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Attenuation of 6-hydroxydopamine-induced dopaminergic nigrostriatal lesions in superoxide dismutase transgenic mice
Pergamon
PII: S0306-4522(97)00665-9
Neuroscience Vol. 85, No. 3, pp. 907–917, 1998 IBRO Published by Elsevier Science Ltd Printed in Great Britain
ATTENUATION OF 6-HYDROXYDOPAMINE-INDUCED DOPAMINERGIC NIGROSTRIATAL LESIONS IN SUPEROXIDE DISMUTASE TRANSGENIC MICE M. ASANUMA, H. HIRATA and J. L. CADET* Molecular Neuropsychiatry Section, NIH/NIDA, Division of Intramural Research, Baltimore, MD 21224, U.S.A. Abstract––6-Hydroxydopamine is a neurotoxin that produces degeneration of the nigrostriatal dopaminergic pathway in rodents. Its toxicity is thought to involve the generation of superoxide anion secondary to its autoxidation. To examine the effects of the overexpression of Cu,Zn-superoxide dismutase activity on 6-hydroxydopamine-induced dopaminergic neuronal damage, we have measured the effects of 6-hydroxydopamine on striatal and nigral dopamine transporters and nigral tyrosine hydroxylaseimmunoreactive neurons in Cu,Zn-superoxide dismutase transgenic mice. Intracerebroventricular injection of 6-hydroxydopamine (50 µg) in non-transgenic mice produced reductions in the size of striatal area and an enlargement of the cerebral ventricle on both sides of the brains of mice killed two weeks after the injection. In addition, 6-hydroxydopamine caused marked decreases in striatal and nigral [125I]RTI-121labelled dopamine transporters not only on the injected side but also on the non-injected side of non-transgenic mice; this was associated with decreased cell number and size of tyrosine hydroxylaseimmunoreactive dopamine neurons in the substantia nigra pars compacta on both sides in these mice. In contrast, superoxide dismutase transgenic mice were protected against these neurotoxic effects of 6-hydroxydopamine, with the homozygous transgenic mice showing almost complete protection. These results provide further support for a role of superoxide anion in the toxic effects of 6-hydroxydopamine. They also provide further evidence that reactive oxygen species may be the main determining factors in the neurodegenerative effects of catecholamines. Key words: 6-hydroxydopamine, superoxide dismutase, transgenic mice, superoxide anion, dopamine transporter, Parkinson’s disease.
Parkinson’s disease is a neurodegenerative disorder that is characterized by dopamine (DA) depletion in the basal ganglia and DA cell death in the substantia nigra (SN). Although the mechanism for the development of the disorder has not been completely clarified, oxygen-based free radicals are thought to play an important role in the pathogenesis of Parkinson’s disease.1,18,25,26,37 This idea is supported by evidence showing increases in lipid peroxidation and decreases in the levels of free radical scavenging enzymes such as glutathione peroxidase and catalase in the brains of patients with Parkinson’s disease.2,29 It is also supported by the presence of increased iron deposits in the SN pars compacta (SNpc),22,39 because iron can lead to the formation of free radicals via interaction with hydrogen peroxide *To whom correspondence should be addressed. Abbreviations: DA, dopamine; DAB, 3,3 -diaminobenzidine; DAT, dopamine transporter; GBR-12909, 1-[2[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylprophyl)piperazine; O2, superoxide anion; •OH, hydroxyl radical; 6-OHDA, 6-hydroxydopamine; RTI-121, 3â-[4(trimethylstannyl)phenyl]-tropan-2â-carboxylic acid isopropyl ester; SN, substantia nigra; SNpc, SN pars compacta; SOD, superoxide dismutase; TBS, Trisbuffered saline; TH, tyrosine hydroxylase.
(H2O2).1,18,25,37 Other supporting evidence for a role of free radicals in the developmental course of Parkinson’s disease includes abnormal activity of mitochondrial complex I.6,32 Furthermore, several toxins used to create animal models of Parkinson’s disease might produce their lesions through the production of free radicals.16,19,27,41 One such toxin is 6-hydroxydopamine (6-OHDA) which has provided useful animal models of the destruction of nigrostriatal DA systems.44 6-OHDA is a neurotoxin that produces degeneration of DA neurons in the SNpc and damage of their nerve endings in the striatum.5,8,44 This can be accomplished by intranigral44 or intrastriatal injections5,13 in rats or intracerebroventricular (i.c.v.) injections in mice.8,30,35 The toxic effects of 6-OHDA have also been examined in vitro using dopaminergic cell cultures.31 The accumulated evidence both in vitro and in vivo suggests that the toxicity of 6-OHDA involves the generation of reactive oxygen species, since 6-OHDA can autoxidize to form semiquinone and superoxide anion (O2) which can subsequently be converted to the more cytotoxic hydroxyl radical (•OH) through interaction with H2O2.19,27 The role of free radicals in 6-OHDAinduced toxicity is also supported by reports that
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Fig. 1. Representative photomicrographs of Haematoxylin–Eosin-stained striatal sections of nontransgenic (A, B), heterozygous (C, D) and homozygous (E, F) SOD-transgenic mice. Animals received intracerebroventricular injection of saline (A, C, E) or 50 µg of 6-OHDA (B, D, F). 6-OHDA-injected non-transgenic mice had marked enlargement of lateral cerebral ventricles and severely shrunken striatal area two weeks after the injection (B). These 6-OHDA-induced atrophic changes were attenuated in the heterozygous (D) and the homozygous (F) SOD-transgenic mice.
in vitro treatment with reactive oxygen species-related enzymes protected cells against 6-OHDA-induced cell death43 and that in vivo treatment with iron chelator inhibited the 6-OHDA-induced DA depletion in the striatum.4 In order to assess the possible role of O2 in the in vivo toxic effects of 6-OHDA, we have made use of heterozygous and homozygous Cu,Zn-superoxide dismutase (SOD) transgenic mice and examined its effects on striatal and nigral dopamine transporters
(DAT) and on nigral tyrosine hydroxylase (TH)-positive cells following i.c.v. injection of the toxin. EXPERIMENTAL PROCEDURES
Chemicals [125I]RTI-121, radioiodinated 3â-[4-(trimethylstannyl)phenyl]-tropan-2â-carboxylic acid isopropyl ester was purchased from New England Nuclear (Boston, MA).
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Fig. 2. Effects of the intracerebroventricular injection of 6-OHDA on striatal area of mice. Striatal area (relative unit) was measured on the Haematoxylin–Eosin-stained sections as described under Experimental Procedures. The value is shown as mean percentage of each saline-injected groupS.E.M. of five animals per each group. **P<0.01 compared with the same side of each saline-treated group. #P<0.05, ##P<0.01 compared with the same side of 6-OHDA-injected non-transgenic (Tg) group (Mann–Whitney U-test).
GBR-12909, 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3phenyl-prophyl)piperazine dihydrochloride was purchased from Research Biochemicals International (Natick, MA). 6-OHDA hydrobromide salt and desipramine were obtained from Sigma (St Louis, MO). Other chemicals were purchased from Mallinckrodt Specialty Chemicals (Paris, KY). Animals and 6-hydroxydopamine lesions Male heterozygous and homozygous SOD-transgenic mice of strain 218/3 (12–14-weeks-old) carrying the complete human Cu,Zn-SOD gene were used in the present experiment; they show respective increases of 3.6- and 5.0-fold in striatal Cu,Zn-SOD enzyme activity when compared to non-transgenic controls (J. Subramaniam and J. L. Cadet, unpublished observation). The production of these animals has been previously described.23 All animal use procedures were according to the NIH Guide for the Care and Use of Laboratory Animals and were approved by the local Animal Care Committee. Thirty minutes prior to 6-OHDA injection, all animals were administered with desipramine (25 mg/kg, i.p.) to protect noradrenaline-containing neurons.36 Under anaesthesia with pentobarbital (50 mg/kg, i.p.), mice were injected with 50 µg of 6-OHDA (dissolved in 2 µl physiological saline containing 0.05% ascorbic acid) or the same volume of saline with ascorbic acid into the right cerebral ventricle via a 5-µl Hamilton microsyringe. The following coordinates were used: AP, +0.3 mm; ML, 1.0 mm; DV, 3.0 mm from bregma. Tissue preparation Two weeks after the injection, mice were killed by decapitation. This time was chosen because previous experiments have shown that the effects of 6-OHDA in mice are most prominent around one to two weeks after i.c.v. injection of similar doses of the toxin.35,36 Their brains were quickly removed, frozen in isopentane on dry ice, and stored at 70C until use. Coronal sections (20 µm-thick) were cut in a cryostat at 20C and thaw-mounted onto gelatin-coated glass slides, dried under negative pressure and then stored at 70C until used in the studies described below. To assess the regional distribution of the lesion, sections were taken at the level of the mid-striatum (+1.0+0.6 mm from
bregma) and the SN (2.83.0 mm from bregma) of each brain.42 For measurement of striatal area, Haematoxylin–Eosin (HE) staining was performed. Autoradiographic binding assay Binding assays for DAT were performed according to our previously reported method28 with minor modifications of a previous protocol7 that uses the cocaine analogue, [125I]RTI-121. This compound selectively binds to DAT but not to other monoamine transporters in the striatum.7 Briefly, slide-mounted sections were incubated for 60 min at room temperature with 100,000 c.p.m./ml of [125I]RTI-121 (specific activity, 2200 Ci/mmol) using a binding buffer consisting 137.0 mM NaCl, 2.7 mM KCl, 10.14 mM Na2HPO4, 1.76 mM KH2PO4 and 10.0 mM NaI. Specific binding was determined in the presence of 10 µM GBR12909 and represented greater than 90% of total binding. At the end of the incubation, the slides were washed twice in fresh binding buffer for 20 min at room temperature, dipped in ice-cold distilled water and dried under cool air stream. The slides were then apposed to radiosensitive films (Hyperfilm âmax, Amersham, Arlington Heights, IL) with a plastic standard (125I microscales, Amersham, U.K.) for three days at 4C. Immunohistochemistry Immunohistochemistry for TH was performed using nontransgenic and homozygous SOD-transgenic mice according to standard techniques. Slides were warmed up to room temperature and fixed in 4% paraformaldehyde (in 0.1 M phosphate buffer) for 10 min. Sections were then washed 415 min in 50 mM Tris-buffered saline (TBS; pH 7.6) before being incubated in 1% H2O2 and 100% methanol for 10 min. After washing with TBS (45 min), the slidemounted sections were incubated in 3% normal goat serum and 0.2% Triton X-100 in TBS for 1 h, and then exposed to a rabbit polyclonal antibody (diluted 1:20,000 in TBS containing 1% normal goat serum) specific for TH (Eugene Tech International, Ridgefield Park, NJ) for 18 h at room temperature. After incubation with the primary antibody, sections were washed for 35 min in 50 mM TBS before being incubated with biotinylated goat anti-rabbit secondary IgG (diluted 1:200 in TBS; Vector Laboratories, Burlingame, CA) for 1.5 h at room temperature. Following
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Fig. 3. Representative autoradiograms of striatal [125I]RTI-121-labelled DAT of non-transgenic (A, B), heterozygous (C, D) and homozygous (E, F) SOD-transgenic mice injected with saline (A, C, E) or 6-OHDA (B, D, F). Striatal [125I]RTI-121 binding on both sides was markedly decreased two weeks after the injection of 6-OHDA in the non-transgenic mice (B). These 6-OHDA-induced reductions of DAT levels were attenuated in the heterozygous SOD-transgenic mice (D) and almost completely prevented in the homozygous SOD-transgenic mice (F). Quantitative data are given in Fig. 4. washes in TBS (415 min), the sections were incubated with ABC reagents (Vector Laboratories) for 2 h. THimmunopositive neurons were visualized by 3,3 diaminobenzidine (DAB), nickel and H2O2 using a commercially available kit (DAB substrate kit, Vector Laboratories). The counterstaining of nuclei was then performed with 1% Methyl Green. Negative controls were run using normal goat serum instead of the primary antibody. Semiquantitative image analyses Striatal area on the HE-stained slides was measured by an image analysis software (Image 1.56, NIH) run on a Macintosh computer. For each coronal slice, each side of the striatum on the digitized image was separately outlined with a screen cursor driven by a hand-held mouse and relative size units were measured. The autoradiograms for [125I]RTI-121 binding were analysed using a Macintosh computer-based image analysis
system (Image 1.56, NIH). Within each slice, the striatum and SN were defined according to a mouse brain atlas.42 Each region on the injected side (right) or the non-injected side (left) was outlined with a screen cursor driven by a hand-held mouse as described above. Optical densities in the striatum and SN were quantified and converted to nCi/mg tissue using standard curve (125I microscales) exposed at the same time with the brain sections. TH-immunoreactive neurons in the SNpc were counted manually on two to three slices per each animal (n=6 mice/group) using a Zeiss microscope at a magnification of 400 with a superimposed grid. The boundary between the SNpc and ventral tegmental area was defined as a line extending dorsally from the most medial boundary of the cerebral peduncle. Counting was performed blindly. Cell size and relative density of the TH-positive neurons in the SNpc were assessed on six to nine TH-positive cells per each animal (n=6 mice/group) using the microscope at a magnification of 630 and a Macintosh computer-based image
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Fig. 4. Changes in striatal DAT levels in SOD-transgenic (Tg) mice following the intracerebroventricular injection with 6-OHDA (50 µg). Striatal [125I]RTI-121 binding (nCi/mg tissue) was measured as described under Experimental Procedures. The values represent mean percentages of each saline-injected groupS.E.M. (four or five animals/group). There were no significant differences between the salineinjected mice of the three strains. *P<0.05 compared with the same side from each saline-treated group. # P<0.05 compared with the same side from non-transgenic+6-OHDA group (Mann–Whitney U-test).
analysis system (Image 1.56, NIH). Only TH-positive neurons with a complete nucleus in the section were assessed and outlined with the screen cursor driven by a hand-held mouse. This allowed for the measurement of neuron size and relative density of TH-staining. Relative density of TH-immunoreactive fibres was also measured in two representative upper areas of the striatum (500 µm500 µm) per each mouse (n=6 mice/group) using the microscope at a magnification of 400 and Image 1.56, NIH. Statistical analyses For each group, raw data on the injected side and the non-injected side were compared using a two-tailed Student’s t-test for paired samples. Statistical analyses for these indices expressed as percentages were done using Mann–Whitney U-test for two paired group. The values for number or size of TH-positive neurons in the SNpc of each side were analysed using one-way ANOVA followed by Fisher’s PLSD post hoc test. RESULTS
Effects of 6-hydroxydopamine on striatal area in mice Representative striatal sections for HE staining are shown in Fig. 1. In non-transgenic mice, i.c.v. injection of 50 µg of 6-OHDA caused marked reductions in the size of the striatum with associated enlargement of the lateral cerebroventricle not only on the injected side but on the non-injected side as well (Fig. 1B). These numbers in the non-transgenic mice correspond to 75.136.26% and 77.023.54% of control on the injected and non-injected side, respectively (Fig. 2). Neither the heterozygous nor the homozygous SOD-transgenic mice showed any significant decreases (Figs 1D, F, 2). The homozygous SOD-transgenic animals were almost completely protected against 6-OHDA-induced striatal atrophy (Fig. 2).
Effects of 6-hydroxydopamine injection on striatal and nigral dopamine transporter 6-OHDA caused significant decreases in [125I]RTI121-labelled DAT in the striatum (Figs 3, 4). For example, in the non-transgenic mice, [125I]RTI-121 binding on both sides of the striatum was markedly decreased two weeks after the injection of 6-OHDA; these consisted of decreases of 37.61% and 35.34% on the injected and non-injected sides, respectively (Figs 3A, B, 4). These reductions of striatal DAT were significantly attenuated on the non-injected side but not on the injected side (29.12%) of the heterozygous SOD-transgenic mice (Figs 3D, 4), whereas they were significantly attenuated on both sides in the homozygous SODtransgenic mice (Figs 3F, 4). DAT levels in the SN were also decreased two weeks after the 6-OHDA injection (Figs 5, 6). These decreases in nigral [125I]RTI-121 binding were observed on both sides (51.37% and 37.41% for the injected and non-injected sides, respectively) of the brains of non-transgenic mice (Figs 5B, 6). DAT levels were also significantly decreased (40.44%) on the injected side of the brains of heterozygous SOD-transgenic mice (Figs 5D, 6). The SNpc of homozygous SOD-transgenic mice were not affected by 6-OHDA (Figs 5F, 6). Tyrosine hydroxylase immunohistochemistry in substantia nigra pars compacta Figure 7 shows representative photomicrographs of TH-immunoreactive neurons in the SNpc of nontransgenic and homozygous SOD-transgenic mice. The quantitative data are shown in Table 1. 6-OHDA
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Fig. 5. Representative autoradiograms of [125I]RTI-121-labelled DAT in the SN of non-transgenic (A, B), heterozygous (C, D) and homozygous (E, F) SOD-transgenic mice two weeks after the injection with saline (A, C, E) or 6-OHDA (B, D, F). The 6-OHDA injection caused decreases in [125I]RTI-121 binding in the SN of the non-transgenic (B) and the heterozygous SOD-transgenic (D) mice, whereas no apparent reductions of DAT in the SN were observed in the homozygous SOD-transgenic mice after the 6-OHDA injection (F). Quantitative data are given in Fig. 6.
injection significantly reduced the number of THpositive neurons in the SNpc of the non-transgenic mice on both sides of the brain (29.94% of salineinjected group on the injected side; 41.14% of saline controls on the non-injected side; Fig. 7B, Table 1). The size of TH-positive neurons in the SNpc was also reduced on both sides (68.06% and 68.79% of salineinjected group on the injected and non-injected sides, respectively) of the brains of non-transgenic mice by the i.c.v. injection of 6-OHDA (Table 1). Homozygous SOD-transgenic mice were almost completely protected from these effects of 6-OHDA (Fig. 7D, Table 1). 6-OHDA also caused small but statisti-
cally significant increases in the relative density of TH-staining in the SNpc on both sides (+6.65% and +7.50% for the injected and non-injected sides, respectively) of the brains of non-transgenic mice, whereas there was no change on either side in the homozygous SOD-transgenic mice. In non-transgenic mice treated with 6-OHDA injection, significant decreases in relative density of striatal TH-immunoreactive fibres were observed on both sides (13.96% and 7.97% for the injected and non-injected sides, respectively), whereas no significant changes were observed on neither side of the brains of homozygous SOD-transgenic mice.
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Fig. 6. Effects of the 6-OHDA injection on DAT levels in the SN of SOD-transgenic (Tg) mice. [125I]RTI-121 binding (nCi/mg tissue) in the SN was measured as described above. The values represent mean percentages of each saline-injected groupS.E.M. of four or five animals per each group. There were no significant differences in the saline-injected mice of the three strains. *P<0.05 compared with the same side from each saline-treated group. #P<0.05 compared with the same side from non-transgenic+ 6-OHDA group (Mann–Whitney U-test).
DISCUSSION
The main findings of this paper are that (i) SODtransgenic mice are protected against atrophic changes and the reduction of TH-positive fibres caused by 6-OHDA in mice striata; (ii) they show less decreases in striatal and nigral DAT levels caused by the toxin; and (iii) they are also protected against the loss and shrinkage of nigral TH-positive cell bodies. The occurrence of these changes on both sides of the striatum is consistent with the marked bilateral decreases in DA levels previously reported one to two weeks after a similar i.c.v. injections of 6-OHDA (20– 40 µg) to mice.8,30,35,36 These data are also consistent with dose-dependent reductions of [3H]mazindollabelled DAT in the striatum after unilateral intrastriatal injection of 6-OHDA to rats.15,38 In the present study, the changes in all indices examined on the injected side were not significantly different from those of the contralateral side, thus suggesting that 6-OHDA injected on one side of the ventricle had diffused to the contralateral side. The attenuation observed in the heterozygous SOD-transgenic and the almost complete protection seen in the homozygous SOD-transgenic mice suggest that O2 production must play an important role in 6-OHDA-induced toxicity in vivo. This role of O2 in the toxic effects of 6-OHDA is also supported by the previous report that i.c.v. injection of 6-OHDA in mice resulted in acute but transient increases in Cu,Zn-SOD activity and lipid peroxidation in the striatum.34 This discussion is also supported by the observation that administration of the DA receptor agonist, pergolide, which caused increases in striatal Cu,Zn-SOD activity17 was also neuroprotective against the toxic effects caused by the i.c.v. injection of 6-OHDA.3 Further support for this idea is pro-
vided by the observation that pretreatment with the DA receptor agonist bromocriptine which possesses •OH radical scavenging properties could also provide complete protection against i.c.v. 6-OHDA injections.36 When taken together with our present report, these data indicate that O2 produced after 6-OHDA might interact with H2O2 to produce the more cytotoxic •OH1,25 with subsequent degeneration of the nigrostriatal dopaminergic pathway. This discussion may also be relevant to the toxic effects of methamphetamine14,20,21,24,45 and related drugs11,12 on monoaminergic systems. For example, it has been reported that high doses of methamphetamine can produce 6-OHDA from endogenous DA in the striatum.40 This mechanism is thought to be responsible, in part, for methamphetamine-induced neurotoxic effects9,10,19 These suggestions were recently supported by our demonstration that the toxic effects of methamphetamine and of related drugs on striatal DA levels and DAT were attenuated in heterozygous and homozygous Cu,Zn-SODtransgenic mice.11,12,14,28 Thus, our present results provide further evidence for a high degree of similarities between methamphetamine-induced and 6-OHDA-induced degeneration of brain DA systems as suggested elsewhere.10 The changes in nigral TH-positive cell bodies are consistent with previous reports that 6-OHDA can cause retrograde degeneration of nigrostriatal neurons after intrastriatal 6-OHDA injection in rats.5,13 The bilateral decreases in nigral cell bodies are consistent with the bilateral effects on striatal and nigral DAT levels described above. The reduction in cell size suggests that these cells might be going through apoptotic cell death, since cells undergoing that process are known to shrink in size.33,38,46 This
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Fig. 7. Representative photomicrographs of TH-immunoreactive neurons in the SNpc of non-transgenic (A, B) and homozygous SOD-transgenic (C, D) mice after the injection with saline (A, C) or 6-OHDA (B, D). TH-positive neurons in the SNpc was markedly reduced on both sides of the 6-OHDA-injected non-transgenic mice (B) but not in the homozygous SOD-transgenic mice (D). Quantitative data are given in Table 1. Scale bar=100 µm.
remains to be demonstrated after administration of 6-OHDA to mice. The protection afforded by increased Cu,Zn-SOD activity in the SOD-transgenic mice supports the view that similar mechanisms are
involved in the toxic effects of 6-OHDA at both terminal and cell body regions. It is important to recognize that these studies were done at one time-point using only one known toxic
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Table 1. Effects of intracerebroventricular injection of 6-hydroxydopamine on the number and size of tyrosine hydroxylase-immunoreactive cells in the substantia nigra pars compacta of mice
Cell number Non-transgenic+saline Non-transgenic+6-OHDA Homozygous SOD-transgenic+saline Homozygous SOD-transgenic+6-OHDA Cell size (µm2) Non-transgenic+saline Non-transgenic+6-OHDA Homozygous SOD-transgenic+saline Homozygous SOD-transgenic+6-OHDA
Injected side (right)
Non-injected side (left)
106.883.02 32.001.93* 100.441.53 86.363.81*†
102.433.43 42.143.60* 105.061.91 86.364.94*†
187.194.59 127.414.01* 191.134.08 168.943.39*†
207.205.71 142.544.70* 189.064.70 181.474.95*†
TH-immunopositive neurons were counted and measured as described under Experimental Procedures. For cell number, the values represent meansS.E.M. for 14–16 slices from six mice per each group. For cell size, the values represent meansS.E.M. (µm2) for 40–50 neurons from six mice per each group. *P<0.01 vs the same side of non-transgenic+saline group. †P<0.01 vs the same side of non-transgenic+6-OHDA group (one-way ANOVA followed by Fisher’s PLSD test).
dose of 6-OHDA.35,36 It is, thus, possible to suggest that the SOD-transgenic mice might not be able to provide protection against a much higher concentration of the toxin. This argument would only imply that a very high degree of oxidative stress could overwhelm any endogenous protective antioxidant system in these mice. This is consistent with the small decreases in cell number and size observed in the homozygous SOD-transgenic mice that received the 6-OHDA injection. It could also be argued that further deterioration could be observed if the animals were studied at later time-points i.e. four weeks instead of the two weeks used in the present study. This suggestion would not have been consistent with previous reports in which it was shown that the most dramatic effects of 6-OHDA occurred one to two weeks after injection of the toxin, followed by some degree of recovery thereafter.35,36 Thus, SOD-transgenic mice are able to provide protection against 6-OHDA at a time when the toxin is known to produce its most profound neurotoxic effects.
CONCLUSIONS
In the present study, we have examined the protective effects of high Cu,Zn-SOD activity against 6-OHDA-induced neurotoxicity in the nigrostriatal DA system of mice. The dopaminergic nigrostriatal lesions induced by the i.c.v. injection of 6-OHDA were attenuated in Cu,Zn-SOD-transgenic mice, in a gene-dose dependent fashion. The protection afforded by high SOD activity provides support for a role of O2 in the neurotoxicity of 6-OHDA. These results provide further evidence that reactive oxygen species may be the main determining factors in the neurodegenerative effects of catecholamines. Acknowledgements—The authors thank the staff of Animal Care Facility at Division of Intramural Research of NIH/ NIDA for the impeccable care of the animals. We also thank Mr B. Ladenheim for technical assistance. The authors are very grateful to C. J. Epstein and E. Carlson for initially providing breeding pairs of SOD-transgenic mice.
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(1994) Attenuation of methamphetamineinduced neurotoxicity in copper/zinc superoxide dismutase transgenic mice. J. Neurochem. 62, 380–383. Cadet J. L. and Zhu S. M. (1992) The intrastriatal 6-hydroxydopamine model of hemiparkinsonism: quantitative receptor autoradiographic evidence of correlation between circling behavior and presynaptic as well as postsynaptic nigrostriatal markers in the rat. Brain Res. 595, 316–326. Chacon J. N., Chedekel M. R., Land E. J. and Truscott T. G. (1987) Chemically induced Parkinson’s disease: intermediates in the oxidation of 1-methyl-4-phenylpyridinium ion. Biochem. biophys. Res. Commun. 144, 957–964. Clow A., Hussain T., Glover V., Sandler M., Walker M. and Dexter D. (1992) Pergolide can induce soluble superoxide dismutase in rat striata. J. neural Transm. 90, 27–31. Cohen G. (1986) Monoamine oxidase, hydrogen peroxide, and Parkinson’s disease. Adv. Neurol. 45, 119–125. Cohen G. and Heikkila R. E. (1974) The generation of hydrogen peroxide, superoxide radical, and hydroxyl radical by 6-hydroxydopamine, dialuric acid, and related cytotoxic agents. J. biol. Chem. 249, 2447–2452. Cubells J. F., Rayport S., Rajndron G. and Sulzer D. (1994) Methamphetamine neurotoxicity involves vacuolation of endocytic organelles and dopamine-dependent intracellular oxidative stress. J. Neurosci. 14, 2260–2271. De Vito M. J. and Wagner G. C. (1989) Methamphetamine-induced neuronal damage: a possible role for free radicals. Neuropharmacology 28, 1145–1150. Dexter D. T., Wells F. R., Lees A. J., Agid Y., Jenner P. and Marsden C. D. (1989) Increase nigral iron content and alterations in other metal ions occurring in brain in Parkinson’s disease. J. Neurochem. 52, 1830–1836. Epstein C. J., Avraham K. B., Lovett M., Smith S., Elroy-Stein O., Rotman G., Bry C. and Groner Y. (1987) Transgenic mice with increased Cu/Zn-superoxide dismutase activity: animal model of dosage effects in Down syndrome. Proc. natn. Acad. Sci. U.S.A. 84, 8044–8048. Giovanni A., Liang L. P., Hastings T. G. and Zigmond M. J. (1995) Estimating hydroxyl radical content in rat brain using systemic and intraventricular salicylate: impact of methamphetamine. J. Neurochem. 64, 1819–1825. Halliwell B. (1992) Reactive oxygen species and the central nervous system. J. Neurochem. 59, 1609–1623. Halliwell B. and Gutteridge J. M. C. (1985) Oxygen radicals and nervous system. Trends Neurosci. 8, 22–26. Heikkila R. E. and Cohen G. (1973) 6-Hydroxydopamine: evidence for superoxide radical as an oxidative intermediate. Science 181, 456–457. Hirata H., Ladenheim B., Carlson E., Epstein C. and Cadet J. L. (1996) Autoradiographic evidence for methamphetamine induced striatal dopaminergic loss in mouse brain: attenuation in CuZn-superoxide dismutase transgenic mice. Brain Res. 714, 95–103. Kish S. J., Morito C. and Hornykiewicz O. (1985) Glutathione peroxidase activity in Parkinson’s disease. Neurosci. Lett. 58, 343–346. Kostrzewa R. and Jacobowitz D. (1974) Pharmacological actions of 6-hydroxydopamine. Pharmac. Rev. 26, 199–288. Michel P. P. and Hefti F. (1990) Toxicity of 6-hydroxydopamine and dopamine for dopaminergic neurons in culture. J. Neurosci. Res. 26, 428–435. Mizuno Y., Ohta S., Tanaka M., Takamiya S., Suzuki K., Sato T., Oya H., Ozawa T. and Kagawa Y. (1989) Deficiencies in complex I subunits of the respiratory chain in Parkinson’s disease. Biochem. biophys. Res. Commun. 163, 1450–1455. Mochizuki H., Nakamura N., Nishi K. and Mizuno Y. (1994) Apoptosis is induced by 1-methyl-4-phenylpyridinium ion (MPP+) in ventral mesencephalic-striatal co-culture in rat. Neurosci. Lett. 170, 191–194. Ogawa N., Asanuma M., Kondo Y., Hirata H., Nishibayashi S. and Mori A. (1994) Changes in lipid peroxidation, Cu/Zn-superoxide dismutase and its mRNA following an intracerebroventricular injection of 6-hydroxydopamine in mice. Brain Res. 646, 337–340. Ogawa N., Asanuma M., Kondo Y., Kawada Y., Yamamoto M. and Mori A. (1994) Differential effects of chronic L-DOPA treatment on lipid peroxydation in the mouse brain with or without pretreatment with 6-hydroxydopamine. Neurosci. Lett. 171, 55–58. Ogawa N., Tanaka K., Asanuma M., Kawai M., Masumizu T., Kohno M. and Mori A. (1994) Bromocriptine protects mice against 6-hydroxydopamine and scavenges hydroxyl free radicals in vitro. Brain Res. 657, 207–213. Olanow C. W. and Cohen G. (1992) The pathogenesis of Parkinson’s disease. In The Scientific Basis for the Treatment of Parkinson’s Disease (eds Olanow C. W. and Lieberman A. N.), pp. 59–76. The Parthenon Publishing Group, Lancashire, U.K. Przedborski S., Levivier M., Jiang H., Ferreira M., Jackson-Lewis V., Donaldson D. and Togasaki D. M. (1995) Dose-dependent lesions of the dopaminergic nigrostriatal pathway induced by intrastriatal injection of 6-hydroxydopamine. Neuroscience 67, 631–647. Riederer P., Sofic E., Rausch W. D., Schmidt B., Reynolds G. P., Kellinger K. and Youdim M. B. H. (1989) Transition metals, ferritin, glutathione, and ascorbic acid in parkinsonian brains. J. Neurochem. 52, 515–520. Seiden L. S. and Vosmer G. (1984) Formation of 6-hydroxydopamine in caudate nucleus of the rat brain after a single large dose of methylamphetamine. Pharmac. Biochem. Behav. 21, 29–31. Sinha B. K., Singh Y. and Krishna G. (1986) Formation of superoxide and hydroxyl radicals from 1-methyl-4phenylpyridinium ion (MPP+): reductive activation by NADPH cytochrome P450 reductase. Biochem. biophys. Res. Commun. 135, 583–588.
SOD transgenic mice attenuate 6-OHDA-induced lesions 42. 43. 44. 45. 46.
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Slotnick B. M. and Leonard C. M. (1975) A Stereotaxic Atlas of the Albino Mouse Forebrain. DHEW Publications, Rockville, MD. Tiffani-Castiglioni E., Saneto R. P., Proctor P. H. and Perez-Polo J. R. (1982) Participation of active oxygen species in 6-hydroxydopamine toxicity to a human neuroblastoma cell line. Biochem. Pharmac. 31, 181–188. Ungerstedt U. (1968) 6-Hydroxydopamine induced degeneration of central monoamine neurons. Eur. J. Pharmac. 5, 107–110. Wagner G. C., Carelli R. M. and Jarvis M. F. (1985) Pretreatment with ascorbic acid attenuates the neurotoxic effects of methamphetamine in rats. Res. Commun. chem. Pathol. Pharmac. 47, 221–228. Ziv I., Melamed E., Nardi N., Luria D., Achiron A., Offen D. and Barzilai A. (1994) Dopamine induces apoptosis-like cell death in cultured chick sympathetic neurons—a possible novel pathogenetic mechanism in Parkinson’s disease. Neurosci. Lett. 170, 136–140. (Accepted 5 December 1997)
PII: S0306-4522(97)00665-9
Neuroscience Vol. 85, No. 3, pp. 907–917, 1998 IBRO Published by Elsevier Science Ltd Printed in Great Britain
ATTENUATION OF 6-HYDROXYDOPAMINE-INDUCED DOPAMINERGIC NIGROSTRIATAL LESIONS IN SUPEROXIDE DISMUTASE TRANSGENIC MICE M. ASANUMA, H. HIRATA and J. L. CADET* Molecular Neuropsychiatry Section, NIH/NIDA, Division of Intramural Research, Baltimore, MD 21224, U.S.A. Abstract––6-Hydroxydopamine is a neurotoxin that produces degeneration of the nigrostriatal dopaminergic pathway in rodents. Its toxicity is thought to involve the generation of superoxide anion secondary to its autoxidation. To examine the effects of the overexpression of Cu,Zn-superoxide dismutase activity on 6-hydroxydopamine-induced dopaminergic neuronal damage, we have measured the effects of 6-hydroxydopamine on striatal and nigral dopamine transporters and nigral tyrosine hydroxylaseimmunoreactive neurons in Cu,Zn-superoxide dismutase transgenic mice. Intracerebroventricular injection of 6-hydroxydopamine (50 µg) in non-transgenic mice produced reductions in the size of striatal area and an enlargement of the cerebral ventricle on both sides of the brains of mice killed two weeks after the injection. In addition, 6-hydroxydopamine caused marked decreases in striatal and nigral [125I]RTI-121labelled dopamine transporters not only on the injected side but also on the non-injected side of non-transgenic mice; this was associated with decreased cell number and size of tyrosine hydroxylaseimmunoreactive dopamine neurons in the substantia nigra pars compacta on both sides in these mice. In contrast, superoxide dismutase transgenic mice were protected against these neurotoxic effects of 6-hydroxydopamine, with the homozygous transgenic mice showing almost complete protection. These results provide further support for a role of superoxide anion in the toxic effects of 6-hydroxydopamine. They also provide further evidence that reactive oxygen species may be the main determining factors in the neurodegenerative effects of catecholamines. Key words: 6-hydroxydopamine, superoxide dismutase, transgenic mice, superoxide anion, dopamine transporter, Parkinson’s disease.
Parkinson’s disease is a neurodegenerative disorder that is characterized by dopamine (DA) depletion in the basal ganglia and DA cell death in the substantia nigra (SN). Although the mechanism for the development of the disorder has not been completely clarified, oxygen-based free radicals are thought to play an important role in the pathogenesis of Parkinson’s disease.1,18,25,26,37 This idea is supported by evidence showing increases in lipid peroxidation and decreases in the levels of free radical scavenging enzymes such as glutathione peroxidase and catalase in the brains of patients with Parkinson’s disease.2,29 It is also supported by the presence of increased iron deposits in the SN pars compacta (SNpc),22,39 because iron can lead to the formation of free radicals via interaction with hydrogen peroxide *To whom correspondence should be addressed. Abbreviations: DA, dopamine; DAB, 3,3 -diaminobenzidine; DAT, dopamine transporter; GBR-12909, 1-[2[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylprophyl)piperazine; O2, superoxide anion; •OH, hydroxyl radical; 6-OHDA, 6-hydroxydopamine; RTI-121, 3â-[4(trimethylstannyl)phenyl]-tropan-2â-carboxylic acid isopropyl ester; SN, substantia nigra; SNpc, SN pars compacta; SOD, superoxide dismutase; TBS, Trisbuffered saline; TH, tyrosine hydroxylase.
(H2O2).1,18,25,37 Other supporting evidence for a role of free radicals in the developmental course of Parkinson’s disease includes abnormal activity of mitochondrial complex I.6,32 Furthermore, several toxins used to create animal models of Parkinson’s disease might produce their lesions through the production of free radicals.16,19,27,41 One such toxin is 6-hydroxydopamine (6-OHDA) which has provided useful animal models of the destruction of nigrostriatal DA systems.44 6-OHDA is a neurotoxin that produces degeneration of DA neurons in the SNpc and damage of their nerve endings in the striatum.5,8,44 This can be accomplished by intranigral44 or intrastriatal injections5,13 in rats or intracerebroventricular (i.c.v.) injections in mice.8,30,35 The toxic effects of 6-OHDA have also been examined in vitro using dopaminergic cell cultures.31 The accumulated evidence both in vitro and in vivo suggests that the toxicity of 6-OHDA involves the generation of reactive oxygen species, since 6-OHDA can autoxidize to form semiquinone and superoxide anion (O2) which can subsequently be converted to the more cytotoxic hydroxyl radical (•OH) through interaction with H2O2.19,27 The role of free radicals in 6-OHDAinduced toxicity is also supported by reports that
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Fig. 1. Representative photomicrographs of Haematoxylin–Eosin-stained striatal sections of nontransgenic (A, B), heterozygous (C, D) and homozygous (E, F) SOD-transgenic mice. Animals received intracerebroventricular injection of saline (A, C, E) or 50 µg of 6-OHDA (B, D, F). 6-OHDA-injected non-transgenic mice had marked enlargement of lateral cerebral ventricles and severely shrunken striatal area two weeks after the injection (B). These 6-OHDA-induced atrophic changes were attenuated in the heterozygous (D) and the homozygous (F) SOD-transgenic mice.
in vitro treatment with reactive oxygen species-related enzymes protected cells against 6-OHDA-induced cell death43 and that in vivo treatment with iron chelator inhibited the 6-OHDA-induced DA depletion in the striatum.4 In order to assess the possible role of O2 in the in vivo toxic effects of 6-OHDA, we have made use of heterozygous and homozygous Cu,Zn-superoxide dismutase (SOD) transgenic mice and examined its effects on striatal and nigral dopamine transporters
(DAT) and on nigral tyrosine hydroxylase (TH)-positive cells following i.c.v. injection of the toxin. EXPERIMENTAL PROCEDURES
Chemicals [125I]RTI-121, radioiodinated 3â-[4-(trimethylstannyl)phenyl]-tropan-2â-carboxylic acid isopropyl ester was purchased from New England Nuclear (Boston, MA).
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Fig. 2. Effects of the intracerebroventricular injection of 6-OHDA on striatal area of mice. Striatal area (relative unit) was measured on the Haematoxylin–Eosin-stained sections as described under Experimental Procedures. The value is shown as mean percentage of each saline-injected groupS.E.M. of five animals per each group. **P<0.01 compared with the same side of each saline-treated group. #P<0.05, ##P<0.01 compared with the same side of 6-OHDA-injected non-transgenic (Tg) group (Mann–Whitney U-test).
GBR-12909, 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3phenyl-prophyl)piperazine dihydrochloride was purchased from Research Biochemicals International (Natick, MA). 6-OHDA hydrobromide salt and desipramine were obtained from Sigma (St Louis, MO). Other chemicals were purchased from Mallinckrodt Specialty Chemicals (Paris, KY). Animals and 6-hydroxydopamine lesions Male heterozygous and homozygous SOD-transgenic mice of strain 218/3 (12–14-weeks-old) carrying the complete human Cu,Zn-SOD gene were used in the present experiment; they show respective increases of 3.6- and 5.0-fold in striatal Cu,Zn-SOD enzyme activity when compared to non-transgenic controls (J. Subramaniam and J. L. Cadet, unpublished observation). The production of these animals has been previously described.23 All animal use procedures were according to the NIH Guide for the Care and Use of Laboratory Animals and were approved by the local Animal Care Committee. Thirty minutes prior to 6-OHDA injection, all animals were administered with desipramine (25 mg/kg, i.p.) to protect noradrenaline-containing neurons.36 Under anaesthesia with pentobarbital (50 mg/kg, i.p.), mice were injected with 50 µg of 6-OHDA (dissolved in 2 µl physiological saline containing 0.05% ascorbic acid) or the same volume of saline with ascorbic acid into the right cerebral ventricle via a 5-µl Hamilton microsyringe. The following coordinates were used: AP, +0.3 mm; ML, 1.0 mm; DV, 3.0 mm from bregma. Tissue preparation Two weeks after the injection, mice were killed by decapitation. This time was chosen because previous experiments have shown that the effects of 6-OHDA in mice are most prominent around one to two weeks after i.c.v. injection of similar doses of the toxin.35,36 Their brains were quickly removed, frozen in isopentane on dry ice, and stored at 70C until use. Coronal sections (20 µm-thick) were cut in a cryostat at 20C and thaw-mounted onto gelatin-coated glass slides, dried under negative pressure and then stored at 70C until used in the studies described below. To assess the regional distribution of the lesion, sections were taken at the level of the mid-striatum (+1.0+0.6 mm from
bregma) and the SN (2.83.0 mm from bregma) of each brain.42 For measurement of striatal area, Haematoxylin–Eosin (HE) staining was performed. Autoradiographic binding assay Binding assays for DAT were performed according to our previously reported method28 with minor modifications of a previous protocol7 that uses the cocaine analogue, [125I]RTI-121. This compound selectively binds to DAT but not to other monoamine transporters in the striatum.7 Briefly, slide-mounted sections were incubated for 60 min at room temperature with 100,000 c.p.m./ml of [125I]RTI-121 (specific activity, 2200 Ci/mmol) using a binding buffer consisting 137.0 mM NaCl, 2.7 mM KCl, 10.14 mM Na2HPO4, 1.76 mM KH2PO4 and 10.0 mM NaI. Specific binding was determined in the presence of 10 µM GBR12909 and represented greater than 90% of total binding. At the end of the incubation, the slides were washed twice in fresh binding buffer for 20 min at room temperature, dipped in ice-cold distilled water and dried under cool air stream. The slides were then apposed to radiosensitive films (Hyperfilm âmax, Amersham, Arlington Heights, IL) with a plastic standard (125I microscales, Amersham, U.K.) for three days at 4C. Immunohistochemistry Immunohistochemistry for TH was performed using nontransgenic and homozygous SOD-transgenic mice according to standard techniques. Slides were warmed up to room temperature and fixed in 4% paraformaldehyde (in 0.1 M phosphate buffer) for 10 min. Sections were then washed 415 min in 50 mM Tris-buffered saline (TBS; pH 7.6) before being incubated in 1% H2O2 and 100% methanol for 10 min. After washing with TBS (45 min), the slidemounted sections were incubated in 3% normal goat serum and 0.2% Triton X-100 in TBS for 1 h, and then exposed to a rabbit polyclonal antibody (diluted 1:20,000 in TBS containing 1% normal goat serum) specific for TH (Eugene Tech International, Ridgefield Park, NJ) for 18 h at room temperature. After incubation with the primary antibody, sections were washed for 35 min in 50 mM TBS before being incubated with biotinylated goat anti-rabbit secondary IgG (diluted 1:200 in TBS; Vector Laboratories, Burlingame, CA) for 1.5 h at room temperature. Following
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Fig. 3. Representative autoradiograms of striatal [125I]RTI-121-labelled DAT of non-transgenic (A, B), heterozygous (C, D) and homozygous (E, F) SOD-transgenic mice injected with saline (A, C, E) or 6-OHDA (B, D, F). Striatal [125I]RTI-121 binding on both sides was markedly decreased two weeks after the injection of 6-OHDA in the non-transgenic mice (B). These 6-OHDA-induced reductions of DAT levels were attenuated in the heterozygous SOD-transgenic mice (D) and almost completely prevented in the homozygous SOD-transgenic mice (F). Quantitative data are given in Fig. 4. washes in TBS (415 min), the sections were incubated with ABC reagents (Vector Laboratories) for 2 h. THimmunopositive neurons were visualized by 3,3 diaminobenzidine (DAB), nickel and H2O2 using a commercially available kit (DAB substrate kit, Vector Laboratories). The counterstaining of nuclei was then performed with 1% Methyl Green. Negative controls were run using normal goat serum instead of the primary antibody. Semiquantitative image analyses Striatal area on the HE-stained slides was measured by an image analysis software (Image 1.56, NIH) run on a Macintosh computer. For each coronal slice, each side of the striatum on the digitized image was separately outlined with a screen cursor driven by a hand-held mouse and relative size units were measured. The autoradiograms for [125I]RTI-121 binding were analysed using a Macintosh computer-based image analysis
system (Image 1.56, NIH). Within each slice, the striatum and SN were defined according to a mouse brain atlas.42 Each region on the injected side (right) or the non-injected side (left) was outlined with a screen cursor driven by a hand-held mouse as described above. Optical densities in the striatum and SN were quantified and converted to nCi/mg tissue using standard curve (125I microscales) exposed at the same time with the brain sections. TH-immunoreactive neurons in the SNpc were counted manually on two to three slices per each animal (n=6 mice/group) using a Zeiss microscope at a magnification of 400 with a superimposed grid. The boundary between the SNpc and ventral tegmental area was defined as a line extending dorsally from the most medial boundary of the cerebral peduncle. Counting was performed blindly. Cell size and relative density of the TH-positive neurons in the SNpc were assessed on six to nine TH-positive cells per each animal (n=6 mice/group) using the microscope at a magnification of 630 and a Macintosh computer-based image
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Fig. 4. Changes in striatal DAT levels in SOD-transgenic (Tg) mice following the intracerebroventricular injection with 6-OHDA (50 µg). Striatal [125I]RTI-121 binding (nCi/mg tissue) was measured as described under Experimental Procedures. The values represent mean percentages of each saline-injected groupS.E.M. (four or five animals/group). There were no significant differences between the salineinjected mice of the three strains. *P<0.05 compared with the same side from each saline-treated group. # P<0.05 compared with the same side from non-transgenic+6-OHDA group (Mann–Whitney U-test).
analysis system (Image 1.56, NIH). Only TH-positive neurons with a complete nucleus in the section were assessed and outlined with the screen cursor driven by a hand-held mouse. This allowed for the measurement of neuron size and relative density of TH-staining. Relative density of TH-immunoreactive fibres was also measured in two representative upper areas of the striatum (500 µm500 µm) per each mouse (n=6 mice/group) using the microscope at a magnification of 400 and Image 1.56, NIH. Statistical analyses For each group, raw data on the injected side and the non-injected side were compared using a two-tailed Student’s t-test for paired samples. Statistical analyses for these indices expressed as percentages were done using Mann–Whitney U-test for two paired group. The values for number or size of TH-positive neurons in the SNpc of each side were analysed using one-way ANOVA followed by Fisher’s PLSD post hoc test. RESULTS
Effects of 6-hydroxydopamine on striatal area in mice Representative striatal sections for HE staining are shown in Fig. 1. In non-transgenic mice, i.c.v. injection of 50 µg of 6-OHDA caused marked reductions in the size of the striatum with associated enlargement of the lateral cerebroventricle not only on the injected side but on the non-injected side as well (Fig. 1B). These numbers in the non-transgenic mice correspond to 75.136.26% and 77.023.54% of control on the injected and non-injected side, respectively (Fig. 2). Neither the heterozygous nor the homozygous SOD-transgenic mice showed any significant decreases (Figs 1D, F, 2). The homozygous SOD-transgenic animals were almost completely protected against 6-OHDA-induced striatal atrophy (Fig. 2).
Effects of 6-hydroxydopamine injection on striatal and nigral dopamine transporter 6-OHDA caused significant decreases in [125I]RTI121-labelled DAT in the striatum (Figs 3, 4). For example, in the non-transgenic mice, [125I]RTI-121 binding on both sides of the striatum was markedly decreased two weeks after the injection of 6-OHDA; these consisted of decreases of 37.61% and 35.34% on the injected and non-injected sides, respectively (Figs 3A, B, 4). These reductions of striatal DAT were significantly attenuated on the non-injected side but not on the injected side (29.12%) of the heterozygous SOD-transgenic mice (Figs 3D, 4), whereas they were significantly attenuated on both sides in the homozygous SODtransgenic mice (Figs 3F, 4). DAT levels in the SN were also decreased two weeks after the 6-OHDA injection (Figs 5, 6). These decreases in nigral [125I]RTI-121 binding were observed on both sides (51.37% and 37.41% for the injected and non-injected sides, respectively) of the brains of non-transgenic mice (Figs 5B, 6). DAT levels were also significantly decreased (40.44%) on the injected side of the brains of heterozygous SOD-transgenic mice (Figs 5D, 6). The SNpc of homozygous SOD-transgenic mice were not affected by 6-OHDA (Figs 5F, 6). Tyrosine hydroxylase immunohistochemistry in substantia nigra pars compacta Figure 7 shows representative photomicrographs of TH-immunoreactive neurons in the SNpc of nontransgenic and homozygous SOD-transgenic mice. The quantitative data are shown in Table 1. 6-OHDA
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Fig. 5. Representative autoradiograms of [125I]RTI-121-labelled DAT in the SN of non-transgenic (A, B), heterozygous (C, D) and homozygous (E, F) SOD-transgenic mice two weeks after the injection with saline (A, C, E) or 6-OHDA (B, D, F). The 6-OHDA injection caused decreases in [125I]RTI-121 binding in the SN of the non-transgenic (B) and the heterozygous SOD-transgenic (D) mice, whereas no apparent reductions of DAT in the SN were observed in the homozygous SOD-transgenic mice after the 6-OHDA injection (F). Quantitative data are given in Fig. 6.
injection significantly reduced the number of THpositive neurons in the SNpc of the non-transgenic mice on both sides of the brain (29.94% of salineinjected group on the injected side; 41.14% of saline controls on the non-injected side; Fig. 7B, Table 1). The size of TH-positive neurons in the SNpc was also reduced on both sides (68.06% and 68.79% of salineinjected group on the injected and non-injected sides, respectively) of the brains of non-transgenic mice by the i.c.v. injection of 6-OHDA (Table 1). Homozygous SOD-transgenic mice were almost completely protected from these effects of 6-OHDA (Fig. 7D, Table 1). 6-OHDA also caused small but statisti-
cally significant increases in the relative density of TH-staining in the SNpc on both sides (+6.65% and +7.50% for the injected and non-injected sides, respectively) of the brains of non-transgenic mice, whereas there was no change on either side in the homozygous SOD-transgenic mice. In non-transgenic mice treated with 6-OHDA injection, significant decreases in relative density of striatal TH-immunoreactive fibres were observed on both sides (13.96% and 7.97% for the injected and non-injected sides, respectively), whereas no significant changes were observed on neither side of the brains of homozygous SOD-transgenic mice.
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Fig. 6. Effects of the 6-OHDA injection on DAT levels in the SN of SOD-transgenic (Tg) mice. [125I]RTI-121 binding (nCi/mg tissue) in the SN was measured as described above. The values represent mean percentages of each saline-injected groupS.E.M. of four or five animals per each group. There were no significant differences in the saline-injected mice of the three strains. *P<0.05 compared with the same side from each saline-treated group. #P<0.05 compared with the same side from non-transgenic+ 6-OHDA group (Mann–Whitney U-test).
DISCUSSION
The main findings of this paper are that (i) SODtransgenic mice are protected against atrophic changes and the reduction of TH-positive fibres caused by 6-OHDA in mice striata; (ii) they show less decreases in striatal and nigral DAT levels caused by the toxin; and (iii) they are also protected against the loss and shrinkage of nigral TH-positive cell bodies. The occurrence of these changes on both sides of the striatum is consistent with the marked bilateral decreases in DA levels previously reported one to two weeks after a similar i.c.v. injections of 6-OHDA (20– 40 µg) to mice.8,30,35,36 These data are also consistent with dose-dependent reductions of [3H]mazindollabelled DAT in the striatum after unilateral intrastriatal injection of 6-OHDA to rats.15,38 In the present study, the changes in all indices examined on the injected side were not significantly different from those of the contralateral side, thus suggesting that 6-OHDA injected on one side of the ventricle had diffused to the contralateral side. The attenuation observed in the heterozygous SOD-transgenic and the almost complete protection seen in the homozygous SOD-transgenic mice suggest that O2 production must play an important role in 6-OHDA-induced toxicity in vivo. This role of O2 in the toxic effects of 6-OHDA is also supported by the previous report that i.c.v. injection of 6-OHDA in mice resulted in acute but transient increases in Cu,Zn-SOD activity and lipid peroxidation in the striatum.34 This discussion is also supported by the observation that administration of the DA receptor agonist, pergolide, which caused increases in striatal Cu,Zn-SOD activity17 was also neuroprotective against the toxic effects caused by the i.c.v. injection of 6-OHDA.3 Further support for this idea is pro-
vided by the observation that pretreatment with the DA receptor agonist bromocriptine which possesses •OH radical scavenging properties could also provide complete protection against i.c.v. 6-OHDA injections.36 When taken together with our present report, these data indicate that O2 produced after 6-OHDA might interact with H2O2 to produce the more cytotoxic •OH1,25 with subsequent degeneration of the nigrostriatal dopaminergic pathway. This discussion may also be relevant to the toxic effects of methamphetamine14,20,21,24,45 and related drugs11,12 on monoaminergic systems. For example, it has been reported that high doses of methamphetamine can produce 6-OHDA from endogenous DA in the striatum.40 This mechanism is thought to be responsible, in part, for methamphetamine-induced neurotoxic effects9,10,19 These suggestions were recently supported by our demonstration that the toxic effects of methamphetamine and of related drugs on striatal DA levels and DAT were attenuated in heterozygous and homozygous Cu,Zn-SODtransgenic mice.11,12,14,28 Thus, our present results provide further evidence for a high degree of similarities between methamphetamine-induced and 6-OHDA-induced degeneration of brain DA systems as suggested elsewhere.10 The changes in nigral TH-positive cell bodies are consistent with previous reports that 6-OHDA can cause retrograde degeneration of nigrostriatal neurons after intrastriatal 6-OHDA injection in rats.5,13 The bilateral decreases in nigral cell bodies are consistent with the bilateral effects on striatal and nigral DAT levels described above. The reduction in cell size suggests that these cells might be going through apoptotic cell death, since cells undergoing that process are known to shrink in size.33,38,46 This
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Fig. 7. Representative photomicrographs of TH-immunoreactive neurons in the SNpc of non-transgenic (A, B) and homozygous SOD-transgenic (C, D) mice after the injection with saline (A, C) or 6-OHDA (B, D). TH-positive neurons in the SNpc was markedly reduced on both sides of the 6-OHDA-injected non-transgenic mice (B) but not in the homozygous SOD-transgenic mice (D). Quantitative data are given in Table 1. Scale bar=100 µm.
remains to be demonstrated after administration of 6-OHDA to mice. The protection afforded by increased Cu,Zn-SOD activity in the SOD-transgenic mice supports the view that similar mechanisms are
involved in the toxic effects of 6-OHDA at both terminal and cell body regions. It is important to recognize that these studies were done at one time-point using only one known toxic
SOD transgenic mice attenuate 6-OHDA-induced lesions
915
Table 1. Effects of intracerebroventricular injection of 6-hydroxydopamine on the number and size of tyrosine hydroxylase-immunoreactive cells in the substantia nigra pars compacta of mice
Cell number Non-transgenic+saline Non-transgenic+6-OHDA Homozygous SOD-transgenic+saline Homozygous SOD-transgenic+6-OHDA Cell size (µm2) Non-transgenic+saline Non-transgenic+6-OHDA Homozygous SOD-transgenic+saline Homozygous SOD-transgenic+6-OHDA
Injected side (right)
Non-injected side (left)
106.883.02 32.001.93* 100.441.53 86.363.81*†
102.433.43 42.143.60* 105.061.91 86.364.94*†
187.194.59 127.414.01* 191.134.08 168.943.39*†
207.205.71 142.544.70* 189.064.70 181.474.95*†
TH-immunopositive neurons were counted and measured as described under Experimental Procedures. For cell number, the values represent meansS.E.M. for 14–16 slices from six mice per each group. For cell size, the values represent meansS.E.M. (µm2) for 40–50 neurons from six mice per each group. *P<0.01 vs the same side of non-transgenic+saline group. †P<0.01 vs the same side of non-transgenic+6-OHDA group (one-way ANOVA followed by Fisher’s PLSD test).
dose of 6-OHDA.35,36 It is, thus, possible to suggest that the SOD-transgenic mice might not be able to provide protection against a much higher concentration of the toxin. This argument would only imply that a very high degree of oxidative stress could overwhelm any endogenous protective antioxidant system in these mice. This is consistent with the small decreases in cell number and size observed in the homozygous SOD-transgenic mice that received the 6-OHDA injection. It could also be argued that further deterioration could be observed if the animals were studied at later time-points i.e. four weeks instead of the two weeks used in the present study. This suggestion would not have been consistent with previous reports in which it was shown that the most dramatic effects of 6-OHDA occurred one to two weeks after injection of the toxin, followed by some degree of recovery thereafter.35,36 Thus, SOD-transgenic mice are able to provide protection against 6-OHDA at a time when the toxin is known to produce its most profound neurotoxic effects.
CONCLUSIONS
In the present study, we have examined the protective effects of high Cu,Zn-SOD activity against 6-OHDA-induced neurotoxicity in the nigrostriatal DA system of mice. The dopaminergic nigrostriatal lesions induced by the i.c.v. injection of 6-OHDA were attenuated in Cu,Zn-SOD-transgenic mice, in a gene-dose dependent fashion. The protection afforded by high SOD activity provides support for a role of O2 in the neurotoxicity of 6-OHDA. These results provide further evidence that reactive oxygen species may be the main determining factors in the neurodegenerative effects of catecholamines. Acknowledgements—The authors thank the staff of Animal Care Facility at Division of Intramural Research of NIH/ NIDA for the impeccable care of the animals. We also thank Mr B. Ladenheim for technical assistance. The authors are very grateful to C. J. Epstein and E. Carlson for initially providing breeding pairs of SOD-transgenic mice.
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