. Assessment Form For Medical Graduates During The
Internship Posting. Malaysian Medical Council.
February 2008.
ENSURING THE STANDARD OF MEDICAL GRADUATES IN MALAYSIA
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AN OVERVIEW OF BONE CELLS AND THEIR REGULATING
FACTORS OF DIFFERENTIATION
Alizae Marny Mohamed
Department of Orthodontic,
Faculty of Dentistry, Jalan Raja Muda Abdul Aziz,
50300 Kuala Lumpur, Malaysia
Bone is a specialised connective tissue and together with cartilage forms the strong
and rigid endoskeleton. These tissues serve three main functions: scaffold for muscle
attachment for locomotion, protection for vital organs and soft tissues and reservoir
of ions for the entire organism especially calcium and phosphate. One of the most
unique and important properties of bone is its ability to constantly undergo
remodelling even after growth and modelling of the skeleton have been completed.
Remodelling processes enable the bone to respond and adapt to changing functional
situations. Bone is composed of various types of cells and collagenous extracellular
organic matrix, which is predominantly type I collagen (85-95%) called osteoid
that becomes mineralised by the deposition of calcium hydroxyapatite. The non-
collagenous constituents are composed of proteins and proteoglycans, which are
specific to bone and the dental hard connective tissues. Maintenance of appropriate
bone mass depends upon the precise balance of bone formation and bone resorption
which is facilitated by the ability of osteoblastic cells to regulate the rate of both
differentiation and activity of osteoclasts as well as to form new bone. An overview
of genetics and molecular mechanisms that involved in the differentiation of
osteoblast and osteoclast is discussed.
Key words :Bone cells, osteoblasts, osteoclasts, regulations
Introduction
Bone is rigid and its architecture arranged to
provide maximum strength for the least weight. Most
bones have a dense rigid outer shell of compact bone,the cortex and the central medullary or cancellous
zone of thin interconnecting narrow bone trabeculae.
The space in the medullary bone between trabeculae
is occupied by haemopoietic bone marrow.
Bone extracellular matrix comprises of both
mineral and organic phases. About 60% of bone net
weight is inorganic material, 25% organic material
and 5% water. By volume, bone comprises of 36%
inorganic, 36% organic and 28% water.
The inorganic/mineral component comprises
of calcium and phosphate in the form of needle-like
or thin plates of hydroxyapatite crystals
[Ca10
(PO4)
6(OH)
2]. These are conjugated to a small
proportion of magnesium carbonate, sodium and
potassium ions. The organic matrix of bone is
composed of collagen and non-collagenous organic
materials. Collagen comprises about 90% of the
organic bone matrix. Type I collagen is the most
abundant form of intrinsic collagen found in the bonethat is secreted by osteoblasts. Most of the non-
collagenous organic materials are endogenous
proteins produced by the bone cells. One group of
non-collagenous proteins is the proteoglycans. This
incorporates chondroitin sulphate and heparan
sulphate glycosaminoglycans. As the proteoglycans
bind to collagen, they may help regulate collagen
fibril diameters and may play a role in
mineralisation. Other components include
osteocalcin (Gla protein), involved in binding
calcium during the mineralisation process,
osteonectin which may serve some bridging function
between collagen and the mineral component,
sialoproteins (rich in sialic acid) and certain proteins
Submitted : 6.03.2007, Accepted : 30.12.2007
Malaysian Journal of Medical Sciences, Vol. 15, No. 1, January 2008 (4-12)
REVIEW ARTICLE
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which appear to be concentrated from plasma.
Bone also contains exogenously derived
proteins that may circulate in the blood and become
locked up in the bone matrix itself. It is a rich source
of cytokines (such as interleukin, tumour necrosis
factor and colony-stimulating factors) and growth
factors (such as transforming growth factors,
fibroblast growth factors, platelet-derived growth
factors and insulin-like growth factors) produced by
variety of cells associated with bone. These proteins
play an important role in biological activity of bone
cells. When present within the bone, they are inactive
but may become mobilised when bone is being
resorbed by osteoclasts.
Bone is composed of four different cell types;
osteoblasts, osteocytes, osteoclasts and bone lining
cells. Osteoblasts, bone lining cells and osteoclasts
are present on bone surfaces and are derived from
local mesenchymal cells called progenitor cells.
Osteocytes permeate the interior of the bone and are
produced from the fusion of mononuclear blood-
borne precursor cells.
Bone Lining Cells And Osteocytes
When bone surfaces are neither in the
formative nor resorptive phase, the bone surface is
completely lined by a layer of flattened and
elongated cells termed bone-lining cells. These show
little sign of synthetic activity as evidenced by their
organelle content. They are regarded as post
proliferative osteoblasts. By covering the bone
surface, they protect it from any osteoclast resorptive
activity. They may be reactivated to form osteoblasts.
Osteocytes are cells lying within the bone
itself and are entrapped osteoblasts. They are post-
proliferative, representing the most mature
differentiation state of osteoblast lineage. There are
about 25,000 osteocytes per mm3 of bone. The
osteocytes occupy lacunae, which are regularly
distributed, and many fine canals called canaliculi
radiate from them in all directions. The canaliculi
allow the diffusion of substances through the bone.
Numerous cell processes from the osteocytes run in
the canaliculi in all directions. The canaliculi of
osteocytes are arranged in a more perpendicular than
parallel direction to the bone surface direction.
As a result of their widespread distribution
and interconnections osteocytes are obviouscandidates to detect stresses induced in bone and
are therefore regarded as the main mechanoreceptors
AN OVERVIEW OF BONE CELLS AND THEIR REGULATING FACTORS OF DIFFERENTIATION
Figure 1. Relationship of OPG/RANK/RANKL ; The control of osteoclastogenesis that emerged in
the relationship of OPG/RANK/RANKL. RANKL, expressed on the surface of preosteoblastic/
stromal cells. M-CSF, which binds to its receptor, c-fms, on preosteoclastic cells, appears
to be necessary for osteoclast development because it is the primary determinant of the
pool of these precursor cells. RANKL, however is critical for the differentiation, fusion into
multinucleated cells, activation and survival of osteoclastic cells. OPG put a break on theentire system by blocking the effects of RANKL. Khosla, 2001 (55).
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of bone. It has been shown that mechanical stress
can be sensed by osteocytes and these cells secreteparacrine factors such as insulin-like growth factor-
I (IGF-I) and express c-fos in response to mechanical
forces (1).
At the structural level, the appearance of the
osteocyte may vary according to its position in
relation to the surface layer. Osteocytes which are
newly incorporated into bone matrix from the
osteoblast layer have high organelle content, similar
to osteoblasts. However, as they become more
deeply situated with continued bone formation, they
appear to be less active. The cell is then seen to havea nucleus with a thin ring of cytoplasmic processes
extending from the osteocyte into the canaliculi in
the matrix.
The processes of one cell are joined to those
of another by gap junctions. These allow cell-to-
cell communication and co-ordination of activity.
In this feature, they are lack of processes and are
isolated. A pericellular space (which might represent
a shrinkage artefact) is usually seen to intervene
between the cell membrane and the surrounding
bone and contains unmineralised matrix and a few
collagen fibrils. Osteocytes are also in
communication with osteoblasts at the surface.
Osteoblasts
Osteoblasts are specialised fibroblast-likecells of primitive mesenchymal origin called
osteoprogenitor cell that originate from pluripotent
mesenchymal stem cells of the bone marrow. The
evidence of mesenchymal stem cells as precursors
for osteoblasts is based on the capacity of bone to
regenerate itself both in vivo and in vitro by using
cell populations (2). It has been shown that the bone
marrow stroma have the capacity to differentiate into
osteoblasts, chondroblasts, fibroblasts, adipocytes
and myoblasts (3).
In active form, osteoblasts are cuboidal inshape and found on a bone surface where there is
active bone formation. Osteoblasts are in contact
with each other by means of adherens and gap
junctions. These are functionally connected to
microfilaments and enzymes (such as protein kinase)
associated with intracellular secondary messenger
systems. This complex arrangement provides for
intercellular adhesion and cell to cell
communication.
The principle function of osteoblasts is to
synthesize the components that constitute the
extracellular matrix of bone. These include structural
macromolecules, such as type I collagen, which
Alizae Marny Mohamed
Figure 2. Bone Remodelling Process ; Remodelling process is accomplished by cycles of resorption
of old bone by osteoclasts and the subsequent formation of bone by osteoblasts. Modified
from Manolagas and Jilka, 1995 (57).
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accounts for about 90% of the organic matrix, as
well as numerous proteoglycans, non-collagenous
and cell attachment proteins.
Osteoblasts also promote mineralisation of the
organic matrix by matrix vesicles, extracellular
organelles found in osteoid and associated withmatrix calcification (4). Matrix vesicles contain
alkaline phosphatase, adenosine triphosphatase
(ATPase) and inorganic pyrophosphatase as well as
proteinases such as plasminogen activator. They act
as seeding sites for hydroxyapatite crystal formation
through localized enzymatic accumulation of
calcium and phosphate (5). Crystal growth proceeds
from these initial foci in matrix vesicles to form
spheroids, which gradually coalesce to form a
network of apatite crystals. Type I collagen provides
an additional mineralisation mechanism by bindingand orientating proteins, such as osteonectin, that
also nucleate hydroxyapatite.
Regulation of osteoblast differentiation
The systematic and logical study of many
mouse mutants generated led to establishment of
genetic control in osteoblast differentiation. Many
genes have been identified as regulators of cell
differentiation.
A. Transcriptional factor
1. Core-binding factor alpha-1
Core-binding factor alpha-1 (Cbfa-1) is an
osteoblast-specific gene whose expression is
essential for osteoblast differentiation and skeletal
patterning (6-8). Deletion of Cbfa-1 in mice leads
to mutant animals in which the skeleton comprises
only of chondrocytes producing a typical
cartilaginous matrix without evidence of bone
formation (6, 8, 9). Even, patients with Cbfa-1
mutations develop cleidocranial dysplasia (10).
Cbfa-1 function is not only limited to osteoblast celldifferentiation.In vivo study has shown that Cbfa-1
also acts as a maintenance factor for differentiated
osteoblasts by regulating the level of bone matrix
deposited by already differentiated osteoblasts (11).
B. Secreted molecules factor
1. Bone Morphogenetic Proteins(BMPs)
Osteoblasts are cells responsible for the
secretion and deposition of bone morphogenetic
proteins (BMPs) into the extracellular matrix duringbone formation. BMPs, except BMP-1, belong to
the transforming growth factor- (TGF-)
superfamily, members of which are known to
regulate the proliferation, differentiation and death
of cells in various tissues (12).
The unique activity of BMPs suggests that
they regulate osteoblast and chondrocyte
differentiation during skeletal development.Identification of skeletal abnormalities in animals
and patients with mutations in BMPs genes has been
reported (13, 14). However, it is still unclear whether
BMPs are involved in bone and cartilage formation
after birth. The biological effects of recombinant
BMP proteins on osteoblast differentiation have been
studied in vitro using cell lines.
In cultures of osteoblast lineage cells,
Yamaguchiet al., 1991 (15) determined differential
effects of BMP-2 on osteoblasts at various stages of
differentiation in vitro. They indicated that BMP-2preferentially stimulates proliferation and
differentiation of osteoprogenitor cells into mature
osteoblasts with the ability to synthesize osteocalcin.
In MC3T3-E1 cells, BMP-2 and BMP-4 enhance
the expression of alkaline phosphatase activity (16,
17). BMP-2 and BMP-3 were significantly found to
stimulate collagen synthesis (16).
In mesenchymal cell lines, cultures of
C3H10T1/2 cells were used to investigate the role
of BMPs. Studies indicated that BMP-2 and BMP-
7 enhanced osteoblast-related markers in C3H10T1/
2 cells (18, 8). On the other hand, in bone marrow
stromal cell cultures, Yamaguchi et al., 1996 (19)
demonstrated the effects of BMP-2 on osteoblastic
differentiation differ among cell types. The
osteogenic potency of each BMP might depend on
the cell lineage, the stage of differentiation of the
cells and the dose of each BMP.
BMPs originally were identified as an activity
that induces ectopic bone formation in muscular
tissue, suggesting that BMPs regulate the pathway
of differentiation of myogenic cells. Katagiri et al.,
1994 (20) examined this and found that BMP-2inhibited myogenic differentiation of C2C12
myoblasts, and converted their differentiation
pathway into osteoblasts.
2. Ihh
Indian hedgehog (Ihh) is one member of the
Hedgehog family of growth factors that is expressed
in the developing skeleton (21). St Jacques et al.,
1999 (22) reported that Ihh mutant mice that
survived after birth had a markedly reduced
proliferation of chondrocytes result in a failure ofosteoblast development in endochondral bones.
There was no cortical or trabecular structures in the
AN OVERVIEW OF BONE CELLS AND THEIR REGULATING FACTORS OF DIFFERENTIATION
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long bones could be detected histologically and there
was no detectable osteocalcin expressed. Thus,Ihh
signalling is essential for maturation of the
chondrocyte. However, there is no evidence whether
this is a direct or indirect consequence of the absence
ofIhh signalling in regulation of osteoblastdifferentiation.
Osteoclasts
Osteoclasts are large multinucleated
phagocytic cells derived from the macrophage-
monocyte cell lineage (23). They migrate from bone
marrow to a specific skeletal site. They may fuse
either with existing multinucleate osteoclasts or with
each other to form de novo multinucleate osteoclasts,
or remain as mononuclear cells to constitute a
precursor pool for future recruitment.The bone microenvironment plays an
important role in osteoclast formation and function
and is dependent upon local signals from other cells
and growth factors sequestrated in the bone matrix.
Osteoclasts express the enzyme tartrate resistant acid
phosphatase (TRAP), calcitonin receptors, vacuolar
proton ATPase and vitronectin receptors (24).
Osteoclasts are involved in bone resorption
that contributes to bone remodelling in response to
growth or changing mechanical stresses upon the
skeleton. Osteoclasts also participate in the long-
term maintenance of blood calcium homeostasis.
During bone resorption, the osteoclasts resorb the
bone surface forming depressions known as
Howships lacunae.
Resorbing osteoclasts are highly polarized
cells containing four structurally and functionally
distinct membrane domains.In vitro studies revealed
the domains are the ruffled border, the sealing zone,
the basal membrane and a new functional plasma
membrane domain (25, 26). At sites of ac tive
resorption the organic and inorganic components of
bone are endocytosed at the ruffled border,transcytosed through the cell in vesicles and liberated
into the extracellular space via the plasma membrane
domain (25, 26). The ruffled border secretes several
organic acids by maintaining sufficiently low pH in
the microenvironment at the bone surface, which
dissolves the mineral component. The organic matrix
is degraded by lysosomal proteolytic enzymes,
especially the matrix metalloproteinases (MMPs)
including collagenase and gelatinase B and cysteine
proteinases (CPs) such as Cathepsin B, L and K (27-
29) These extensive exchanges between the cell andbone are effectively sealed off from the extracellular
environment by the sealing zone (30).
Regulation of osteoclast differentiation
The systematic and logical study of many
mouse mutants generated led to the establishment
of genetic control in osteoclast differentiation. Many
genes have been identified as regulators of cell
differentiation.
A. Transcriptional control
1. op/op
Osteopetrosis (op) is a skeletal condition
where there is failure of bone resorption to keep in
balance with bone formation. This results in an
excessive amount of mineralised bone. Osteopetrotic
(op/op) is the classical mouse mutation that controls
osteoclast differentiation (31). Mice homozygous for
this recessive mutation lack osteoclasts andmacrophages. The osteopetrotic phenotype of these
mice is not cured by bone marrow transplantation.
2. PU.1
Specific DNA binding proteins regulate the
transcription of eukaryotic gene. Many of these DNA
binding proteins are unique in their expression and
probably serve a general role in gene transcription.
Others are restricted in their expression to one or a
few cell types. PU box revealed a region containing
a purine-rich sequence (5-GAGGAA-3). PU.1 is
a binding protein, that code for this specific DNA
enhancer activity. PU.1 belongs to the member of
the family proteins that exhibit tyrosine-specific (ets)
domain-containing transcription factor that is
expressed specifically in the macrophage and B
lymphoid lineages (32). Deletion of PU.1 results in
a multilineage defect in the generation of progenitors
for B and T lymphocytes, monocytes, and
granulocytes (33).
3. c-fos
Another transcription factor that plays acritical role during osteoclast differentiation is c-fos.
This factor is the cellular homolog of the v-fos
oncogene and is a major component of the AP-1
transcription factor. Deletion ofc-fos in mice led to
an early arrest of osteoclast differentiation without
any overt consequences on osteoblast differentiation
(34).Grigoriadis et al., 1994 (35) also showed that
mice lacking c-fos factor develop osteopetrosis but
have normal macrophage differentiation.
4. Nuclear factor kappa BNuclear factor kappa B (NF-B) is a
transcription factor that is composed of five
Alizae Marny Mohamed
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polypeptide subunits; p50, p52, p65, c-Rel, and RelB
(36). Mice deficient with both p50 and p52 subunits
of NF-B have impaired macrophages functions thatfailed to generate mature osteoclasts and B cells and
developed osteopetrosis (37). NF-B plays a critical
role in expression of a variety of cytokines involvedin early osteoclast differentiation, including
interleukin-1 (IL-1), tumour necrosis factor-(TNF-), interleukin-6(IL-6) and other growth factors.
5. c-Src
c-Src plays a critical role in the activation of
quiescent osteoclasts to become bone-resorbing
osteoclasts. Animals lacking this gene developed
osteopetrosis although the osteoclast formation was
normal. However, it has shown that mature
osteoclasts could not form a ruffled border andtherefore failed to resorb bone (38).
6. Microphthalmia
This transcription factor was identified by
searching for the gene mutated in the
microphthalmia (mi) mouse. Heterozygous mi mice
have the following defects; loss of pigmentation,
reduced eye size and failure of secondary bone
resorption (osteopetrosis). In mi mice, osteoclasts
differentiate normally, but they fail to resorb bones
(39).
B. Secreted molecules factor
1. Macrophage colony-stimulating factor
The gene mutated in osteopetrotic (op/op)
mice encodes the growth factor, macrophage colony-
stimulating factor (M-CSF). M-CSF plays an
important role in osteoclast development. Mutation
in M-CSF gene showed a severe osteopetrosis due
to absence of osteoclasts (40).Fulleret al., 1993 (41)
also identified the role of M-CSF in maintaining the
survival and chemotactic behaviour of matureosteoclasts. They showed that M-CSF prevented
apoptosis of osteoclasts, enhanced osteoclast
motility and inhibited bone resorption.
2. Osteoprotegerin
Simonet et al., 1997 (42) identified a protein
which belongs to a member of the tumour necrosis
factor (TNF) receptor superfamily that regulated
osteoclast differentiation. This molecule,
osteoprotegerin (OPG) contained no hydrophobic
transmembrane-spanning sequence, indicating thatit is a soluble factor. This molecule is identical to
osteoclastogenesis inhibitory factor (OCIF). It
strongly inhibits osteoclast formation in vitro and
in vivo (43).
The OPG/OCIF-deficient mice develop
osteoporosis due to an increase in osteoclast number
(44, 45). Recombinant of OPG/OCIF blocks
osteoclast differentiation from precursor cells invitro; due to its ability to bind and neutralize
osteoprotegerin ligand (OPGL) produced by
activated osteoblasts or stromal cells (43).
Recombinant OPG has been used to screen
for OPGL on the surface of various cell lines. OPGL
has been shown to directly stimulate bone resorption
dose-dependently in vitro, and OPG blocked its
action in vitro and in vivo (46). Previously, this
protein (47) had been cloned and found to be
identical to tumour necrosis factor (TNF)-related
activation-induced cytokine (TRANCE), RANK-ligand (RANKL) or osteoclast differentiation factor
(ODF) (48-49).
3. Receptor activator of NF-B and its ligandReceptor activator of NF-B (RANK) is a
membrane bound receptor found on the osteoclast
membrane and T cells (48, 50). Transgenic mice
expressing RANK develop an osteopetrosis.
The presence of RANK on osteoclasts and
their precursors suggested that osteoclast-
differentiating factor, residing on stromal cells, may
be RANK-ligand (RANKL). RANKL and RANK
are members of the TNF and TNF-receptor
superfamilies, respectively.
RANKL is present on the membrane of the
osteoblast progenitor but also can be found as soluble
molecules in the bone microenvironment. The
membrane-bound of this protein could be a reservoir
of the active molecule.In vitro this protein has all
the attributes of a real osteoclast differentiation
factor. It favours osteoclast differentiation in
conjunction with M-CSF, it bypasses the need for
stromal cells and 1, 25 (OH)2 vitamin D3 to induceosteoclast differentiation, and it activates mature
osteoclasts to resorb mineralised bone (50).
RANKL is also expressed in abundance by
activated T cells, cells that can, in vitro, induce
osteoclastogenesis (51, 52). These cells can directly
trigger osteoclastogenesis and are probably pivotal
to the joint destruction. Indeed, it is the balance
between the expression of the stimulator of
osteoclastogenesis, RANKL, and of the inhibitor
OPG, that dictates the quantity of bone resorbed (53).
RANKL has been shown to activate matureosteoclasts to resorb bone in vitro (46). RANKL-
deficient mice lack osteoclasts and develop a severe
osteopetrosis and immunological defect (54).
AN OVERVIEW OF BONE CELLS AND THEIR REGULATING FACTORS OF DIFFERENTIATION
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It is possible to summarize the role of OPG-
RANK-RANKL in this signal transduction pathway.
(Figure 1)
Osteoclast-Osteoblast Relationship
Termination of bone resorption and theinitiation of bone formation in the resorption lacunae
occur through a coupling mechanism (56). This
coupling mechanism ensures that the amount of bone
laid down is equivalent to the bone removed during
the resorption phase. A model illustrating this
coupling process is shown in Figure 2.
During resorption the osteoclasts release local
factors from the bone which result in two effects;
inhibition of osteoclast function and stimulation of
osteoblast activity. Finally, when the osteoclast
completes its resorptive cycle, it secretes proteinsthat serve as a substrate for osteoblast attachment
(58).
Conclusion
Bone remodelling is required to preserve the
functional capacity of bone. The process of bone
remodelling involves the resorption of bone by the
activity of osteoclasts on a particular surface,
followed by a phase of bone formation by osteoblast.
The status of the bone represents the net result of abalance between these two processes. Normally
during growth there is a balance between bone
resorption and formation. In the normal adult
skeleton, bone formation equals resorption and this
is a constant dynamic process throughout life.
Corresponding Author :
Dr. Alizae Marny Fadzlin Syed Mohamed
BDS (Malaya) MSc in Orth. (London) MOrth RCS
(Edinburgh)
Department of Orthodontic, Faculty of Dentistry,
Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur
Malaysia
Tel: + 603-92897588
Fax: +603-92897856
Email: [email protected]
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ORIGINAL ARTICLE
PROFOUND SWIM STRESS-INDUCED ANALGESIA WITH
KETAMINE
Asma Hayati Ahmad, Zalina Ismail**, Myo Than***, Azhar Ahmad*
Department of Physiology, *Department of Chemical Pathology,
**Deputy Deans Office, School of Health Sciences, School of Medical Sciences,
Universiti Sains Malaysia, Health Campus
16150 Kubang Kerian, Kelantan, Malaysia
***Department of Anatomy, Perak College of Medicine, 30450 Ipoh, Perak, Malaysia
The potential of ketamine, an N-methyl D-aspartate (NMDA) receptor antagonist,
in preventing central sensitization has led to numerous studies. Ketamine is
increasingly used in the clinical setting to provide analgesia and prevent the
development of central sensitization at subanaesthetic doses. However, few studies
have looked into the potential of ketamine in combination with stress-induced
analgesia. This study looks at the effects of swim stress, which is mediated by
opioid receptor, on ketamine analgesia using formalin test. Morphine is used as
the standard analgesic for comparison. Adult male Sprague-Dawley rats were
assigned to 6 groups: 3 groups (stressed groups) were given saline 1ml/kg
intraperitoneally (ip), morphine 10mg/kg ip or ketamine 5mg/kg ip and subjected
to swim stress; 3 more groups (non-stressed groups) were given the same drugs
without swim stress. Formalin test, which involved formalin injection as the painstimulus and the pain score recorded over time, was performed on all rats ten
minutes after cessation of swimming or 30 minutes after injection of drugs.
Combination of swim stress and ketamine resulted in complete analgesia in the
formalin test which was significantly different from ketamine alone (p
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pain suppression systems (7) which are activated
by noxious stimulation (8). It is also the basis for
stress-induced analgesia (SIA) (9), whereby different
forms of stress can produce potent analgesia (10).
The factors involved in the induction of SIA include
intensity of the stress stimulus, duration, and
temporal aspects i.e. whether the stimulus is applied
continuously or intermittently (11). SIA plays an
important role in the survival of animals especially
in fight-or-flight situations (9). This phenomenon is
particularly difficult to study in humans (12) but its
existence is confirmed by various studies (13, 14).
Among the earliest reports of SIA in humans are
observations done by Beecher, as reported by Koltyn
(15), who found that soldiers severely wounded in
battle reported little pain and required considerably
less analgesic medication compared with civilians
undergoing similar surgery.
Assessment of analgesia in experimental
animals employs the use of pain tests such as the
tail flick test, the hot plate test or the formalin test.
Formalin test is widely used to assess analgesia
produced by various stressors, including swim stress(16). It has a peculiar two-phase response produced
by different mechanisms which makes it an ideal
instrument in pain research (17). Ultimately, there
is involvement of the NMDA receptor (18) as a result
of repetitive peripheral nociceptive impulses
mediated through C fibres resulting in increased
central excitability of dorsal horn neurons (19). With
NMDA receptor involvement, the formalin test
inevitably causes induction of c-fos mRNA and
subsequently Fos protein expression which allows
quantification of the pain response (20; 21).
In this study, experimental animals were
subjected to swim stress to produce SIA, and the
resultant analgesia is measured using formalin test
as the pain test. Morphine, the gold standard for
analgesics (22), and low dose ketamine were given
prior to stress-induced analgesia. Both these drugs
are widely used in clinical practice as analgesics and/
or for the prevention of neuroplasticity and central
sensitization (23, 4). The objective of this study is
to assess the analgesia produced by a subanaesthetic
dose of ketamine alone and in combination with
swim stress in the rat formalin test.
Materials & Methods
Animals
Figure 1: Mean formalin test scores in non-stressed groups against
time. n=8 for all groups. Values are means S.E.M. *
p
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Adult male Sprague-Dawley rats, weighing
between 230-350g, were maintained in a 12-h light
dark cycle and allowed free access to food and water.
Rats obtained from the Animal House were housed
in individual cages and allowed adaptation for at
least four days in the Department of Physiology
laboratory. Each animal was used only once.
Experiments were performed between 0800 and
1600 in the same departments laboratory. This study
was approved by the Animal Ethics Committee and
Research Committee of Universiti Sains Malaysia.
Vehicle Used in Experiment
All drugs and saline controls were
administered as pretreatment i.e. before the swim
stress and formalin test procedures. Saline 0.9%
(Sigma) was used as vehicle to dissolve the drugs.
The drugs used were:
1) Ketamine (Gedeon Richter Ltd.) 5mg/kg,
intraperitoneal2) Morphine (Duopharma (M) S/B) 10mg/kg,
intraperitoneal
3) Saline (Sigma) 0.9% as control
The dosage used for ketamine were a
subanaesthetic dose (24, 25, 26) whereby the rats
would experience loss of righting reflex for about
five minutes only and would have recovered fully
before undergoing swim stress. The dosage for
morphine was one that gave analgesic in the rat
formalin test (27, 28). Morphine was the gold
standard against which the analgesic or
antinociceptive activities of other compounds were
compared (29).
Experimental Groups
Rats were allocated to one of six experimental
groups with eight animals in each group. The
experimental group A (non-stressed group) consisted
of one group of rats pretreated with ketamine, second
group of rats pretreated with morphine and the third
group of rats pretreated with saline. Formalin test
was carried out 30 minutes after pre treatment to
allow time for the action of each drug to reach its
peak (30-31, 28).The experimental group B (stressed group)
consisted of the first group of rats pretreated with
ketamine, second group of group rats pretreated with
Figure 2 : Mean formalin test scores in stressed groups against
time. Values are means S.E.M. *p
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morphine and third group of rats pretreated with
salineAnimals in this group received similar
pretreatment as Group A. Fifteen minutes after
pretreatment (30, 31) they were subjected to three
minutes (32, 33) of swim stress. Ten minutes after
cessation of swimming, formalin test was performed
on all the rats. Ten minutes is the time of peak
antinociception following swim stress (32). The
timing is set thus so as to equalize the time interval
between drugs administration and pain stimulation
for both the stressed and the non stressed groups.
Acute Swim Stress Procedure
A container measuring 92 cm x 46 cm x 46
cm high containing 20 cm of water (30; 32; 25) at
20C (30, 33) was used for this purpose. Rats wereplaced in the water individually and left to swim for
three minutes before being removed (32; 34).
Formalin Test
Formalin test was performed 10 minutes after
cessation of acute swim-stress. Diluted (1%)
formalin (35) was prepared freshly from 37%formaldehyde with 0.9% normal saline before use
(36), 50 l was injected subcutaneously into theplantar surface of the right hindpaw using a 27-gauge
needle (28). The rat was then placed in a perspex
testing chamber measuring 26cm x 20cm x 20cm.A mirror was placed below the floor of the chamber
at 45 angle to allow an unobstructed view of the
rats paws (27, 37, 38). The amount of time spent in
each of four behavioural categories, 0-3, was
recorded with a videocam (39) starting from the time
of injection until the end of one hour. The tape was
later viewed by two observers blinded to the
treatment of each rat and the formalin test score was
tabulated every minute and averaged at 5-minute
intervals (35). The quantification was based on the
total time spent in 4 behavioural categories (27). Thecategories were:
0 - the injected paw was not favoured (i.e. foot flat
on the floor with toes splayed) indicating
insignificant or no pain felt
1 - the injected paw had little or no weight on it with
no toe splaying indicating mild pain felt
2 - the injected paw was elevated and the heel was
not in contact with any surface indicating
moderate pain3 - the injected paw was licked, bitten or shaken
indicating severe pain All rats were used only
Figure 3 : A comparison of mean formalin test scores during phase 1 of non-stressed
and stressed groups. n=8 for all groups. Values are means S.E.M. *
p
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once and sacrificed after experiment.
Statistical analysis
Pain behaviour scores by formalin test were
analyzed using repeated measures analysis of
variance (ANOVA) with post hoc Scheffs test.One-way ANOVA was used to calculate significant
differences at each time point, as well as effects of
Phase 1 formalin test (mean score at 5 minutes) and
Phase 2 (mean of scores from 10 to 60 minutes) (17).
Significance was accepted atp
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Formalin test results in non-stressed groups
Formalin produced the typical biphasic pain
response in the saline group (Figure 1). The first
phase includes a burst of activity within 30 seconds
of formalin injection. This phase lasted for about 5
minutes and was followed by a 5 to 10 minutes ofreduced response i.e. the rats showed very little
nociceptive behaviour, and then by a second phase
of activity that lasts for at least 60 minutes after the
formalin injection.
For both the morphine and ketamine groups
of rats, the biphasic response was markedly
attenuated compared to the saline group signifying
analgesia. This attenuation was marked at 10 minutes
until 35 minutes post-formalin injection, after which
the formalin scores for both treatment groups started
to increase. From the graph, morphine showedgreater analgesic effect compared to ketamine
although comparison between morphine and
ketamine groups did not show significant differences
except for one instance at 40 minutes post-formalin.
Formalin test results in stressed groups
For the stressed groups, morphine and saline
groups showed biphasic pattern but the second phase
of the formalin test was depressed (Figure 2). While
for the ketamine group, the second phase was
completely suppressed, obliterating the biphasic
pattern. At 5 minutes post-formalin, which is
equivalent to phase 1, ketamine demonstrated the
lowest score which was significantly (p
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counting the incidence of flinching. This study
shows that a ketamine dose as low as 5mg/kg is
antinociceptive in the rat formalin test. This is
consistent with the findings from previous studies
(47; 46). Studies done with other NMDA antagonists
such as dextromethorphan and memantine (45) andMK-801 (48) also showed similar pattern of Phase
2 inhibition. The fact that ketamine produced
preemptive analgesia by preventing central
sensitization during Phase 1 as shown by Gilron et
al (47) is supported by clinical data suggesting
preemptive analgesia with ketamine (5, 49), by
electrophysiological study demonstrating inhibition
of dorsal horn neuronal firing by ketamine after
noxious stimulation (50), and by another behavioural
study in a different model of persistent pain (51).
Following systemic administration ofketamine, several mechanisms have been proposed
to be involved in producing the analgesia. The first
one reflects actions on mechanisms within the spinal
cord involving central sensitization (52). Other
mechanisms include supraspinal actions, either by
inhibiting NMDA receptors at, for example, thalamic
sites (54), or activation of descending pain inhibitory
mechanisms involving biogenic amines (54). Active
metabolites such as norketamine also contribute to
systemic actions of ketamine (55). It has also been
shown that antagonists of NMDA receptors
modulate elevated discharge of spinal nociceptive
dorsal horn neurons that manifests as suppression
of the second phase of the formalin test (28). Benrath
et al (56), in an in vivo experiment, demonstrated
that low-dose S(+)-ketamine does not affect C-fibre-
evoked potentials alone but blocks long term
potentiation induction in pain pathways. Long term
potentiation was one of the resulting effects of
central sensitization whereby there was long lasting
increase in the efficacy of synaptic transmission (3).
Swim stress, as expected, reduced formalin
nociceptive response during the second phase.Previous studies using similar swim stress paradigm
also produced similar result (40). The
neuroanatomical locus underlying this opioid-
mediated stress-induced response has been shown
to be the ventral tegmental area which has both and receptors (57).
The analgesia produced by this swim stress
paradigm has been shown to be mediated by-opioidreceptor (40). However another study by Vaccarino
et al (30) showed that subjecting mice to the same
swim-stress paradigm produced a non-opioidanalgesia in the formalin test. These researchers
demonstrated that another NMDA antagonist, MK-
801 (dizocilpine maleate), blocked the analgesia
produced by swim stress. Another more recent study
also demonstrated blockade of stress-induced
analgesia by MK-801 (33). This is in contrast with
this study which showed enhancement of stress-
induced analgesia by ketamine. However, Vaccarinoet al (30) only measured formalin-induced
nociceptive response during the initial 10 minutes
following formalin injection i.e. equivalent to the
first phase. Therefore, the NMDA mediation of the
swim stress may be involved only during the first
phase. However, in this study, ketamine inhibited
the first phase after swim stress i.e. producing
analgesia instead of blocking it so some other
explanation may be likely for this discrepancy (40).
Deutsch et al (58) proposed that swim stress altered
or diminished NMDA-mediated neural transmission.Further studies are needed to look at the molecular
mechanism that results following administration of
ketamine such as determining the expression of c-
fos gene, which is mediated through the NMDA
receptor.
In conclusion, this study provides evidence
that low dose ketamine is antinociceptive in the rat
formalin test and this antinociception is enhanced
by swim stress. Taking the finding further into the
clinical setting, it suggests that under stressful
situations such as operative stress, ketamine is
capable of producing profound analgesia at a
subanaesthetic dose (59). Further studies need to be
done to determine the underlying mechanism for this
synergistic effect of ketamine and stress-induced
analgesia.
Acknowledgements
This study was approved by the USM Animal
Ethic . Number 304/PPSP/6131130
Corresponding Author :
Dr Asma Hayati Ahmad MBBS, MSc (Physiology)
Department of Physiology
School of Medical Sciences
Universiti Sains Malaysia, Health Campus,
16150 Kubang Kerian, Kelantan, Malaysia
Tel: + 609 766 4908
Fax: + 609766 3370
Email: [email protected]
PROFOUND SWIM STRESS-INDUCED ANALGESIA WITH KETAMINE
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ORIGINAL ARTICLE
HISTOPATHOLOGICAL STUDIES OF CARDIAC LESIONS AFTER AN
ACUTE HIGH DOSE ADMINISTRATION OF METHAMPHETAMINE
Arthur Kong Sn Molh, Lai Chin Ting, Jesmine Khan, Al-Jashamy K*, Hasnan Jaafar*, Mohammed
Nasimul Islam
School of Health Sciences, *School of Medical Science, Universiti Sains Malaysia, Health Campus
16150 Kubang Kerian, Kelantan, Malaysia
Eighteen male Wistar rats aged six weeks were divided equally into
Methamphetamine (MA), Placebo and Control group. MA group were injected
with 50mg/kg body weight of Methamphetamine hydrochloride (MAHCl) in normalsaline, Placebo group were injected with normal saline only, while Control group
not injected with anything. Five MA group rats died within four hours of injection
and their hearts collected on the same day. Another MA group rat was sacrificed
two days after injection. Placebo and control group were sacrificed at similar
intervals. Collected hearts were studied for cardiac lesions under light microscopy
using special staining and immunohistochemistry. Microscopic examination of the
myocardium of the rats that died on the first day of injection showed loss of nuclei
in some myocytes, indicating cell death. Some areas in the sub-endocardium region
showed internalization and enlargement of myocyte nuclei, consistent with
regeneration of cells. There were very few foci of necrosis observed in these samples.
The heart samples from the single rat that survived injection for two days showed
foci of infiltration of macrophage-like cells that were later revealed to beregenerating myocytes. There were also spindle-like fibroblasts, macrophages and
a few leucocytes found within these foci. The overall appearance of the myocardium
did not indicate any inflammatory response, and the expected signs of necrosis
were not observed. These results suggest a need to re-evaluate the toxic and lethal
dosages of MA for use in animals testing. Cause of death was suspected to be due
to failure of other major organs from acute administration of MA. Death occurred
within a time period where significant changes due to necrosis may not be evident
in the myocardium. Further investigations of other organs are necessary to help
detect death due to acute dosage of MA.
Key words :MA, acute dose administration, cardiac lesions, myocardium.
Introduction
The use of MA along with other designer
drugs have seen a dramatic increase beginning from
the 1990s, as more drug abusers seek cheaper, more
potent alternatives to the traditional stimulants
such as cocaine (13). The stimulant and euphoric
effect of MA is similar to cocaine, bringing about
similar behaviour in animal tests of MA and cocaine.
MA in the form of hydrochloride crystals are volatile
and smokeable, bringing an immediate euphoria that
lasts longer than cocaine (1, 4). Cardiovascular
symptoms related to MA toxicity include chest pain,
palpitations, dyspnoea, hypertension, tachycardia,
atrial and ventricular arrhythmias, and myocardial
ischaemia (1, 49). MA abusers often go through a
repeated pattern of frequent drug administrations
(binge) followedby a period of abstinence. This
pattern of chronic MA abuse can significantly alter
cardiovascular function and cardiovascularreflex
function and produce serious cardiacpathology (10).
However, tachyphylaxis occurs with MA abuse, with
long-term abusers being able to tolerate higher doses
with fewer symptoms. MA has been known to cause
Submitted-20-02-2007, Accepted-03-12-07
Malaysian Journal of Medical Sciences, Vol. 15, No. 1, January 2008 (23-30)
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death at an ingested dose as low as 1.5 mg per kg
body weight, while long-time abusers developing
drug tolerance may use as much as 5,000 to 15,000
mg per day (1). MA sold in the streets is usually
mixed with other stimulants such as cocaine,
phenypropanolamine hydrochloride, D-amphetamine, ephedrine, or pseudoephedrine, and
also with other adulterants such as lead, caffeine and
baking soda (1). This discrepancy in the purity of
MA available leads to the question whether the
abuser may be taking high dosages far too toxic to
the body, which may result in sudden death of the
abusers. Given the pattern of MA abuse, previous
studies have focused largely upon the chronic effect
of MA intake to major organs, such as the brains
and the heart, by using animal testing (6, 9, 1113).
However, there is a lack of research into the effectsof acute dose intake of MA, especially pertaining to
the heart. Sudden death due to acute MA intoxication
has been suggested to be similar to acute myocardial
infarction, where pathological changes to the
myocardium generally are hard to detect, even under
light microscopy (14). Thus, there is a need to review
the effects of acute dosages of MA intake to the heart
through microscopic studies in rats, which can help
medical examiners differentiate myocardium
changes due to acute MA intake from those of other
cardiovascular diseases.
Materials and Methods
Eighteen male Wistar rats aged of six weeks
were reared in the animal house of Universiti Sains
Malaysia, Kubang Kerian, Kelantan under standard
atmospheric conditions in three 12 (w) X 24 (l) X 8
(h) inch cage. Each cage was labelled according to
the three groups the rats were divided into, namely
the Control, Placebo, and MA injected groups. The
weight of the rats ranged from 102.6 123.1 grams.
Control Group
The six rats in this group were kept under
normal rearing condition, fed with standard
laboratory chow and tap water ad libitum until six
weeks of age. The rats were fasted for 24 hours
before being sacrificed according to similar time
intervals as the MA-injected group, and their hearts
were collected.
Placebo GroupThe six rats in this group were kept under
normal rearing condition, fed with standard
laboratory chow and tap water ad libitum until six
weeks of age. Each rat was then injected
intraperitoneally with 0.3ml of 0.9% (w/v) saline
each. The rats were then fasted for 24 hours after
injection before being sacrificed at similar time
intervals as the MA-injected group and their hearts
were collected.
MA Injected Group
The six rats in this group were kept under
normal rearing condition, fed with standard
laboratory chow and tap water ad libitum until six
weeks of age. Each rat was then given an
intraperitoneal injection of MAHCl dissolved in
Arthur Kong Sn Molh, Lai Chin Ting et. al
Figure 1 : Foci of cellular infiltration in the sub-endocardium region at
400X magnification
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0.9% (w/v) saline, the volume of which was adjusted
according to body weight so that the final dosage
received by each rat was approximately 50mg/kg.
The rats were fasted for 24 hours before being
sacrificed and their hearts collected for pathological
observation.A total amount of 50 milligrams MAHCl used
in this experiment was obtained from the Department
of Chemistry Malaysia (JKM), Petaling Jaya, as
MAHCl is a restricted substance classified under
Section 39 (B) of the Dangerous Drugs Act 1952 in
Malaysia, whereby possession, import or sale of the
substance is strictly prohibited and punishable by
Malaysian law. Moreover, this is an export forbidden
item. As such, only the JKM is authorized by the
Malaysian government to provide chemicals
classified as restricted substance under Malaysianlaw for use in laboratory and scientific studies. The
purity of the MAHCl obtained has been assayed and
certified as to be of a minimum 99% pure, as stated
in the certification report provided by the JKM.
The dosage of MA given was calculated based
on previous studies (15) so as to induce observable
effects on the rats and to let the rats survive for at
least 24 hours after injection. However, rats No.3,
4, 5, and 6 of MA group died after two hours of
injection while rat No.2 died four hours after
injection. The hearts of these rats were collected on
the same day. Rat No.1 survived for 48 hours after
injection before being sacrificed. The rats in the
Control and Placebo groups were also sacrificed at
similar intervals as the deaths that occur in the MA
injected group rats.
The rats were sacrificed by confining them
in a glass chamber saturated with chloroform (except
the rats from the MA injected group that died a few
hours after injection). A small sample of the free
upper left ventricle walls from each heart was takenand preserved in 0.9% (w/v) saline for future use in
electron microscopy methods. A section of the upper
levels of both ventricles from each heart were
collected and preserved in 10% (w/v) formalin for
paraffin embedding while the adjoining section was
harvested for frozen sectioning. The sections of
ventricles preserved in 10% (w/v) formalin were
then processed in a tissue processor and embedded
in standard paraffin blocks.
The frozen sectioned ventricle samples were
stained with Hematoxylin and Eosin (H&E) stain(commercial kit from Sigma Aldrich) for observation
under light microscopy. The consecutive sections
of paraffin embedded samples were stained using
H&E, Massons Trichrome Stain (MTS)
(commercial kit from Sigma Aldrich) and
immunohistochemistry staining using rabbit anti-
myosin (commercial kit from Calbiochem). For
immunohistochemistry, the heart samples were
treated with rabbit anti-myosin as the primary
antibody, which was then reacted with biotinylated
anti-rabbit immunoglobulin G (IgG) secondary
antibody. Biotinylated horseradish peroxidise, avidin
dehydrogenase, and hydrogen peroxide were then
used to provide sites for binding of
diaminobenzidine tetrahydrochloride (DAB) dye to
HISTOPATHOLOGICAL STUDIES OF CARDIAC LESIONS AFTER AN ACUTE HIGH DOSE ADMINISTRATION OF METHAMPHETAMINE