Rotenone induced neurotoxicity in rat brain areas: A histopathological study

Neuroscience Letters 501 (2011) 123–127

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Rotenone induced neurotoxicity in rat brain areas: A histopathological study Supriya Swarnkar a , Sarika Singh a , Sharad Sharma a , Ramesh Mathur b , Ishan K. Patro b , Chandishwar Nath a,∗ a b

Division of Toxicology, Central Drug Research Institute (CSIR), Lucknow 226001, U.P., India School of Studies in Neuroscience, Jiwaji University, Gwalior 474011, M.P., India

a r t i c l e

i n f o

Article history: Received 26 November 2010 Received in revised form 1 March 2011 Accepted 10 March 2011 Keywords: Rotenone Neurotoxicity Histopathology Oxidative stress Neuromuscular coordination

a b s t r a c t Rotenone a pesticide is known to induce neurotoxicity. In earlier study we correlated rotenone induced biochemical changes and cerebral damage in brain areas with neuromuscular coordination in rats. The present study involves investigation of rotenone induced histopathological changes in the brain areas, viz. striatum (STR) and substantia nigra (SN) using HE (hematoxylin and eosin) and CV (Cresyl Violet) staining after 1, 7, and 14 day of unilateral intranigral administration of rotenone (3, 6 and 12 ␮g/5 ␮l) in adult male SD rats. Significant morphological changes in cell area or shape were shown by HE staining. The neuronal degeneration was shown by distorted neuronal cells, shrinkage of nuclei, dark staining in the regions of rotenone treated animals by CV staining. Rota rod test demonstrated significant impairment in motor coordination after 14 days of treatment along with decreased GSH and increased MDA in STR and mid brain (MB). The study inferred rotenone causes neuronal damage which is evident by histopathological changes, impaired neuromuscular coordination and biochemical changes. The pattern of histopathological alterations corresponds with behavioral and biochemical manifestations. © 2011 Elsevier Ireland Ltd. All rights reserved.

Rotenone is a pesticide derived from the plant roots of leguminosae family and known to cause neurotoxicity [1]. Reports are available suggesting the considerable role of environmental toxins in neurological and other disorders [3]. Neurodegeneration is attributed to several mechanisms viz. imbalance of redox system/antioxidant levels and mitochondrial impairment [15]. Uversky [20] has reported that rotenone induces neuronal injury through multiple pathophysiological mechanisms viz. complex I inhibition, oxidative damage, microglial activation, production of ROS, apoptosis and ␣-synuclein aggregation. The detailed knowledge of long term effect of single intranigral administration of rotenone on brain is not well explored. However, behavioral, pathological and biochemical lesions were reported after chronic intrajugular administration of rotenone [2]. Our earlier studies, in brain homogenates [18] and in vivo [19] suggested that rotenone induces neuronal damage differentially among the brain areas as mid-brain (MB) and striatum (STR) were found more affected as compared to other brain regions. But studies involving histopathological evidences for rotenone induced toxicity in brain regions are lacking. Therefore, the present study was undertaken to investi-

∗ Corresponding author at: Scientist G & Head, Division of Toxicology, Central Drug Research Institute, P.O. Box 173, Lucknow 226001, India. Tel.: +91 522 2212411 18 4434; fax: +91 522 2623405. E-mail address: [email protected] (C. Nath). CDRI Manuscript no. 8041. 0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2011.03.036

gate the histopathological changes caused by rotenone in STR and MB. Thus, we investigated correlation of morphological changes with neuromuscular coordination and oxidative stress based on dose and time course. Histopathological analysis was done at three time points to evaluate microscopic changes. Further 14 days after rotenone administration, impairment in motor coordination assessed by Rota rod test and biochemical estimation of GSH and MDA level was done in STR and MB. The experiments were carried out with adult male Sprague– Dawley rats (180–200 g). The animals were kept in polyacrylic cage and maintained under standard housing conditions (room temperature 22 ± 1 ◦ C and humidity 60–65%) with 12 h light and dark cycle (lights on at 6:00 a.m.). The food in form of dry chow pellets and water were available ad libitum. The animals were procured from the Laboratory Animal service Division of Central Drug Research Institute and experiments were performed according to internationally followed ethical standards and approved by Institutional Animal Ethics Committee of Central Drug Research Institute, Lucknow, India. The rats were anesthetized with chloral hydrate (300 mg/kg, i.p.) and placed on a stereotaxic frame (Stoelting, USA), for intranigral administration of rotenone. Rotenone was dissolved in DMSO and infused (5 ␮l) into the right SN. The stereotaxic coordinates for SN were AP: 5.3 mm; L: 2.0 mm; DV: 7.8 mm, from the Bregma point [14]. The needle was kept in position for about 5 min to prevent outflow. After the injection, burr hole was sealed with bone wax and antibiotic powder (neosporin) sprayed at incision

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wound prior to suturing. The animals were then kept in a cage for recovery with free access to food and water. The proper postoperative care was done till the animals recovered completely. Animals were divided in five groups viz. control (without any treatment), vehicle (DMSO), rotenone (3, 6 and 12 ␮g/5 ␮l) treatment. Animals were sacrificed after defined point of time and brain were isolated, dissected and proceeded as per assay requirement. Rats were perfused intracardially with ice-cold 0.1 M phosphate-buffered saline (PBS) followed by cold paraformaldehyde (4%, w/v) in 0.1 M PBS. Animals decapitated and the brains were removed and processed for paraffin embedded sectioning. Five-micrometer thick MB region containing substantia nigra (SN) and STR (Paxinos and Watson, 1988) sections were cut on a Microtome (Leica) and collected on poly-l-lysine coated slides to process for HE (hematoxylin and eosin) and CV (Cresyl Violet) staining. Image analysis (100× magnification) was done using Qwin V3 Software (Leica) and results represented in terms of ratio of total cell area (␮m2 ) to total number of cells.

Neuromuscular coordination of rats was tested on rota rod [16]. Animal was pre-trained at 5–10 rpm for a maximum of 120 s on the Rota rod and then tested after 14 days of treatment. The total time of stay of each rat was recorded; its mean value was calculated for each group. Biochemical markers of oxidative stress GSH and MDA in the brain were estimated after 14 days of rotenone treatment. Rats were anesthetized with mild ether anesthesia and intracardially perfused with pre chilled 0.9% NaCl. After perfusion animal was sacrificed by decapitation. The whole brain was removed carefully and was kept quickly in petridish placed over ice. The brain was dissected into STR and MB [5]. A 10% (w/v) homogenate of different regions of brain samples in sodium phosphate buffer (30 mmol/l, pH 7.0) were prepared using Ultra-Turrax T25 (USA) homogenizer at a speed of 9500 rpm. The homogenate was collected and immediately assayed for estimation of GSH and MDA level. Reduced glutathione (GSH) level as a marker of oxidative stress was estimated spectrophotometrically [6]. Homogenates

Fig. 1. H&E staining after treatment with rotenone (3, 6, and 12 ␮g) at time points 1day, 7 days and 14 days in STR (a) and SN (b). Values are expressed as means ± S.E.M. **p < 0.01 and *p < 0.01 vehicle vs treated groups.

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were deproteinised using 1% Trichloroacetic acid (TCA), centrifuged for 5 min at 5000 rpm at 4 ◦ C using centrifuge (Biofuge Fresco, Heraeus, Germany). Supernatant was collected and used in reaction with 5,5 -dithiobis-2-nitrobenzoic acid, potassium phosphate buffer (0.1 N, pH 8.4) and distilled water. The reaction developed yellow colour and the absorbance was read immediately at 412 nm by ELISA plate reader (BIO-TEK Instruments). The assay was done in duplicate sample. Concentration of GSH (␮g/mg protein) in the samples was extrapolated from the standard curve obtained by plotting the OD of the standard GSH. The results were expressed in terms of % change in GSH concentration as compared to control. MDA level (marker of lipid peroxidation) was measured spectrophotometrically [4]. Homogenate was deproteinised with 30% TCA and 5 N HCl, followed by 2% thiobarbituric acid in 0.5 N NaOH. The reaction mixture was heated on a water bath at 90 ◦ C for 15 min and centrifugation (Biofuge Fresco, Heraeus, Germany) at 5000 rpm for 10 min and supernatant was collected. The Absorbance was measured at 532 nm, by ELISA plate reader (BIO-TEK Instru-

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ments). The assay for each sample was run in duplicate. The MDA concentration (nmol/mg protein) in the samples was extrapolated from the standard curve obtained by plotting the OD of the standard concentrations of tetraethoxypropane. Results were expressed as % change in MDA concentration as compared to control. The protein concentration was estimated by using bovine serum albumin (BSA) 0.01–0.1 mg/ml as standard [11]. The data was analyzed by one way analysis of variance (ANOVA) followed by post hoc Dunnett’s Multiple Comparison test. Results are expressed as the mean ± SEM, value of p < 0.05 was considered as level of significance. There were dose dependent histopathological changes in STR and SN following rotenone treatment. Histopathological observations HE staining: Rotenone (12 ␮g) showed significant decrease in neuronal integrity, represented by decreased ratio of total area of cells (␮m2 ) to total number of cells in the STR at all the three time points (1, 7 and 14 days) while 6 ␮g dose of rotenone resulted in significant cell shrinkage/morphological alteration in STR only after 7

Fig. 2. CV Staining after treatment with rotenone (3, 6, and 12 ␮g) at time points 1day, 7 days and 14 days in STR (a) and SN (b). Values are expressed as mean ± S.E.M. **p < 0.01 and *p < 0.01 vehicle vs treated groups.

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Table 1 Locomotor activities after treatment with rotenone (3, 6, and 12 ␮g) after 14 days, values are expressed as mean ± S.E.M. Significance w.r.t. vehicle vs treated groups. Time of stay (s)

Mean ± S.E.M. Pretreatment

Control Vehicle 3 ␮g Rotenone 6 ␮g Rotenone 12 ␮g Rotenone

120 120 120 120 120

± ± ± ± ±

0 0 0 0 0

Significance Postreatment 120 111.7 69.36 7.32 8.88

± ± ± ± ±

0 8.28 14.8 1.736 1.482

*p < 0.05 **p < 0.001 **p < 0.001

and 14 days of treatment. However, 3 ␮g dose of rotenone did not caused any significant alteration at any time points (Fig. 1a). All the doses of rotenone (3, 6 and 12 ␮g) showed significant degeneration of cellular constituents indicated by decrease in cell size (shrinkage) and cell number in the SN regions at all the three time points (1 day, 7 days and 14 days) (Fig. 1b). Histopathological observations CV staining: Rotenone (6 and 12 ␮g) showed significant degeneration of neuronal cells in terms of decrease in cell size/area and number, represented by ratio of total area of cells (␮m2 ) to total number of cells. Significant morphological changes were found in STR with all the doses of rotenone after 7 and 14 days. However, 1 day after treatment 6 and 12 ␮g doses of rotenone affected neuronal damage (Fig. 2a). Rotenone (3, 6 and 12 ␮g) showed concentration dependent significant decrease in morphological changes in the SN at all the three time points (1 day, 7 days and 14 days) (Fig. 2b). There was a significant reduction in duration of stay in the rota rod test after 14 days of treatment with rotenone as compared to vehicle control group (Table 1). Significantly lowered GSH level in MB was found with all the three doses (3, 6 and 12 ␮g) of rotenone

in comparison to vehicle. GSH level was decreased with only 6 and 12 ␮g of rotenone in STR [Fig. 3a]. Rotenone (3, 6 and 12 ␮g) caused significant rise in MDA level in STR as well as MB after 14 days as compared to vehicle. However, increase in MDA level in MB was lesser than that in STR [Fig. 3b]. Present study demonstrated the dose and time dependent severity of rotenone induced neurotoxicity by histopathological changes in rat brain areas. Rotenone caused more pronounced changes in nigral region as compared to STR. The similar pattern of cerebral damage based on TTC staining was also observed after 1 and 7 days of rotenone toxicity [19]. Microscopic changes in cytostructure were evaluated by HE and CV staining that displays a broad range of cytoplasmic, nuclear, and extracellular matrix features. The assessment of morphological or structural changes was done by quantifying the cell area and number of cells after staining. HE stains nuclear and non-nuclear cell components and thereby provides morphological information about cell [10]. The morphological changes seen after rotenone treatment were suggestive of neuronal damage. These alterations were in the size, shape and staining of cellular perikarya, and vacuolation (sponginess) of the neuropil. Neurons contain Nissl substance which can be identified through CV staining as bluish and colors cell bodies a brilliant violet [12]. Therefore CV was used to evaluate the adverse changes on neuronal cells after administration of rotenone. The CV staining of the nigral neurons in MB of the control animals showed optimally sized cells with a prominent nucleus and pigmented neurons. The rotenone treated animals showed loss of pigmented neurons indicating extensive neuronal damage. The neurons appear to be smaller and shrunken along with formation of Nissl granules compared to the control cells, suggestive of neuronal damage. CV staining demonstrated the neuron specific changes in both STR and nigral neurons in MB in rotenone treated rats.

Fig. 3. (a) GSH assay in homogenates of different rat brain areas – STR (striatum), MB (mid-brain) after treatment with rotenone (3, 6, and 12 ␮g) at time point 14 days. Values are expressed as mean ± S.E.M. **p < 0.001 and *p < 0.01 vehicle vs treated groups. (b) MDA assay in homogenates of different rat brain areas – STR (striatum), MB (mid-brain) after treatment with rotenone (3, 6, and 12 ␮g) at 14 days. Values are expressed as mean ± S.E.M. **p < 0.001 and *p < 0.01 vehicle vs treated groups.

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The quantitative assessment of morphological changes in terms of ratio of cell area to cell number was done using Qwin V3 software. HE staining showed dose dependent neurotoxicity in STR on 7 and 14 days after treatment with rotenone. Only highest dose caused toxicity on 1 day after treatment, pointing towards quick effect of high dose. CV staining was found more sensitive to detect neuronal specific damage as compared to HE staining. CV staining revealed significant neuronal changes by mid dose of rotenone in STR from 1 day and even the lowest dose which was found ineffective in HE staining showed significant damage to striatal neurons after 7 and 14 days of treatment. It seems that neuronal damage precedes morphological changes in rotenone induced neurotoxicity. In contrast to STR, both the staining in SN region indicated similar pattern of histological changes in dose dependent manner at all the time points. This is suggestive of earlier occurrence of neuronal damage in SN as compared to STR. This could be attributed to selective toxicity of rotenone on DA-ergic cells as found in, in vitro and in vivo studies [13,17]. SN contains less GSH peroxidase and tends to bind to redox active metals, which make SN neurons vulnerable to free radical generation from their easily oxidizable melanin complement [7]. Thus, the neurons in SN appear to sustain an active level of oxidation even under normal condition [8,9]. SN is the richest site of microglia cells. It is well documented that microgliosis induced by neurotoxins involves ROS generation. This might be the possible reason for SN being more affected in comparison to STR by rotenone. Rotenone caused maximal histological changes in both STR and SN after 14 days of treatment in a dose responsive manner. Thus, 14 days time point was selected for assessment of biochemical changes and motor coordination to correlate with histopathological changes. Rota rod test showed significant deficit in motor coordination with all the three doses of rotenone. It appears that histological changes in SN is more linked to locomotor impairment than STR because 1 day after treatment the lowest dose caused significant changes only in SN as observed in the present study and same dose also impaired neuromuscular coordination after 1 day [19]. Interesting observation of present study was similarity in morphological changes by HE staining and biochemical changes in antioxidant enzyme, GSH both in STR and SN after 14 days of treatment by higher doses. Also, the result pattern of neuronal changes indicated by CV staining was found more similar to biochemical changes in terms of lipid peroxidation, MDA both in STR and SN after 14 days of treatment by all the doses. The lowering in GSH in MB by all the doses of rotenone was accompanied by elevated MDA level. However, the lowest dose of rotenone elevated MDA level without changing GSH level in STR. This indicates that brain areas may respond differently to oxidative stress caused by rotenone. Variable vulnerability of brain areas to rotenone induced oxidative stress has also been demonstrated in the in vitro study [18]. The study provided histological evidences for rotenone induced neuronal damage in SN and STR. It has emerged in the study that SN is more vulnerable to damage by rotenone that may be attributed to higher susceptibility of SN for free radicals. Histological findings

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in SN were more correlated with impaired motor coordination responses along with biochemical evidences. Acknowledgments Authors are thankful to Mr. Vishal Singh and Mrs. Poonam Goswami for their help in histological studies and supporting this work. Author S.S. gratefully acknowledges the Council of Scientific and Industrial Research (CSIR), India for research fellowship. References [1] M. Alam, W.J. Schmidt, Rotenone destroys dopaminergic neurons and induces parkinsonian symptoms in rats, Behav. Brain Res. 136 (2002) 317–324. [2] R. Betarbet, T.B. Sherer, G. MacKenzie, M.G. Osuna, V.A. Pavanov, T.J. Greenamyre, Chronic systemic pesticide exposure reproduces features of Parkinson’s disease, Nat. Neurosci. 3 (2000) 1301–1306. [3] A. Caban-Holt, M. Mattingly, G. Cooper, F.A. Schmitt, Neurodegenerative memory disorders, a potential role of environmental toxins, Neurol. Clin. 23 (2005) 485–521. [4] M.I. Colado, E. O’Shea, R. Granados, A. Misra, T.K. Murray, A.R. Green, A study of the neurotoxic effect of MDMA (“ectasy”) on 5-HT neurons in the brains of mothers and neonates following administration of the drug during pregnancy, Brit. J. Pharmacol. 121 (1997) 827–833. [5] J. Glowinski, L.L. Iversen, Regional studies of catecholamines in the rat brain. The disposition of [3H] norepinephrine [3H] dopamine and [3H] DOPA in various regions of the brain, J. Neurochem. 13 (1966) 655–670. [6] Y.K. Gupta, M. Sharma, Effect of alpha lipoic acid on intracerebroventricular streptozotocin model of cognitive impairment in rats, Eur. Neuropsychol. Pharmacol. 13 (2003) 241–247. [7] R.A.S. Hemat, Orthomolecularism: Principles and Practice, first ed., Author House, Nutrients, 2004, p. 405. [8] E.C. Hirsch, A.M. Graybiel, Y. Agid, Melanized dopaminergic neurons are differentially susceptible to degeneration in Parkinson’s disease, Nature 334 (1988) 345–348. [9] A. Kastner, E.C. Hirsch, O. Lejeune, F. Javoy-Agid, O. Rascol, Y. Agid, Is the vulnerability of neurons in the substantia nigra of patients with Parkinson’s disease related to their neuromelanin content? J. Neurochem. 59 (1992) 1080–1089. [10] Y. Li, C. Powers, N. Jiang, M. Chopp, Intact, injured, necrotic and apoptotic cells after focal cerebral ischemia in the rat, J. Neurological. Sci. 156 (1998) 119–132. [11] O.H. Lowry, N.J. Rosebrough, A.I. Farr, R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193 (1951) 265–275. [12] N. Ludvig, L.G. Sheffield, H.M. Tang, S.L. Baptiste, O. Devinsky, R.I. Kuzniecky, Histological evidence for drug diffusion across the cerebral meninges into the underlying neocortex in rats, Brain Res. 1188 (2008) 228–232. [13] Marey Semper, M. Gelman, M. Levi Strauss, A selective toxicity toward cultured mesencephalic dopaminergic neurons is induced by the synergistic effects of energetic metabolism impairment and NMDA receptor activation, J. Neurosci. 15 (1995) 5912–5918. [14] G. Paxinos, C. Watson, The Rat Brain in Sterotaxic Coordinates, Academic Press, New York, 1988, pp. 1–120. [15] C. Rice-Evans, R. Burdon, Free radical–lipid interactions and their pathological consequences, Prog. Lipid Res. 32 (1993) 71–110. [16] G. Rozas, J.L. Labandeira-Garcˇııa, Drug-free evaluation of rat models of parkinsonism and nigral grafts using a new automated rotarod test, Brain Res. 749 (1997) 188–199. [17] K.S. Saravanan, K.M. Sindhu, K.P. Mohanakumar, Acute intranigral infusion of rotenone in rats causes progressive biochemical lesions in the striatum similar to Parkinson’s disease, Brain Res. 1049 (2005) 147–155. [18] S. Swarnkar, E. Tyagi, R. Agrawal, M.P. Singh, C. Nath, A comparative study on oxidative stress induced by LPS and rotenone in homogenates of rat brain areas, Environ. Toxicol. Pharmacol. 27 (2009) 219–224. [19] S. Swarnkar, S. Singh, R. Mathur, I.K. Patro, C. Nath, A study to correlate rotenone induced biochemical changes and cerebral damage in brain areas with neuromuscular coordination in rats, Toxicology 272 (2010) 17–22. [20] V.N. Uversky, Neurotoxicant induced animal models of Parkinson’s disease: understanding the role of rotenone, maneb and paraquat in neurodegeneration, Cell Tissue Res. 318 (2004) 225–241.