His to Pathological Studies of Cardiac Lesions After An

8/14/2019 His to Pathological Studies of Cardiac Lesions After An

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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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5

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


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



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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



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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).



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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.


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


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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


***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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16

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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17

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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19

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


Dr Asma Hayati Ahmad MBBS, MSc (Physiology)

Department of Physiology

School of Medical Sciences

Universiti Sains Malaysia, Health Campus,


Tel: + 609 766 4908

Fax: + 609766 3370


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


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



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



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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25

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

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