Forensic Science, 11 (1978) 139 - 146 0 Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands

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ELECTRON MICROSCOPIC STUDY OF THE EFFECTS OF THAL- LIUM POISONING ON THE RAT CEREBELLUM

MAHDI HASAN

Brain Research Laboratory, Jawaharkd Nehru Medical College, Aligarh Muslim Univer- sity, Aligarh (India)

IQBAL ASHRAF

Department of Forensic Medicine, Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh (India)

V. K. BAJPAI

Electron Microscope Section, C.D.R.I., Lucknow (India)

(Received March 7, 1977;in revised form October 5, 1977; accepted January 30, 1978)

Summary

Rats were given thallium acetate (5 mg elemental thallium/kg body weight) intra- peritoneally daily for 7 days. The brain was fiied by perfusion-fixation and small pieces of the cerebellum were processed for electron microscopy. Variegated mito- chondrial profiles, increased incidence of electron - dense bodies and proliferation of Golgi zones were observed in the thallium - poisoned rat cerebellum. Multilamellar cytoplasmic bodies were discernible in the cerebellar cortex of the thallium-treated rats.

Introduction

Salts of thallium have been associated with accidental, occupational homicidal as well as suicidal poisoning [l] . In Europe, this heavy metal is claimed to be superseding arsenic as a homicidal agent [ 21. Reports of thallitoxicosis still appear frequently from different parts of the world [3-S] . The initial signs and symptoms of thallium poisoning are frequently neurologic and include ataxia, tremors, and peripheral neuropathy [3]. In the words of Canavagh et al. [4], “Only further ultrastructural studies on thallium poisoned animals will show whether the swelling noted in the preterminal nerve fibres was due to alterations in mitochondria or to some other organelle”. The mechanism of action of thallium on the nervous system and pathogenesis are, as yet, poorly understood [4] . Since ataxia and tremor are known to be associated with cerebellar lesions, the main aim of the present investigation is to find out whether experimental thallium poisoning leads to ultrastructural alterations in the rat cerebellum.

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Methods

Twenty male albino rats, weighing 150 f 20 g, obtained from the Central Drug Research Institute, Lucknow (Charles Foster strain), were used in this study. They were permitted free access to pellet diet (Hind Lever Labo- ratory Feeds, India) and tap water up to the time of experiment. The experimental and control groups consisted of an equal number of animals. Ten rats of the experimental group were injected with thallous acetate solu- tion (containing 5 mg elemental thallium per kg body weight) intraperito- neally daily for seven days. The total dose administered was 35 mg thallium/kg, corresponding to the LD 50 dose of Brewer and Haggerty [9] . The control group of animals (10) were treated in an identical manner with equal volumes of sodium acetate solution of the same molar concentration.

Two rats were anaesthetized at a time (one experimental and one control) with sodium pentobarbital and their brains fixed by perfusion- fixation. They were perfused through the left ventricle of the heart with phosphate-buffered glutara.ldehyde/paraformaldehyde solution prepared along the lines recommended by Kamovsky [lo]. The brains were removed from the cranium taking care to avoid trauma. Small pieces of the cerebel- lum were dissected out. They were quickly immersed in the same fixative for 3 hours at 4 “C. The tissue was then rinsed in 0.1 M phosphate buffer (pH 7.3) and post-fixed in 1% osmotically adjusted osmium tetroxide for 1-2 hours. After dehydration in graded series of alcohol the material was embedded in a mixture of Araldite 502 and Epon 812. An LKB Ultratome model 3 was used to cut thin, 600-700 8, sections which were stained with uranyl acetate [ 111 and lead citrate [ 121, and examined with an Hitachi HU 11 E electron microscope at an accelerating voltage of 75 kV.

Results

Most of the cerebellar neurones of the thallium-treated rats exhibited variegated, often bizarre shaped, mitochondrial profiles (Fig. 1). Electron- dense bodies, at times showing electron-luscent vacuoles, were frequently encountered in neuronal perikarya of the molecular and Purkinje cell layers (Fig. 2). Well developed Golgi zones were prominently seen, at times three or four in the same field (Fig. 3). Additionally, multilamellar bodies (finger-print pattern) were also visualised in the vicinity of the Golgi zone of the neurones in the molecular and Purkinje cell layers of 6 out of 10 experimental rats (Fig. 4). On higher magnification, the stacked lamellae of spherical or oval membranous cytoplasmic 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 concentrically. In the centre of many of the circular forms was a homogeneous or finely granular zone. One similar oval body was observed within a neighbouring mitochondrial profile (Fig.

Fig. 1. Electron micrograph of a part of the perikaryon of cerebellar neurone of a thal- lium-poisoned rat. Note the bizarre shaped mitochondria (arrow) (~39,000).

5). The appearance of these abnormal organelles was unique. The above- mentioned ultrastructural findings were negative in the control rats.

The lone earlier ultrastructural investigation of poisoning [ 131, with which the present findings can overabundance of stacked cristae mitochondriale in

experimental thallium be compared, revealed the deep white matter

of the cerebellum. Also, lipofuscin bodies were often found to be numerous in the cytoplasm of neurones. Although electron-dense, often vacuolated, bodies were commonly encountered in the perikarya of cerebellar neurons in the present study as well, it is difficult to interpret them as lipofuscin. Lipofuscin-like pigments in man and animals, either experimentally produc- ed in the latter or primarily associated with specific local or environmental factors, are generally classified under the heading “ceroid” 1141. This term is now commonly applied to lipo-pigments induced by a number of experi- mental conditions [ 151. According to Kerenyi et al. [ 161 these pigment masses are irreversibly injured lysosomes and are likely to develop under the influence of a specific stress on the nervous system. Glees and Hasan

Discussion and conclusion

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Fig. 2. Electron micrograph of a part of cerebellar neurone showing a segment of the nucleus (N) and electron-dense bodies (arrow) in the immediate viscinity of the nucleus (x 28,800).

[ 151, on the basis of available experimental evidence, concluded that neu- rones subjected to enhanced or reduced metabolic activity respond in the final stages by lipofuscin/ceroid formation. Furthermore, they have shown that the main material for these electron-dense bodies stems from degenerating mitochondria. It is likely that thallium intoxication, acting as a specific stress on the nervous system, triggers off a chain of activity in the Golgi zone-endoplasmic reticulum-lysosomes and mitochondria, culminat- ing in the excessive deposition of lipofuscin/ceroid in the neuronal peri- karya.

The most interesting finding of the present study was the occurrence of peculiar finger-print-like multilamellar cytoplasmic membranous bodies in the cerebellar neurones. They did not conform to the description of “myelin-figures”, as observed by Somlyo et al. [7] in response to the anti- biotic X 537 A. In the neighbourhood of the independently situated “multilamellar body”, one similar profile was visualized within a mito- chondrion.

Earlier, Terry and Korey [ 191 have reported membranous cytoplasmic granules in cases of infantile amauortic idiocy. The membranous cyto- plasmic bodies of Tay-Sachs disease, according to Terry and Korey, probably attain their structure as a result of the general tendency of lipids

Fig. 3. Electron micrograph of a part of cerebellar neurone of a Tl+-intoxicated rat. Three Golgi zones (arrows) exhibiting dilated disternae are shown (~40,000).

Fig. 4. Electron micrograph of cerebellar neurone of a Tl+-intoxicated rat. Note the well- defined Golgi zone in the centre and multilamellar bodies (arrows) (X 36,000).

Fig. 5. Electron micrograph (higher magnification) showing multilamellar bodies (arrows) and a vacuolated dense body. Note that one multilamellar body is within a mitochon- drion (double arrow) (X 120,000).

to become aligned into membranes. Furthermore, Sell et al. [ZO] induced neurofibrillary spheroids in the rabbit dorsal root ganglia by exposure to aluminium phosphate. But in aluminium encephalomyelopathy the lesion consists of compact ordered bundles of 100 .4 filaments, each with a poorly defined lumen [ 211.

It is apparent that membranous cytoplasmic bodies are not only encountered in gangliosidosis but can also be experimentally induced. In the present study, the intimate relationship of multilamellar membranous cyto- plasmic bodies with mitochondrial profiles has been demonstrated. Interestingly, not only are histomorphological alterations of mitochondria correlated with thallium intoxication, but also significant disturbances of mi- tochondrial function are induced by thallium [22]. It is conceivable that thallium-induced alterations in the mitochondrion might be associated with the genesis of the multilamellar body.

Acknowledgements

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

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Shipstone, Incharge, Electron Microscope Section of C.D.R.I., for providing facilities for this work. Thanks are also due to Mr. S. F. Ali and Mr. S. Mujir for skilful technical assistance.

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