His To Pathological Studies Of Cardiac Lesions After An

Malaysian Journal of Medical Sciences, Vol. 15, No. 1, January 2008 (1-3)

EDITORIAL ENSURING THE STANDARD OF MEDICAL GRADUATES IN MALAYSIA Mafauzy Mohamed Director, Health Campus, Universiti Sains Malaysia, Health Campus 16150 Kubang Kerian, Kelantan, Malaysia Key words : Medical graduates, Malaysia.

In Malaysia, the Malaysian Medical Council (MMC) is assumed to be the body responsible for ensuring that medical graduates are of a sufficient standard to be safe, and competent medical practitioners. Under the Medical Act 1971, MMC is responsible for recognizing medical schools for the purpose of licensing the graduates to practice in Malaysia. This means that it should set standards and certify the achievement of the standards of Medical degree programmes awarded by all medical schools within and outside Malaysia. Unfortunately, this is akin to a final product inspection before approval and any pre-emptive remedial action prior to the finished product (ie. the process) is not possible. The teaching, training and curriculum is determined by the respective universities whereby the standards are loosely set by the Ministry of Education and since 2004, by the Ministry of Higher Education (MOHE). With the mushrooming of private medical colleges, the task of ensuring the standard was entrusted upon the National Accreditation Board (Lembaga Akreditasi Negara – LAN) which was set-up in 1996. LAN assured the quality by accrediting the basic medical programmes of private medical schools as governed by the LAN Act. This, unfortunately, gave rise to 2 sets of standards – the public medical schools as determined by MOHE and the private medical schools as determined by LAN. This anomaly was resolved when LAN was dissolved and replaced by the Malaysian Qualifying Agency (MQA) under the MQA Act 2007 which is now authorized to accredit both public and private medical schools thus ensuring a single, uniform standard. In the accreditation process of medical programmes, the MQA is advised by a Joint Technical Committee

comprising of MMC, MOHE and the Public Services Department (PSD). The PSD is involved by virtue of the fact that all medical graduates must do their compulsory service in government hospitals or clinics for 3 years and PSD is the body that appoints them in government service. The Joint Technical Committee is also responsible for approval of medical programmes, constituting the evaluation panels, studying the report of the accrediting teams and submitting the recommendations on accreditation for approval by MQA, MMC and PSD. The Committee also conduct training courses for accreditors who can be from public or private universities, Ministry of Health and members of MMC to ensure that evaluating and accrediting teams sent out to the various medical schools are trained for the tasks. Accreditation of medical schools is done based on a set of criteria, standards and procedures which were first formulated in 1998. In 2000, the format of the accreditation guidelines was reviewed and the format of the World Federation for Medical Education (WFME) adopted. The WFME had developed ‘International Standards in Medical Education’ which specified basic and quality development standards that served as performance indicators for quality assurance in medical education. In 2006, a review was made of the Guidelines based on feedbacks and comments of those involved in the accreditation process. The new edition of the guidelines was adopted in 2007. The key changes include a ruling on continuous assessments whereby it should either be used as a pre-requisite for sitting for the final or professional examination or contribute not more that 40% to the final examination score. If used as a pre-requisite, 1

Rahmattullah Khan bin Abdul Wahab Khan

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 be more than 60%. Staff-student ratio is also stipulated for all the various teaching-learning activities such as tutorialsnot 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 teachingnot 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 purpose much 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 school’s 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 been overcome. 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 good medical graduate as the graduate’s own personal traits and behaviour would play a large bearing on

2

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 of Higher 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.

ENSURING THE STANDARD OF MEDICAL GRADUATES IN MALAYSIA

3.

Assessment Form For Medical Graduates During The Internship Posting. Malaysian Medical Council. February 2008.

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Malaysian Journal of Medical Sciences, Vol. 15, No. 1, January 2008 (4-12)

REVIEW ARTICLE 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 noncollagenous 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 Submitted : 6.03.2007, Accepted : 30.12.2007

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 4

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 bone that is secreted by osteoblasts. Most of the noncollagenous 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

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 the entire system by blocking the effects of RANKL. Khosla, 2001 (55).

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 bloodborne 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 postproliferative, representing the most mature differentiation state of osteoblast lineage. There are about 25,000 osteocytes per mm 3 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 obvious candidates to detect stresses induced in bone and are therefore regarded as the main mechanoreceptors 5

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).

of bone. It has been shown that mechanical stress can be sensed by osteocytes and these cells secrete paracrine factors such as insulin-like growth factorI (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 have a 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-tocell 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.

6

Osteoblasts Osteoblasts are specialised fibroblast-like cells 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 in shape 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

AN OVERVIEW OF BONE CELLS AND THEIR REGULATING FACTORS OF DIFFERENTIATION

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 with matrix 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 binding and 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 cell differentiation. 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).

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, Yamaguchi et al., 1991 (15) determined differential effects of BMP-2 on osteoblasts at various stages of differentiation in vitro. They indicated that BMP-2 preferentially 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 BMP7 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-2 inhibited myogenic differentiation of C2C12 myoblasts, and converted their differentiation pathway into osteoblasts. 2.

B. 1.

Secreted molecules factor

Bone Morphogenetic Proteins (BMPs) Osteoblasts are cells responsible for the secretion and deposition of bone morphogenetic proteins (BMPs) into the extracellular matrix during bone formation. BMPs, except BMP-1, belong to the transforming growth factor- β (TGF- β)

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 of osteoblast development in endochondral bones. There was no cortical or trabecular structures in the

7

Alizae Marny Mohamed

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 of Ihh signalling in regulation of osteoblast differentiation.

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.

Osteoclasts Osteoclasts are large multinucleated phagocytic cells derived from the macrophagemonocyte 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 longterm maintenance of blood calcium homeostasis. During bone resorption, the osteoclasts resorb the bone surface forming depressions known as Howship’s 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 active 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 (2729) These extensive exchanges between the cell and bone are effectively sealed off from the extracellular environment by the sealing zone (30).

8

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 and macrophages. 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 a critical 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 of c-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 B Nuclear factor kappa B (NF- κB) is a transcription factor that is composed of five

AN OVERVIEW OF BONE CELLS AND THEIR REGULATING FACTORS OF DIFFERENTIATION

polypeptide subunits; p50, p52, p65, c-Rel, and RelB (36). Mice deficient with both p50 and p52 subunits of NF-κB have impaired macrophages functions that failed to generate mature osteoclasts and B cells and developed osteopetrosis (37). NF-κB plays a critical role in expression of a variety of cytokines involved in early osteoclast differentiation, including interleukin-1 (IL-1), tumour necrosis factor-α(TNFα), 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 and therefore failed to resorb bone (38). 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).

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 in vitro; 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), RANKligand (RANKL) or osteoclast differentiation factor (ODF) (48-49).

6.

B.

Secreted molecules factor

1.

Macrophage colony-stimulating factor The gene mutated in osteopetrotic (op/op) mice encodes the growth factor, macrophage colonystimulating 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). Fuller et al., 1993 (41) also identified the role of M-CSF in maintaining the survival and chemotactic behaviour of mature osteoclasts. 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 that it is a soluble factor. This molecule is identical to osteoclastogenesis inhibitory factor (OCIF). It

3.

Receptor activator of NF-κB and its ligand Receptor activator of NF-κB (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 osteoclastdifferentiating 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 induce osteoclast 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 mature osteoclasts to resorb bone in vitro (46). RANKLdeficient mice lack osteoclasts and develop a severe osteopetrosis and immunological defect (54). 9

Alizae Marny Mohamed

It is possible to summarize the role of OPGRANK-RANKL in this signal transduction pathway. (Figure 1)

2.

Stein GS, Lian JB. Molecular mechanisms mediating proliferation/differentiation interrelationships during progressive development of the osteoblast phenotype. Endocrine Review 1993; 14: 424-442.

Osteoclast-Osteoblast Relationship Termination of bone resorption and the initiation 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 proteins that serve as a substrate for osteoblast attachment (58).

3.

Friedenstein AJ. Precursor cells of mechanocytes. International Review of Cytology 1976; 47: 327-359.

4.

Anderson HC. Vesicles associated with calcification in the matrix of epiphyseal cartilage Journal of Cell Biology 1969; 41: 59-72.

5.

Anderson HC, Reynolds JJ. Pyrophosphate stimulation of calcium uptake into cultured embryonic bones. Fine structure of matrix vescles and their role in calcification. Developmental Biology 1973; 34: 211227

6.

Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K, Deguchi K, Shimizu Y, Bronson RT, Gao YH, Inada M, Sato M, Okamoto R, Kitamura Y, Yoshiki S, Kishimoto T. Targeted distruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 1997; 89: 755764.

7.

Otto F, Thornell AP, Crompton T, Denzel A, Gilmour KC, Rosewell IA, Stamp GWH, Beddington RSP, Mundlos S, Olsen BR, Selby PB, Owen MJ. Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell 1997; 89: 765-771.

8.

Ducy P, Zhang R, Geoffroy V, Ridall AL, Karsenty G. Osf2/Cbfa1: A transcriptional activator of osteoblast differentiation. Cell 1997; 89: 747-754.

9.

Kim IS, Otto F, Zabel B, Mundlos S. Regulation of chondrocyte differentiation by Cbfa1. Mechanisms of Development 1999; 80: 159-170.

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 a balance 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]

References 1.

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Lean JM, Mackay A, Chow J, Chambers T. Osteocytic expression of mRNA for c-fos and IGF-I; an immediate early gene response to an osteogenic stimulus. American Journal of Physiology 1996; 270: 937-945.

10. Lee B, Thirunavukkarasu K, Zhou L, Pastore L, Baldini A, Hecht J, Geoffroy V, Ducy P, Karsenty G. Missense mutations abolishing DNA binding OSF2/CBFA1 in patients affected with cleidocranial dysplasia. Nature Genetics 1997; 16: 307-310. 11. Ducy P, Starbuck M, Priemel M, Shen J, Pinero G, Geoffroy V, Amling M, Karsenty G. A Cbfa1dependent genetic pathway controls bone formation beyond embryonic development. Genes and Development 1999; 13: 1025-1036. 12. Hogan BL. Bone morphogenetic proteins: multifunctional regulators of vertebrate development. Genes and Development 1996; 10: 1580-1594. 13. Kingsley DM, Bland AE, Grubber JM, Marker PC, Russell LB, Copeland NG, Jenkins NA. The mouse short ear skeletal morphogenesis locus is associated with defects in a bone morphogenetic member of the TGFβ superfamily. Cell 1992; 71: 399-410. 14. Thomas JT, Kilpatrick MW, Lin K, Erlacher L, Lembessis P, Costa T, Tsipouras P, Luyten FP. Distruption of human limb morphogenesis by a dominant negative mutation in CDMP1. Nature Genetics 1997; 17: 58-64.

AN OVERVIEW OF BONE CELLS AND THEIR REGULATING FACTORS OF DIFFERENTIATION

15. Yamaguchi A, Katagiri T, Ikeda T, Wozney JM, Rosen V, Wang EA, Kahn AJ, Suda T, Yoshiki S. Recombinant human bone morphogenetic protein-2 stimulates osteoblastic maturation and inhibits myogenic differentiation in vitro. Journal of Cell Biology 1991; 113: 681-687. 16. Takuwa Y, Ohse C, Wang EA, Wozney JM, Yamashita K. Bone morphogenetic protein-2 stimulates alkaline phosphatase activity and collagen synthesis in cultured osteoblastic cells, MC3T3-E1. Biochemical and Biophysical Research Communications 1991; 174: 96101. 17. Nakase T, Takaoka K, Masuhara K, Shimizu K, Yoshikawa H, Ochi T. Interleukin-1β enhances and tumour necrosis factor-α inhibits bone morpogenetic protein-2 induce alkaline phosphatase activity in MC3T3-E1 osteoblastic cells. Bone 1997; 11: 17-21. 18. Katagiri T, Yamaguchi , Ikeda T, Yoshiki S, Wozney JM, Rosen V, Wang EA, Tanaka H Omura S, Suda T. The non-osteogenic mouse pluripotent cell line, C3H1OT1/2 is induced to differentiate into osteoblastic cells by recombinant human bone morphogenetic protein-2. Biochemical and Biophysical Research Communications 1990; 172: 295-299 19. Yamaguchi A, Ishizuya T, Kintou N, Wada Y, Katagiri T, Wozney JM, Rosen V, Yoshiki S. Effects of BMP-2, BMP-4 and BMP-6 on osteoblastic differentiation of bone marrow-derived stromal cell lines, ST2 and MC3T3-G2/PA6. Biochemical and Biophysical Research Communications 1996; 220: 366-371. 20. Katagiri T, Yamaguchi A, Komaki M, Abe E, Takashi N, Ikeda T, Rosen V, Wozney JM, Fujisawa-Sehara A, Suda T. Bone morphogenetic protein-2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage. Journal of Cell Biology 1994; 127: 1755-1766. 21. Bitgood MJ and McMahon AP. Hedgehog and Bmp genes are coexpressed at many diverse sites of cellcell interaction in the mouse embryo. Developmental Biology 1995; 172: 126-138. 22. St-Jacques B, Hammerschmidt M, McMahon AP. Indian hedgehog signaling regulates proliferation and differentiation of chondrocytes and is essential for bone formation. Genes and Development 1999; 13: 20722086. 23. Walker DG. Osteoporosis cured by temporary parabiosis. Science 1973; 180: 875 24. Lee SK, Goldring SR, Lorenzo JA. Expression of the calcitonin receptor in bone marrow cell cultures and in bone: a specific marker of the differentiated osteoclast that is regulated by calcitonin. Endocrinology 1995; 136: 4572-4581. 25. Salo J, Lehenkari P, Mulari M, Metsikkö K, Väänänen HK. Removal of osteoclast bone resorption products by transcytosis. Science 1997; 276: 270-273. 26. Blair H, Teitelbaum SL, Ghiselli R and Gluck S. Osteoclastic bone resorption by a polarised vacuolar proton pump. Science 1989; 245: 855-857.

27. Hill PA, Docherty A, Bottomley K, O’Connell JP, Morphy JR, Reynolds SJ, Meikle MC. Inhibition of bone resorption in vitro by selective inhibitors of gelatinase and collagenase. Biochemical Journal 1995; 308: 167-175. 28. Hill PA, Buttle D, Jones S, Boyde A, Murata M, Reynolds JJ, Meikle MC. Inhibition of bone resorption by selective inactivators of cysteine proteinases. Journal of Cellular Biochemistry 1994; 56: 118-130. 29. Drake FH, Robert AD, James IE, Conver JR, Debouck CC, Richardson S, Lee-Rykaczewski E, Coleman L, Rieman D , Barthlow R, Hastings G, Gowen M. Cathepsin K but not Cathepsins B, L or S is abundantly expressed in human osteoclasts. Journal of Biological Chemistry 1996; 271: 12511-12516. 30. Silver IA, Murrills RJ, Etherington DJ. Microelectrode studies on the acid microenvironment beneath adherent macrophages and osteoclasts. Experimental Cell Research 1988; 175: 266-276. 31. Marks SCJ, Lane PW. Osteopetrosis, a new recessive skeletal mutation on chromosome 12 of the mouse. Journal of Heredity 1976; 67: 11-18. 32. Klemsz MJ, McKercher SR, Celada A, Van Beveren C, Maki RA. The macrophage and B cell-specific transcription factor PU.1 is related to the ets oncogene. Cell 1990; 61: 113-124. 33. Scott EW, Simon MC, Anastasi J, Singh H. Requirement of transcription factor PU.1 in the development of multiple hematopoietic lineages. Science 1994; 265: 1573-1577. 34. Johnson RS, Spiegelman BM, Papaioannou V. Pleitropic effects of a null mutation in the c-fos protooncogene. Cell 1992; 71: 577-586. 35. Grigoriadis AE, Wang ZQ, Cecchini MG, Hofstetter W, Felix R, Fleisch HA, Wagner EF. c-Fos: a key regulator of osteoclast-macrophage lineage determination and bone remodelling. Science 1994; 266: 443-448. 36. Verma IM, Stevenson JK, Schwarz EM, Van Antwerp D, Miyamoto S. Rel/NF-κB/IκB family: intimate tales of association and dissociation. Genes and Development 1995; 9: 2723-2735. 37. Franzoso G, Carlson L, Xing L, Poljak L, Shores EW, Brown KD, Leonardi A, Tran T, Boyce BF, Siebenlist U. Requirement for NF-κB in osteoclast and B-cell development. Genes and Development 1997; 11: 34823496. 38. Boyce BF, Yoneda T, Lowe C, Soriano P, Mundy GR. Requirement of pp60c-src expression for osteoclasts to form ruffled borders and resorb bone in mice. Journal of Clinical Investigation 1992; 90: 1622-1627. 39. Hodgkinson CA, Moore KJ, Nakayama A, Steingrimsson Copelan NG, Jenkins NA, Arnheiter H. Mutations at the mouse micropthalmia are associated with defects in a gene encoding a novel basic-helixloop-helix-zipper protein. Cell 1993; 74: 395-404.

11

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40. Yoshida H, Hayashi SI, Kunisada T, Ogawa M, Nishikawa S, Okamura H, Sudo T, Shultz LD, Nishikawa SI. The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene. Nature 1990; 345: 442-444. 41. Fuller K, Owens JM, Jagger CJ, Wilson A, Moss R, Chambers TJ. Macrophage colony-stimulating factor stimulates survival and chemotactic behavior in isolated osteoclasts. Journal of Experimental Medicine 1993; 178: 1733-1744. 42. Simonet WS, Lacey DL, Dunstan CR, Kelley M, Chang MS, Lüthy R, Nguyen HQ, Wooden S, Bennett L, Boone T, Shimamoto G, DeRose M, Elliot R, Colombero A, Tan HL, Trail G, Sullivan J, Davy E, Bucay N, Renshaw-Gegg L, Hughes TM, Hill D, Pattison W, Campbell P, Sander S, Van G, Tarpley J, Derby P, Lee R, Amgen EST Program, Boyle WJ. Osteoprogeterin: a novel secreted protein involved in the regulation of bone density. Cell 1997; 89: 309-319. 43. Yasuda H, Shima N, Nakagawa N, Mochizuki SI, Yano K, Fujise N, Sato Y, Goto M, Yamaguchi K, Kuriyama M, Kanno T, Murakami A, Tsuda E, Morinaga T, Higashio K. Identity of osteoclastogenesis inhibitory factor (OCIF) and osteoprotegerin (OPG): A mechanism, by which OPG/OCIF inhibits osteoclastogenesis in vitro. Endocrinology 1998; 139: 1329-1337.

the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function. Nature 1997; 390: 175179. 49. Yasuda H, Shima N, Nakagawa N, Yamaguchi K, Kinosaki M, Mochizuki SI, Tomoyasu A, Yano K, Goto M, Murakami A, Tsuda E, Morinaga T, Higashio K, Udagawa N, Takahashi N, Suda T. Osteoclast differentiation factor is a ligand for osteoprotegerin/ osteoclastogenesis-inhibitor factor and is identical to TRANCE/RANKL. Proceedings of the National Academy of Sciences of the United States of America 1998; 95: 3597-3602. 50. Burgess TL, Qian YX, Kaufman S, Ring BD, Van G, Capparelli C, Kelley M, Hsu H, Boyle WJ, Dunstan CR, Hu S, Lacey DL. The ligand for osteoprotegerin (OPGL) directly activates mature osteoclasts. The Journal of Cell Biology 1999; 14: 527-538. 51. Horwood NJ, Kartsogiannis V, Quinn JM, Romas E, Martin TJ, Gillespie MT. Activated T lymphocytes support osteoclast formation in vitro. Biochemical and Biophysical Research Communications 1999; 265: 144-150. 52. Rifas L, Arackal S, Weitzmann MN. Inflammatory T cells rapidly induce differentiation of human bone marrow stromal cells into mature osteoblasts. Journal of Cellular Biochemistry 2003; 88: 650-659.

44. Bucay N, Sarosi I, Dunstan CR, Morony S, Tarpley J, Capparelli C, Scully S, Tan HL, Xu W, Lacey DL, Boyle WJ, Simonet WS. Osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification. Genes and Development 1998; 12: 12601268.

53. Hofbauer LC, Gori F, Riggs LR, Lacey DL, Dunstan CR, Spelsberg TC, Khosla S. Stimulation of osteoprotegerin ligand and inhibition of osteoprotegerin production by glucocorticoids in human osteoblastic lineage cells: potential paracrine mechanisms of glucocorticoid-induced osteoporosis. Endocrinology 1999; 140: 4382-4389.

45. Mizuno A, Amizuka N, Irie K, Murakami A, Fujise N, Kanno T, Sato Y, Nakagawa N, Yasuda H, Mochizuki S, Gomibuchi T, Yano K, Shima N, Washida N, Tsuda E, Morinaga T, Higashio K, Ozawa H. Severe osteoporosis in mice lacking osteoclastogenesis inhibitory factor /osteoprotegerin. Biochemical and Biophysical Research Communications 1998; 247: 610-615.

54. Kong YY, Yoshida H, Sarosi I, Tan HL, Timms E, Caparelli C, Morony S, Oliveira-dos-Santos AJ, Van G, Itie A, Khoo W, Wakeham A, Dunstan CR, Lacey DL, Mak TW, Boyle WJ, Penniger JM. OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature 1999; 397: 315-323.

46. Lacey DL, Timms E, Tan HL, Kelley MJ, Dunstan CR, Burgess T, Elliot R, Colombero A, Elliott G, Scully S, Hsu H, Sullivan J, Hawkins N, Davy E, Capparelli C, Eli A, Qian YX, Kaufman S, Sarosi I, Shalhoub V, Senaldi G, Guo J, Delaney J, Boyle WJ. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 1998; 93: 165-176.

56. Parfitt AM. The coupling of bone formation to bone resorption: a critical analysis of the concept and of its relevance to the pathogenesis of osteoporosis. Metabolic Bone Disease and Related Research 1982; 4: 1-6.

47. Wong BR, Josien R, Lee Sy, Sauter B, Li HL, Steinman RM, Choi Y. TRANCE [Tumor Necrosis Factor (TNF)related activation-induced cytokine], a new TNF family member predominantly expressed in T cells, is a dendritic cell-specific survival factor. Journal of Experimental Medicine 1997; 186: 2075-2080. 48. Anderson DM, Maraskovsky E, Billingsley WL, Dougall WC, Tometsko ME, Roux ER, Teepe MC, Dubose RF, Cosman D, Galibert L. A homologue of

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55. Khosla S. Minireview: The OPG/RANKL/RANK system. Endocrinology 2001; 142: 5050-5055.

57. Manolagas SC, Jilka RL. Bone marrow, cytokines, and bone remodelling. Emerging insights into the pathophysiology of osteoporosis. New England Journal of Medicine 1995; 332: 305-311. 58. McKee MD, Farach-Carson MC, Butler WT, Hauschka PV, Nanci A. Ultrastructural immunolocalization of non-collagenous (osteopontin and osteocalcin) and plasma (albumin and α2HS-Glycoprotein) proteins in rat bone. Journal of Bone and Mineral Research 1993; 8: 485-496.

Malaysian Journal of Medical Sciences, Vol. 15, No. 1, January 2008 (13-22)

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 Dean’s 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 pain stimulus 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<0.05) and saline with stress (p<0.01). There is no significant difference between ketamine stressed and morphine stressed. These results indicate that ketamine and swim stress act synergistically to produce profound analgesia in the formalin test. This suggests that in the clinical setting, under stressful situations such as operative stress, ketamine is capable of producing profound analgesia at a subanaesthetic dose. Key words : ketamine, morphine, formalin test, swim stress. Submitted : 20-02-2007, Accepted : 03-12-07

Introduction Studies have identified the pathway of pain commencing from the site of stimulation to the spinal cord and subsequently the brain. One breakthrough finding is the study which showed that noxious stimulation in the periphery is able to cause the expression of a gene named c- fos in the corresponding spinal cord segment mediated via the N-Methyl-D-Aspartate (NMDA) receptor (1). The importance of c-fos is that it is implicated in the

development of central sensitization (2), a phenomenon brought about by changes in the pain pathway due to neuroplasticity in the central nervous system (3). One drug that has the potential to reduce the development of central sensitization is ketamine, an NMDA receptor antagonist (4). Low dose ketamine is increasingly used to provide analgesia without producing side effects usually produced by its normal anaesthetic dose (5) and it is also widely used in preemptive analgesia (6). The central nervous system possesses intrinsic 13

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Figure 1:

Mean formalin test scores in non-stressed groups against time. n=8 for all groups. Values are means ± S.E.M. * p<0.05, ** p<0.01, *** p<0.001 for comparison between morphine and saline. # p<0.05, ## p<0.01, # # # p<0.001 for comparison between ketamine and saline. + p<0.05 for comparison between morphine and ketamine.

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 14

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

PROFOUND SWIM STRESS-INDUCED ANALGESIA WITH KETAMINE

Figure 2 :

Mean formalin test scores in stressed groups against time. Values are means ± S.E.M. * p<0.05, ** p<0.01, *** p<0.001 for comparison between morphine and saline. # p<0.05, ## p<0.01, ### p<0.001 for comparison between ketamine and saline.

3) Saline (Sigma) 0.9% as control 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 department’s 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, intraperitoneal 2) Morphine (Duopharma (M) S/B) 10mg/kg, intraperitoneal

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 15

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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<0.05 compared with saline stressed.

morphine and third group of rats pretreated with saline Animals 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 20°C (30, 33) was used for this purpose. Rats were placed 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 µl was injected subcutaneously into the plantar surface of the right hindpaw using a 27-gauge 16

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 rat’s 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). The categories 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 pain 3 - the injected paw was licked, bitten or shaken indicating severe pain All rats were used only

PROFOUND SWIM STRESS-INDUCED ANALGESIA WITH KETAMINE

Figure 4 :

A comparison of mean formalin test scores during phase 2 of non-stressed and stressed groups. n=8 for all groups. Values are means ± S.E.M. **p<0.01, *** p<0.001 compared with saline non-stressed ++ p<0.01, +++ p<0.001compared with saline stressed # p<0.05 compared with ketamine non-stressed

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 Scheffé’s 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 at p<0.05.

Results Effects of ketamine and morphine on rats’ behaviour Observation of the rats immediately after injection of drugs showed that rats receiving ketamine 5mg/kg demonstrated loss of righting

reflex for about 2 to 3 minutes and recovered fully before being subjected to swim stress. This showed that the dosage used only had transient anaesthetic effect and did not affect performance during the swim stress procedure. Rats receiving morphine 10mg/kg, however, displayed no apparent changes in behaviour. Formalin test results The overall results demonstrated that there were significant (p<0.05) ‘within subject’ (i.e. the differences in time variable for each group) and ‘between subject’ (i.e. the differences between groups) effects as shown by repeated measures ANOVA. This meanst that significant differences exist in the formalin test score over time and the pattern of changes is not the same for the nonstressed and stressed groups as well as the different treatment groups. 17

Asma Hayati Ahmad, Zalina Ismail et. al

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 of reduced 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 showed greater 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<0.05) lower than saline. After 5 minutes, however, all three groups showed marked attenuation of the formalin score. For saline this attenuation lasted until 35 minutes post-formalin when the score started to steadily increase again. Morphine showed slight increase in the score after 35 minutes but the scores were still significantly (p<0.01) lower than the saline group. Ketamine, however, demonstrated the greatest analgesia by complete attenuation of the formalin score until 60 minutes post-formalin. Formalin test results during phase 1 Comparing both the non-stressed and the stressed groups during phase 1 (5 minutes postformalin injection), there were no significant differences among the three non-stressed groups. Among the stressed groups, the only significant difference was between saline stressed and ketamine stressed (p<0.05) (Figure 3). Analyzing the formalin scores for each drug, it was demonstrated that swim stress did not cause any significant differences in the formalin scores for

18

saline, morphine and ketamine, i.e. there were no significant differences between the formalin scores of non-stressed and stressed groups during phase 1. Formalin test results during phase 2 During phase 2 (10 to 60 minutes postformalin injection), among the non- stressed groups, both morphine and ketamine showed significantly lower formalin scores than saline ( p<0.001) indicating analgesia (Figure 4). All the stressed groups, including saline stressed, had significantly lower scores when compared to the saline nonstressed. Among the stressed groups, both morphine stressed and ketamine stressed showed significantly lower scores than saline stressed (morphine: p<0.01; ketamine: p<0.001). No significant differences were seen between morphine stressed and non-stressed. Formalin score for ketamine stressed was significantly lower than ketamine non-stressed (p<0.05) and saline stressed ( p<0.001). No significant differences were seen between ketamine stressed and morphine stressed. Summary of formalin test results In the non-stressed groups, both morphine 10mg/kg and ketamine 5mg/kg produced analgesia in the formalin test. All three stressed groups demonstrated stress-induced analgesia during phase 2. This analgesia was enhanced by prior treatment with morphine or ketamine. In the morphine group, stress did not enhance the analgesia produced by morphine alone. In the ketamine group, stress significantly enhanced the analgesia produced by ketamine alone.

Discussion The biphasic response due to formalin injection as shown by previous studies (27; 40; 41; 42) was reproducible in this study. Formalin test was used because it provides a valid model for clinical pain (43). The first phase of the formalin test is due to direct chemical stimulation of nociceptors (27) and involved substance P and bradykinin (44). The second phase involved local inflammatory processes (44) as well as processes in the spinal cord (17). The finding that ketamine inhibited phase two but not phase one of the formalin test in this study is consistent with previous studies that used even higher doses of ketamine (45). Lee & Lee (46) demonstrated suppression of both phases but the quantification of pain behaviour was only by

PROFOUND SWIM STRESS-INDUCED ANALGESIA WITH KETAMINE

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) and MK-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 of ketamine, 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-fibreevoked 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 opioidmediated stress-induced response has been shown to be the ventral tegmental area which has both µ and δ receptors (57). The analgesia produced by this swim stress paradigm has been shown to be mediated by δ-opioid receptor (40). However another study by Vaccarino et al (30) showed that subjecting mice to the same swim-stress paradigm produced a non-opioid analgesia 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 stressinduced analgesia by ketamine. However, Vaccarino et 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 cfos 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]

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References 1.

Hunt, S.P., Pini, A. & Evan, G. Induction of c-fos-like protein in spinal cord neurons following sensory stimulation. Nature 1987; 328: 632 – 634.

2.

Woolf, C.J. Generation of acute pain: Central mechanisms. Br Med Bull 1991; 47(3): 523–533.

3.

Pockett, S. Spinal cord synaptic plasticity and chronic pain. Anesth Analg 1995; 80: 173–179.

4.

Kohrs, R and Durieux, M.E. Ketamine: Teaching an old drug new tricks. Anesth Analg 1998; 87(5): 11861193.

5.

Annetta , M.G., Iemma, D., Garisto, C., Tafani, C., Proietti, R. Ketamine: new indications for an old drug. Curr Drug Targets 2005; 6(7): 789-94.

6.

Aida, S. The challenge of preemptive analgesia. Pain 2005; 13(2): 1-4.

7.

8.

9.

Willis, W.D. Jr. Temperature perception and pain. In Greger, R and Windhorst, U eds. Comprehensive Human Physiology. From Cellular Mechanisms to Integration. 1996; Vol 1. Berlin: Springer: 677-696. Fields & Basbaum. Central nervous system mechanisms of pain modulation. In Wall & Melzack eds. Textbook of Pain. 3rd edn. London : Churchill Livingstone, 1994: 243-254. Amit, Z. & Galina, Z.H. Stress-induced analgesia: Adaptive pain suppression. Physiol Rev 1986; 66(4): 1091–1120.

10. Watkins, L.R. The Pain Of Being Sick: Implications of immune-to-brain communication for understanding pain. Annu Rev Psychol 2000; www. AnnualReviews. org. 11. Grau, J.W., Hyson, R.L., Maier, S.F., Madden IV, J., Barchas, J.D. Long-term stress-induced analgesia and activation of the opiate system. Science 1981; 213: 1409– 411. 12. Davis, G.C.. Endorphins and Pain. Psychiatr Clin North Am 1983; 6(3): 473–487. 13. Paustian, E. Conditioned stress-induced analgesia in humans. Clinical Psychology – Research Projects. 2000 Pg. 1 – 3 at http://www.psychologie.hu.berlin.de/ kli/kliko3.htm 14. Washington, L.L., Gibson, S.J., Helme, R.D.. Agerelated differences in the endogenous analgesic response to repeated cold water immersion in human volunteers. Pain 2000; 89(1): 89–96. 15. Koltyn, K.F. Analgesia following exercise: A Review. Sports Med 2000; 29(2): 85–98. 16. Quintero, L., Cuesta, M.C., Silva, J.A., Arcaya, J.L., Pinerua-Suhaibar, L., Maixner, W., Suarez-Roca, H. Repeated swim stress increases pain-induced expression of c-Fos in the rat lumbar cord. Brain Res 2003; 965: 259–268.

20

17. Tjølsen, A., Berge, O., Hunskaar, S., Rosland, J. H., Hole, K. The formalin test: an evaluation of the method. Pain 1992; 51: 5-17. 18. Fukuda, T., Nishimoto, C., Hisano, S., Miyabe, M., Toyooka, H. The analgesic effect of xenon on the formalin test in rats: A Comparison with nitrous oxide. Anesth Analg 2002; 95: 1300-1304. 19. Dickenson, A.H. & Sullivan, A.F. Evidence for a role of the NMDA receptor in the frequency dependent potentiation of deep rat dorsal horn nociceptive neurons following C fibre stimulation. Neuropharmacology 1987; 26(8): 1235-1238. 20. Buritova, J., Larrue, S., Aliaga, M., Besson, J.M., Colpaert, F. Effects of the high-efficacy 5-HT1A receptor agonist, F 13640 in the formalin pain model: a c-Fos study. Eur J Pharmacol 2005; 514(2-3): 12130. 21. Fukuda, T., Watanabe, K., Hisano, S., Toyooka, H. Licking and c-fos expression in the dorsal horn of the spinal cord after the formalin test. Anesth Analg 2006; 102(3): 811-4. 22. Martin, B. (1994). Opioid and non-opioid analgesics. In Modern Pharmacology , 4 th edn. (Craig & Stitzel eds.), p. 431-444. Boston: Little, Brown and Company. 23. Aida, S., Yamakura, T., Baba, H., Iaga, K., Fukuda, S., Shimaji, K. Preemptive analgesia by intravenous lowdose ketamine and epidural morphine in gastrectomy: A randomized double-blind study. Anesthesiology 2000; 92(6): 1624-1630. 24. Irifune, M., Shimizu, T., Nomoto, M. Ketamineinduced hyperlocomotion associated with alteration of presynaptic components of dopamine neurons in the nucleus accumbens of mice. Pharmacol Biochem Behav 1991; 40(2): 399-407. 25. Suarez-Roca, H., Silva, J.A., Arcava, J.L., Quintero, L., Maixner, W., Pinerua-Shuhaibar, L. Role of muopioid and NMDA receptors in the development and maintenance of repeated swim stress-induced thermal hyperalgesia. Behav Brain Res 2006; 167(2): 205-11. 26. UCSF Animal Care & Use Program. www.iacuc.ucsf.edu/Proc/awA&A_DS.asp 27. Dubuisson, D. & Dennis, S.G. The formalin test: A quantitative study of the analgesic effects of morphine, meperidine, and brain stem stimulation in rats and cats. Pain 1977; 4: 161-174. 28. Sevostianova, N., Danysz, W., Bespalov, A.Y. Analgesic effects of morphine and loperamide in the rat formalin test: Interactions with NMDA receptor antagonists. Eur J Pharmacol 2005; 525(1-3): 83-90. 29. Zheng, M., McErlane, K.M., Ong, M.C. Identification and synthesis of norhydromorphone, and determination of antinociceptive activities in the rat formalin test. Life Sci 2004; 75(26): 3129-46. 30. Vaccarino, A. L., Marek, P., Sternberg, W., Liebeskind, J. C. NMDA receptor antagonist MK-801 blocks nonopioid stress-induced analgesia in the formalin test. Pain 1992; 50: 119-123.

PROFOUND SWIM STRESS-INDUCED ANALGESIA WITH KETAMINE

31. Smith, D.J., Bouchal, R.L., DeSanctis, C.A. Properties of the interaction between ketamine and opiate binding sites in vivo and in vitro. Neuropharmacology 1987; 26(9): 1253-1260. 32. Vanderah, T.W., Wild, K.D., Takemori, A.E., Sultana, M., Portoghese, P.S., Bowen, W.D., Mosberg, H.I. and Porreca, F. Mediation of swim-stress antinociception by the opioid delta2 receptor in the mouse. J Pharmacol Exp Ther 1992; 262(1): 190-197. 33. Vendruscolo, L.F. & Takahashi, R.N. Synergistic interaction between mazindol, an anorectic drug, and swim-stress on analgesic responses in the formalin test in mice. Neurosci Lett 2004; 355(1-2): 13-16. 34. Fazli-Tabaei, S., Yahyavi, S.H., Alagheband, P., Samie, H.R., Safari, S., Rastegar, F., Zarrindast, M.R. Crosstolerance between antinociception induced by swimstress and morphine in formalin test. Behav Pharmacol 2005; 16(8): 613-9. 35. Sun, W.Z., Shyu, B.C. & Shieh, J.Y. Nitrous oxide or halothane, or both, fail to suppress c-fos expression in rat spinal cord dorsal horn neurones after subcutaneous formalin. Br J Anaes 1996; 76: 99-105. 36. Lee, I-O., Kong, M-H., Kim, N-S., Choi, Y-S., Lim, S-H., Lee, M-K. Effects of different concentrations and volumes of formalin on pain response in rats. Acta Anaesthesiol Sin 2000; 38: 59-64. 37. Vaccarino, A. L., Tasker, R. A. R. and Melzack, R. Analgesia produced by normal doses of opioid antagonists alone and in combination with morphine. Pain 1989; 36: 103-109. 38. Gogas, K.R., Cho, H.J., Botchkina, G.I., Levine, J.D., Basbaum, A.I. Inhibition of noxious stimulus-evoked pain behaviors and neuronal fos-like immunoreactivity in the spinal cord of the rat by supraspinal morphine. Pain 1996; 65: 9-15. 39. Sawamura, S., Fujinaga, M., Kingery, W.S., Belanger, N., Davies, M.F., Maze, M. Opioidergic and adrenergic modulation of formalin-evoked spinal c-fos mRNA expression and nocifensive behavior in the rat. Eur J Pharmacol 1999; 379(2-3): 141-149. 40. Kamei, J., Hitosugi, H., Misawa, M., Nagase, H., Kasuya, Y. δ-Opioid receptor-mediated forced swimming stress-induced antinociception in the formalin test. Psychopharmacology 1993; 113: 15-18. 41. Shannon, H.E. & Lutz, E.A. Comparison of the peripheral and central effects of the opioid agonists loperamide and morphine in the formalin test in rats. Neuropharmacology 2002; 42: 253–261. 42. Oliveira, A.R. & Barros, H.M. Ultrasonic rat vocalizations during the formalin test: a measure of the affective dimension of pain? Anesth Analg 2006; 102(3): 832-9. 43. Chen, A.C.N., Dworkin, S.F., Haug, J., Gehrig, J. Human pain responsivity in a tonic pain model: psychological determinants. Pain 1989; 37: 143-160.

44. Shibata, M., Ohkubo, T., Takahashi, H., Inoki, R. Modified formalin test: characteristic biphasic pain response. Pain 1989; 38: 347-352. 45. Sawynok, J. & Reid, A. Modulation of formalininduced behaviors and edema by local and systemic administration of dextrometorphan, memantine and ketamine. Eur J Pharmacol 2002; 450: 153–162. 46. Lee, I. & Lee, I. Systemic, but not intrathecal, ketamine produces preemptive analgesia in the rat formalin model. Acta Anaesthesiol Sin 2001; 39: 123-127. 47. Gilron I., Quirion, R., Coderre, T.J. Pre-versus postinjury effects of intravenous GABAergic anesthetics on formalin-induced fos immunoreactivity in the rat spinal cord. Anesth Analg 1999; 88: 414-20. 48. Yamamoto, T. & Yaksh, T.L. Comparison of the antinociceptive effects of pre- and post-treatment with intrathecal morphine and MK-801, an NMDA antagonist, on the formalin test in the rat. Anesthesiology 1992; 77: 757-63. 49. Fu, E.S., Miguel, R., Scharf, J.E. Preemptive Ketamine Decreases Postoperative Narcotic Requirements in Patients Undergoing Abdominal Surgery. Anesth Analg 1997; 84: 1086–90. 50. Hao, J.X., Sjolund, B.H., Wiesenfeld-Hallin, Z. Electrophysiological evidence for an antinociceptive effect of ketamine in the rat spinal cord. Acta Anaesthesiol Scand 1998; 42(4): 435-441. 51. Hartrick, C.T., Wise, J.J., Patterson, J.S. Preemptive intrathecal ketamine delays mechanical hyperalgesia in the neuropathic rat. Anesth Analg 1998; 86(3): 557– 60. 52. Chaplan, S.R., Malmberg, A.B., Yaksh, T.L. (1997). Efficacy of spinal NMDA receptor antagonism in formalin hyperalgesia and nerve injury evoked allodynia in the rat. J Pharmacol Exp Ther 280, 829– 838. 53. Kolhekar, R., Murphy, S., Gebhart, G.F. Thalamic NMDA receptors modulate inflammation-produced hyperalgesia in the rat. Pain 1997; 71: 31– 40. 54. Kawamata, T., Omote, K., Sonoda, H., Kawamata, M., Namiki, A. Analgesic mechanisms of ketamine in the presence and absence of peripheral inflammation. Anesthesiology 2000; 93(2): 520–528. 55. Shimoyama, M., Shimoyama, N., Gorman, A.L., Elliott, K.J., Inturrisi, C.E. Oral ketamine is antinociceptive in the rat formalin test: role of the metabolite, norketamine. Pain 1999; 81: 85–93. 56. Benrath, J., Brechtel, C., Stark, J., Sandkuhler, J. Low dose of S+-ketamine prevents long-term potentiation in pain pathways under strong opioid analgesia in the rat spinal cord in vivo. Br J Anaesth 2005; 95(4): 51823. 57. Altier, N. & Stewart, J. Opioid receptors in the ventral tegmental area contribute to stress-induced analgesia in the formalin test for tonic pain. Brain Res 1996; 718: 203-206.

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58. Deutsch, S.I., Mastropaolo, J., Riggs, R.L., Rosse, R.B. The antiseizure efficacies of MK-801, phencyclidine, ketamine, and memantine are altered selectively by stress. Pharmacol Biochem Behav 1997; 58(3): 70912.

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59. Schmid, R.I., Sandler, A.N., Katz, J. Use and efficacy of low-dose ketamine in the management of acute postoperative pain: a review of current techniques and outcomes. Pain 1999; 82: 111-125.

Malaysian Journal of Medical Sciences, Vol. 15, No. 1, January 2008 (23-30)

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 normal saline, 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 be regenerating 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. Submitted-20-02-2007, Accepted-03-12-07

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 (1–3). 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, 4–9). MA abusers often go through a repeated pattern of frequent drug administrations (binge) followed by a period of abstinence. This pattern of chronic MA abuse can significantly alter cardiovascular function and cardiovascular reflex function and produce serious cardiac pathology (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 23

Arthur Kong Sn Molh, Lai Chin Ting et. al

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, Damphetamine, 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, 11–13). However, there is a lack of research into the effects of 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 Figure 1 :

24

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 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 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

Foci of cellular infiltration in the sub-endocardium region at 400X magnification

HISTOPATHOLOGICAL STUDIES OF CARDIAC LESIONS AFTER AN ACUTE HIGH DOSE ADMINISTRATION OF METHAMPHETAMINE

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 Malaysian law 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 Figure 2 :

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 taken and 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, Masson’s Trichrome Stain (MTS) (commercial kit from Sigma Aldrich) and immunohistochemistry staining using rabbit antimyosin (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

Loss of nuclei and large, internalised nuclei in rat no.2 viewed under 400X magnification

25

Arthur Kong Sn Molh, Lai Chin Ting et. al

the myosin molecules, and the nuclei of cells in the samples were counter-stained with Harris Hematoxylin. Any loss of staining of tissue using immunohistochemistry would indicate injury due to myocardial infarcts (16). The procedures for staining were sourced from the manuals of the commercial kits for each stain. The light microscopy assessments of the samples were conducted blindly by two observers to observe pathological changes highlighted in previous studies. Findings were confirmed while excluding artefacts through comparison with histochemical slides. The parameters observed for included eosinophilic changes, cellular infiltration, and contraction band necrosis. Comparison of H&E and immunohistochemistry results were used to assess distribution of myoglobin, while MTS was used to confirm any signs of fibrosis. The parameters of pathological changes observed were tabulated and graded using a scale of 0–4, where “0” signifies the absence of the parameters in the samples, or healthy myocardium; “1” indicating observed presence and distribution of the parameter up to 25% of the area examined; “2” indicating observed presence and distribution of the parameter between 26% to 50% of the area examined; “3” indicating observed presence and distribution of the parameter between 51% to 75% of the area examined; and “4” indicating observed presence and distribution of the parameter in more than 75% of the area examined. The Kruskall-Wallis test was used for statistical analysis in the SPSS Figure 3 :

26

version 11.5 software and a P value of <0.05 was considered significant. Procedure Fifty milligrams of MAHCl was dissolved in 2.5ml 0.9% (w/v) saline for the injection of the test group rats. The volume of MA infused saline injected into each rat was adjusted with the calculated concentration of MAHCl injected for each rat to be approximately 50 mg/kg body wt.

Results Rat no.1 was injected with MAHCl earlier to observe the effect of the concentration of MA and the acceptability of the dose. This rat was subsequently sacrificed, 48 hours after the injection. The other rats were all injected with methamphetamine a day later. Rats no.3, 4, 5, and 6, died within two hours of injection, while rat no.2 died after four hours of injection. The hearts of these rats were collected on the same day after injection. Gross The hearts of the MA-injected rats were normal in size, without any signs of necrosis throughout the musculature. The hearts of the rats that died within 4 hours of injection were congested. The heart weight to body weight ratios were similar in all three groups. The hearts of the MA-injected rats had patent coronary arteries that were free of

Foci of cellular infiltration in rat no.1, DAB stained cells indicated as regenerating myocytes, viewed under 400X magnification

HISTOPATHOLOGICAL STUDIES OF CARDIAC LESIONS AFTER AN ACUTE HIGH DOSE ADMINISTRATION OF METHAMPHETAMINE

Table 1: Results of histopathological observation. MA group rat no.

Cell infiltration

Eosinophilic changes

1

1

0

0

1

1

2

0

0

0

3

2

3

0

0

0

2

1

4

0

0

0

2

1

5

0

1

0

2

1

6

0

0

0

2

1

Pathological parameters observed

Enlargement and Contraction band necrosis internalisation of nuclei

Loss of nuclei

* Legend – “0” = Parameter not present “1” = Parameter present in up to 25% of area examined “2” = Parameter present in between 26% to 50% of area examined “3” = Parameter present in between 51% to 75% of area examined “4” = Parameter present in more than 75% of area examined atheroma and their heart valves were normal. Light microscopy examination Hematoxylin and Eosin Staining (H&E) The sample from rat No.1 of MA group, which survived for 48 hours, displayed distinct foci of cellular infiltration (Figure 1), with clusters of macrophage-like cells having large nuclei and little cytoplasm evident in the sub-endocardium region. There were also presences of few leucocytes and fibroblast-like spindle cells. Certain areas also show loss of cross-striation and general disruption of the cellular ultra-structure. The samples from rat No.2 showed loss of nuclei in certain sub-endocardium regions, indicating dead cells, while other areas showed internalisation of large nuclei (Figure 2). The internalisations of the large nuclei are indicative of the myocytes’ regeneration process. In rats No.3, 4, 5, and 6, the heart samples showed little difference from control samples, except for the presence of large, internalised nuclei, indicative of regenerative cells. In rat No.5, the sub-endocardium also showed a few foci of necrosis with eosinophilic changes. Masson’s Trichrome Stain (MTS) MTS was used to highlight any fibrous changes to the heart tissue of the rats, which may occur due to healing and scarring of myocardium after an infarct or similar injury. The samples from MA group rats do not show any noticeable fibrous tissue changes compared to the control samples using MTS for observation under light microscopy.

Immunohistochemistry In rat No.1 of the MA group, the foci of cellular infiltration were found to be stained similarly to its surrounding area (Figure 3), thus indicating the clusters of cells were actually regenerating myocytes. However, there may still be a few leucocytes or macrophages present within these foci of cellular infiltration, and further investigation with other anti-leucocyte or anti-macrophage antibodies can be used to elucidate their presence. In all the MA group rats, the myocardium samples were stained in almost uniform intensity, with several areas of differentiated staining intensity of DAB. Thus, the presences of cellular death within the samples were confirmed Table 1 summarizes the observed pathological changes in the samples of the MA group, in relation to the type of change observed and the extent of spread of each type of pathological change. Although the expected parameters of eosinophilic changes and contraction band necrosis were not observed in most of the samples, the presence of enlarged, internalised nuclei, and the absence of nuclei in certain myocytes showed a statistically significant relationship (P value of <0.05 using Kruskal-Wallis test) between the MA group rats and the control group rats.

Discussion The acute dose of MA injected to the rats in this experiment were determined so that the rats would be able to show effects of acute MA intake and be able to survive for a day (13, 15). However, 27

Arthur Kong Sn Molh, Lai Chin Ting et. al

a complication to the design of this study arose when four of the MA group rats died within two hours after the MA injection, while a fifth rat died within four hours of injection. The deaths of the rats occurring within such a short time resulted in certain histopathological changes that were indicated in previous studies were found to be absent in the MA group rat samples. The difference in the length of time where each rat had survived after MA injection also could not be determined. The histopathological changes observed in the MA group rats’ samples include cellular infiltration in rat No.1; enlargement and internalisation of nuclei in all samples; loss of nuclei in certain parts of all samples; and eosinophilic changes in a few foci of necrosis in rat No.5. Previous studies in the acute dose intake of MA had indicated that eosinophilic changes can be noticeable in rats after 2.5 hours of acute MA administration, while cellular infiltration were noticeable after 18 hours of MA administration (15). The eosinophilic changes in rat No.5 and the cellular infiltration in rat no.1 were in agreement with the findings of the previous study (15). The cellular infiltration in rat No.1 was found in the sub-endocardium, particularly near the left ventricle free wall region. The sub-endocardial location of the cellular infiltration may be due to transmural gradient in blood flow, which resulted in the inner layer of the wall being more severely affected than its outer layer. In a typical transmural infarct the corresponding gradient in ischaemic injury initially produces rapid coagulative necrosis of the inner layer of the wall and lethal injury, subsequently propagates itself towards the epicardial surface to an extent dependent on both time and the characteristics of the vascular bed involved (17). The cells found in the foci of cellular infiltration in rat no.1 were identified as regenerating myocytes, with the presence of a few macrophages and leucocytes, but there were no fibrous tissue observed in these areas. There were no signs of inflammatory reaction in the tissue, even though death of cells was evident in scattered areas of the myocardium. The regenerating myocytes indicated that the affected tissue were trying to repair themselves by producing new myocytes to compensate for the dead cells. Macrophages and leucocytes were suspected to be present at these sites of regenerating myocytes, mainly because the macrophages function to remove dead cells through phagocytosis, thus eliminating the toxic substances from being released by dead cells in the damaged tissue area and preventing further tissue damage.

28

Although the observation indicated certain cells in the foci of cellular infiltration were macrophages or leucocytes, further staining using anti-macrophage or anti-leucocyte antibodies is needed to have a positive confirmation of the presence of these cells. However, one major difference between the results of this study and previous studies is the absence of contraction band necrosis, a characteristic pattern of myocardial cell alteration that was present in experimentally induced acute regional myocardial infarcts of 2 hours or more duration (15, 17). Contraction band necrosis was reported to be frequently encountered in recent acute myocardial infarcts, and its presence facilitate the definitive diagnosis of sudden cardiac death (17). As such, the short duration of which the rats in the MA group survived in this study should be adequate to show contraction band necrosis. The absence of contraction band necrosis, however, indicates that the toxic effects of MA on other major organs apart from the heart may have caused the early deaths of the rats in MA group, before the changes to the myocardium had become noticeable. The disposition of MA and its metabolite amphetamine is highest in kidneys, followed by spleen, brain, liver, heart and serum (12). It was also observed that high MA concentrations in the brain occurred immediately after intravenous bolus dosing, suggesting that there is essentially no hindrance to passage of MA at the blood brain barrier, and MA distributes very rapidly to all tissues studied except the spleen (12). As such, there is a high probability that the deaths that occurred in the MA group rats in this study were due to the toxic effects of MA in other major organs, most probably the brain, and not due to toxic effects on the heart. However, the scope of the study limited the choice of exploring the effects of acute high dose administration of MA on the other major organs as well. Loss of staining with antibodies to myoglobin is the most sensitive and reliable diagnosis of early myocardial ischaemic damage (16). The results of the immunohistochemistry staining show that most of the samples have varying intensities of brown coloured staining by diaminobenzidine tetrahydrochloride (DAB). Thus, it can be concluded that the myocardium may have suffered some form of ischaemic damage to the tissue, which could have been due to the direct effect of MA in the blood or by other pathways of pathological damage that had yet to be explained. The deaths of rats within four hours of acute

HISTOPATHOLOGICAL STUDIES OF CARDIAC LESIONS AFTER AN ACUTE HIGH DOSE ADMINISTRATION OF METHAMPHETAMINE

MA administration on a dose designed for the rats to survive for 24 hours indicate that there may be a need to re-evaluate the toxic and lethal dosages of MA for use in animals testing. However, the exact mechanism of MA toxicity that leads to sudden death is still unknown (3), thus further investigation into this issue should be done to provide a helpful guide for medical examiners when dealing with cases involving sudden deaths where MA abuse may be suspected. Further testing procedures such as examination of changes in other major organs and detection of serum levels of MA may be necessary to help detect deaths due to acute intake of MA. The use of electron microscopy in conjunction with light microscopy is also recommended, as they would allow clearer differentiation of the necrotic changes in myocardium or other major organs. It is undeniable, however, that an acute dose intake of MA will lead to serious damage of the heart or other major organs that may possibly lead to death, and as such, drug abusers and the society in general should be aware of the dangers of abusing this drug or other harmful drugs.

2.

Jacobs W. Fatal amphetamine-associated cardiotoxicity and its medicolegal implications. Am. Journal of Forensic Medicine & Pathology 2006; 27 (2): 156 – 160

3.

Wijetunga M, Seto T, Lindsay J and Schatz I. Crystal methamphetamine-associated cardiomyopathy: Tip of the iceberg? J. of Toxi: Cl. Toxicology 2003; 41 (7): 981 – 986

4.

Ellinwood EH, King G and Lee TH. Chronic amphetamine use and abuse. Psychopharmacology: The Fourth Generation of Progress. 2000. Website http://www.acnp.org/G4/GN401000166/CH162.htm, accessed 2.2.2007

5.

He SY1, Matoba R, Sodesaki K, Fujitani N and Ito Y. Morphological and morphometric investigation of cardiac lesions after chronic administration of methamphetamine in rats. Nihon Hoigaku Zasshi 1996; 50 (2): 63 – 71

6.

Maeno Y, Iwasa M, Inoue H, Koyama H, Matoba R and Nagao M. Direct effects of methamphetamine on hypertrophy and microtubules in cultured adult rat ventricular myocytes. Forensic Science International 2000; 113: 239 – 243

7.

Matoba R. Cardiac lesions in methamphetamine abusers. Nihon Hoigaku Zasshi 2001; 55(3): 321 – 330

8.

Rostagno C, Caciolli S, Felici M, Gori F and Serneri GGN. Dilated cardiomyopathy associated with chronic consumption of phendimetrazine. Am. Heart J. 1996; 131: 407 – 9

9.

Yu Q, Montes S, Larson DF and Watson RR. Effects of chronic methamphetamine exposure on heart function in uninfected and retrovirus-infected mice. Life Sciences 2002; 71: 953 – 965

Acknowledgements The authors wish to thank the Department of Chemistry, Malaysia for supplying the MAHCl used in the experiment, as well as the staff of the Pathology Laboratory of Hospital Universiti Sains Malaysia, and Biomedicine Laboratory, School of Health Sciences, USM. This research was conducted under IRPA grant no. 06-02-05-00021-EAR

Corresponding Author : Assoc. Prof. Dr. Mohammed Nasimul Islam (MBBS, MCPS, DLM, LLB, PhD) School of Health Sciences Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia Tel: + 609-766 3915 Fax: +609-764 7884 Email: [email protected]

References 1.

Derlet RW and Heischober B. Methamphetamine – Stimulant of the 1990s? West J. Med. 1990; 153: 625 – 628

10. Varner KJ, Ogden BA, Delcarpio J and Meleg-Smith S. Cardiovascular responses elicited by the “binge” administration of methamphetamine. The Journal of Pharmacology and Experimental Therapeutics 2001; 301: 152 – 159 11. Islam MN, Kuroki H, Bai H, Ogura Y, Kawaguchi N, Onishi S and Wakasugi C. Cardiac lesions and their reversibility after long term administration of methamphetamine. Forensic Science International 1995; 75: 29 – 43 12. Riviere GJ, Brooks G and Owens M. Disposition of methamphetamine and its metabolite amphetamine in brain and other tissues in rats after intravenous administration. The Journal of Pharmacology and Experimental Therapeutics 2000; 292 (3): 1042 – 1047 13. Shaw KP, Pu CE, Lin DL, Liu JC and Fong JM. A comparative study of inhalation and self-administration methamphetamine-induced toxicities in rats. Advances in Forensic Sciences 1995; 5: 264 – 273 14. Smith HJ, Roche AHG, Jagusch MF and Herdson PB. Cardiomyopathy associated with amphetamine administration. American Heart Journal 1976; 91 (6): 792 – 797

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15. Maruta T, Nihira M and Tomita Y. Histopathological study on acute poisoning of methamphetamine, morphine or cocaine. Jpn. J. Alcohol & Drug Dependence 1997; 32 (2): 122 – 138 16. Stefan J and Toupalik P. Our experience with immunocytochemical staining of myoglobin in myocardium. The Journal of Czecho-Slovak Forensic Medicine 1994; 39 (4): 39 – 42

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17. Armiger LC and Smeeton WMI. Contraction-band necrosis: Patterns of distribution in the myocardium and their diagnostic usefulness in sudden cardiac death. Pathology 1986; 18: 289 – 295

Malaysian Journal of Medical Sciences, Vol. 15, No. 1, January 2008 (31-36)

ORIGINAL ARTICLE BODY COMPOSITION AND HAND GRIP STRENGTH IN MALE BRICK-FIELD WORKERS Amit Bandyopadhyay School of Health Sciences Universiti Sains Malaysia, Health Campus 16150 Kubang Kerian, Kelantan, Malaysia

Ninety two male brickfield workers and sixty sedentary individuals of 25-45 years were randomly selected from brickfields of West Bengal, India, to evaluate the body composition and hand grip strength among male brickfield workers and to compare the data with their sedentary counterparts. Assessment of body composition by skinfold measurements and determination of hand grip strength (HGS) by hand grip dynamometer indicated significantly higher (p<0.001) fatness, skinfold values and body mass index (BMI) among the sedentary individuals though HGS and hand grip endurance were significantly higher (p<0.001) among brickfield workers. BMI and %fat values indicated that the subjects were non-obese and non-overweight and regression norms for prediction of %fat from BMI in both the groups were computed as follows : Control Group : Y = 1.647 X – 22.789 (r = 0.92, p<0.001, SEE = 1.01), Brick-field Workers : Y = 0.747 X – 8.398 (r = 0.78, p<0.001, SEE = 1.34). Percentage of lean body mass (%LBM) was significantly higher (p<0.001) among brickfield workers whereas absolute LBM value had insignificant variation because of significant difference (p<0.001) in body mass between the groups. The present investigation revealed that the daily labour of the brick-field workers hindered the accumulation of body fat and extensive use of their hand and finger muscles enabled them to achieve greater arm strength. The proposed norms for prediction of %fat from BMI will also provide a firsthand impression about the body composition in the studied population. Key words : Brickfield workers, %fat, hand grip strength, BMI Submitted : 20-02-2007, Accepted : 03-12-07

Introduction The occupational health of workers refers to the state of body which should remain free of physical and mental stress at the time of work not only for maximum productivity but also to reduce the risks of accidents and proneness to injuries (1). Health hazards in the working environment primarily arrive out of the amount of work load imposed on an individual, either because of industrial need or due to postural distress (2). Anthropometric measurements and body composition are important tools for evaluating the health status as well as nutritional pattern of individuals (2, 3, 4). Moreover, different components

of body composition not only reflect various energy balances with relation to the functional and metabolic parameters of the subject, but are also highly correlated with the cardiorespiratory fitness (4, 5, 6, 7). Skeletal muscle function is influenced by nutrition as well as cardiorespiratory fitness (8) and hand grip strength (HGS) measurement determines motor fitness or skill-related fitness of the entire skeletal musculature of the arm (9). Several small scale industries are in existence in India of which brick-making industry is one. Despite its immense importance to mankind and society, the other parameters to judge the health and fitness status of the workers engaged in brick making

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Amit Bandyopadhyay

Table 1 :

Physical parameters, anthropometric measurements and hand grip endurance of brick-field workers and control subjects.

jobs in India and abroad have not yet been examined except for pulmonary function testing (11, 12). The present study was therefore conducted to evaluate the body composition and hand grip strength among male brick-field workers and to compare the data with their sedentary or control counterparts.

to an accuracy of + 0.25 kg and height to an accuracy of + 0.5 cm. Body surface area (BSA), body mass index (BMI) and Reciprocal of Ponderal Index (RPI) were calculated from the equations of DuBois and DuBois (12), Meltzer et al. (13) and Sloan (14), respectively.

Materials and Methods

Determination of body composition Body composition was determined from skinfold measurements. Skinfolds were measured by Holtain Skinfold Caliper with constant tension (Holtain Ltd., UK) and different components of body composition were measured by using standardised equations (2, 15). The TF or total fat determines the body’s fat content in absolute value while the %fat determines the body’s fat content as a percentage of body weight.

Subjects Ninety two male brick-field workers and sixty sedentary subjects (as control group) of 25 – 45 years of age were selected for the study from different brick-fields of Hooghly district, West Bengal, India. Control subjects were randomly selected from the administrative staff of those brick-fields from which the male brick-field workers were randomly sampled. The ethical committee and managements of the brick-fields gave necessary written permission to conduct the study. All the subjects gave informed written consent to act as volunteers after being thoroughly explained about the experimental protocol to allay their apprehension and ensure maximum co-operation from them. Age of the participants was calculated from the date of birth as recorded in their office register and body mass and body height were measured by a weighing machine fitted with height measuring rod ( Avery India Ltd., India ). Body mass was measured 32

Determination of Hand grip strength Hand grip strength (HGS) was determined by using a hand grip dynamometer (Inco, Ambala, India) to test the maximum voluntary contraction (8). The best of three trials was accepted with three minutes rest in-between (10). Hand grip endurance (HGE) was determined by asking the subject to maintain 1/3 rd of the maximum HGS score for as long as the subject could (8). All the hand grip measurements were taken with the arm straight, i.e.,

BODY COMPOSITION AND HAND GRIP STRENGTH IN MALE BRICK-FIELD WORKERS

Table 2 :

Skinfolds and body composition of brick-field workers and control subjects.

at 00 elbow-angle (8,9). Statistical Analysis Unpaired two tail t-test was performed to compute the level of significance of difference between the mean values obtained in two groups. Pearson’s product moment correlation and linear regression analysis were also adopted to test the relationship between different parameters and to compute regression norms, respectively.

group as well as in the brick-field workers. Significantly lower values (p<0.001) of skinfolds with lesser fat content have been depicted among the brick-field workers, though the total muscle content (LBM) did not show any significant intergroup variation. BMI indicated significant (p<0.001) positive correlation with %fat in both the groups and corresponding prediction norms are presented in figure 2.

Results

Discussion

The physical parameters and hand grip endurance of control subjects and male brick-field workers are presented in Table 1 which depicts statistically insignificant inter-group variation in age, body height and RPI though body mass, BMI, BSA and hand grip endurance were significantly (p<0.001 and p<0.01) different between these two groups. Hand grip strengths measured from early in the morning, to the end of the day’s work in the respective groups are presented in Fig 1. Actually the brick-field workers start working from 5am to avoid heat stress and work up to 5pm in the evening with recess periods from 7am to 9am and 12noon to 2pm. But the office workers, i.e., the control subjects (who are sedentary subjects but occasionally participate in recreational sports) of the study, work from 10am to 5pm with lunch break from 12noon to 1pm. Table 2 shows the skinfolds and different components of body composition in the control

BMI, an accepted index for detection of obesity (4) is within the normal range in all the groups. Observed BMI values indicated that the subjects were non-obese and non-overweight (17) as per the available classification (13). All the skinfolds were significantly higher (p<0.001) in the control group indicating that control subjects have a greater quantity of subcutaneous fat. This is also reflected in the occurrence of significantly higher (p<0.001) value of %fat among control subjects than the brick-field workers. Though there was a significant inter-group difference (p<0.001) in the BMI value, the inter-group variation in RPI (which indicates the linearity of an individual’s stature) was not significant. This finding further indicated that brick-field workers have a lower extent of fat accumulation per unit of their body mass and that is perhaps the cause for existence of significant positive correlation (p<0.001) between BMI and %fat in both the groups (Fig 2). Regression 33

Amit Bandyopadhyay

Figure 1 :

Hand grip strength among brick-field workers and control subjects during different times of day’s work (all the subjects were right-handed)

norms could be computed for prediction of %fat from BMI in the studied population with considerably low values of standard error of estimate (SEE). Though %LBM was significantly higher (p<0.001) among brick-field workers, the absolute value of LBM did not show any inter-group variation Figure 2 :

34

because of significant inter-group difference (p<0.001) in the body mass parameter. Fluctuations in the HGS score has been depicted in both the groups (Fig 1) in day-long recording of hand muscle power by hand grip dynamometer. The brick-field workers have

Regression norms for prediction of % fat from BMI in male brick-field workers and control group

BODY COMPOSITION AND HAND GRIP STRENGTH IN MALE BRICK-FIELD WORKERS

significantly higher values of hand grip strength (HGS) in both the hands through out the day, i.e., during pre-working condition as well as during working schedule (Fig 1). The HGS decreased gradually with working period (either during household work or in the brick-field jobs or office work) but increased after recess periods (7am to 9am and 12noon to 2pm in case of brick-field workers and 12 noon to 1pm in case of control group, i.e., office workers). Not only HGS but also hand grip endurance was also significantly higher (p<0.001) among the workers. This is not surprising because of the extensive use of hand muscles as well as finger muscles for the working activities in brick-field workers. This is probably because of the fact that brick-field workers have higher percentage of LBM; the more the LBM the greater will be the energy output and the endurance capacity (2, 5, 17, 18). The HGS score in both the groups are higher than those reported for Italian males (19), Indian underweight, overweight and normal weight adolescent (8) and adolescent sportspersons of India (8, 9). The existence of a greater percentage of muscularity among these workers than their sedentary counterparts was clearly because of their regular labour that prevented the accumulation of fat in the body. The working habits are equivalent to training schedules and available reports indicate that mild to vigorous training programmmes significantly reduce the fat weight (2, 19 - 21). These observations reflected higher fatness among sedentary individuals than brick-field workers although variation in body linearity was insignificant. Hand grip strength and hand grip endurance were also higher among the workers involved in the brick building jobs. Moreover, the proposed norms for prediction for %fat from BMI will easily provide a first hand impression about the body composition in the studied population.

Corresponding Author : Dr. Amit Bandyopadhyay M.Sc.(Calcutta), Ph.D. (Calcutta), FICN. School of Health Sciences Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia Tel: + 609 7663982 Fax: + 609 7647884 Email: [email protected]

References 1.

Park JE, Park K. Environment and Health. In Hamilton P, ed Text book of Preventive and Social Medicine. 12th Ed. New Delhi, Banarsidas Bhanot, 1989: 357-372.

2.

Bandyopadhyay A, Chatterjee S. Body composition, morphological characteristics and their relationship with cardiorespiratory fitness. Ergonomics SA 2003; 15: 19-27.

3.

Kruger HS, Margetts BM, Vorster HH. Nutrition. Evidence for relatively greater subcutaneous fat deposition in stunted girls in the North West Province, South Africa, as compared with non-stunted girls. South Afr J Sci 2004; 20: 564-569.

4.

Chatterjee S, Chatterjee P, Bandyopadhyay A. Skinfold thickness, body fat percentage and body mass index in obese and non-obese Indian boys. Asia Pac J Clin Nutr 2006; 15: 232-235.

5.

Chatterjee S, Chatterjee P, Bandyopadhyay A. Cardiorespiratory fitness of obese boys. Indian J Physiol & Pharmacol 2005; 49: 353-357.

6.

Gilliam TV, Katch VL, Thorland W, Weltman A. Prevalence of coronary heart disease risk factors in active children, 7-12 years of age. Med Sci Sports 1977; 9: 21-25.

7.

Parizkova J, Bunc V, Sprynarova S, Mackova E, Heller J. Body composition, aerobic capacity, ventilatory threshold, and food intake in different sports. Ann Sports Med 1987; 3: 171-177.

8.

Ravishankar P, Madanmohan, Udupa K, Prakash ES. Correlation between body mass index and blood pressure indices, hand grip strength and hand grip endurance in underweight, normal weight and overweight adolescent. Indian J Physiol and Pharmacol 2005, 49: 455-461.

9.

Chatterjee P, Debnath P, Chatterjee P, Das P. Motor fitness qualities in junior badminton players of Kolkata. Indian J Physiol & Allied Sci 2005; 59: 5257.

Acknowledgements The author is indebted to the brick-field authorities and all the subjects for their tremendous assistance and co-operation for the successful completion of the study.

10. Chien VC, Chai SK, Hai DN, Takaro T, Checkoway H, Keifer M, Son PH, Trunge le V, Barnhart S. Pneumoconiosis among workers in a Vietnamese refractory brick facility. Am J Ind Med 2002, 42: 397-402.

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11. Keverenchkhiladze RG, Saakadze VP, Rekhviashvili VA. Working conditions and health status of women employed in clay brick industry (in subtropical climate). Med Tr Prom Ecol 1993, 11: 16-18.

17. Watanabe K, Nakadomo F, Maeda K. Relationship between body composition and cardiorespiratory fitness in Japanese junior high school boys and girls. Ann Physiol Anthropol 1994; 13: 167-174.

12. DuBois D, DuBois EF. A formula to estimate approximate body surface area if height and weight be known. Arch Int Med 1916; 17: 863.

18. Huttunen NP, Knip M, Paavilainen T. Physical activity and fitness in obese children. Int J Obes 1986; 10: 519525.

13. Meltzer A, Mueller W, Annegers J, Grimes B, Albright D. Weight history and hypertension. J Clin Epidemiol 1988; 41: 867-874.

19. Sayer AA, Syddall HE, Martin HJ, Dennison EM, Roberts HC, and Cooper C. Is grip strength associated with health related quality of life? Finding from Hertfordshire Cohort study. Age Aging 2006; 11: 409415.

14. Sloan AW. Physical fitness and body build of young men and women. Ergonomics 1969; 12: 25-32. 15. Jackson AS, Pollock ML. Generalized equations for predicting body density of men. Br J Nutr 1978; 40: 497-504. 16. Robergs RA, Roberts SO. Fundamental principles of Exercise Physiology for Fitness, Performance and Health Exercise, Performance and Clinical Applications. Mexico City: McGraw-Hill, 2000.

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20. Chatterjee P, Chatterjee S, Mukherjee PS, Bandyopadhyay A. Evaluation and inter-relationship of body mass index, percentage of body fat, skinfolds and girth measurements in boys of 10-16 years. Biomedicine 2002; 22: 9-16. 21. Buskirk E, Taylor HL. Maximal oxygen intake and its relation to body composition, with special reference to chronic physical activity and obesity.J Appl Physiol 1957; 11: 72-78.

Malaysian Journal of Medical Sciences, Vol. 15, No. 1, January 2008 (37-41)

ORIGINAL ARTICLE AN APPROACH FOR ASSESSMENT OF TUMOR VOLUME FROM MAMMOGRAPHY IN LOCALLY ADVANCED BREAST CANCER Gupreet Singh Medical Physics Unit, Department of Radiodiagnosis Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, New Delhi-110029, India.

Tumor size is an important independent indicator in patients with carcinoma of the breast. Repeated size measurements during primary systemic therapy produce detailed information about response that could be used to select the most effective treatment regimen and to estimate the patient’s prognosis. Measurement of tumor burden with ultrasonography and computed tomography is being used with increasing frequency to assess the effectiveness of cytotoxic anticancer drugs. Standardization of assessment and results reporting are important steps that aim at increasing the amount of usable therapeutic information at the physician’s disposal. The purpose of our study is to calculate the tumor volume by mammography after demagnification and compare the tumor volume measured from this method with ultrasonography volumes. Key words : Mammography, tumor volume, magnification, locally advanced breast cancer, prognosis. Submitted-20-02-2007, Accepted-03-12-07

Introduction Breast cancer is reported to be commonest Table 1 :

cancer with an annual age adjusted incidence rate of 22-28 per 100,000 women per year in urban areas and 6 per 100,000 women per year in rural areas.

Comparison of tumor volume measurement by ultrasound (US), Mammagraphy, with vernier calipers and Water Displacement methods

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Gupreet Singh

Table 2 :

Analysis of breast tumor volumes (c.c.) by ultrasound and mammography

Over 75,000 new cases of breast cancer are reported to occur in India per year. Majority of breast cancers in India (50-70%) present with locally advanced disease. In the coming year at current growth it will surpass the cervix cancer [1]. Currently neoadjuvant chemotherapy is a common approach which gives an opportunity to asses the tumor response where the benefits are mostly concentrated in a small number of patients who achieved optimal tumor burden reduction. A change in organ size is a predominant feature of a disease process or a manifestation of pathology elsewhere. Various radiological and clinical techniques have been attempted in the past to estimate tumor or organ volumes, but have been of limited utility due to unacceptable accuracy, poor reproducibility or difficulty in obtaining suitable images for volume measurements [2]-[4]. Quantification of tumor burden with ultrasonography and computed tomography is being used with increasing frequency to assess the effectiveness of cytotoxic anticancer drugs. Standardization of assessment and results reporting is an important step that aims at increasing the amount of usable therapeutic information at the disposal of the physician. Therefore it is necessary to develop a ‘common language’ to describe the results of cancer treatment and to agree upon internationally acceptable general principles for Figure 1 :

38

reporting and assessing data [5]-[7]. Since 1979, World Health Organization (WHO) used the radiological tumor response evaluation criteria. Whereby, the tumor is measured in two dimensions its maximum diameter (width) in the translation plane and its largest perpendicular diameter on same image (thickness) [8]. The purpose of our study is to potentially calculate the tumor volume by mammography after demagnifying the images and compare the tumor volume measured from this method with ultrasonography volumes. Measuring a mass is a tedious task for most radiologists. For the three dimensional measurement approaches, the criteria for partial treatment response is defined as greater than 65% reduction in tumor volume, stable disease being the size between that for partial response and that for progressive disease and disease progression is greater than 44% increase in volume [8]. The criterion for complete response was total tumor disappearance. Tumor size is an important independent indicator of prognosis in the patients with carcinoma of breast. Repeated size measurements during primary systemic therapy produce detailed information about response that can be used to select the most effective treatment regimen and to better estimate patient’s prognosis.

Materials and Methods

Plot of volume determined by observer 1 and observer 2 in Mammography (n=25)

AN APPROACH FOR ASSESSMENT OF TUMOR VOLUME FROM MAMMOGRAPHY IN LOCALLY ADVANCED BREAST CANCER

Figure 2 :

Plot of volume calculated by mammography (Mean of volume measured by observer 1 and observer 2) and ultrasound (n=25)

The oncology data base was reviewed for all female patients with biopsy proven breast carcinomas that were treated with standard chemotherapeutic protocols. Inclusion criteria required that the mass lesion should be clearly visible with that of mammography and on ultrasound. Delineation of tumor sizes from the mammograms as well as from the ultrasound was performed by senior radiologists. Tumors ranged from the clearly marginated to poorly marginated. Those that were judged by senior radiologists as not being measurable lesions were not included in the study. The sizes were measured with mammography and ultrasound. Tumor volumes were calculated using the formula of an ellipsoid. In mammography two views were used to measure tumor diameter which were in the craniocaudal view and the mediolateral view. In ultrasonography a 5 MHz linear array probe was used. The transducer was placed in transverse direction on the breast tumor, the width and depth are measured and then the transducer was placed in longitudinal direction which gives the another dimension of the breast tumor. The three greatest dimensions (referred to as a, b and c) were then used to calculate several estimates of tumor volume using the equations for the volume of a sphere ([4π/3]r3), Here r is average of a, b and c the volume of an ellipsoid V= (abcπ)/6. Where a and b are the maximum dimensions of tumor in cm from the craniocaudal view as well as mediolateral view respectively and c was maximum dimension measured from the mediolateral view. Spherical volume calculations were based on the average radius across all three axes. The comparisons were done among the volumes measured by mammography and ultrasound. The percentage variations between the two modalities were calculated. Demagnifying volume was obtained by dividing each dimension of mammography image

by magnification factor. The volume measurement method by vernier calipers, ultrasound (US) and mammography method calibrated using tumor mimicking substances (like Potato, Pumpkin). These objects dipped in water were subjected to volume estimation by the above methods. Standard was taken as volume estimated by water displacement method.

Result Figure 1 show the tumor volume determined by observer 1 plotted against the volume measured by observer 2. Regressing on linear scale shows the high correlation (r = 0.99) with the slope of 1.018. Figure 2 shows the tumor volume calculated by mammography (Mean volume measured by observer 1 and observer 2) plotted against the volume determined by ultrasonography. In this plot there is high correlation of tumor volumes (r = 0.99) and slope of 0.99. Estimates based on volume equations of an ellipse, a sphere of mammography differed from the ultrasound volume by a mean of -33.58% (95% confidence interval (CI) from -22.78 to 22.11), 40.68% (95% CI from -22.85 to 22.04) respectively.

Discussion Pharmaceutical companies have developed an approval process for anticancer drugs on the basis of tumor shrinkage. Volumetric measurements would overcome difficulty estimating the size of lesions that are irregular in shapes and sizes. The tumor volumes measured in breast cancer cases by US may be taken as standard since calibration experiment (Table I) showed that US is very accurate in measuring the volume of an irregular shaped

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Gupreet Singh

subject (less than 3% error). The percentage difference between ultrasound and mammography volumes decreases after demagnification as shown in Table III. So mammography can also be used for accurate measurement of the volume and can help the radiologists screen the breast cancer patients used with volume. The maximum tumor volume was 88.71 cm3 and minimum tumor volume was 2.84 cm3. According to response evaluation criteria in breast tumors 4% patients shows the complete response, 25% shows the partial response, 63 % patients the stable disease and 8% shows the disease progression after chemotherapy in the present study. Volumetric measurement can help significantly for evaluating therapeutic response compared to unidimensional and bidimensional measurements. The ability to accurately calculate the in vivo volume of tumors also has clinical potential especially in oncology. Tumor volumes could be used to assess the progression of disease and success of therapy, such as response to irradiation or chemotherapy. Tumor volume using ellipsoid formula is preferred over other cubical and spherical methods. As most of the tumors are of irregular shapes so spherical formulae are not used and the cubical formulae overestimate the volume. For treatment evaluation stress is on reproducibility and accuracy of serial measurements. The rule for obtaining bidimensional measurement of a tumor is to find its longest diameter (length) and the dimension which is perpendicular to length. This method provides a fixed set of values that are not altered so producing reproducibility as right angle law is followed. Here is the first study to calculate the tumor volume using mammograms. However, manual measurement of tumor volume is time consuming and estimates based on simple geometric shapes are inaccurate. The errors, whenever they occur, are due to error in marking the tumor on the mammography film, which become significant in case of speculated, vague and diffused tumor mass instead of solid and well defined one. In routine reporting of regression or progression of tumor from the imaging film radiologists either use scale or try to infer the tumor size by visual assessment of the irregular tumor part appearing in the images. In such a situation a change of tumor sizes up to 20% may allude by visual perception [9-11]. A careful statistical analysis of the measurements of 16 experienced oncologists showed the false positive response rate in unchanged masses as measured by same investigator at 25% when 25% regression criteria (product of two diameters) was employed

40

[12]. Even volumes by diameters measurement by calipers are reported to have large errors and measured the three mutually perpendicular diameters in mouse tumors by calipers and calculated tumor volume by the product of these diameters [13]. Tumors were excised and volumes were measured by Archimedes’s principle. Their correlation showed an error of 50% on mean diameter measurement at a given volume. Area (product of two perpendicular diameters), volume and diameter of tumor mass are frequently used in practice to indicate the size, growth or regression.

CONCLUSION Tumor volume is a more representative quantity of three dimensional tumor mass which is generally of irregular shape and size. Thus, it is desirable to use mammography based tumor volume as response indicator which is not only accurate but better representative of tumor mass. This approach of assessment of tumor volume can be used for development of mammography computed tomography 3-D scans.

Corresponding Author : Dr. Pratik Kumar (Ph.D) Medical Physics Unit, Department of Radiodiagnosis Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, New Delhi-110029, India. Tel: + 01126594448 Fax: + 91-11-26588663 Email: [email protected]

References 1.

Chopra R. The Indian scene Journal of clinical oncology 2001; 15(suppl 8): 106-111.

2.

Richard S, Breiman, John W. Beck, Melvyn Korobkin et al. Volume determinations using computed tomography AJR 1982; 138: 329-333.

3.

Koga T, Morikawa Y. Ultrasonographic determination of the splenic size and its clinical usefulness in various liver diseases Radiology 1975; 115: 157-161.

4.

Riemenschneider RA, Ehalen JA. The relative accuracy of estimation of enlargement of the liver and spleen by radiologic and clinical methods AJR 1965; 94: 462-468.

5.

A.B. Miller, B. Hoogstraten et al. reporting results of cancer treatment Cancer 1981; 47: 207-214.

AN APPROACH FOR ASSESSMENT OF TUMOR VOLUME FROM MAMMOGRAPHY IN LOCALLY ADVANCED BREAST CANCER

6.

Hayward Jl, Carbone PP, Heuson JC et al. Assessment of response to therapy in advanced breast cancer Cancer 1977; 39: 1289-1294.

7.

Karnofsky DA, Abelman WH et al. The use of nitrogen mustards in the palliative treatment of carcinoma Cancer 1948; 1: 634-656.

8.

9.

Therasse P, Arbuk SG, Eisenhauer EA, et al. New guidelines to evaluate response to treatment in solid tumors J Natl Cancer Inst 2000; 92: 205-16. Moertel CG, Hanley JA. The effect of measuring error on the results of therapeutic trials in advanced cancer. Cancer 1976; 38: 388-394.

11. Kumar P, Rehani MM, Anand, Raina V, Rao K. Assessment of lymphnode tumor mass from CT scans: How good is diameter and visual assessment J Med Phy 1995; 3: 20-24. 12. Watson JV. The cell proliferation kinetics of the EMT 6/M/AC mouse tumors at four volumes during unperturbed growth in vivo Cell Tissue Kinetic 1976, 9: 147-154. 13. Spears CP. Volume doubling measurement of spherical and ellipsoidal tumors Med Pedai Oncol 1984; 4: 212220.

10. Aydin H, Richter E, Feyerbend T, Bohndorf W. In vivo determination of tumor volume: what does a CT calculation mean in recurrences of rectal carcinoma Strahlenther onkol 1990; 3: 204-210.

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Malaysian Journal of Medical Sciences, Vol. 15, No. 1, January 2008 (42-43)

CASE REPORT LASER POSTERIOR CORDECTOMY FOR BILATERAL ABDUCTOR VOCAL CORD PALSY : A CASE REPORT Irfan Mohamad, Wan Shah Jihan, Hazama Mohamad, Baharuddin Abdullah Department of Otorhinolaryngology-Head & Neck Surgery School of Medical Sciences, Universiti Sains Malaysia, Health Campus 16150 Kubang Kerian, Kelantan, Malaysia Bilateral abductor vocal cord palsy is comparatively a rare vocal cord lesion, especially in a patient with no history of neck mass, previous surgery or trauma. Many patients are not stridulous. A patient presenting with stridor may need emergency airway management before the other treatment is commenced. We report a case of bilateral abductor palsy which required an emergency tracheostomy and subsequently a laser posterior cordectomy. Key words : Bilateral abductor palsy, posterior cordectomy, laser Submitted-20-02-2005, Accepted-20-03-07

Case Summary A 63 year old Malay female presented with history of increasing stridor for the past six months. Initially she described the noisy breathing only occurred during sleep, started about one year ago. Occasionally, her breathing stopped during sleep. She also complained of reduced effort tolerance and sometimes wheezing. There was no history of loss of weight or reduced appetite. No history of dysphagia, neck mass, previous surgery or trauma to the neck region documented. On examination, she was tachypnoeic but not cyanosed. There was a biphasic stridor. No neck mass was palpable. On 70o laryngoscopy, both vocal cords were in median position, leaving only a small gap at the posterior commisure. There was no other mass seen. Investigations were unremarkable. Chest xray was normal and computed tomography (CT scan) revealed a normal brain study and a small left solitary thyroid nodule. In view of the worsening stridor, she was subjected to emergency tracheostomy under local anaesthesia. Few days later, laser posterior cordectomy was performed to the left vocal cord. The patient was doing well post-operatively and the tracheostomy was decannulated few weeks after the procedure. She was able to talk with

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reasonable voice and no episodes of aspiration documented.

Discussion A paralyzed cord, especially the bilateral vocal cords involvement will compromise the airway. It also increases the risk of aspiration pneumonia. Unilateral palsy usually get compensated from the opposite cord, where as the bilateral non-functioning cords, depending on the final site they were rested will determine whether the remaining airway is too wide (such as in uncompensated adductor palsy) that lead to aspiration or too narrow (abductor palsy) that will compromise the respiratory tract. Bilateral vocal cord palsy can present as acute emergency condition to otolaryngologist. Although majority of the bilateral abductor palsy patients were non-stridorous, a superimposed upper respiratory tract infection can lead to a life-threatening condition. An oedema to the paralyzed cord will very much compromise the airway. Our patient was having this noisy breathing for the past six months, and she can live with the symptom quite comfortably except a slight reduced in effort tolerance. The worsening of symptom was only noted few days prior to admission. As there was no room for an oral intubation, tracheostomy needs to be performed. In some centers

LASER POSTERIOR CORDECTOMY FOR BILATERAL ABDUCTOR VOCAL CORD PALSY : A CASE REPORT

where there was no laser equipment, permanent tracheostomy is the best treatment that can be offered. However, the patient with permanent tracheostomy tube needs to be well educated on how to manage the tube at home which includes regular cleaning, humidification, tube change, suction and others. The other disadvantage is the patient have to take extra effort to enable him to talk (to occlude the tube externally or use a speaking valve). Considering all this factors, and with the laser equipment and well trained expertise available, a laser assisted posterior cordectomy was performed. The tracheostomy tube was first changed to the laserresistance metal tube. The posterior cordectomy procedure was first proposed by Kashima and Dennis in 1989. With time, this procedure has become the treatment of choice because complications are rare, effective and easily repeatable in case of recurrence (1). In this procedure, the posterior third of a vocal cord, in this patient, the left side was ablated with 20 watt Sharplan laser. By using the micro manipulator, the targeted part was ablated with carbon dioxide laser until a desired size of airway created. Care should be taken to avoid over ablation that will lead to a wide space inducing aspiration. That was also the reason why some surgeon advocated doing only a unilateral cordectomy in each sitting, and not a simultaneous bilateral procedure although some centers proved it to be safe. For example, in a series of 22 bilateral vocal cord palsy patients who were treated with simultaneous bilateral posterior cordectomy in Egypt (Khlifa, 2005), the success rate was up to 92 % with good airway and voice quality. Problems related to deglutition, aspiration, or granuloma formation were not reported (2). In a well trained hand, the minimal risk of unnecessary burn or ablation to other undesired part can be avoided. The advantage of this laser procedure is that it will not bleed and thus reduce the risk of blood aspiration into the lung, intra or post operatively. It is because the mechanism of this destructive procedure is by tissue ablation unlike cutting with cold instruments. The disadvantages were rarely observed and reported. When compared with other techniques, the advantages offered by the posterior cordectomy included rapidity and simplicity in concept, reliability of outcome, short hospitalization, low risk of complications, and the possibility for revision when necessary (3). With a course of steroid cover postoperatively, the patient was managed well in the ORL general ward. She was decannulated from

tracheostomy the next week. The procedure can be repeated to the opposite vocal cord if the space created was found to be inadequate. Although it is a rare condition, the otolaryngolgists in centers equipped with laser facilities should be able to offer the best treatment to the patient. Laser posterior cordectomy has been widely performed in Western countries. This case demonstrates to us the ability to perform such procedures in local setting, Hospital Universiti Sains Malaysia.

Corresponding Author : Dr. Irfan Mohamad (MD USM) Department of Otorhinolaryngology-Head & Neck Surgery, School of Medical Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia Tel: + 609 -766 4498 Fax: + 609- 765 3370 Email: [email protected]

References 1.

Joel A Ernster. Vocal Fold Paralysis, Bilateral. eMedicine. Updated March 2006.

2.

Khlifa M C. Simultaneous bilateral posterior cordectomy in bilateral vocal fold paralysis . Otolaryngol Head Neck Surg. 2005; 132(2): 249-50.

3.

Segas J, Stavroulakis P, Manolopoulos L, Adamopoulus G. Management of bilateral vocal fold paralysis: experience at the University of Athens. Otolaryngol Head Neck Surg. 2001; 124(1): 68-71.

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Malaysian Journal of Medical Sciences, Vol. 15, No. 1, January 2008 (44-48)

CASE REPORT A RARE CASE OF PAEDIATRIC PONTINE GLIOBLASTOMA PRESENTING AS A CEREBELLOPONTINE ANGLE OTOGENIC ABSCESS Kantha Rasalingam, Jafri Malin Abdullah, Zamzuri Idris, Hillol Kanti Pal, Nasser Wahab, Effat Omar* and Salma@Win Mar** Department of Neurosciences, *Department of Pathology, **Department of Radiology School of Medical Sciences, Universiti Sains Malaysia, Health Campus 16150 Kubang Kerian, Kelantan, Malaysia

We describe rare case of a 9-year old boy who presented with a two- week history of right ear discharge and mild fever. Contrast enhanced CT scan of the brain showed a lesion in the right cerebellopontine angle with mild enhancement mimicking early abscess formation. Involvement of the mastoid air cells pointing towards a radiological diagnosis of mastoiditis reinforced the diagnosis of an abscess. A magnetic resonance imaging (MRI) was planned for the patient but his conscious level deteriorated and patient slipped into coma warranting immediate surgical intervention. Intraoperatively, about 90% of the tumour was removed and the appearance of the tumour resembled that of an acoustic schwannoma but histopathology confirmed the diagnosis of a glioblastoma multiforme (GBM). MRI done post operatively showed lesion in the pons confirming the diagnosis of an exophytic pontine glioblastoma multiforme. Key words : Maxillary molar abscess, cavernous sinus thrombosis, superior ophthalmic vein thrombosis. Submitted-20-02-2005, Accepted-03-12-07

Introduction Paediatric high-grade gliomas include a heterogeneous group of tumors with different sites of origin and histologic aspects, and they affect children of different ages. These tumors can originate from any site in the central nervous system (CNS), particularly the supratentorial region and the brainstem. They rarely originate from the spinal cord or the cerebellum.(1) When arising from the brainstem, these high-grade gliomas are usually called diffuse brainstem gliomas. Histologically, most of these neoplasms contain only an astrocytic component and are called either anaplastic astrocytoma (AA; World Health Organization [WHO] grade III) or glioblastoma multiforme (GBM; WHO grade IV), depending on their grade. Tumors with mixed or nonastrocytic phenotypes are uncommon in pediatric patients. The number of children with high-grade gliomas is much smaller than the number of adults

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with these neoplasms. Whereas 40%–50% of all paediatric CNS tumours are gliomas, supratentorial high-grade astrocytomas constitute only 6%–12% of all primary paediatric brain tumors, and diffuse brainstem gliomas constitute only 3%–9%. (1, 2) Brainstem tumours are perhaps the most dreaded of cancers in children due to its poor prognosis, yet they remain an area of intense research. Brainstem gliomas account for about 10% of all brain and spinal childhood tumours.(1,3) Peak incidence for this tumour occurs around age 6 to 9 years. The term glioma encompasses various tumour types such as ganglioglioma, pilocytic astrocytoma, fibrillary astrocytoma, anaplastic astrocytoma and glioblastoma multiforme.(1) They have historically been some of the most difficult paediatric cancers to treat.

Case Report We present a 9-year-old boy who was referred

A RARE CASE OF PAEDIATRIC PONTINE GLIOBLASTOMA PRESENTING AS A CEREBELLOPONTINE ANGLE OTOGENIC ABSCESS

Figure 1 :

CECT of the brain showing a lesion of mixed density in the region of the right Cerebello pontine angle

to our centre after complaining of double vision and ataxia of 2 weeks duration. He complained of severe headache 48 hours prior to admission which was retro-auricular in location associated with a yellowish discharge from the right ear, and fever . On examination in our centre, he was drowsy but arousable. Fundoscopy revealed bilateral papilloedema. Further examination showed a right 6th nerve palsy with horizontal nystagmus and mild cerebellar signs localized to the right. Otoscopic examination revealed pus in the right external external auditory canal. A CT scan of the brain ( Figure 1) was performed showed an ill defined Figure 2 :

isodense lesion in the right cerebellopontine angle measuring 2.4 x 2.3 x 3.8cm with moderate enhancement and acute obstructive hydrocephalus. Fluid collection was noted in the right mastoid air cells and middle ear cavity. The impression obtained at this stage with the clinical evidence of mastoiditis and ear discharge was that of a cerebral abscess. The total white cell count was not raised and there was no clinical evidence of bacteraemia. The patient was immediately planned for a MRI on the same day of admission, but while waiting for it his conscious level deteriorated and emergency surgery was undertaken. The aetiology

Pathological slide of the patient showings areas of necrosis and nuclear pleomorphism.

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Kantha Rasalingam, Jafri Malin Abdullah et. al

Figure 3 :

Axial T1 weighted MRI shows diffuse expansion of the pons measuring 3.2cm x 2.6 cm x 3.4cm.

for the deterioration was both due to his hydrocephalus and the compression of the brain stem by the lesion. A right retromastoid sub occipital craniectomy was performed immediately. A right parietal burr hole was made and ventriculostomy gave clear cerebrospinal fluid (CSF) under high pressure.Transdural aspiration from the suspected right cerebellar lesion revealed no pus. Durotomy was performed which did not relieve the tense right cerebellum. The removal of the lateral one third of the right cerebellar hemisphere had to be done due to herniation which did not response to medical andtreatment. It revealed a right cerebellopontine angle mass which was huge and enveloped by a layer of arachnoid mater. The tumour was reddish grey, noncapsulated, firm, solid with a central area of necrosis containing 3-4 ml of dirty greyish fluid. The right lower cranial nerves were protected along with the VIIth nerve and VIIIth nerve. Intraoperatively it seemed to be arising from the extracanicular portion of the vestibular nerve. The posterior lip of the porus acousticus was intact. There were no hemodynamic changes during surgery. Subtotal tumor removal was achieved with a small remnant of the tumor attached to the pons left in situ. Pathological Examination Fragments of tissue were composed of sheets of neoplastic glial cells with numerous small capillaries in between them.(Figure 2) The tumor was cellular with some pleomorphism and some cells 46

undergoing mitosis were noted. Endothelial proliferation and areas of necrosis were noted. The tumor cells stained positive for glial fibilary acid protein (GFAP). The tumor also expressed vementin. In conclusion, the histological diagnosis was that of a glioblastoma multiforme. Post operative progress Post operatively the patient was kept on ventilatory support for 24 hours. Once conscious, he was able to move all his limbs. He had a mild right sided 7th lower motor neuron palsy and a right sixth nerve palsy with associated nystagmus and mild right sided cerebellar signs. In view of the histopathology of glioblastoma multiforme, a brain MRI was ordered and the findings are consistent with that of a diffuse pontine glioma. (Figure 3). The patient was managed in the ward with a hospital acquired infection. Two months after surgery the patient was taken home by the parents against doctor’s advice and died on the same day.

Discussion This case is presented as there was a discrepancy in the clinico-radiological diagnosis with that of the operative and histopatholigical diagnosis. We are presenting this case because of its subacute presentation (with a history of less than 2 weeks) mimicking that of an otogenic cerebellar abscess with obstructive hydrocephalus. The radiological investigation available on admission (

A RARE CASE OF PAEDIATRIC PONTINE GLIOBLASTOMA PRESENTING AS A CEREBELLOPONTINE ANGLE OTOGENIC ABSCESS

plain and CECT scan brain) suggested a rapidly advancing lesion in the right side in the posterior fossa. The peripheral vein enhancement suggested an inflammatory pathology. At surgery however, a solid mass with a nectrotic central area encased by arachnoid layer in close proximity and anterior to the VII th and VIII th nerves suggested an acoustic neuroma. The site of origin of this tumour could be the cerebellum, root entry zone or the brainstem. Post operative MRI however confirmed the diagnosis to be that of a diffuse brainstem glioma. The histopathological diagnosis of this patient was that of a glioblastoma multiforme. With this diagnosis, it was necessary to determine the site of origin of the tumor which could be localized to 3 main regions mainly the pons, the cerebellum or the nerve root entry zone. MRI done post operatively however established the site of origin to be pons. As the tumor originated from the pons and majority of the tumour appeared to be residing outside of the pons the diagnosis of this tumor is a exophytic pontine glioma. In 1985 Epstein et al reported classification schemes based on CT appearance of brainstem tumors.(5) These classification schemes described surgically treatable subgroups of gliomas. A simple way to classify these tumours is into two categories: typical brain stem gliomas and atypical brain stem gliomas. They are now recognized as a heterogeneous group of tumors and have been broadly classified by Epstein in four categories: diffuse, focal, exophytic and cervicomedullary Exophytic brainstem gliomas arise from the subependymal glial tissues and expand outside the pons. However, the area of extension is mainly into the fourth ventricle unlike our patient where the area of extension was mainly the cerebellopontine angle. The clinical presentation of our patient was rather subacute with minimal cranial nerve involvement unlike that presentation of an exophytic glioma. The pathology of exophytic brainstem tumours are mainly that of a low grade glioma however as established our histopathological diagnosis was that of a glioblastoma multiforme. Unlike the majority of brainstem gliomas which are malignant tumors, most exophytic brainstem gliomas are low grade and this alters the management for the patient. Surgery is only potentially beneficial for patients with focal, exophytic and cervicomedullary tumors and not diffuse brainstem tumors. The goal of surgery should be to reduce the tumour size and not aim for complete resection as an attempt to do so might

prove catastrophic. The diagnosis of a malignant glioma provided a poor prognosis as GBM carries a median survival rate of less than 12 months. The other possible origin for this tumour could be the nerve root entry zone or the cerebellum. With the exception of Neurofibromatosis type II, paediatric extraparenchymal cerebellopontine angle (CPA) tumors is very rare.(4) Most gliomas encountered in the cerebellopontine angle involve the CPA angle as exophytic extension of primary brainstem and/or cerebellar tumors. A CPA glioma is exteremly rare and so rare there have only been 7 cases reported. The rarity of this location and with the MRI findings however, we have ruled this out as a possible site or origin. In conclusion, brainstem gliomas are a common but difficult tumour to treat. From this patient’s clinical presentation, radiological and pathological findings, we can conclude that they need not follow the normal pattern as described by previous literature. Nevertheless, all brainstem gliomas need to be classified carefully as their management will depend on its location. With the advancement of gene therapy, it is hoped that futher development in the management of brainstem gliomas is achieved.

Corresponding Author : Dr. Kantha Rasalingam MBBS, Master Surg. (Neurosurgery) USM Department of Neurosciences, School of Medical Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia Tel: + 609 -766 4240 Fax: + 609- 764 8613 Email: [email protected]

References 1.

John Ragheb,MD:Fred J.Epstein,MD. The surgical Classification and management of Brainstem Tumors in Children: International Pediatrics 2000; Vol. 15/No. 1: 15.

2.

Alberto Broniscer, Amar Gajjar .Supratentorial High Grade Astrocytoma and Diffuse Brainstem Glioma: Two challenges for the Pediatric oncologist: The oncologist, April 2004; Vol 9-No.2: 197-206,

3.

David A Walker, Jonathan A G Punt, Michael Sokal. Clinical management of brain stem gliomas: Archives of Disease in Childhood: Jun 1999: 80,6

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Kantha Rasalingam, Jafri Malin Abdullah et. al

4.

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Arnautovic, Husain MM, Linskey ME. Cranial nerve root entry primary cerebellopontine angle gliomas a rare and poorly recognized set of extre parenchymal tumors. J Neurooncol 2000; 49: 205-12.

5.

Epstein, F. (1985). A staging system for brain stem glioma. Cancer 56 (7 Suppl): 1804-6.

Malaysian Journal of Medical Sciences, Vol. 15, No. 1, January 2008 (49-51)

SHORT COMMUNICATION CARDIOPULMONARY RESUSCITATION : THE SHORT COMINGS IN MALAYSIA Chew Keng Sheng, Mohd Idzwan Zakaria, Nik Hisamuddin Nik Abdul Rahman, Kamaruddin Jaalam*, Wan Aasim Wan Adnan* Department of Emergency Medicine, * Department of Anesthesiology, School of Medical Sciences, Universiti Sains Malaysia, Health Campus 16150 Kubang Kerian, Kelantan, Malaysia This short review explores the current status of cardiopulmonary resuscitation in Malaysia and highlights some of the factors that have a negative impact on its rate of success. Absence of a unifying body such as a national resuscitation council results in non-uniformity in the practice and teaching of cardiopulmonary resuscitation. In the out-of-hospital setting, there is the lack of basic skills and knowledge in performing bystander cardiopulmonary resuscitation as well as using an automated external defibrillator among the Malaysian public. The ambulance response time is also a significant negative factor. In the in-hospital setting, often times, resuscitation is first attended by junior doctors or nurses lacking in the skill and experience needed. Resuscitation trolleys were often inadequately equipped. Key words : Cardiopulmonary resuscitation, defibrillation, chain of survival, out-of-hospital CPR, in-hospital CPR, Malaysia Submitted-20-02-2005, Accepted-03-12-07

Modern cardiopulmonary resuscitation (CPR) is, without a shadow of doubt, closely linked with the name of Peter Safar (1924-2003). The enormity of his contributions since the 1950s, including his landmark paper of mouth-to-mouth ventilation (1), earned him titles such as “Father of Modern Resuscitation” (2) and “Father of CPR” (3, 4). About the same time (around 1960s), Kouwenhowen, together with his colleagues Knickerbocker and Jude, were experimenting with defibrillation and rediscovered the efficacy of external chest compression to produce a passable circulation, first in canine models, then in humans (1, 2). Ever since then, the technique of resuscitation is progressively improving. The American Heart Association (AHA) has since made various recommendations on CPR and emergency cardiovascular care (ECC) and the European Resuscitation Council (ERC) has also produced its own resuscitation guidelines. In many countries like the United Kingdom, Australia, New Zealand, Malta, Southern Africa and Serbia and Montenegro, there are respective national resuscitation councils that serve as an integration body to coordinate the teaching and practice of resuscitation. Unfortunately

at the present moment, there is no such resuscitation council in Malaysia to serve as a unifying body to promote the excellence and consistency of resuscitation teaching and practice for the whole of Malaysia. The lack of a national resuscitation council in Malaysia also results in a great variation of syllabus and teaching contents of basic life support for the public and advanced life support for trained healthcare providers. This great variation is due to the fact that there are various agencies in Malaysia that are conducting basic life support courses such as Hospital Universiti Sains Malaysia, the Malaysian Association of Emergency Medicine (MAEM), National Heart Institute, Hospital Universiti Kebangsaan Malaysia, Hospital Kuala Lumpur, the Malaysian Society of Trauma and Emergency Medicine (MASTEM), the St. John Ambulance Malaysia and the Red Crescent Society Malaysia (5). This is compounded by the fact that there are agencies which are using outdated guidelines and not the guidelines produced in 2005. Cardiac arrests can be divided into out-ofhospital cardiac arrests or in-hospital cardiac arrests (6). This distinction is important because the chance 49

Chew Keng Sheng, Mohd Idzwan Zakaria et. al

of survival of out-of-hospital cardiac arrest is dependent on the prompt initiation of sequentially linked actions known as the chain of survival (7). The links in this chain are early recognition of signs of cardiac arrest, early activation of emergency medical services (EMS), early initiation of basic cardiopulmonary resuscitation, early defibrillation and early initiation of advanced cardiac life support (7). The first three chains in an out-ofhospital setting depend very much on the initiation of bystanders to call and activate the emergency medical services, to start CPR while awaiting the arrival of the ambulance and to be able to competently use the automated external defibrillator (AED) to analyse and start defibrillation if necessary. Every chain is important and weakness in any link would lessen the chance of survival of out-ofhospital cardiac arrests (7). Early access to the EMS is extremely important, more so in a case of suspected acute coronary event or stroke. Unfortunately until recently, there was no universal access number for the Malaysian public to activate the EMS. Previously Civil Defence Department Malaysia had ‘991’ as their access number; the Fire and Rescue Department had ‘994’, but the Emergency Medical Services in Malaysia did not have a universal access number. Different healthcare agencies were using different communication system resulting in the lack of inter-agencies communication and at times, overlapping of resources with more resources available at the site of incident than was actually needed. Realising these nagging problems, the government has recently re-introduced a single emergency number for the entire nation for all types of emergencies regardless of whether it is health related or non-health related. This project is called “One Nation, One Number” (8). From July 2007 onwards, Malaysian public need only to call the number 999. This single ‘999’ entry point will be answered by a trained telephone operator, who will direct the call to either the fire and rescue, the police or the nearest appropriate hospital depending on the type of emergencies. Nevertheless, how effective this new system will be is yet to be seen at this time. Furthermore, the emergency access number in Malaysia has always been plagued with prank calls in up to 98-99% of all calls (8, 9). These prank calls have contributed adversely to wastage of resources as well as hindering the speedy and effective delivery of EMS to those who are in true medical emergency conditions.

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Besides that, the Malaysian public too, are not well educated in communicating essential and correct information to the call centre operator. Often, the exact locations of the incidents are not easily found. In addition, there are various problems in the area of pre-hospital care in Malaysia that hamper the delivery of rapid and effective emergency medical services to a cardiac arrest victim. The mean ambulance response time in Malaysia, varies from places to places; in Kota Bharu, for example it is about 15.2 minutes and in Kuala Lumpur, it is about 21.1 minutes (10). The mean ambulance response time is the time taken from the time of call received to the time of arrival at the scene (10). In both examples quoted above, however, it is far longer than the critical first five minutes where the chance of survival of a cardiac arrest victim is the greatest (11). The long ambulance response time, besides due to the problem of poor communication, is also compounded by problems of traffic congestion on major roads in big cities in Malaysia (10). In other words, the chance of survival of a cardiac arrest victim during these critical first few minutes before the arrival of ambulance depends very much on the proficiency of the Malaysian public to perform bystander CPR. The Malaysian public needs to be taught the importance of prompt, proper bystander CPR and the importance of placing AEDs in strategic places as well as proper use of the AEDs. This is illustrated in the tragic death of the late Malaysian actor Hani Mohsin, the host of a popular television game show, who complained of sudden chest pain while waiting for his flight at the Low Cost Carrier Terminal before he collapsed (12). This was a typical example where effective CPR and the use of AED could have been extremely vital. He was rushed to a nearby hospital where he was pronounced dead. The better way should be to rush the “hospital” to him, namely to rush the AED and the resuscitation bags with the essential drugs and airway equipments to the scene. As for in-hospital cardiac arrest, there are still not many studies done in Malaysia. Suffice to say, the majority of the cardiac arrest cases that occur in the wards would first be attended by the house officers or the junior medical officers. These junior doctors often lack the experience and skill needed in resuscitation. They would then call the senior doctors or specialists for help. Often the time taken by these senior doctors to arrive is substantially longer than the crucial initial first few minutes for the patient’s survival.

CARDIOPULMONARY RESUSCITATION : THE SHORT COMINGS IN MALAYSIA

A study done in 1997 to look into the outcomes of CPR performed in six Malaysian district hospitals found that up to almost 60% of cases were inadequately resuscitated (14). Many reasons were cited, including staff nurses who failed to initiate chest compression and to provide positive pressure ventilation through bag-valve-mask, inadequate duration of resuscitation as well as incomplete resuscitation trolleys (14). In short, much is needed to be done in the area of cardiopulmonary resuscitation in Malaysia. Firstly, there is a need for a coordination body, much like a national resuscitation council that can serve as a platform for discussion and training of the various governmental and non-governmental agencies involved in this area. The public needs to be educated regarding the importance and the skill to perform timely and effective CPR as well as the use of an AED. The public needs to be educated regarding the proper ways of conveying vital information when calling for ambulance. There is also a need to educate the public regarding the proper use of the emergency numbers and the complications of playing prank calls. Lastly, there is a great need to update the knowledge and skills of staff nurses and junior doctors in performing CPR.

5.

Lee, H.T. and Low, B.T. Education of the public on cardio-pulmonary resuscitation: issues and challenges. Med J Malaysia 1999; 54(2): 167-8.

6.

Jacobs, I., Nadkarni, V., Bahr, J., Berg, R.A., Billi, J.E., Bossaert, L., Cassan, P., Coovadia, A., D’este, K., Finn, J., Halperin, H., Handley, A., Herlitz, J., Hickey, R., Idris, A., Kloeck, W., Larkin, G.L., Mancini, M.E., Mason, P., Mears, G., Monsieurs, K., Montgomery, W., Morley, P., Nichol, G., Nolan, J., Okada, K., Perlman, J., Shuster, M., Steen, P. A., Sterz, F., Tibballs, J., Timerman, S., Truitt, T. & Zideman, D. Cardiac arrest and cardiopulmonary resuscitation outcome reports: update and simplification of the Utstein templates for resuscitation registries. A statement for healthcare professionals from a task force of the international liaison committee on resuscitation (American Heart Association, European Resuscitation Council, Australian Resuscitation Council, New Zealand Resuscitation Council, Heart and Stroke Foundation of Canada, InterAmerican Heart Foundation, Resuscitation Council of Southern Africa). Resuscitation 2004; 63: 233-249.

7.

Cummins, R.O., Ornato, J.P., Thies, W.H. & Pepe, P.E. Improving survival from sudden cardiac arrest: the „chain of survival“ concept. A statement for health professionals from the Advanced Cardiac Life Support Subcommittee and the Emergency Cardiac Care Committee, American Heart Association. Circulation 1991; 83(5): 1832-47.

8.

Yeoh, C. 999: One Nation, One Number. In: The Star. 2007 October 25.

9.

Rajah, D. Dial 999 for all emergencies. In: The Star. 2007 October 1.

Corresponding Author : Dr. Chew Keng Sheng MD (USM), MMed (USM) Lecturer/Emergency Physician, Department of Emergency Medicine, School of Medical Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia Tel: + 609 -766 3204 Fax: + 609- 765 9360 Email: [email protected]

References 1.

Safar P. Ventilatory efficacy of mouth-to-mouth artificial respiration; airway obstruction during manual and mouth-to-mouth artificial respiration. J Am Med Assoc 1958; 167: 335-41.

2.

Baskett PJF. Obituary Peter J. Safar. Resuscitation 2003; 59(1): 3-5.

3.

Oransky I. Obituary Peter Safar. The Lancet 2003; 362 (9385): 749.

4.

Mosesso VNJ, Paris PM. In Memoriam A tribute to Peter Safar, MD: physician, researcher, mentor, visionary, humanist. Prehospital Emergency Care 2003; 8: 76-79.

10. Hisamuddin N.A., Hamzah M.S. & Holliman C.J. Prehospital emergency medical services in Malaysia. J Emerg Med 2007; 32(4): 415-21. 11. American Heart Association (AHA) 2005 American Heart Association Guidelines for Cardiopulmonary Resuscitation and Emergency Cardiovascular Care. Circulation 2005; 112: IV1-203. 12. Zulkifli A.R. Roda Impian’s Hani Mohsin dies at 43. In: The Star. 2006 July 26. 13. Vaillancourt C., Stiell I.G. Cardiac arrest care and emergency medical services in Canada. Can J Cardiol 2004; 20(11): 1081-90. 14. Chan S.C. Active Resuscitation In Malaysian District Hospitals - Is It Adequate? Med J Malaysia 1997; 52(3): 244-50.

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