histopathological studies of cardiac lesions …

23

ORIGINAL ARTICLE

HISTOPATHOLOGICAL STUDIES OF CARDIAC LESIONS AFTER ANACUTE HIGH DOSE ADMINISTRATION OF METHAMPHETAMINE

Arthur Kong Sn Molh, Lai Chin Ting, Jesmine Khan, Al-Jashamy K*, Hasnan Jaafar*, MohammedNasimul Islam

School of Health Sciences, *School of Medical Science, Universiti Sains Malaysia, Health Campus16150 Kubang Kerian, Kelantan, Malaysia

Eighteen male Wistar rats aged six weeks were divided equally intoMethamphetamine (MA), Placebo and Control group. MA group were injectedwith 50mg/kg body weight of Methamphetamine hydrochloride (MAHCl) in normalsaline, Placebo group were injected with normal saline only, while Control groupnot injected with anything. Five MA group rats died within four hours of injectionand their hearts collected on the same day. Another MA group rat was sacrificedtwo days after injection. Placebo and control group were sacrificed at similarintervals. Collected hearts were studied for cardiac lesions under light microscopyusing special staining and immunohistochemistry. Microscopic examination of themyocardium of the rats that died on the first day of injection showed loss of nucleiin some myocytes, indicating cell death. Some areas in the sub-endocardium regionshowed internalization and enlargement of myocyte nuclei, consistent withregeneration 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 showedfoci of infiltration of macrophage-like cells that were later revealed to beregenerating myocytes. There were also spindle-like fibroblasts, macrophages anda few leucocytes found within these foci. The overall appearance of the myocardiumdid not indicate any inflammatory response, and the expected signs of necrosiswere not observed. These results suggest a need to re-evaluate the toxic and lethaldosages of MA for use in animals testing. Cause of death was suspected to be dueto failure of other major organs from acute administration of MA. Death occurredwithin a time period where significant changes due to necrosis may not be evidentin the myocardium. Further investigations of other organs are necessary to helpdetect death due to acute dosage of MA.

Key words : MA, acute dose administration, cardiac lesions, myocardium.

Introduction

The use of MA along with other “designer”drugs have seen a dramatic increase beginning fromthe 1990s, as more drug abusers seek cheaper, morepotent alternatives to the “traditional” stimulantssuch as cocaine (1–3). The stimulant and euphoriceffect of MA is similar to cocaine, bringing aboutsimilar behaviour in animal tests of MA and cocaine.MA in the form of hydrochloride crystals are volatileand smokeable, bringing an immediate euphoria thatlasts longer than cocaine (1, 4). Cardiovascular

symptoms related to MA toxicity include chest pain,palpitations, dyspnoea, hypertension, tachycardia,atrial and ventricular arrhythmias, and myocardialischaemia (1, 4–9). MA abusers often go through arepeated pattern of frequent drug administrations(binge) followed by a period of abstinence. Thispattern of chronic MA abuse can significantly altercardiovascular function and cardiovascular reflexfunction and produce serious cardiac pathology (10).However, tachyphylaxis occurs with MA abuse, withlong-term abusers being able to tolerate higher doseswith fewer symptoms. MA has been known to cause

Submitted-20-02-2007, Accepted-03-12-07

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

24

death at an ingested dose as low as 1.5 mg per kgbody weight, while long-time abusers developingdrug tolerance may use as much as 5,000 to 15,000mg per day (1). MA sold in the streets is usuallymixed with other stimulants such as cocaine,phenypropanolamine hydrochloride, D-amphetamine, ephedrine, or pseudoephedrine, andalso with other adulterants such as lead, caffeine andbaking soda (1). This discrepancy in the purity ofMA available leads to the question whether theabuser may be taking high dosages far too toxic tothe body, which may result in sudden death of theabusers. Given the pattern of MA abuse, previousstudies have focused largely upon the chronic effectof MA intake to major organs, such as the brainsand the heart, by using animal testing (6, 9, 11–13).However, there is a lack of research into the effectsof acute dose intake of MA, especially pertaining tothe heart. Sudden death due to acute MA intoxicationhas been suggested to be similar to acute myocardialinfarction, where pathological changes to themyocardium generally are hard to detect, even underlight microscopy (14). Thus, there is a need to reviewthe effects of acute dosages of MA intake to the heartthrough microscopic studies in rats, which can helpmedical examiners differentiate myocardiumchanges due to acute MA intake from those of othercardiovascular diseases.

Materials and Methods

Eighteen male Wistar rats aged of six weekswere reared in the animal house of Universiti Sains

Malaysia, Kubang Kerian, Kelantan under standardatmospheric conditions in three 12 (w) X 24 (l) X 8(h) inch cage. Each cage was labelled according tothe three groups the rats were divided into, namelythe Control, Placebo, and MA injected groups. Theweight of the rats ranged from 102.6 – 123.1 grams.

Control GroupThe six rats in this group were kept under

normal rearing condition, fed with standardlaboratory chow and tap water ad libitum until sixweeks of age. The rats were fasted for 24 hoursbefore being sacrificed according to similar timeintervals as the MA-injected group, and their heartswere collected.

Placebo GroupThe six rats in this group were kept under

normal rearing condition, fed with standardlaboratory chow and tap water ad libitum until sixweeks of age. Each rat was then injectedintraperitoneally with 0.3ml of 0.9% (w/v) salineeach. The rats were then fasted for 24 hours afterinjection before being sacrificed at similar timeintervals as the MA-injected group and their heartswere collected.

MA Injected GroupThe six rats in this group were kept under

normal rearing condition, fed with standardlaboratory chow and tap water ad libitum until sixweeks of age. Each rat was then given anintraperitoneal injection of MAHCl dissolved in

Arthur Kong Sn Molh, Lai Chin Ting et. al

Figure 1 : Foci of cellular infiltration in the sub-endocardium region at400X magnification

25

0.9% (w/v) saline, the volume of which was adjustedaccording to body weight so that the final dosagereceived by each rat was approximately 50mg/kg.The rats were fasted for 24 hours before beingsacrificed and their hearts collected for pathologicalobservation.

A total amount of 50 milligrams MAHCl usedin this experiment was obtained from the Departmentof Chemistry Malaysia (JKM), Petaling Jaya, asMAHCl is a restricted substance classified underSection 39 (B) of the Dangerous Drugs Act 1952 inMalaysia, whereby possession, import or sale of thesubstance is strictly prohibited and punishable byMalaysian law. Moreover, this is an export forbiddenitem. As such, only the JKM is authorized by theMalaysian government to provide chemicalsclassified as restricted substance under Malaysianlaw for use in laboratory and scientific studies. Thepurity of the MAHCl obtained has been assayed andcertified as to be of a minimum 99% pure, as statedin the certification report provided by the JKM.

The dosage of MA given was calculated basedon previous studies (15) so as to induce observableeffects on the rats and to let the rats survive for atleast 24 hours after injection. However, rats No.3,4, 5, and 6 of MA group died after two hours ofinjection while rat No.2 died four hours afterinjection. The hearts of these rats were collected onthe same day. Rat No.1 survived for 48 hours afterinjection before being sacrificed. The rats in theControl and Placebo groups were also sacrificed atsimilar intervals as the deaths that occur in the MA

injected group rats.The rats were sacrificed by confining them

in a glass chamber saturated with chloroform (exceptthe rats from the MA injected group that died a fewhours after injection). A small sample of the freeupper left ventricle walls from each heart was takenand preserved in 0.9% (w/v) saline for future use inelectron microscopy methods. A section of the upperlevels of both ventricles from each heart werecollected and preserved in 10% (w/v) formalin forparaffin embedding while the adjoining section washarvested for frozen sectioning. The sections ofventricles preserved in 10% (w/v) formalin werethen processed in a tissue processor and embeddedin standard paraffin blocks.

The frozen sectioned ventricle samples werestained with Hematoxylin and Eosin (H&E) stain(commercial kit from Sigma Aldrich) for observationunder light microscopy. The consecutive sectionsof paraffin embedded samples were stained usingH&E, Masson’s Trichrome Stain (MTS)(commercial kit from Sigma Aldrich) andimmunohistochemistry staining using rabbit anti-myosin (commercial kit from Calbiochem). Forimmunohistochemistry, the heart samples weretreated with rabbit anti-myosin as the primaryantibody, which was then reacted with biotinylatedanti-rabbit immunoglobulin G (IgG) secondaryantibody. Biotinylated horseradish peroxidise, avidindehydrogenase, and hydrogen peroxide were thenused to provide sites for binding ofdiaminobenzidine tetrahydrochloride (DAB) dye to

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

Figure 2 : Loss of nuclei and large, internalised nuclei in rat no.2 viewed under400X magnification

26

the myosin molecules, and the nuclei of cells in thesamples were counter-stained with HarrisHematoxylin. Any loss of staining of tissue usingimmunohistochemistry would indicate injury due tomyocardial infarcts (16). The procedures for stainingwere sourced from the manuals of the commercialkits for each stain.

The light microscopy assessments of thesamples were conducted blindly by two observersto observe pathological changes highlighted inprevious studies. Findings were confirmed whileexcluding artefacts through comparison withhistochemical slides. The parameters observed forincluded eosinophilic changes, cellular infiltration,and contraction band necrosis. Comparison of H&Eand immunohistochemistry results were used toassess distribution of myoglobin, while MTS wasused to confirm any signs of fibrosis.

The parameters of pathological changesobserved were tabulated and graded using a scaleof 0–4, where “0” signifies the absence of theparameters in the samples, or healthy myocardium;“1” indicating observed presence and distributionof the parameter up to 25% of the area examined;“2” indicating observed presence and distributionof the parameter between 26% to 50% of the areaexamined; “3” indicating observed presence anddistribution of the parameter between 51% to 75%of the area examined; and “4” indicating observedpresence and distribution of the parameter in morethan 75% of the area examined. The Kruskall-Wallistest was used for statistical analysis in the SPSS

version 11.5 software and a P value of <0.05 wasconsidered significant.

ProcedureFifty milligrams of MAHCl was dissolved in

2.5ml 0.9% (w/v) saline for the injection of the testgroup rats. The volume of MA infused saline injectedinto each rat was adjusted with the calculatedconcentration of MAHCl injected for each rat to beapproximately 50 mg/kg body wt.

Results

Rat no.1 was injected with MAHCl earlier toobserve the effect of the concentration of MA andthe acceptability of the dose. This rat wassubsequently sacrificed, 48 hours after the injection.The other rats were all injected withmethamphetamine a day later. Rats no.3, 4, 5, and6, died within two hours of injection, while rat no.2died after four hours of injection. The hearts of theserats were collected on the same day after injection.

GrossThe hearts of the MA-injected rats were

normal in size, without any signs of necrosisthroughout the musculature. The hearts of the ratsthat died within 4 hours of injection were congested.The heart weight to body weight ratios were similarin all three groups. The hearts of the MA-injectedrats had patent coronary arteries that were free of

Figure 3 : Foci of cellular infiltration in rat no.1, DAB stained cells indicated asregenerating myocytes, viewed under 400X magnification


27

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 fociof cellular infiltration (Figure 1), with clusters ofmacrophage-like cells having large nuclei and littlecytoplasm evident in the sub-endocardium region.There were also presences of few leucocytes andfibroblast-like spindle cells. Certain areas also showloss of cross-striation and general disruption of thecellular ultra-structure. The samples from rat No.2showed loss of nuclei in certain sub-endocardiumregions, indicating dead cells, while other areasshowed internalisation of large nuclei (Figure 2).The internalisations of the large nuclei are indicativeof the myocytes’ regeneration process. In rats No.3,4, 5, and 6, the heart samples showed little differencefrom control samples, except for the presence oflarge, internalised nuclei, indicative of regenerativecells. In rat No.5, the sub-endocardium also showeda 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 mayoccur due to healing and scarring of myocardiumafter an infarct or similar injury. The samples fromMA group rats do not show any noticeable fibroustissue changes compared to the control samplesusing MTS for observation under light microscopy.

ImmunohistochemistryIn rat No.1 of the MA group, the foci of

cellular infiltration were found to be stained similarlyto its surrounding area (Figure 3), thus indicatingthe clusters of cells were actually regeneratingmyocytes. However, there may still be a fewleucocytes or macrophages present within these fociof cellular infiltration, and further investigation withother anti-leucocyte or anti-macrophage antibodiescan be used to elucidate their presence. In all theMA group rats, the myocardium samples werestained in almost uniform intensity, with severalareas of differentiated staining intensity of DAB.Thus, the presences of cellular death within thesamples were confirmed

Table 1 summarizes the observed pathologicalchanges in the samples of the MA group, in relationto the type of change observed and the extent ofspread of each type of pathological change. Althoughthe expected parameters of eosinophilic changes andcontraction band necrosis were not observed in mostof the samples, the presence of enlarged, internalisednuclei, and the absence of nuclei in certain myocytesshowed a statistically significant relationship (Pvalue of <0.05 using Kruskal-Wallis test) betweenthe MA group rats and the control group rats.

Discussion

The acute dose of MA injected to the rats inthis experiment were determined so that the ratswould be able to show effects of acute MA intakeand be able to survive for a day (13, 15). However,


* 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

MAgrouprat no.

Pathological parameters observed

Cell infiltration

Eosinophilicchanges

Contractionband necrosis

Enlargement andinternalisation of nuclei

Loss of nuclei

1

2

3

4

5

6

1

0

0

0

0

0

0

0

0

0

1

0

0

0

0

0

0

0

1

3

2

2

2

2

1

2

1

1

1

1

Table 1: Results of histopathological observation.

28

a complication to the design of this study arose whenfour of the MA group rats died within two hoursafter the MA injection, while a fifth rat died withinfour hours of injection. The deaths of the ratsoccurring within such a short time resulted in certainhistopathological changes that were indicated inprevious studies were found to be absent in the MAgroup rat samples. The difference in the length oftime where each rat had survived after MA injectionalso could not be determined.

The histopathological changes observed in theMA group rats’ samples include cellular infiltrationin rat No.1; enlargement and internalisation of nucleiin all samples; loss of nuclei in certain parts of allsamples; and eosinophilic changes in a few foci ofnecrosis in rat No.5. Previous studies in the acutedose intake of MA had indicated that eosinophilicchanges can be noticeable in rats after 2.5 hours ofacute MA administration, while cellular infiltrationwere noticeable after 18 hours of MA administration(15). The eosinophilic changes in rat No.5 and thecellular infiltration in rat no.1 were in agreementwith the findings of the previous study (15).

The cellular infiltration in rat No.1 was foundin the sub-endocardium, particularly near the leftventricle free wall region. The sub-endocardiallocation of the cellular infiltration may be due totransmural gradient in blood flow, which resultedin the inner layer of the wall being more severelyaffected than its outer layer. In a typical transmuralinfarct the corresponding gradient in ischaemicinjury initially produces rapid coagulative necrosisof the inner layer of the wall and lethal injury,subsequently propagates itself towards the epicardialsurface to an extent dependent on both time and thecharacteristics of the vascular bed involved (17).

The cells found in the foci of cellularinfiltration in rat no.1 were identified as regeneratingmyocytes, with the presence of a few macrophagesand leucocytes, but there were no fibrous tissueobserved in these areas. There were no signs ofinflammatory reaction in the tissue, even thoughdeath of cells was evident in scattered areas of themyocardium. The regenerating myocytes indicatedthat the affected tissue were trying to repairthemselves by producing new myocytes tocompensate for the dead cells. Macrophages andleucocytes were suspected to be present at these sitesof regenerating myocytes, mainly because themacrophages function to remove dead cells throughphagocytosis, thus eliminating the toxic substancesfrom being released by dead cells in the damagedtissue area and preventing further tissue damage.

Although the observation indicated certain cells inthe foci of cellular infiltration were macrophages orleucocytes, further staining using anti-macrophageor anti-leucocyte antibodies is needed to have apositive confirmation of the presence of these cells.

However, one major difference between theresults of this study and previous studies is theabsence of contraction band necrosis, a characteristicpattern of myocardial cell alteration that was presentin experimentally induced acute regional myocardialinfarcts of 2 hours or more duration (15, 17).Contraction band necrosis was reported to befrequently encountered in recent acute myocardialinfarcts, and its presence facilitate the definitivediagnosis of sudden cardiac death (17). As such, theshort duration of which the rats in the MA groupsurvived in this study should be adequate to showcontraction band necrosis. The absence ofcontraction band necrosis, however, indicates thatthe toxic effects of MA on other major organs apartfrom the heart may have caused the early deaths ofthe rats in MA group, before the changes to themyocardium had become noticeable.

The disposition of MA and its metaboliteamphetamine is highest in kidneys, followed byspleen, brain, liver, heart and serum (12). It was alsoobserved that high MA concentrations in the brainoccurred immediately after intravenous bolusdosing, suggesting that there is essentially nohindrance to passage of MA at the blood brainbarrier, and MA distributes very rapidly to all tissuesstudied except the spleen (12). As such, there is ahigh probability that the deaths that occurred in theMA group rats in this study were due to the toxiceffects of MA in other major organs, most probablythe brain, and not due to toxic effects on the heart.However, the scope of the study limited the choiceof exploring the effects of acute high doseadministration of MA on the other major organs aswell.

Loss of staining with antibodies to myoglobinis the most sensitive and reliable diagnosis of earlymyocardial ischaemic damage (16). The results ofthe immunohistochemistry staining show that mostof the samples have varying intensities of browncoloured staining by diaminobenzidinetetrahydrochloride (DAB). Thus, it can be concludedthat the myocardium may have suffered some formof ischaemic damage to the tissue, which could havebeen due to the direct effect of MA in the blood orby other pathways of pathological damage that hadyet to be explained.

The deaths of rats within four hours of acute


29

MA administration on a dose designed for the ratsto survive for 24 hours indicate that there may be aneed to re-evaluate the toxic and lethal dosages ofMA for use in animals testing. However, the exactmechanism of MA toxicity that leads to sudden deathis still unknown (3), thus further investigation intothis issue should be done to provide a helpful guidefor medical examiners when dealing with casesinvolving sudden deaths where MA abuse may besuspected.

Further testing procedures such asexamination of changes in other major organs anddetection of serum levels of MA may be necessaryto help detect deaths due to acute intake of MA. Theuse of electron microscopy in conjunction with lightmicroscopy is also recommended, as they wouldallow clearer differentiation of the necrotic changesin myocardium or other major organs. It isundeniable, however, that an acute dose intake ofMA will lead to serious damage of the heart or othermajor organs that may possibly lead to death, andas such, drug abusers and the society in generalshould be aware of the dangers of abusing this drugor other harmful drugs.

Acknowledgements

The authors wish to thank the Department ofChemistry, Malaysia for supplying the MAHCl usedin the experiment, as well as the staff of thePathology Laboratory of Hospital Universiti SainsMalaysia, and Biomedicine Laboratory, School ofHealth Sciences, USM. This research was conductedunder IRPA grant no. 06-02-05-00021-EAR

Corresponding Author :

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

References

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

2. Jacobs W. Fatal amphetamine-associatedcardiotoxicity 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. Crystalmethamphetamine-associated cardiomyopathy: Tip ofthe iceberg? J. of Toxi: Cl. Toxicology 2003; 41 (7):981 – 986

4. Ellinwood EH, King G and Lee TH. Chronicamphetamine use and abuse. Psychopharmacology:The Fourth Generation of Progress. 2000. Websitehttp://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 ofcardiac lesions after chronic administration ofmethamphetamine in rats. Nihon Hoigaku Zasshi 1996;50 (2): 63 – 71

6. Maeno Y, Iwasa M, Inoue H, Koyama H, Matoba Rand Nagao M. Direct effects of methamphetamine onhypertrophy and microtubules in cultured adult ratventricular myocytes. Forensic Science International2000; 113: 239 – 243

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

8. Rostagno C, Caciolli S, Felici M, Gori F and SerneriGGN. Dilated cardiomyopathy associated with chronicconsumption of phendimetrazine. Am. Heart J. 1996;131: 407 – 9

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

10. Varner KJ, Ogden BA, Delcarpio J and Meleg-SmithS. Cardiovascular responses elicited by the “binge”administration of methamphetamine. The Journal ofPharmacology 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 theirreversibility after long term administration ofmethamphetamine. Forensic Science International1995; 75: 29 – 43

12. Riviere GJ, Brooks G and Owens M. Disposition ofmethamphetamine and its metabolite amphetamine inbrain and other tissues in rats after intravenousadministration. The Journal of Pharmacology andExperimental Therapeutics 2000; 292 (3): 1042 – 1047

13. Shaw KP, Pu CE, Lin DL, Liu JC and Fong JM. Acomparative study of inhalation and self-administrationmethamphetamine-induced toxicities in rats. Advancesin Forensic Sciences 1995; 5: 264 – 273

14. Smith HJ, Roche AHG, Jagusch MF and Herdson PB.Cardiomyopathy associated with amphetamineadministration. American Heart Journal 1976; 91 (6):792 – 797


30

15. Maruta T, Nihira M and Tomita Y. Histopathologicalstudy on acute poisoning of methamphetamine,morphine or cocaine. Jpn. J. Alcohol & DrugDependence 1997; 32 (2): 122 – 138

16. Stefan J and Toupalik P. Our experience withimmunocytochemical staining of myoglobin inmyocardium. The Journal of Czecho-Slovak ForensicMedicine 1994; 39 (4): 39 – 42

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


histopathological studies of cardiac lesions …

Documents