Forenz~e Science, 11 (1978) 139 - 1,1~6 ~ 39 © Elsevier Sequom S A , Lausanne -- Pnnted m the Netherlamts

ELECTRON MICROSCOPIC ,STUDY OF T H E EFFECTS OF Ti}{AL- LIUM POISONING ON THE RAT CEREBELLUM

MAHDI HASAN

Brazn Research Laboratory, J~weherlal Nehru Med~ea/ Co~ege, Ahgarh Ml~hrn Ur.~cr~ ~;Ry, AhgaW~ (India)

IQBAL A S H R A F

D.epcrtment of Forensm Medm;ne, I Jaw lh¢~r~ Nehra Medical College, Ahgarh Id~hm U~wem~ty. Ahgarh (Indm)

V K BAJPAI

Efectrorz Mrcroscop¢ Secnon, C.D R I , Luchr~ouJ (Indfa)

(Reeeaved M a ~ h 7 , 1 9 7 7 , m teemed form October 5, 1977 . accepted January 30, 1978)

S u m m a r y

Rats were g~ven tha lhum acetat,: (5 mg elemental tha~hum/kg body weight) iatra- p~ntoneal ly dmiy for 7 days The brmn was f'~xed by perfus ion-f i~a t lon ~nd ~mall p~eees of the cerebellum were processed for electron talc o~,copy. Variegated ra~to- vhondrml profiles, increased incidence of electron - - dense bodms and prohferat~on of Golg~ zones were observed m the tha lhum - - poisoned r ~ v~rebenum. Mult~lamcllar cytoplasmic bodies were dlscermble in the cerebellar ~cr~ex ,~v the thai[hum-treated rats

Introduetlon

S~ts of ~haUium hmre been assoclated xauth aceldental, occupetmnal homicldal as well as smeldal poisoning [I]. In Enrope, this heavy me~fl Is c!almed to be superseding arsemc as a homlcid~U agent [2]. Repor~ of th~htomleo~s still appear frequently ~rom dfffl,<~ant parks el the world [3-8] The m~tml s~ns and symptoms of ~h.~/lium pozs~mng ~e frequently neuro]o~c and :nc~ude ata~m, t~emo~, and pempherm neurcpa~hy [o]. In the words of Cannv~gh ct a~. [~], 'On!y lurther u/~a¢.~actu~a[ s~ad~es on lhalhum poisoned 2mm~ls ~m:l] show whether 'hhe swelling ne,~ed ~n the pretermm~/ nerve flbres ",v~ due ~o dierntlons m Imltochondna or ,~o some ohher organe]~eC The mechanism of ac~on of t~s~A~um on ~he nervous system and pathogenes~s ~re. ~ yet, poorly ~nd~,I%iood [4]. ~Ince ~.%<u~:z and tremor ~ lmown to be as=oc~aied w~ih eerebelkRr ]es~,~ns, @he m m n mm of ~he present anve~:gat..on ~ %0 hnd ou% v~h~,hez ~:perm~enzal thulium po~sonm~ lends to ul~,rastm~u~l ~-./l,era~mns in ih~ re% cerebellum.

140

Methods

Twenty male albino rats, weighing 150 -+ 20 g, obtmned from the Central Drug Research Institute, Lucknow (Charles Foster strmn), were used an this study. They were permitted free access to pellet diet (Hind Lever Labo- ratury Feeds, India) and tap water up to the time of experiment The experimental and control groups consls ed of an equal number of animals. Ten rats of the experimental group were injected with thallous acetate solu- tmn (containing 5 mg elemental thallitvu per kg body weight) mtrapento- neally dally for seven days. The total dose admlmstered was 35 mg thalhum/kg, corresponding to the LD 50 dose of Brewer and Haggerty [9] The control group of animals (10) were treated an an identical manner ~nth equal volumes of sodium acetate solulnon of the same molar concentration.

Two rats were anaesthetized at a time (one experimental and one control) with sodium pentobarbltal and thetr b"mns fixed by perfusion- fLxation. They were perfused through the left ventricle of the heart with phosphate-buffered glutaraldehyde/pvmformaldehyde solution prepared along the hnes recommended by Ka1,~ovsky [10]. The brmns were removed from the cranium taking care to avoid trauma. Small pieces of the cerebel- lum were dtssected out. They were qmckly immersed m the same fzxatlve for 3 horn-r, at 4 °C The tissue was then nnsed in 0 1 M phosphate buffer (pH 7.3) ~ad post-fixed m 1% osmotically adjusted osmium tetroxlde for l - 2 hours. After dehydration an graded series of alcohol the material was embedded ra a n~cture of Aralchte 502 and Epon 812. An LKB Ultratome mode[ 3 w~,~ used to cut than, 600-700 A, sections which were stained with uranyl acetate [11] and lead citrate [12], and examined with an Hitachi HU 11 E electron microscope at an accelerating voltage of 75 kV.

Resets

Most of the cerebenar neurones oI the thalhum-treated rats exhibited ~anegated, often bizarre shaped, mltochondmal profiles (Fag. 1). Electron- dense bodies, at tunes showing electr,:n-luscent vacuoles, were frequently encountered m neuronal penkarya (,f the molecular and Parkmle cell layers (Fig. 2). Weli deveIoped Golgi z~ nes were prominently seen, at tames three or four m the sam~ field (Fig. ~ ~. Additionally, multalamellar bodies (finger-p,~nt pattern) were also wsualls.~d m the vmmlty of the Golgl zone of the neurones in the molecular and ?urkmje cell layers of 6 out of 10 experimented rats (Fig. 4). On tugher magmf~cahon, the stacked lamellae ,)f spherical or oval membranous eytaplasmm bodies revealed regular periodi- city of 50 to 60 A (Fig 5). They were composed of close-packed, electron- dense membranes arranged in a strikingly regular, finger-print fashion. Most often, the layers were arranged concentnciflly In the centre of many of the circular forms was a homogeneous ,)r finely granular zone One similar oval body was observed within a nelghbounng mltochondmal profile (Fig.

141

F~g 1 Electron rnmrograph of a part of the pe~karyo,: of cerebeltar neurone of a thai- hum-poisoned r~ Note the bizarre shaped m~tochondtda (~-wcow) (×.~9,000)

5). The appearance of these abnormal organelles was umque The above- mentmned ultrastructural finchngs were negative in the control rats.

Discussmn and conclusmn

The lone earher uli,rastructural investigation of expellmentaI thall,um pmsonmg [13J, ~nth whmh the present tmdmgs ,Man be cc,mpared, reve~ed overabundance of stacked cnstae mltochondnale m the deep white matte~ of the cerebellum Also, hpofuscm boding, were o'ften found to be numerc*us m the cytoplasm of neurones Although electrorL-dense, often vacuolated, boches were commonly encountered in the penkarya of cerebellar neurons in the present study as well, it is chfficlflt to m~erpret them as hpofuscm Llpofuscm-hke pl,~aents m man and anmmls, either experimentally produc- ed m the latter or pnmanly associated w:th specific local or enwronmental factors, are generally cl~sffmd unde," the heading "ceroid" [14]. This term is now commonly applied to hpo-plg~laen,~s reduced by a number of experi- mental conchtlons [15]. According to Keren~i et cl [16] these pigment masses are trl~ ~emlbly injured lysosomas and are hkely to develop under the 1,1[luence ol a specific stress on the nervous system. Glees and Hasan

142

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[15], on the basts of avmlable experimental evidence, concluded that neu- rones subjected to enhanced or reduced metabohc activity r~spond m the final stages by hpofuscm/cerold formation. Furthermore, they hawe shown that the mmn material for these electron-dense bothes stems from degeneratang mltochondna I t is hkely that thalhum lntoxicatmn, acting as a specific stress on the nervous system, triggers of£ a chain of actlwl,y m the Golgl zone-endoplasmlc reticulum-lysosomes and mztochondna, c,~lmmat- mg m the excessive deposition of hpofuscm/cermd m the neuronal pen- karya.

The most interesting finding of the present study was the occurrence of peculiar finger-pnnt-hke multflamellar cytoplasmm membranous bodies m the ce~ebellar neutones. They thd noL conform to t~he description of "myehn-flgures', as observed by Somlyo e ta ] [7] in response to 1,he anti- blo~l~ X 537 A In the nelghbourhood of the independently situated "multllameliar body", one sanflar profile was visualized within a mito- chondnon

Earher, Ten5' and Korey [19] have reported membranous cytoplasmic granules in cases of infantile amauortm idiocy. The membranous cyto- plasnnc bodies of Tay-Sachs disease, accozdmg to Terry and Korey, probably attam thmr ~tructure as a result of the general tendency of hplds

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F~g 3 Electron m~crograph of a part of cerebellar neurone of a Tl÷-mtox]cated rat Three Golg~ zones (arrows) exlnb~tmg dilated d~steroae ar~ shown (X 40,000).

Fig 4 Electron mzcrog~aph of cerabeRar neurone of a Tl*-lntoRl( ated rat Note the weE- defined Golgl zone m the centre and multdameHar bodies (arrows) (× 36,000).

144

Fig 5 ]~ectron mlCrOgraph (higher magniflcatlon)showlngmult|laraellar bodies (arrows) and a vacuolal,ed dense body Note that one multdame~ar body ~s within a mltochon- dnon (double ,trrow) (× 120,000)

to become ahgned into membranes Fu~hermore, Sell et al [20] reduced neuroflbrillaxy ~phermds in the rabbit dorsal root ganglia by exposure to alummmm phosphate. But in alumimum encephalomyelopathy the lesion consists of compact ordered bundles of 100 A filaments, each with a poorly defined lumen [21].

It is apparent tha~ membranous cytoplasmic bodms are not only encountered m ganghos]dosls but can also be erpenmentally reduced In the present study, the intimate relationship of mull~Iamellar membranous cyto- plasmic bedms vnth mltochondnal proflIe~ has been demonstrated Interestingly, not only are histomorphologmal alteratmns of mltochondna correlated wltb thalhum mtoxmataon, bJ t ~lso slt,~ufacant disturbances of ml- tochondnal funchon axe reduced by thallmm F',~2] It is conceivable that t~alhum-mdu,ced alteratmns m the rmtochondrmn might be assoemted vath the genesis of the multllamellar body.

Acknowledgements

The authors are grateful to :Dr. S. H. Zmdl, Director, I T R C, to Professor B. N Dhawan, Deputy Director, C.D R.1, Lucknow, and to Dr A. C

145

S m p s t o n e , I n c h a r g e , E l e c t r o n M i c r o s c o p e S e c t i o n o f C . D . R , I . , f o r p r o v i d i n g f a c l h t ] e s f o r th~s w o r k . T h a n k s a r e a l s o d u e t o M r . S . F . A l l , rod M r . S. Muj~r f o r skaHtfl t e c h n i c a l a s s i s t a n c e .

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