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Increased iron in the substantia nigra of 6-OHDA induced parkinsonian rats: a nuclear microscopy study
BRAIN RESEARCH ELSEVIER
Brain Research 735 (1996) 149-153
Short communication
Increased iron in the substantia nigra of 6-OHDA induced parkinsonian rats: a nuclear microscopy study Y. H e a, P . S . P . T h o n g b, T. L e e a, * , S . K . L e o n g c, C . Y . S h i d, P . T . H . W o n g
e, S . Y . Y u a n a, F. W a t t b
a Department of Surgery, National University of Singapore, 10 Kent Ridge Crescent, Singapore 0511, Singapore b Department of Physics, National Universi~ of Singapore, 10 Kent Ridge Crescent, Singapore 0511, Singapore c Department of Anatomy, National University of Singapore, 10 Kent Ridge Crescent, Singapore 0511, Singapore d Department of Community, Occupational and Family Medicine, National University of Singapore, 10 Kent Ridge Crescent, Singapore 0511, Singapore e Department of Pharmacology, National University of Singapore, 10 Kent Ridge Crescent, Singapore 0511, Singapore Accepted 5 March 1996
Abstract
The trace elemental concentrations, including iron, in the substantia nigra (SN) of a 6-OHDA induced rat model of Parkinson's disease were measured using nuclear microscopy. Only rats that exhibited amphetamine induced rotation of more than 7 turns/min were used. The results showed that the iron levels were significantly increased in the 6-OHDA lesioned SN, compared with the intact contralateral SN, and the SN of normal control rats injected with ascorbic acid, which showed no significant difference in iron levels between injected and non-injected sides. In both 6-OHDA lesioned and ascorbic acid injected SN, there were no alterations in the levels of calcium, magnesium, copper and zinc. In the 6-OHDA lesioned SN there was an almost complete loss of tyrosine hydroxylase positive cells in the SN. These results suggested that the 6-OHDA induced dopaminergic cell death may be related to the increased iron. Keywords: Iron; Substantia nigra; 6-Hydroxydopamine; Parkinson's disease; Rat
The pathognomonic feature of Parkinson's disease (PD) is the destruction of the pigmented substantia nigra (SN), particularly the pars compacta (SNc). The cause of dopaminergic cell death in the SN is still unknown. Recent studies on the pathogenesis of PD have centred on the involvement of environmental and endogenous toxins. Oxidative stress and altered iron content are also thought to be important factors [2,3]. A variety of techniques have been used to study the iron concentration in the SN of patients with PD. Some authors demonstrated an increased iron level in the SN of parkinsonian brain with spectrophotometric method and ferrocyanide iron reaction (Perl's stain) [14], inductively coupled plasma spectroscopy [5], magnetic resonance imaging (MRI) [9] or laser microprobe mass analysis [7]. In contrast, some other authors found no significant iron increase in the SN of parkinsonian brains [12,13,16]. In addition to iron, zinc and other trace elements have also been implicated in neurodegenerative diseases [4] and
* Corresponding author. Fax: (65) 777-8427.
conflicting results have been obtained regarding their levels in parkinsonian brain. In view of the above controversies, the present study employed nuclear microscopy to detect some trace metals, especially iron, in the SN of rats injected with 6-hydroxydopamine (6-OHDA), which has long been known to produce an animal model of Parkinson's disease [17]. Nuclear microscopy was used because of its multielemental analytical capability, its high sensitivity to trace elements and its high degree of quantitative accuracy compared with chemical staining and other trace analysis methods [ 18,19]. Male Sprague-Dawley rats(weighing 150-200 g) were anesthetized by 7% chloral hydrate (0.4 m g / g i.p.) injected intraperitoneally. Four Ixl of 0.2% 6-OHDA (Sigma) dissolved in 0.02% ascorbic acid solution was then stereotaxically injected into the right SN at AP - 4 mm, ML - 1 ram, DV - 7 . 5 rnm from bregma, according to the atlas of Paxinos and Watson [10]. Control rats received 4 txl of 0.02% ascorbic acid into the right SN. Two weeks after operation, the rats receiving 6-OHDA or vehicle were tested for rotational response to amphetamine (Sigma, 5 m g / k g , i.p.). Amphetamine induced
0006-8993/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PH S 0 0 0 6 - 8 9 9 3 ( 9 6 ) 0 0 3 1 3-7
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K He et al. / Brain Research 7_{5 (1996) 149-153
Table 1 Trace metal concentrations (parts per million, dry weight) in the substantia nigra (SN) of 6-OHDA lesioned and control rats Rats
Concentration of selected trace metals in SN (ppm/dry weight, mean ± S.E.M.) iron
Lesioned Control
*
right (lesioned) left (unlesioned) right (injected) left (uninjected)
294 234 242 235
± + ± ±
27.6 21.2 26.9 24.9
magnesium
calcium
copper
429 415 602 532
271 266 345 317
23.9 20.2 21.0 21.2
+ ± ± ±
45 35 28 30
+ ± ± +
22.7 30.2 9. I 12.6
+ ± ± +
zinc 1. I 2.7 1.6 1.6
48.0 56.4 67.1 64.9
+ + ± +
4.2 4.8 2.6 3.2
Iron content was significantly increased in the 6-OHDA lesioned right SN. compared with the intact left SN (P < 0.05).
rotations were continuously measured with an automatic counting system (Rota-Count-8, Columbus instruments) for 45 min, and subsequently analyzed by computer. 6-
OHDA lesioned rats showing rotation of more than 7 turns/rain were kept as the successful PD model [4] and control rats were tested once only to demonstrate that no
Fig. 1. PIXE elemental maps showing iron distribution in the substantia nigra (SN) on the lesioned (A) and non-lesioned (B) side. Phosphorus images (C, D) are also included, from which the pinhole markers used to identify the boundaries of the SN can be clearly observed. The images are depicted in false colour, where blue/green represents low concentrations and red/yellow higher concentrations. Scan size is 2 mm × 2 ram.
K He et al./Brain Research 735 (1996) 149-153
rotation was observed. The amphetamine tests were repeated every 3 - 4 months for the turning rats. After survival for 1 year, 6 - O H D A lesioned (n = 7) and control (n = 8) rats were sacrificed by decapitation. The brains were quickly removed and frozen on dry ice and
151
subsequently kept in a freezer at - 7 0 ° C . They were then mounted in a cryostat (Jung Firgocut 2800E) operating at -16°C, and sectioned until the SN. was exposed. Its outline was marked with three pin-holes (using French 30 needles) to facilitate the identification of its boundaries
Fig. 2. Photomicrographs showing an almost complete loss of TH-positive cells in the 6-OHDA lesioned (A, C) substantia nigra (SN), compared with the intact contralateral SN (A, B).
152
Y. He et al./Brain Research 735 (1996) 149-153
during nuclear microscopic analysis (the boundaries of SN were also confirmed on tyrosine hydroxylase-immunostained sections). The sections encompassing both the right and left SN were cut at 20 txm thickness, picked up on freshly made submicron pioloform films mounted on nuclear microscope target holders, and freeze dried. Adjacent sections of 20 Ixm thickness were taken for immunohistochemical studies. The regions of the left and right SN outlined by the pin-holes on each section were scanned, using the NUS Nuclear Microscope operating with a 2 MeV proton beam [18]. The complementary techniques of particle induced X-ray emission (PIXE), Rutherford backscattering (RBS) and scanning transmission ion microscopy (STIM) were simultaneously employed in the analysis. PIXE was used for simultaneous multi-elemental analysis for sodium and upwards in the Periodic Table at the parts per million (ppm) level. RBS was used to provide information on the matrix composition (carbon, nitrogen and oxygen), thickness and density to facilitate normalisation of elemental concentrations. Off-axis STIM was used for structural imaging and fast positioning of the sample. The NUS XYZ manipulator was set at 45 ° with respect to the beam axis in order to maximise the X-ray detection efficiency, and a 400 Ixm perspex filter with 1 mm central hole was used with the 60 mm 2 Si(Li) X-ray detector to optimise the system for the detection of trace elements from iron upwards in the Periodic Table [19]. Multiparameter data were recorded, using listmode data handling techniques built into the PC based OMDAQ data acquisition system [6]. This enabled quantitative analytical data to be extracted from any region of the section, for example, the area encompassing the SN. Sections adjacent to those obtained for iron measurement in the nuclear microscope were processed for immunohistochemistry. The sections were fixed with Stefanini's solution (consisting of a mixture of 2% paraformaldehyde and saturated picric acid, adjusted to pH 7.4) for 15 rain immediately after cutting, washed three times for 5 min in phosphate-buffered saline (PBS) and incubated with a monoclonal mouse anti-tyrosine hydroxylase (TH) antibody (Boehringer Mannheim Biochemica) at a dilution of 1:50 in PBS overnight at room temperature. Thereafter the sections were treated with a biotinylated anti-mouse IgG (Vector) at a dilution of 1:200 in PBS for 1 h at room temperature and subsequently incubated with avidin-biotinylated horseradish peroxidase complex for 1.5 h. The peroxidase was revealed by incubation with a solution of 3,3'-diaminobenzidine tetrahydrochloride (1 m g / 1 ml) containing 0.015% hydrogen peroxide. Trace elemental concentrations (parts per million, dry weight) were extracted from the entire SN in the sections analysed and Table 1 presents the results for selected elements of interest. The results showed that the iron levels were significantly increased in the 6-OHDA lesioned right SN, compared with the intact left SN (paired t-test, P <
0.05). In control animals, there was no significant difference (paired t-test, P > 0.05) in iron levels between the right (injected with ascorbic acid) and left SN. In 6-OHDA lesioned rats there were no differences ( P > 0.05) in the levels of calcium, magnesium, copper and zinc, between the right and left SN. Fig. 1 shows phosphorus images of the lesioned and non-lesioned sides of the brain, from which the pinhole markers outlining the boundaries of the SN are easily identified. Iron images from the same scan are also shown and indicate a slight increase in total iron in the lesioned SN compared with the non-lesioned side. In the 6-OHDA lesioned SN there was an almost complete loss of TH positive cells, compared to the abundance of TH positive cells in the unlesioned SN (Fig. 2). Compared with other conventional methods used in the detection of iron in the brain, nuclear microscopy is a sensitive and efficient way of detecting trace metals and provide quantitative data [19]. Also, the specimens need not be fixed or homogenized. This circumvents the problem of perfusion which may affect the water soluble metal complexes in the brain and the possibility of iron contamination by the perfusate. In addition, both lesioned and control sides in the same section can be studied and compared at the same time. Lastly, the brain prepared for nuclear microscopy can be used for immunohistochemical staining. Using the technique of nuclear microscopy and tyrosine hydroxylase immunohistochemistry, the present study showed a definite increase in the level of iron and a loss of dopaminergic cells in the 6-OHDA lesioned SN. This is in agreement with the results of previous works [5,7,8,14,15] but not those of some others [12,13,16] who could not demonstrate any significant increase in iron content in parkinsonian brains. Oestreicher et al. [8] demonstrated an increase in iron level and a loss of neurons in the SN of rats three weeks after exposure to 6-OHDA. The present results showed that the increase of iron in the SN still existed and the TH-positive cells in the SN were lost almost completely one year after exposure to 6-OHDA. There are suggestions that the death of dopaminergic neurons in the SN of PD may be due to an active toxic process involving highly reactive oxygen species. Oxidative stress has been demonstrated by an increase in basal levels of malondialdehyde, an intermediate index of lipid peroxidation, in the SN of postmortem parkinsonian brains [3], and by the decreased nigral content of reduced glutathione (GSH), a very important antioxidant [ 11]. Iron has been shown to facilitate the production of lipid peroxides and formation of a variety of reactive species, including hydroxyl (the Fenton reaction) and superoxide radicals and hydrogen peroxide, leading to cellular damage [1]. Hence, an increased level of iron in parkinsonian brain may lead to the release of ferric ions which would then stimulate free radical reactions. Dexter and colleagues [4,5] demonstrated reduced copper levels, elevated zinc levels and unchanged manganese
Y. He et al./Brain Research 735 (1996) 149-153
and lead levels, in the S N o f f r o z e n p o s t m o r t e m brain f r o m patients with P a r k i n s o n ' s disease by i n d u c t i v e l y c o u p l e d p l a s m a spectroscopy. Uitti et al. [16], using atomic absorption and atomic e m i s s i o n spectroscopy, s h o w e d reduced c o p p e r levels and u n c h a n g e d zinc levels in the S N o f f o r m a l i n - f i x e d h u m a n parkinsonian brain. The present study, h o w e v e r , s h o w e d no alternations in the levels o f m a g n e s i u m , calcium, c o p p e r and zinc in the 6 - O H D A l e s i o n e d S N o f rats. The different results obtained for iron, zinc, copper, m a g n e s i u m and c a l c i u m levels in the a b o v e studies m a y be due to the different detecting m e t h o d s used and other parameters like the different d e g r e e o f d a m a g e in S N o f parkinsonian brains [8]. It w o u l d appear f r o m the present investigation that a m o n g the various trace metals e x a m i n e d , only increased iron is related to degeneration o f d o p a m i n e r g i c cells in the 6 - O H D A injected SN. Further w o r k needs to be d o n e to establish w h e t h e r the iron a c c u m u l a t i o n in S N is an early or late e v e n t and w h e t h e r the increased iron and iron induced free radical reactions are the cause o f the neurodegeneration or the c o n s e q u e n c e o f a pathological process.
References [1] Ben-Shachar, D., Riederer, P. and Youdim, M.B.H., Iron-melanin interaction and lipid peroxidations: implications for Parkinson's disease, J. Neurochem., 57 (1991) 1609-1614. [2] Ben-Shachar, D., Eshel, G., Finberg, J.P.M. and Youdim, M.B.H., The iron chelator desferrioxamine (Desferal) retards 6-hydroxydopamine-induced degeneration of nigrostriatal dopamine neurons, J. Neurochem., 56 (1991) 1441-1444. [3] Dexter, D.T., Carter, C.J., Wells, F.R., Javoy-Agid, Y., Lees, A., Jenner, P. and Marsden, C.D., Basal liquid peroxidation in substania nigra is increased in Parkinson's disease, J. Neurochem., 52 (1989) 381-389. [4] Dexter, D.T., Wells, F.R., Lees, A.J., Agid, Y., Jenner, P. and Marsden, C.D., Increased nigral iron content and alterations in other metal ions occurring in brain in Parkinson's disease, J. Neurochem., 52 (1989) 1830-1836. [5] Dexter, D.T., Carayon, A., Javoy-Agid, F., Agid Y., Wells, F.R., Daniel, S.E., Lees A.J., Jenner, P. and Marsden, C.D., Alterations in the levels of iron, ferritin and other trace metals in Parkinson's disease and other neurodegenerative diseases affecting the basal ganglia, Brain, 114 (1991) 1953-1975. [6] Grime, G.W., Dawson, M., A PC-based data acquisition package for
[7]
[8]
[9]
[10] [11]
[12]
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nuclear microbeam systems, Nucl. Instr. Methods, B89 (1994) 223228. Good, P.F., Olanow, C.W. and Perl, D.P., Neuromelanin-containing neurons of the substantia nigra accumulate iron and aluminum in Parkinson's disease: a LAMMA study, Brain Res., 593 (1992) 343-346. Oestreicher, E., Sengstock, G.J., Riederer, P., Olanow, C.W., Dunn, A.J. and Al:endash, G.W., Degeneration of nigrostriatal dopaminergic neurons increases iron within the substantia nigra: a histochemical and neurochemical study, Brain Res., 660 (1994) 8-18. Olanow, C.W. and Drayer, B., Brain iron: MRI studies in Parkinson's syndrome. In S. Fahn, C.D. Marsden, D. Calne and M. Goldstein (Eds.), Recent Developments in Parkinson's Disease, Vol. H, Macmillan Healthcare Information, Florham Park, New Jersey, 1987, pp. 135-143. Paxinos, G. and Watson, C. The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1986. Perry, T.L. and Yong, V.W., Idiopathic Parkinson's disease, progressive supraunclear palsy and glutathione metabolism in the substantia nigra of patients, Neurosci. Lett., 67 (1986) 267-274. Riederer, P., Sofic, E., Rausch, W.D., Kruzik, P. and Youdim, M.B.H., Dopaminforschung heute und morgen-L-DOPA in der Zukunft. In In P. Riederer and P. Umek (Eds.), L-DOPA-Substitution der Parkinson-Krankheit, Geschichte- Gegenwart- Zukunft,
Springer, New York, 1985, pp, 127-144. [13] Riderer, P., Sofic, E., Rausch, W.D., Schmidt, B., Reynolds, G.P., Jellinger, K. and Adam, M.B.H., Transition metals, ferritin, glutathione, and ascorbic acid in parkinsonian brains, J. Neurochem., 52 (1989) 515-520. [14] Sofic, E., Paulus, W., Jellinger, K., Riederer, P. and Youdim, M.B.H., Selective increase of iron in substantia nigra zona compacta of parkinsonian brains, J. Neurochem., 56 (1991) 978-982. [15] Stephen, B.D., Ian, Q.W., Derek, C.R. and Graham, H.J., Dopamine-rich grafts ameliorate whole body motor asymmetry and sensory neglect but not independent limb use in rats with 6-hydroxydopamine lesions, Brain Res., 415 (1987) 63-78. [16] Uitti, R.J., Rajput, A.H., Rozdilsky, B., Bickis, M., Wollin, T. and Yuen, W.K., Regional metal concentrations in Parkinson's disease, other chronic neurological disease, and control brains, Can. J. Neurol. Sci., 16 (1989) 310-314. [17] Ungerstedt, U., 6-Hydroxy-dopamine induced degeneration of central monoamine neurons, Eur. J. PharmacoL, 5 (1968) 107-110. [18] Watt, F., Orlic, I., Loh, K.K., Sow, C.H., Thong, P., Liew, S.C., Osipowicz, T., Choo, T.F., Tang, S.M., The National University of Singapore nuclear microscope facility, Nucl. Instr. Methods, B85 (1994) 708-715. [19] Watt, F. and Grime, G.W., The high energy ion microprobe. In In S.A.E. Johansson, J.L. Campbell and K.G. Malmqvist (Eds.), Particle Induced X-ray Emission Spectrometry, Wiley, New York, 1995, pp. 101-165.
Brain Research 735 (1996) 149-153
Short communication
Increased iron in the substantia nigra of 6-OHDA induced parkinsonian rats: a nuclear microscopy study Y. H e a, P . S . P . T h o n g b, T. L e e a, * , S . K . L e o n g c, C . Y . S h i d, P . T . H . W o n g
e, S . Y . Y u a n a, F. W a t t b
a Department of Surgery, National University of Singapore, 10 Kent Ridge Crescent, Singapore 0511, Singapore b Department of Physics, National Universi~ of Singapore, 10 Kent Ridge Crescent, Singapore 0511, Singapore c Department of Anatomy, National University of Singapore, 10 Kent Ridge Crescent, Singapore 0511, Singapore d Department of Community, Occupational and Family Medicine, National University of Singapore, 10 Kent Ridge Crescent, Singapore 0511, Singapore e Department of Pharmacology, National University of Singapore, 10 Kent Ridge Crescent, Singapore 0511, Singapore Accepted 5 March 1996
Abstract
The trace elemental concentrations, including iron, in the substantia nigra (SN) of a 6-OHDA induced rat model of Parkinson's disease were measured using nuclear microscopy. Only rats that exhibited amphetamine induced rotation of more than 7 turns/min were used. The results showed that the iron levels were significantly increased in the 6-OHDA lesioned SN, compared with the intact contralateral SN, and the SN of normal control rats injected with ascorbic acid, which showed no significant difference in iron levels between injected and non-injected sides. In both 6-OHDA lesioned and ascorbic acid injected SN, there were no alterations in the levels of calcium, magnesium, copper and zinc. In the 6-OHDA lesioned SN there was an almost complete loss of tyrosine hydroxylase positive cells in the SN. These results suggested that the 6-OHDA induced dopaminergic cell death may be related to the increased iron. Keywords: Iron; Substantia nigra; 6-Hydroxydopamine; Parkinson's disease; Rat
The pathognomonic feature of Parkinson's disease (PD) is the destruction of the pigmented substantia nigra (SN), particularly the pars compacta (SNc). The cause of dopaminergic cell death in the SN is still unknown. Recent studies on the pathogenesis of PD have centred on the involvement of environmental and endogenous toxins. Oxidative stress and altered iron content are also thought to be important factors [2,3]. A variety of techniques have been used to study the iron concentration in the SN of patients with PD. Some authors demonstrated an increased iron level in the SN of parkinsonian brain with spectrophotometric method and ferrocyanide iron reaction (Perl's stain) [14], inductively coupled plasma spectroscopy [5], magnetic resonance imaging (MRI) [9] or laser microprobe mass analysis [7]. In contrast, some other authors found no significant iron increase in the SN of parkinsonian brains [12,13,16]. In addition to iron, zinc and other trace elements have also been implicated in neurodegenerative diseases [4] and
* Corresponding author. Fax: (65) 777-8427.
conflicting results have been obtained regarding their levels in parkinsonian brain. In view of the above controversies, the present study employed nuclear microscopy to detect some trace metals, especially iron, in the SN of rats injected with 6-hydroxydopamine (6-OHDA), which has long been known to produce an animal model of Parkinson's disease [17]. Nuclear microscopy was used because of its multielemental analytical capability, its high sensitivity to trace elements and its high degree of quantitative accuracy compared with chemical staining and other trace analysis methods [ 18,19]. Male Sprague-Dawley rats(weighing 150-200 g) were anesthetized by 7% chloral hydrate (0.4 m g / g i.p.) injected intraperitoneally. Four Ixl of 0.2% 6-OHDA (Sigma) dissolved in 0.02% ascorbic acid solution was then stereotaxically injected into the right SN at AP - 4 mm, ML - 1 ram, DV - 7 . 5 rnm from bregma, according to the atlas of Paxinos and Watson [10]. Control rats received 4 txl of 0.02% ascorbic acid into the right SN. Two weeks after operation, the rats receiving 6-OHDA or vehicle were tested for rotational response to amphetamine (Sigma, 5 m g / k g , i.p.). Amphetamine induced
0006-8993/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PH S 0 0 0 6 - 8 9 9 3 ( 9 6 ) 0 0 3 1 3-7
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K He et al. / Brain Research 7_{5 (1996) 149-153
Table 1 Trace metal concentrations (parts per million, dry weight) in the substantia nigra (SN) of 6-OHDA lesioned and control rats Rats
Concentration of selected trace metals in SN (ppm/dry weight, mean ± S.E.M.) iron
Lesioned Control
*
right (lesioned) left (unlesioned) right (injected) left (uninjected)
294 234 242 235
± + ± ±
27.6 21.2 26.9 24.9
magnesium
calcium
copper
429 415 602 532
271 266 345 317
23.9 20.2 21.0 21.2
+ ± ± ±
45 35 28 30
+ ± ± +
22.7 30.2 9. I 12.6
+ ± ± +
zinc 1. I 2.7 1.6 1.6
48.0 56.4 67.1 64.9
+ + ± +
4.2 4.8 2.6 3.2
Iron content was significantly increased in the 6-OHDA lesioned right SN. compared with the intact left SN (P < 0.05).
rotations were continuously measured with an automatic counting system (Rota-Count-8, Columbus instruments) for 45 min, and subsequently analyzed by computer. 6-
OHDA lesioned rats showing rotation of more than 7 turns/rain were kept as the successful PD model [4] and control rats were tested once only to demonstrate that no
Fig. 1. PIXE elemental maps showing iron distribution in the substantia nigra (SN) on the lesioned (A) and non-lesioned (B) side. Phosphorus images (C, D) are also included, from which the pinhole markers used to identify the boundaries of the SN can be clearly observed. The images are depicted in false colour, where blue/green represents low concentrations and red/yellow higher concentrations. Scan size is 2 mm × 2 ram.
K He et al./Brain Research 735 (1996) 149-153
rotation was observed. The amphetamine tests were repeated every 3 - 4 months for the turning rats. After survival for 1 year, 6 - O H D A lesioned (n = 7) and control (n = 8) rats were sacrificed by decapitation. The brains were quickly removed and frozen on dry ice and
151
subsequently kept in a freezer at - 7 0 ° C . They were then mounted in a cryostat (Jung Firgocut 2800E) operating at -16°C, and sectioned until the SN. was exposed. Its outline was marked with three pin-holes (using French 30 needles) to facilitate the identification of its boundaries
Fig. 2. Photomicrographs showing an almost complete loss of TH-positive cells in the 6-OHDA lesioned (A, C) substantia nigra (SN), compared with the intact contralateral SN (A, B).
152
Y. He et al./Brain Research 735 (1996) 149-153
during nuclear microscopic analysis (the boundaries of SN were also confirmed on tyrosine hydroxylase-immunostained sections). The sections encompassing both the right and left SN were cut at 20 txm thickness, picked up on freshly made submicron pioloform films mounted on nuclear microscope target holders, and freeze dried. Adjacent sections of 20 Ixm thickness were taken for immunohistochemical studies. The regions of the left and right SN outlined by the pin-holes on each section were scanned, using the NUS Nuclear Microscope operating with a 2 MeV proton beam [18]. The complementary techniques of particle induced X-ray emission (PIXE), Rutherford backscattering (RBS) and scanning transmission ion microscopy (STIM) were simultaneously employed in the analysis. PIXE was used for simultaneous multi-elemental analysis for sodium and upwards in the Periodic Table at the parts per million (ppm) level. RBS was used to provide information on the matrix composition (carbon, nitrogen and oxygen), thickness and density to facilitate normalisation of elemental concentrations. Off-axis STIM was used for structural imaging and fast positioning of the sample. The NUS XYZ manipulator was set at 45 ° with respect to the beam axis in order to maximise the X-ray detection efficiency, and a 400 Ixm perspex filter with 1 mm central hole was used with the 60 mm 2 Si(Li) X-ray detector to optimise the system for the detection of trace elements from iron upwards in the Periodic Table [19]. Multiparameter data were recorded, using listmode data handling techniques built into the PC based OMDAQ data acquisition system [6]. This enabled quantitative analytical data to be extracted from any region of the section, for example, the area encompassing the SN. Sections adjacent to those obtained for iron measurement in the nuclear microscope were processed for immunohistochemistry. The sections were fixed with Stefanini's solution (consisting of a mixture of 2% paraformaldehyde and saturated picric acid, adjusted to pH 7.4) for 15 rain immediately after cutting, washed three times for 5 min in phosphate-buffered saline (PBS) and incubated with a monoclonal mouse anti-tyrosine hydroxylase (TH) antibody (Boehringer Mannheim Biochemica) at a dilution of 1:50 in PBS overnight at room temperature. Thereafter the sections were treated with a biotinylated anti-mouse IgG (Vector) at a dilution of 1:200 in PBS for 1 h at room temperature and subsequently incubated with avidin-biotinylated horseradish peroxidase complex for 1.5 h. The peroxidase was revealed by incubation with a solution of 3,3'-diaminobenzidine tetrahydrochloride (1 m g / 1 ml) containing 0.015% hydrogen peroxide. Trace elemental concentrations (parts per million, dry weight) were extracted from the entire SN in the sections analysed and Table 1 presents the results for selected elements of interest. The results showed that the iron levels were significantly increased in the 6-OHDA lesioned right SN, compared with the intact left SN (paired t-test, P <
0.05). In control animals, there was no significant difference (paired t-test, P > 0.05) in iron levels between the right (injected with ascorbic acid) and left SN. In 6-OHDA lesioned rats there were no differences ( P > 0.05) in the levels of calcium, magnesium, copper and zinc, between the right and left SN. Fig. 1 shows phosphorus images of the lesioned and non-lesioned sides of the brain, from which the pinhole markers outlining the boundaries of the SN are easily identified. Iron images from the same scan are also shown and indicate a slight increase in total iron in the lesioned SN compared with the non-lesioned side. In the 6-OHDA lesioned SN there was an almost complete loss of TH positive cells, compared to the abundance of TH positive cells in the unlesioned SN (Fig. 2). Compared with other conventional methods used in the detection of iron in the brain, nuclear microscopy is a sensitive and efficient way of detecting trace metals and provide quantitative data [19]. Also, the specimens need not be fixed or homogenized. This circumvents the problem of perfusion which may affect the water soluble metal complexes in the brain and the possibility of iron contamination by the perfusate. In addition, both lesioned and control sides in the same section can be studied and compared at the same time. Lastly, the brain prepared for nuclear microscopy can be used for immunohistochemical staining. Using the technique of nuclear microscopy and tyrosine hydroxylase immunohistochemistry, the present study showed a definite increase in the level of iron and a loss of dopaminergic cells in the 6-OHDA lesioned SN. This is in agreement with the results of previous works [5,7,8,14,15] but not those of some others [12,13,16] who could not demonstrate any significant increase in iron content in parkinsonian brains. Oestreicher et al. [8] demonstrated an increase in iron level and a loss of neurons in the SN of rats three weeks after exposure to 6-OHDA. The present results showed that the increase of iron in the SN still existed and the TH-positive cells in the SN were lost almost completely one year after exposure to 6-OHDA. There are suggestions that the death of dopaminergic neurons in the SN of PD may be due to an active toxic process involving highly reactive oxygen species. Oxidative stress has been demonstrated by an increase in basal levels of malondialdehyde, an intermediate index of lipid peroxidation, in the SN of postmortem parkinsonian brains [3], and by the decreased nigral content of reduced glutathione (GSH), a very important antioxidant [ 11]. Iron has been shown to facilitate the production of lipid peroxides and formation of a variety of reactive species, including hydroxyl (the Fenton reaction) and superoxide radicals and hydrogen peroxide, leading to cellular damage [1]. Hence, an increased level of iron in parkinsonian brain may lead to the release of ferric ions which would then stimulate free radical reactions. Dexter and colleagues [4,5] demonstrated reduced copper levels, elevated zinc levels and unchanged manganese
Y. He et al./Brain Research 735 (1996) 149-153
and lead levels, in the S N o f f r o z e n p o s t m o r t e m brain f r o m patients with P a r k i n s o n ' s disease by i n d u c t i v e l y c o u p l e d p l a s m a spectroscopy. Uitti et al. [16], using atomic absorption and atomic e m i s s i o n spectroscopy, s h o w e d reduced c o p p e r levels and u n c h a n g e d zinc levels in the S N o f f o r m a l i n - f i x e d h u m a n parkinsonian brain. The present study, h o w e v e r , s h o w e d no alternations in the levels o f m a g n e s i u m , calcium, c o p p e r and zinc in the 6 - O H D A l e s i o n e d S N o f rats. The different results obtained for iron, zinc, copper, m a g n e s i u m and c a l c i u m levels in the a b o v e studies m a y be due to the different detecting m e t h o d s used and other parameters like the different d e g r e e o f d a m a g e in S N o f parkinsonian brains [8]. It w o u l d appear f r o m the present investigation that a m o n g the various trace metals e x a m i n e d , only increased iron is related to degeneration o f d o p a m i n e r g i c cells in the 6 - O H D A injected SN. Further w o r k needs to be d o n e to establish w h e t h e r the iron a c c u m u l a t i o n in S N is an early or late e v e n t and w h e t h e r the increased iron and iron induced free radical reactions are the cause o f the neurodegeneration or the c o n s e q u e n c e o f a pathological process.
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