Available online at www.jpsscientificpublications.com Life Science Archives (LSA) ISSN: 2454-1354 Volume 2; Issue - 6; Year 2016; Page: 792 797 DOI: 10.21276/lsa.2016.2.6.2 Research Article ELECTRON MICROSCOPIC STUDY OF ACUTE NEUROTOXICITY OF TOCP (TRIORTHO CRESYL PHOSPHATE) OF SPINAL CORD OF ADULT HEN K. Majeed Saleh* 1, E. Hadi Aula 2, A. Abd Alsamad Methaq 3 and A. Al-Sereah Bahaa 3 1 No. 55 Desborough Road, Hartford- Huntingdom, Cambridgeshire, PE 29 1 SN, England. 2 & 3 Department of Pathology & Poultry Diseases, College of Veterinary Medicine, University of Basrah, Iraq. Abstract In this present study, 2 groups were present untreated control with corn oil (orally), second group treated with 5 mg/kg in corn oil, single dose for 21 days. In acute neurotoxicity of TOCP spinal cord there was few neurotoxic changes in comparison with the more neurotoxic changes in the sciatic nerve. The changes in the spinal cord were restructured to the white matter only in characterized by presence of only few number of degenerate myelinated nerve fibers which characterized by vacuolation of myelin, spheroid body formation and degenerate lamellated myelin. Axoplasm showed presence degenerate lamellated mitochondria oligo dendrocyte occasionally showed presence of degenerate myelin in their cytoplasm. Article History Received : 14.09.2016 Revised : 05.10.2016 Accepted : 01.11.2016 Key words: Acute neurotoxicity, Electron microscope, TOCP, Spinal cord and Hen. 1. Introduction Neurotoxicity study of TOCP in adult hen reported the changes in gray matter of anterior horn associated with sever structural changes in the Boutons terminaux (Bischoff, 1970). The most striking finding in the early stage in massive swolling of the spherical synapses of the axo somatic synapses. Nanda and Tapaswi (1995) did acute neurotoxicity of TOCP study in adult hen using 300 400 mg/kg as acute neurotoxicity of CNS and reported changes after 2 days. Itoh et al. (1984) did acute neurotoxicity of TOCP using 400 mg/kg and reported changes in the spinal cord after 7-35 days, the changes were more prominent after 35 days in axoplasm and myelin sheath. Gupta et al. (2000) did the acute neurotoxicity on spinal cord using Di- * Corresponding author: K. Majeed Saleh isopropyl phosphoro fluoridate reported ataxia and or paralysis. Abou Donia (1995) study the acute neurotoxicity of phenylphos phothione in adult hen in found histopathological changes in spinal cord. Xiu Lan Zhaoa et al. (2006) reported expression changes of neurofilament subunits in the CNS of hens treated with TOCP studying the changes in neurofilaments in axoplasm of nerve fibers in the spinal cord. Abou Donia et al. (1983) study the neurologic manifestation of TOCP as delayed neurotoxicity in cats, reported changes in the spinal cord. Mohamed Abou Donia and Daniel Lapadula (1990) investigated the mechanism of organo phosphorus ester induced delayed neurotoxicity type I and type II and incited ultrastructural changes in spinal cord. A rodent model of organophosphate induced delayed neuropathy: Distribution of central (spinal cord) and peripheral nerve damage Veronesi (1984). The relationship between neurological
K. Majeed Saleh /Life Science Archives (LSA), Volume 2, Issue 6, Page 792 to 797, 2016 793 damage and neurotoxic esterase inhibition in rats acutely exposed to tri-orthocresylphosphatc (Padilla and Veronesi, 1985). Histopathological assessment of triphenyl phosphite neurotoxicity in the hen (Carrington et al., 1988). Al Sereah, B. A and Majeed (2014) studied the sub-chronic ninety days delayed neurotoxicity of tri ortho cresyl phosphate (TOCP) of spinal cord adult hen by orall gavage. Al Sereah and Majeed (2014) studied of acute delayed neurotoxicity of tri ortho cresyl phosphate (TOCP) of spinal cord light microscope of adult hen. Majeed et al. (2014) did electron microscopic study of acute neurotoxicity of TOCP (triortho cresyl phosphate) of sciatic nerve of adult hen. 2. Materials and Methods Forty adult hens were divided into 2 groups for acute delayed neurotoxicity of TOCP, first group 20 untreated control hen dosed by single dose orally with corn oil, second group of 20 hen dosed with 5 mg/kg in corn oil single dose for 21 days. At the end of 21 days both control and treated hen were sacrifized spinal cord was taken and fixed in glutaraldehyde after fixation resin blocks were made and cut by ultra microtome at 1 µg slide were made and stained with toluidine blue for orientation to select the right area for electron microscopy, then Copper grads were made in the stained with urinyl lead acetate for electron microscope examination. 3. Results and Discussion In the acute neurotoxicity of TOCP changes of the spinal cord were minimal in comparison with the sciatic nerve. The electron microscopic changes were restructured to the white matter only and more minmmal agen characterized by presence of degenerate myelin with vacuolation, spheriod body formation and lamellated oviod bodies of degenerate myelin also presence of dark stained lamellated degenerate mitochondria, some myelinated axons showed evidence of demyelination. Occasionally oligo dendrocyte showed dark stained degenerate myelin in their cytoplasm as evidence of autophagocytosis of myelin. Fig - 1: Spinal cord, presynaptic process with degenerate dark stained multilamellar mitochondria, other mitochondria with vesicular degeneration (EM 15000X) Fig - 2: Spinal cord, degeneration mitochondria in presynaptic process (EM 20000X) Fig - 3: Spinal cord, intramitochondrial inclusions in a neurone (EM 10000X)
K. Majeed Saleh /Life Science Archives (LSA), Volume 2, Issue 6, Page 792 to 797, 2016 794 Fig - 4: Spinal cord with degenerate mitochondria characterized by neumerous ovoid lamellar bodies (EM 49000X) Fig - 7: Spinal cord, degenerate nerve fiber with vesiculation, note collapse of axoplasm filled with dark stained structureless materials (EM 12000X) Fig - 5: Spinal cord, degenerate nerve fiber with thin myelin, note axoplasm filled with dark stained structureless materials (EM 40000X) Fig - 8: Spinal cord, disorganized lamellated myelin and depositing of dark stained materials (EM 12000X) Fig - 6: Spinal cord, demyelination with massive numbers of vesicles and ovoid bodies of degenerate myelin (EM 49000 X) Fig - 9: Spinal cord, oligo dendrocyte with intracytoplasmic degenerate lamellated myelin (EM 40000X)
K. Majeed Saleh /Life Science Archives (LSA), Volume 2, Issue 6, Page 792 to 797, 2016 795 Fig - 10: Spinal cord, marked degeneration of myelin characterized by lamellar body formation, vesiculation and vacuolation (EM 52000X) Bischoff (1970) observed the ultrastructural changes in boutons and synapses with swollen spherical synaptic vesicles. The present study agreed with Bischoff (1970) about changes in synapses but the most important change we found was dark stained degenerate lamellated mitochondria more changes in the synaptic vesicles. Nanda and Tapaswi (1995) did acute neurotoxicity of TOCP in adult hen reported clinical changes after 2 days. The present study did acute neurotoxicity in adult hen and observed changes in the CNS after 7-9 days. Itoh et al. (1984) did acute neurotoxicity of TOCP using 400 mg/kg and found changes in the spinal cord especially after 35 days. The present study did acute neurotoxicity with 500 mg/kg but found more electron microscopic studies in the sciatic nerve then the spinal cord, in the acute neurotoxicity of 21 days changes in the spinal cord were minimal characterized by changes in the myelinated nerve fibers and synapses with degenerate dark stained mitochonria. microscopic changes in myelin sheath and axoplasm. Xiu Lan Zhaoa et al. (2006) they investigate the changes of the neurofilament of axoplasm of nerve fibers in the spinal cord. Our study showed by the electron microscopic investigation presence of proliferation of neurofilament in axoplasm associated with dark stained degenerate mitochondria. Abou Donia et al. (1983) in there neurotoxicity study in cats reported histopathological changes in the spinal cord, there finding agreed with us that the acute neurotoxicity of TOCP can induce histopathological changes in the spinal cord, our study was mainly on electron microscopic changes. Mohamed Abou Donia and Daniel Lapadula (1990) reported in there paper ultrastructural changes in the spinal cord mainly associated with proliferation of neurofilament and smooth endoplasmic reticulum in the spinal cord. Our study agreed with presence of electron microscopic pathology changes concentrated on proliferation of microfilament and presence of dark stained degenerate lamellated mitochondria. Veronesi (1984) did neurotoxicity study using a rat as a model and reported histopathological changes in the spinal cord. Our study was mainly on hens but we agreed about the histopathological changes in the spinal cord. In addition, our study was mainly on electron microscopic changes on myelinated nerve fibers. Padilla and Veronesi (1985) reported the changes in the spinal cord deuto neurotoxicity of TOCP in rat. Our study was mainly in adult hen but we found histopathological changes in the spinal cord based on electron microscopy and associated with proliferation and neurofilaments and degenerate mitochondria. Carrington et al. (1988) did histopathological neurotoxicity study of tri phenyl phosphate and reported changes in the spinal cord. In our study, the neurotoxicity was mainly on TOCP and we found electron microscopic changes of axoplasm of myelinated nerve fibers in spinal cord characterized by presence of degenerate mitochondria and proliferation of neurofilament. Al Sereah, B. A and Majeed (2014) showed evidence of recovery of peripheral neuropathy of the sciatic nerve, which was so prominent in case of acute single Gupta et al. (2000) did acute neurotoxicity on di-isopropyl phosphoro fluoridate as acute neurotoxicity reported ataxia and or paralysis. The present study used acute neurotoxicity using 500 mg/kg also observed ataxia and all paralysis in some birds started 7 days upto 21 days. Abou Donia (1995) investigated the neurotoxicity of phenyl phospothioate by acute oral administration in the found histopathological changes in axoplasm and myelin sheath of myelinated nerve fibers in spinal cord. Our finding agreed with Abou Donia (1995) as we found electron dose of neurotoxicity of TOCP and while the
K. Majeed Saleh /Life Science Archives (LSA), Volume 2, Issue 6, Page 792 to 797, 2016 796 peripheral neuropathy of the sciatic nerve was reduced there was progressive increase in severity of the central nervous system neuropathy of the spinal cord with increase in incidence and number of degenerate vacuolated nerve fibers associated with clumps of degenerate myelin in contrast to the lesion of the acute single dose of delayed neurotoxicity in which the severity of the central neuropathy in spinal cord was quite less than the peripheral neuropathy of sciatic nerve. The present study concentrated on electron microscopic changes in the mitochondria induced by neurotoxicity of TOCP. Al Sereah and Majeed (2014) did acute delayed neurotoxicity of spinal cord of adult hens treated with TOCP (triorthocresyl phosphate) as positive control for organophosphorus, histopathology of light microscopy of Toluidine blue stains showed occasional nerve fibers with partial demyelination also nerve fibers with clumps or masses of degenerate myelin. The present paper studied the electron microscopic changes in the mitochondria of the nervous system of adult hen dueto neurotoxicity of TOCP in acute study (Majeed et al., 2014). The result electron microscopy of sciatic nerve showed degeneration of myelin in myelinated nerve fibers characterized by vacculation of myelin, clumping of myelin, spheroid body formation of myelin, lamellated body of degenerate myelin, exoplasm showed increased of neurofilament also present of dark stained lamellated degenerate mitochondria. The present study was concentrated on electron microscopic changes in mitochondria of central nervous system induced by neurotoxicity of TOCP in adult hen. 5. Conclusion Ultrastructural study gave fine information about the changes in different part of the spinal cord. 6. References 1) Abou Donia, M. B. 1979. Delayed neurotoxicity of phenyl phosphonothioate esters. Science, 205 (4407): 713-715. 2) Abou Donia, M. B., Jensen, D. N and Lapadula, D. M. 1983. Neurologic manifestations of tri-o-cresyl phosphate delayed neurotoxicity in cats. Neurobehavioral Toxicology and Teratology, 5 (4): 431-442. 3) Al Sereah, B. A and Majeed, S. K. 2014. Sub chronic ninety days delayed neurotoxicity of tri ortho cresyl phosphate (TOCP) of spinal cord adult hen by orall gavage. Journal of International Academic Research for Multidisciplinary, 2 (9): 90-97. 4) Al Sereah, B. A. and Majeed, S. K. 2014. Study of acute delayed neurotoxicity of tri ortho cresyl phosphate (TOCP) of spinal cord light microscope of adult hen. Journal of International Academic Research for Multidisciplinary, 2 (9): 83-89. 5) Bischoff, A. 1970. Ultrastructure of Tri- Ortho-Cresyl Phosphate poisoning in the chicken. Acta Neuropathologica, 15 (2): 142 155. 6) Carrington, C. D., Brown, H. R and Abou Donia, M. B. 1988. Histopathological assessment of triphenyl phosphite neurotoxicity in the hen. Neurotoxicology, 9: 223-234. 7) Gupta, R. P., Damodaran, T. V and Abou Donia, M. B. 2000. C-fos mrna Induction in the Central and Peripheral Nervous Systems of Diisopropyl Phosphorofluoridate (DFP) - Treated Hens. Neurochemical Research, 25 (3): 327 334. 8) Itoh, H., Kishida, H., Tadokoro, M and Oikawa, K. 1984. Studies on the delayed neurotoxicity of organophosphorus compounds (II). Journal of Toxicological Sciences, 9 (1): 37-50. 9) Majeed, S. K., Al Sereah, B. A and Al Mosawi, O. F. 2014. Electron microscopic study of acute neurotoxicity of TOCP (triortho cresyl phosphate) of sciatic nerve of adult hen. Journal of International Academic Research for Multidisciplinary, 3(4): 488-495. 10) Mohamed, B. Abou Donia and Daniel M. Lapadula. 1990. Mechanism of Organophosphorus ester - induce delayed neurotoxicity: Type I and Type II. Annual Reviews of Pharmacology and Toxicology, 30: 405-440.
K. Majeed Saleh /Life Science Archives (LSA), Volume 2, Issue 6, Page 792 to 797, 2016 797 11) Nanda, S. and Tapaswi, P. K. 1995. Biochemical, neuropathological and behavioral studies in hens induced by acute exposure of tri-ortho-cresyl phosphate. International Journal of Neuroscience, 82 (3-4): 243-254. 12) Padilla, S and Veronesi, B. 1985. The relationship between neurological damage and neurotoxic esterase inhibition in rats acutely exposed to triortho-cresyl phosphatc. Toxicology and Applied Pharmacology,78: 78-87. 13) Veronesi, B. 1984. A rodent model of organophosphate - induced delayed neuropathy: Distribution of central (spinal cord) and peripheral nerve damage. Neuropathology and Applied Neurobiology, 20: 357-368. 14) Xiu Lan Zhaoa, Tian Liang Zhangb, Cui Li Zhanga, Xiao Ying Hanc, Su Fang Yua, Shan Xia Lid and Ning Cuie. 2006. Expression changes of neurofilament subunits in the central nervous system of hens treated with tri-ortho-cresyl phosphate (TOCP). Toxicology, 223 (1-2): 127-135.