Evaluation of Estrogen Neuroprotective Effect on Nigrostriatal Dopaminergic Neurons Following 6-Hydroxydopamine Injection into the Substantia Nigra Pars Compacta or the Medial Forebrain Bundle
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- Ferraz, A.C., Matheussi, F., Szawka, R.E. et al. Neurochem Res (2008) 33: 1238. doi:10.1007/s11064-007-9575-7
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Studies involving estrogen treatment of ovariectomized rats or mice have attributed to this hormone a neuroprotective effect on the substantia nigra pars compacta (SNpc) neurons. We investigated the effect of estradiol replacement in ovariectomized rats on the survival of dopaminergic mesencephalic cell and the integrity of their projections to the striatum after microinjections of 1 μg of 6-hydroxydopamine (6-OHDA) into the right SNpc or medial forebrain bundle (MFB). Estradiol replacement did not prevent the reduction either in the striatal concentrations of DA and metabolites or in the number of nigrostriatal dopaminergic neurons following lesion with 1 μg of 6-OHDA into the SNpc. Nevertheless, estradiol treatment reduced the decrease in striatal DA following injection of 1 μg of 6-OHDA into the MFB. Results suggest therefore that estrogen protect nigrostriatal dopaminergic neurons against a 6-OHDA injury to the MFB but not the SNpc. This may be due to the distinct degree of lesions promoted in these different rat models of Parkinson’s disease.
KeywordsNeuroprotection6-OHDAParkinson’s diseaseEstrogenSubstantia nigra pars compactaMedial forebrain bundleDopaminergic neurons
Parkinson’s disease (PD) is a neurodegenerative disorder that results from a progressive degeneration of striatum-projecting dopamine (DA) neurons in the substantia nigra pars compacta (SNpc) and, to a lesser extent, in the retrorubral field and ventral tegmental area (VTA) [1–5]. Degeneration of the mesencephalic dopaminergic cells causes a progressive decrease of DA content in the corpus striatum leading to a debilitating motor dysfunction. Symptoms include resting tremor, rigidity, slow movement, gait dysfunction and postural instability [6, 7]. PD affects about 1% of the population over 55 years of age [7, 8] and its occurrence is more common in men than in women . Studies using animal models of PD have reported that males are more susceptible than females [10–12]. Reinforcing these data, there is evidence from clinical and basic research suggesting that estrogen plays a neuroprotective role decreasing the risk of neurodegenerative diseases, stroke and seizure-induced hippocampal cell loss [13–16].
6-hydroxydopamine (6-OHDA) lesions at different levels of the nigrostriatal dopaminergic (NSDA) pathway such as at the SNpc, medial forebrain bundle (MFB) or striatum have been widely used as animal models of motor disorders similar to PD [8, 17–22]. Injection of 6-OHDA into the SNpc or MFB leads to rapid cell death whereas its injection into the striatum causes a slow retrograde degeneration of NSDA neurons [23, 24].
Estrogen has been reported to protect against striatal toxicity following 6-OHDA injection into the MFB [19, 25–27] or striatum [28, 29]. For example, ovariectomy increases whereas estrogen treatment decreases striatal DA loss following 6-OHDA lesion of the MFB . In this same 6-OHDA-lesion model, striatal toxicity was reported to change during the rat estrous cycle, being highest when endogenous estrogen is lowest .
Using 6-OHDA injection into the SNpc, we have reported that physiological estrogen treatment in ovariectomized rats was not able to reduce the loss of NSDA neurons occurring 45 days after lesion with 6 μg of 6-OHDA . These data suggested that estrogen may not protect the NSDA neurons from an insult with 6-OHDA directly injected into the SNpc. However, estrogen effect on NSDA neurons survival may have been overwhelmed by the extensive degree of lesion produced in this study. Also, another possibility to be considered is that estrogen might preserve striatal dopamine functionality without affecting cell loss. It remains therefore uncertain whether estrogen is able to protect NSDA neurons in this 6-OHDA-lesion model of PD. Thus, the aim of the present study was to evaluate the effect of physiological estrogen treatment on the survival of NSDA neurons and the activity of striatal DA terminals after administration of 1 μg of 6-OHDA into the SNpc of ovariectomized rats. For comparison, we also determined the effect of the same estrogen treatment on striatal concentrations of DA and metabolites following injection of 1 μg of 6-OHDA into the MFB, an animal model of PD in which neuroprotective effects of estrogen have been reported.
Material and Methods
Adult female Wistar rats from our own breeding stock weighing 240–260 g at the beginning of the experiments were used. The animals were maintained in a temperature-controlled room (22 ± 2°C) on a 12/12 h light cycle (lights on at 7:00 a.m.) and had free access to food and water. All studies involving animals strictly followed the Guide for the Care and Use of Experimental Animals (Canadian Council on Animal Care) and were approved by the Federal University of Paraná Committee of Animal Welfare. All efforts were made to minimize animal use and their suffering in these experiments.
Experiment 1: Estrogen effect on NSDA neurons survival and striatal levels of DA and metabolites after 6-OHDA injection into the SNpc.
Immediately after bilateral ovariectomy, a pellet containing estradiol (E) or corn oil (O) was implanted subcutaneously on the back of the rats. After 5 days, either 1 μg/1 μl of 6-OHDA (group 6-OHDA) or 1 μl of the vehicle, artificial cerebrospinal fluid (aCSF; Sham group) was infused in the right SNpc. Control groups (C) were not submitted to stereotaxic surgery. For the neurochemical study, C, Sham and 6-OHDA rats were evaluated, each of them treated with oil or estradiol: C/O, C/E, Sham/O, Sham/E, 6-OHDA /O and 6-OHDA /E. Ten days after ovariectomy, rats were killed by decapitation. Trunk blood was collected and plasma was stored at -80°C for estradiol measurement. Brain was removed, the striatum tissue was dissected, weighed and assayed for DA and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and 4-hydroxy-3-methoxy-phenylacetic acid (HVA). Six rats were studied in each experimental group. For the immunohistochemical study, following the same protocol of 6-OHDA lesion, Sham/O, Sham/E, 6-OHDA /O and 6-OHDA /E rats were perfused and the brains were processed for tyrosine hydroxylase (TH) immunostaining. The optical density generated by the TH immunostaining and the density of TH positive neurons were evaluated in the SNpc. The same parameters were also evaluated in the VTA to assure that lesion was restricted to the SNpc region. In this analysis, control groups were not studied since we have previously demonstrated that TH immunoreactivity is similar in control and sham-operated rats . As the neurochemical and immunohistochemical studies involve different techniques, another four rats were studied in each experimental group for immunohistochemical study.
Experiment 2: Estrogen effect on striatal levels of DA and metabolites after 6-OHDA injection into the MFB.
Ovariectomy and estradiol treatment was performed as in experiment 1. The same experimental design as in the neurochemical study of experiment 1 was used, but 1 μg of 6-OHDA was injected in the right MFB. Striatal DA, DOPAC and HVA concentrations were determined in rats from the following experimental groups: C/O (n = 7), C/E (n = 7), Sham/O (n = 8), Sham/E (n = 9), 6-OHDA /O (n = 9) and 6-OHDA /E (n = 9).
Ovariectomy and Hormonal Treatment
The ovaries were bilaterally removed under ethyl-ether anesthesia and a 15 mm long Silastic pellet (Down Corning Corp., Midland, MI; inner diameter 1.47 mm, outer diameter 1.96 mm) was implanted subcutaneously on the back of the animal, close to the incision. Pellets contained either 400 μg/8 μl of 17-β estradiol (Sigma, St. Louis, MO, USA) diluted in corn oil or only corn oil. After surgery rats were treated with penicillin G-procain (5,000,000 U/0.1 ml, i.m.).
Five days after ovariectomy and capsule implantation, rats in the 6-OHDA group received atropine sulfate (0.4 mg/kg, i.p.) to suppress salivation and penicillin G-procain (5,000,000 U/0.1 ml, i.m.), and were anesthetized with 3 ml/kg equitesin (1% thiopental, 4.25% chloral hydrate, 2.13% magnesium sulfate, 42.8% propylene glycol, and 3.7% ethanol in water). 6-OHDA HCl (Sigma; 1 μg in 1 μl of aCSF) was unilaterally infused (0.25 μl/min) into the right SNpc (experiment 1) or MFB (experiment 2) through a 30-gauge needle. To avoid the reflow of the drug an interval of 2 min were kept between the ending of infusion and retreat of the needle. In sham-operated rats, 1 μl of aCSF was unilaterally infused into the right SNpc. The coordinates used for SNpc were: AP, −5.0 mm from bregma; ML, −2.1 mm from midline; DV, −7.7 mm from skull, adapted from Paxinos and Watson  and the coordinates used for MPB were: AP, + 1.9 mm from bregma; ML, −1.9 mm from midline; DV, −7.2 mm from skull . The composition of aCSF was as follows: 150 mM NaCl, 2.75 mM KCl, 1.20 mM CaCl2 and 0.85 mM MgCl2.
Rats were rapidly decapitated, brains were rapidly removed and the striatum was dissected, weighed and immediately frozen on dry ice and stored at −80°C until assayed for DA, DOPAC and HVA by high performance liquid chromatography coupled with electrochemical detection (HPLC-ED). The striatum was homogenized in 800 μl of a solution containing 0.2 M perchloric acid (Merck, Darmstadt, Germany), 0.1 mM EDTA (Merck) and 0.45 μM of 3,4-dihydroxybenzylamine (DHBA; Aldrich, Milwaukee, USA) as internal standard. The homogenates were centrifuged for 20 min at 12,000g and the supernatant was filtered through a 0.22 μm filter (Millex PVDF, Millipore, Belford, USA). Ten microliters of each sample were injected by an auto injector (SIL-10Advp; Shimadzu, Kyoto, Japan). Separation was performed by a 250 × 4.6 mm reversed-phase C18 column (Shim-pack VP-ODS, 5 μm; Shimadzu), preceded by a 10 × 4.6 mm C18 guard column (Shim-pack GVP-ODS, 5 μm; Shimadzu). The mobile phase, prepared with Milli-Q water (Simplicity 185, Millipore), consisted of 100 mM sodium dihydrogen phosphate, 10 mM sodium chloride, 0.1 mM EDTA, 0.40 mM sodium 1-octanesulfonic acid (Sigma) and 20% methanol (Omnisolv, EMD Chemical Inc., Gibbstown, USA). pH was adjusted to 3.5 with phosphoric acid. The flow rate was set at 0.9 ml/min, pumped by a dual piston pump (LC-10Advp; Shimadzu). The detector potential was 0.65 V vs. in situ Ag/AgCl (Decade, VT-03 electrochemical flow cell; Antec Leyden, Netherlands). Chromatography data were plotted using Class-VP software (Shimadzu). DA, DOPAC and HVA were identified based on their peak retention times. Quantification was performed by the internal standard method (DHBA as internal standard) based on the area under the peak.
Rats were deeply anesthetized (200 mg/kg sodium thiopental), and transcardially perfused with saline solution followed by a solution of 4% paraformaldehyde in 0.1M phosphate buffer, pH 7.4 (PB). After perfusion the brains were removed from the skulls, post-fixed in the same solution at room temperature for 2 h and cryoprotected by immersion in 30% sucrose solution in PB at 4°C until they sank. Serial coronal sections (50 μm) were obtained in a cryostat (Leitz, Digital 1702) at −20°C and collected in PB. For immunohistochemistry, the free-floating sections were pretreated with 10% methanol diluted in 3% hydrogen peroxidase for 30 min, carefully washed and blocked with 3% normal goat serum (NGS) in 0.1M phosphate buffer, 0.9% NaCl, pH 7.4 (PBS) containing 0.3% Triton X-100 (PBS-Tx, Sigma) for 30 min and incubated with monoclonal TH antibody raised in mice (Sigma) diluted 1:750 with 3% NGS in PBS-Tx for 48 h at 4°C. After washing several times with PBS-Tx, tissue sections were incubated with a biotinylated secondary antibody (Dako Corporation, Carpinteria, CA) diluted 1:200 in PBS-Tx at room temperature for 2 h. Sections were washed again in PBS and incubated with peroxidase-conjugated streptavidin (Dako) diluted 1:100 in PBS for 90 min at room temperature. The immunohistochemical reaction was developed by incubating the sections in a medium containing 0.06% 3,3 diaminobenzidine (DAB, Sigma) dissolved in PBS for 10 min, and then in the same solution containing 1 μM of 3% H2O2 per ml of DAB medium for 10 min. Next, the sections were rinsed with PBS, dehydrated with ethanol, cleared with xylene, and covered with Permount and coverslips. Control sections were prepared by omitting the primary antibody and replacing it with PBS. The brains of the animals were fixed and postfixed for the same time in identical solutions and rigorously processed at the same time, and the sections were incubated in an identical medium for the same period of time. This precaution was taken to avoid overreaction, differences in chromogen reaction, saturation of optical density, and changes in background levels. Coronal sections of the SNpc were selected according to the atlas of Paxinos and Watson  and the readings were made between the coordinates interaural 4.2 mm, bregma −4.8 mm and interaural −2.7 mm, bregma −6.3 mm. In the SNpc and the VTA, the optical density generated by TH-immunoreactive (ir) neurons was evaluated semi-quantitatively by densitometry, and the number of TH-ir neurons per/mm2 was estimated by planar analysis as previously described . All procedures were done using a Nikon Eclipse E-600 (50×) microscope coupled to a Pro-Series High Performance CCD camera and Image Pro Plus Software 4.1 (Media Cybernetics, Carlsbad, CA).
Plasma 17-β estradiol concentration was determined by double antibody radioimmunoassay (RIA) with MAIA kit provided by Biochem Immunosystems (Bologna, Italy). The lower limit for detection was 5 pg/ml. In order to prevent interassay variation, all samples were assayed in the same RIA. The intra-assay coefficient of variation was 4.3%.
Results are presented as the mean ± S.E.M. Data were analyzed by two-way analysis of variance (ANOVA) taking the estrogen and 6-OHDA treatments as dependent variables. Differences between groups were further analyzed by the post-hoc Duncan test.
Plasma 17-β-estradiol concentration in ovariectomized rats treated or not with this hormone
SNpc (1 μg 6-OHDA)
MFB (1 μg 6-OHDA)
8.41 ± 1.52 (n = 6)
5.54 ± 0.66 (n = 7)
30.42 ± 6.39a (n = 6)
39,86 ± 1.45d (n = 7)
8.96 ± 1.06 (n = 6)
5.34 ± 0.62 (n = 8)
28.45 ± 5.67b (n = 6)
39.95 ± 1.94e (n = 9)
8.90 ± 0.97 (n = 6)
5.33 ± 0.55 (n = 9)
25.20 ± 3.21c (n = 6)
39.44 ± 1.63f (n = 9)
Striatal DA and Metabolites Concentrations
Analysis of DA, DOPAC and HVA concentrations in the nonlesioned striatum (left side) revealed no statistical differences among Control, Sham and 6-OHDA groups, regardless of estradiol treatment. In the nonlesioned side of animals in experiment 1, the average striatal DA, DOPAC and HVA concentrations (ng/mg tissue—mean ± S.E.M) were 6.29 ± 0.14, 3.79 ± 0.48 and 0.64 ± 0.04, respectively. Likewise, in experiment 2, the average striatal DA, DOPAC and HVA concentrations in the left side were 6.86 ± 0.17, 3.76 ± 0.13 and 0.70 ± 0.01, respectively. These data validated the use of contralateral values as control in unilaterally lesioned animals. Thus in the present study, DA, DOPAC and HVA concentrations in the lesioned striatum are presented as a percentage of those in the contralateral side.
Our present data showed that physiological estradiol replacement is not able to attenuate either the loss of NSDA neurons or the reduction in striatal DA levels following 6-OHDA microinjection into the SNpc of ovariectomized rats. This indicates that estrogen at physiological levels is not able to preserve the NSDA pathway against 6-OHDA-induced lesion of the SNpc perikarya. Nevertheless, estrogen neuroprotection against 6-OHDA toxicity was found when the same dose of neurotoxin was injected into the MFB. In this case, estrogen was able to attenuate the 6-OHDA-induced depletion in striatal DA, suggesting that 6-OHDA-induced lesion of the SNpc as a rat model of PD is less responsive to the estrogen neuroprotective effects as compared to the 6-OHDA-induced lesion of the MFB. Such a difference between animal models of PD is relevant in the study of estrogen neuroprotection because provides further information about estrogen potential and limitation in protecting against PD.
In order to evaluate the efficacy of estrogen treatment, plasma estradiol concentrations were determined. In the two experiments performed in this study, 10 days after estradiol-containing pellet implantation, rats presented plasma estradiol within the physiological range of a regular estrous cycle, compatible with the ascending levels found on early proestrus. Conversely, in rats implanted with oil-containing pellets plasma estradiol was considerably lower, below the physiological levels of cycling rats .
In the first experiment of the present work, we used 1 μg of 6-OHDA into the SNpc to evaluate whether estrogen replacement would protect NSDA neurons against an injury of smaller degree. Indeed, the damage produced by 1 μg was less severe than that produced by 6 μg of the neurotoxin in our previous study , i.e., decrease of 53% in striatal DA and 30% in SNpc TH-ir neurons. However, estrogen replacement was again ineffective in exerting a neuroprotective effect upon injured NSDA neurons. Thus, our data indicate that physiological estrogen treatment is not able to alter the loss in either TH-ir neurons or striatal DA innervation following 6-OHDA-induced lesion of the SNpc perikarya. This is in accordance with a previous report that estrogen treatment does not attenuate the apomorphine-induced circling behavior after 6-OHDA lesion of the SNpc .
The decrease in optical density measurement and neuronal density of TH-ir neurons after 6-OHDA infusion into the SNpc was restricted to this structure, without affecting the dopaminergic neurons of VTA. This result is in agreement with those of Kondoh et al.  showing that 4 μg of 6-OHDA injected into the SNpc promoted an almost complete disappearance of TH-ir cell bodies within this nucleus without affecting the VTA neurons, ant that only 8 μg or higher doses of 6-OHDA could also lesion the VTA.
In the second experiment, we administered 6-OHDA into the MFB to evaluate the efficiency of estrogen treatment in protecting against the toxicity produced by 6-OHDA injected in NSDA projections, which would conceivably produce a different degree of lesion, as compared to direct lesion of the NSDA perikarya. In fact, we showed in this model that estrogen replacement in physiological levels was effective in exerting a neuroprotective effect upon injured NSDA neurons, attenuating the depletion in striatal DA.
It is presumed that striatal DA depletion reflects loss of DA neurons in the SNpc, but besides the fact that in our previous study  we did not find estrogen protection of SNpc neurons against 6-OHDA, Mac Arthur and colleagues  also reported that estrogen is not able to protect the SNpc neurons when 1 μg of 6-OHDA is injected into the MFB. In addition, estrogen failed to protected DA cells in culture of mesencephalic neurons from 6-OHDA toxicity . Thus, it seems that estrogen effect on striatal DA depletion in 6-OHDA-induced lesion of the MFB occurs independently of cell survival in the SNpc. One possible explanation for estrogen neuroprotective effect on striatal DA terminals, regardless of cell bodies survival, is the occurrence of adaptative mechanisms in DA neurotransmission, such as altered synthesis, release or metabolism of DA in the surviving striatal terminals. This hypothesis has been supported by the demonstration that estrogen may alter the activity of dopaminergic neurons . Besides reduced DA depletion, our data also showed decreased striatal DOPAC levels in the striatum of estradiol-treated rats following 6-OHDA treatment, which suggests that estrogen neuroprotection may involve alteration in DA metabolism.
Taking into account the studies on estrogen neuroprotection using 6-OHDA-lesion models, data from the second experiment are in accordance with previous reports concerning 6-OHDA injection into the striatum or MFB. Murray et al.  have shown consistent protective effects of estrogen against DA depletion induced by infusion of 1 or 3 μg, but not 6 μg, of 6-OHDA into the MFB of ovariectomized rats. Dluzen  and Peinado et al.  reported protective effect of estrogen replacement upon striatal DA following infusion of 6-OHDA into the striatum of ovariectomized rats.
The discrepancies found in estrogen neuroprotection might be explained by differences in the neuronal lesion obtained after 6-OHDA injection in the SNpc or the MFB. Human PD has a progressive nature, whereas the 6-OHDA-lesion model may promote rapid cell death. Therefore, different 6-OHDA-lesion rat models of PD have been developed to study different stages of PD. 6-OHDA injections into the SNpc, MFB or striatum have been widely used as methods of inducing degeneration of NSDA neurons. Nevertheless, depending on the location of the injection site, animals present different time courses of progression and severity of lesion [8, 18, 20, 22, 38]. The MFB and SNpc as targets can promote profound lesions with high degree of dopaminergic cell loss, which would be equivalent to an end stage of PD . Also, some authors agreed that striatal target would be ideal to test neuroprotection, because it promotes lower cell loss [8, 18, 20, 22]. Jeon et al.  showed that, after 6-OHDA injections into the SNpc or MFB, dopaminergic neurons start degenerating within 24 h, but they did not specificity the degree of lesion.
Truong et al.  used the infusion of 16, 8 and 4 μg of 6-OHDA into the MFB to establish a graded rat model of different clinical stages of PD. They reported that the small dose of 4 μg of 6-OHDA induced a 20% decrease in TH-immunoreactive cells in the SNpc, while in experiment 1 of the present work, 1 μg of 6-OHDA infused into the SNpc caused a greater neuronal loss. When the SNpc is the target, it appears that 6-OHDA insult is able to promote extensive lesion. Kondoh et al.  compared lesions induced by 6-OHDA infusion into the SNpc and striatum, monitored by magnetic resonance imaging. Their results showed that intranigral injection of 6-OHDA induced more extensive damage areas and behavioral abnormalities as compared to the intrastriatal one. Likewise, an extensive damage produced by intranigral infusion of 6-OHDA was also observed in our data and in a previous study of Tamás et al. . Even using different doses of 1, 4 and 6 μg of 6-OHDA, both studies reported similar dopaminergic cells loss of about 60% in the SNpc of female rats. Thus, the extensive lesion of NSDA neurons following 6-OHDA infusion into the SNpc has been suggested to reproduce final stages of PD . Moreover, in the present study 6-OHDA lesions were done in the right SNpc, which may have produced an even greater DA depletion, responsiveness to 6-OHDA, with the right side being more susceptible to the neurotoxin .
Our present data showed that injection of 6-OHDA into the SNpc promotes a higher depletion of striatal DA compared with the same dose injected in the MFB. In addition, we found a more dramatic loss in TH-ir neurons following injection of 1 μg of 6-OHDA into the SNpc in comparison with previous studies infusing the neurotoxin in either the striatum or MFB [19, 26, 28]. Thus, the major contribution of the present study is to suggest that estrogen neuroprotection upon striatal DA depends on the location of 6-OHDA injury within the nigrostriatal pathway, which seems to be determinant for the temporal progression and severity of lesion. Estrogen neuroprotection is therefore more likely occur for moderate but not for large lesion of the NSDA pathway.
It is worth noting that toxin injection into the MFB induces dopaminergic cell death retrogradely. Thus, this model may provide a more prolonged temporal window for neuroprotective effects relative to 6-OHDA injection into the SNpc, which probably causes a more rapid and dramatic cell death. However, further studies are necessary to elucidate why estrogen may protect NSDA neurons against 6-OHDA lesion of the MFB but not of the SNpc. Speculatively, one might address the role of dopamine transporter (DAT), because it provides 6-OHDA neuronal uptake  and is apparently modulated by estrogen [27, 44]. It has been suggested a differential distribution of the DAT in midbrain neuronal populations. For example, the particular expression of DAT in tuberoinfundibular dopaminergic neurons is thought to underlie their resistance to be affected in PD .
In summary, the results of our study suggest that estrogen protects striatal DA against 6-OHDA injury of NSDA neurons projections but not perikarya, which may ultimately depend on the severity of lesion. Thus, estrogen neuroprotection appears to be potentially dependent on the severity of lesion promoted in different animal models of PD.
The authors are indebted to Tatiana Nemoto Piccoli Moraes, Renata Lins Fuentes Araújo, Janyana Marcela Doro Deonízio and Renata Vaguetti for their participation in some experiments and to Ruither Oliveira Carolino for technical support. The financial support of CNPq, CAPES and FAPESP is also acknowledged.