Brain Research 943 (2002) 163–173 www.elsevier.com / locate / bres
Research report
Protective effect of melatonin in a chronic experimental model of Parkinson’s disease ´ a , Juan Carlos Mayo a , Rosa Marıa ´ Sainz a,b , Marıa ´ de los Angeles del Brıo ´ a, Isaac Antolın a,b , * ´ a , Carmen Rodrıguez ´ Federico Herrera a , Vanesa Martın a
´ y Biologıa ´ Celular, Facultad de Medicina, Universidad de Oviedo, C / Julıan ´ Claverıa ´ , 33006 Oviedo, Asturias, Spain Departmento de Morfologıa b ´ Instituto Universitario Oncologico del Principado de Asturias ( IUOPA), Facultad de Medicina, Universidad de Oviedo, 33006 Oviedo, Asturias, Spain Accepted 15 January 2002
Abstract Parkinson’s disease is a chronic condition characterized by cell death of dopaminergic neurons mainly in the substantia nigra. Among the several experimental models used in mice for the study of Parkinson’s disease 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine- (MPTP-) induced parkinsonism is perhaps the most commonly used. This neurotoxin has classically been applied acutely or sub-acutely to animals. In this paper we use a chronic experimental model for the study of Parkinson’s disease where a low dose (15 mg / kg bw) of MPTP was administered during 35 days to mice to induce nigral cell death in a non-acute way thus emulating the chronic condition of the disease in humans. Free radical damage has been implicated in the origin of this degeneration. We found that the antioxidant melatonin (500 mg / kg bw) prevents cell death as well as the damage induced by chronic administration of MPTP measured as number of nigral cells, tyrosine hydroxylase levels, and several ultra-structural features. Melatonin, which easily passes the blood–brain barrier and lacks of any relevant side-effect, is proposed as a potential therapy agent to prevent the disease and / or its progression. 2002 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Degenerative disease: Parkinson’s Keywords: Melatonin; Parkinson’s disease; 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP); Chronic experimental model; Mice
1. Introduction Parkinson’s disease is a neurodegenerative disorder characterized by bradykinesia, resting tremor and rigidity. The cause of the disease is the slow and progressive degeneration of dopaminergic neurons in the substantia nigra. Given that clinical symptoms do not show until more than 80% of the said neurons have died [29], it has been suggested that apoptosis, which is not accompanied by inflammation, or another type of non-necrotic cellular
* Corresponding author. Present address: Departamento de Morfologıa ´ ´ Celular, Facultad de Medicina, Universidad de Oviedo, C / y Biologıa ´ ´ 33006 Oviedo, Spain. Tel.: 134-98-510-3057; fax: Julıan Claverıa, 134-98-510-3618. ´ E-mail address:
[email protected] (C. Rodrıguez).
death, is the mechanism involved in this demise, although controversy on this point [26,36] still exists. The etiology of dopaminergic neurons death is not known. However, reported data suggest oxidative stress as the likely candidate to mediate in the original unknown cause. Studies on patients’ brains have given clues in support of this hypothesis. Levels of reduced glutathione are low [44], while the level of the antioxidant enzyme manganese superoxide dismutase is high and is not paralleled by a rise in glutathione peroxidase [49]. Iron level increase has also been reported [18]. Since iron is able to catalyze the Fenton reaction, this implies hydroxyl radical (HO ? ) formation. Radical damage has been demonstrated in lipids [56], proteins [3], and nucleic acids [4] of the substantia nigra of parkinsonian patients. There are reasons for dopaminergic neurons to be more susceptible than other types to exogenous toxins that
0006-8993 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )02551-9
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produce an increase of reactive oxygen species (ROS). The most important is the very use of dopamine as a neurotransmitter. Firstly, catabolic pathways of this amine drive to radical formation [48] and secondly, an end-product of the auto-oxidation of dopamine, the neuromelanin, has been reported to bind iron (that remains reactive) thus enhancing the formation of HO ? through the Fenton reaction [57] and increasing the lipid peroxidation induced by this metal [6]. Clinical trials using antioxidants (vitamin E) in the treatment of Parkinson’s disease have been performed without apparent success (DATATOP study [42,43]). The poor ability of vitamin E to cross the blood–brain barrier [40] should be kept in mind as a possible cause for the lack of effect of this antioxidant in Parkinson’s disease. Melatonin is a neurohormone that was first reported in 1993 by Reiter’s group as an efficient endogenous antioxidant [54]. Since then, evidences showing this molecule to be one of the best physiological antioxidants and in vivo cell protectors has been building up (see Refs. [46,47] for review). What makes melatonin different from other potent antioxidants is (i) its solubility in both lipids [12] and water [51] allowing melatonin to easily enter anywhere in the cell, and (ii) its ability to pass the brain–blood barrier [45] allowing melatonin to enter both glial and neuronal cells [34]. Another hint pointing to melatonin as a possible effective agent in preventing and avoiding neurodegenerative diseases is the fact that this hormone decreases in the body with age (consequently lowering the levels of one important agent of the antioxidant defenses of the organism), thus suggesting a possible concurrent cause of the appearance of neurodegeneration in the elderly. Melatonin has been proven to protect neuronal cells from neurotoxin-induced damage in a wide spectrum of neuronal culture systems serving as experimental models for the study of Parkinson’s disease (for review see Ref. [47]). In vivo experiments are however scarce and have always been done in acute experimental models of the disease. These acute studies show protective effects of melatonin in both the striatum dopaminergic axons [1] and the midbrain neurons [38]. The aim of the present study was to elucidate if the antioxidant melatonin is capable of preventing the dopaminergic cell death induced in a chronic in vivo experimental model of Parkinson’s disease. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been shown to induce parkinsonism in humans [16,28], primates [9] and mice, particularly in the C57b1 / 6 strain [23], showing evidence of oxidative stress in these models [19,24,37,50]. A differential feature that distinguishes the acute MPTP toxicity in mice versus primates is the fact that gradual recovery of dopamine levels and striatal dopaminergic fibers in mice occurs a few days after drug administration, becoming total over longer periods [13,21,35]. This fact could suggest some plasticity at the nigrostriatal system [35], but it has also been proposed that
nigral neurons in mice do not actually die after an acute administration of MPTP as happens in primates. Some authors think that these neurons would be simply functionally damaged as reflected by the temporal loss of striatal dopamine and the recovery of tyrosine hydroxylase (TH) in nigral neurons [25,58]. In the present work we used a chronic experimental model of Parkinson’s disease, formerly reported by some authors [7,55] using MPTP administered daily at low doses (15 mg / kg bw) over a long period of time (35 days). This would lead nigral neurons to progressively die, better mimicking therefore the disease in humans and offering a more relevant experimental model for the testing of potential anti-parkinsonian agents.
2. Materials and methods
2.1. Animals and experimental design A total of 24 male, 8-week-old, C57bl / 6 mice (Charles River, Wilmington, MA, USA) were used in the experiment. Animals were maintained eight per cage with a 12 / 12 light–dark cycle (lights on at 08:00 h) and free access to food and water. The authors have followed the National Research Council’s guide for the care and use of laboratory animals during the experiment. Mice were divided into three groups of eight mice each. The first group received a daily intraperitoneal injection of saline. The second group was daily injected with 15 mg / kg body weight of MPTP in saline. The last group was intraperitoneally administered MPTP (15 mg / kg body weight) plus melatonin (500 mg / kg body weight) 30 min prior to the administration of the MPTP. Treatments were given at 18:00 h. All groups were treated for 35 consecutive days being sacrificed by decapitation. The brain was quickly removed and divided into two halves. Right mid brain of four animals was frozen in liquid nitrogen and stored at 280 8C until used. Left mid brain of four animals was fixed in 4% paraformaldehyde in 0.1 M phosphate buffer for 24 h at 4 8C, dehydrated, and embedded in paraffin for immunohistochemistry studies. The remaining tissue was processed for light and electron microscopy as described in the following section.
2.2. Light and electron microscopy studies Left mid brain of four animals was fixed at 4 8C with ¨ 4% paraformaldehyde (Sigma-Aldrich, Riedel-de Haen, ¨ Seelze, Germany) in 0.1 M Sorensen phosphate buffer, pH 7.2, for light microscopy studies. The right mid brain was fixed with 2.5% paraformaldehyde and 1.5% electron microscope grade glutaraldehyde (Sigma-Aldrich, Fluka, Buchs, Germany) in the same buffer for ultrastructural studies. After 1 h of fixation, a 1 mm-thick slice that included the substantia nigra (SN) was isolated from each
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mid brain and fixation was prolonged overnight in fresh fixative. For light microscopy, slices were dehydrated in a graded series of ethyl alcohol and embedded in Unicryl姠 (BBI, Cardiff, UK) resin with a progressive decrease in temperature (4 to 220 8C). Then, the tissue slices were introduced to BEEM姠 capsules containing fresh resin and placed for 48 h at 220 8C in a polymerization chamber equipped with two 8-W BLB lamps. For electron microscopy, postfixation of the slices in 1% OsO 4 containing 1.25% potassium ferrocyanide was carried out. Afterwards, tissue was dehydrated in a graded series of acetone and embedded in Spurr resin (EMS, Fort Washington, PA, USA). Finally, blocks were polymerized at 70 8C during 48 h. Blocks were cut using a diamond knife placed on a Reicher-Jung Ultracut-E ultramicrotome. Unicryl semithin sections (1 mm) were stained with 0.2% toluidine blue containing 2% borax and photographed in a Leitz Orthoplan light microscope equipped with an Olympus DP-11 digital camera. Spurr ultrathin sections were stained with uranyl acetate and lead citrate and photographed in a Zeiss EM-109 transmission electron microscope equipped with a 35-mm camera. The negatives were digitalized using a Plustek OpticPro UT12 scanner. All digital images were finally captured and processed with Paint Shop Pro 5.0 software.
2.3. Morphometric study Three Unicryl sections (1 mm) per animal representing the mesencephalic coronal planes 335, 343 and 351 of the mice atlas of Sidman et al. [52] were chosen to quantify the number of neurons in the substantia nigra pars compacta (SNpc). Neurons were counted in a Nikon Labophot light microscope (3400) using an ocular with a superimposed grid. Neurons were counted only if they stained above background and they contained a nucleus surrounded by non-shrunken perikarion. The full extent of the structure in each section was examined.
2.4. Immunohistochemistry Deparaffined 5 mm-thick coronal sections corresponding to the mesencephalic coronal plane 351 of the mice atlas of Sidman et al. [52] were incubated with 1% H 2 O 2 and 30% methanol in 0.1 M phosphate buffered saline (PBS) to block endogenous peroxidases. After being treated with 0.05% saponin and incubated with blocking solution containing 3% normal horse serum for 2 h, they were incubated overnight at room temperature with the primary TH-mouse monoclonal antibody (Roche Diagnostics, Mannhein, Germany) diluted 1:500 in PBS containing 0.2% Triton X-100 and 0.2% gelatin. Afterwards, sections were incubated at room temperature with biotinylated horse
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anti-mouse IgG (Roche Diagnostics, Mannhein, Germany) diluted 1:200 for 1 h, followed by streptavidin–biotin– peroxidase complex 1:100 (Vector Laboratories, Burlingame, CA, USA) for 1 h. The reaction was visualized with 0.05% diaminobenzidine (Sigma-Aldrich, Sigma, St. Louis, MO, USA) and 0.005% H 2 O 2 .
2.5. Western blot Slices of brain tissue frozen at 280 8C and containing the substantia nigra (SN) were individually homogenized in RIPA buffer (0.1 M PBS, 1% non-ionic detergent (Igepal), 0.5% deoxycholic acid (sodium salt), 0.1% sodium dodecyl sulfate, 1 mM dithiothreit) containing the protease inhibitors phenyl-methlysulfonyl fluoride (0.1 mM), leupeptin (2 mg / ml), and aprotinin (4.6 mg / ml). After incubation in buffer for 30 min at 4 8C, samples were centrifuged at 10 0003g for 10 min at 4 8C. Supernatants were collected and protein concentration was determined [8]. A 30-mg sample of total protein was denatured at 100 8C for 5 min in sample buffer (0.125 M Tris–HCl, pH 6.8, 4% sodium dodecyl sulfate, 20% glycerol, 10% bmercaptoethanol, and 0.006% bromophenol blue) and electrophoresed onto a 7% SDS–PAGE. After transfer to a Hybond姠 ECL nitrocellulose membrane (Amersham Pharmacia Biotech UK, Buckinghamshire, UK) and in order to verify the quality and equal concentration of the proteins in the filter, staining with Ponceau S was carried out. The blot was afterwards washed in Tris-buffered saline containing 0.05% Tween 20, saturated with 5% bovine serum albumin ´ Vevey, Switzerland) and 5% skim milk (Molico , Nestle, for 1 h and incubated with TH-mouse monoclonal antibody (Roche Diagnostics, Mannhein, Germany) diluted 1:2000 overnight at 4 8C. After washing, blot was incubated with horse anti-mouse IgG linked with peroxidase (Roche Diagnostics, Mannhein, Germany) diluted 1:2000 for 1 h. The reaction was developed using an ECL chemiluminescence method (Amersham Pharmacia Biotech UK, Buckinghamshire, UK). Image Tool 2.0 (downloadable at http: / / www.uthscsa.edu / dig / download.html with ELFO module (available at http: / / www.saske.sk / |tomori / download / gel / gel.htm) were used to quantify Western blot signals after scanning.
2.6. TUNEL DNA nick end labeling in the sites of DNA 39OH breaks with biotinylated deoxyuridine triphosphate (Roche Diagnostics, Mannhein, Germany) was carried out by a terminal deoxynucleotidyl transferase (TdT) (Roche Diagnostics, Mannhein, Germany) and amplified by a streptavidin– peroxidase complex (Vector Laboratories, Burlingame, CA, USA). Unicryl semithin sections (1 mm) were treated as described by Gavrieli et al. [20].
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2.7. Statistical analysis Data were analyzed using one-way analysis of variance (ANOVA) followed by Student-Newman-Keuls post-hoc testing. All values are expressed as the mean6S.E.M.
3. Results
3.1. Long term MPTP treatment at low doses induces nigral dopaminergic cell death which is prevented by melatonin After 35 days of low-dose treatment of mice with MPTP (15 mg / kg bw), cell count in the SNpc is reduced by 52% (Figs. 1 and 2). Tyrosine hydroxylase immunohistochemistry studies show parallel loss of the TH immunoreactivity in the neurons of this area (Fig. 2). Measurement of the amount of the enzyme by Western blot shows similar results, with a drop in TH in the midbrain of MPTP-treated animals (Fig. 1). MPTP-treated mice do not show any loss of nigral cells when simultaneously given melatonin (500 mg / kg bw) (Figs. 1 and 2). TH immunohistochemistry (Fig. 2) and Western blot (Fig. 1) show that melatonin totally prevented the loss of the TH immunoreactivity.
3.2. Surviving neurons after 1 month of chronic MPTP treatment present damage that is prevented by melatonin administration Nigral neurons of non-treated animals show a basophilic perikaryon when stained with toluidine blue (Fig. 3).
Surviving nigral cells of mice treated with MPTP show, besides the normal type, two additional types of neurons having morphological alterations. One type shows light toluidine staining while another one consists of shrunken cells which are darkly stained (Fig. 3). In the group treated with MPTP and melatonin, nigral cells appear elongated and present a basophilia that is similar to that in the controls (Fig. 3). Both light and dark neurons present ultrastructural alterations. Light cells (Fig. 4) show the loss of both the Golgi apparatus, cisternae and Nissl substance. They also present a decrease in the number of mitochondria and show an irregular nuclear outline. Some of them suffer from plasma or nuclear membrane rupture. Dark cells (Fig. 4) present a higher electrondensity in their cytoplasm and the mitochondrial matrix appears to be cleared up. The nucleus also shows increased electrondensity, the chromatin being fragmented into small masses. Integrity of the membranes is nonetheless preserved. Medium electrondense neurons with swollen endoplasmic reticulum cisternae and perinuclear space are also found. Most glial cells observed in the substantia nigra of mice display the morphological features of oligodendrocytes (Fig. 5) with few cells showing the ultrastructural appearance of astrocytes (lightly stained when compared to oligodendrocytes and having the characteristic filaments in the cytoplasm). MPTP-treated mice present alterations in the oligodendrocytes. Some of these cells present some kind of early damage with clearing of the mitochondrial matrix, loss of cristae, fragmentation of the Golgi apparatus cisternae, and enlargement of the perinuclear space (Fig. 5). Others show more advanced damage with ruptured cytoplasmic components and disruption of the membrane (Figs. 4 and 5). Some oligodendrocytes show a higher nuclear density (Fig. 5). Neuropil show swollen axons with damaged myelin sheath (Fig. 5) and with many empty spaces, some of them surrounded by a very thin myelin sheath (Fig. 4). Damage of myelinated and unmyelinated nerve fibers is also observed (Fig. 5). None of the melatonin treated groups showed any of the mentioned alterations (Fig. 5).
3.3. DNA fragmentation is present in the dark cells
Fig. 1. Densitometric measure of the tyrosine hydroxylase Western blot signals (gray bars) and number of neurons as counted in 1-mm sections stained with toluidine blue (black bars) in the substantia nigra of C57bl / 6 mice after 35 days of treatment with one daily i.p. injection of saline (CONTROL), MPTP (15 mg / kg bw) or MPTP plus melatonin (500 mg / kg bw) (n54). *P,0.05 and **P,0.01 vs. MPTP-treated group (A). Signals of a representative tyrosine hydroxylase immunoblot (B).
Although the morphology of the damaged cells observed in the group treated with MPTP shares only some of the typical features of apoptosis, the question of whether the alterations they showed underwent DNA fragmentation soon arose. TUNEL labeling of semithin sections shows some scarce stained cells in both control and melatonintreated groups (Fig. 3). MPTP-treated mice showed a large number of nuclear TUNEL positive cells (Fig. 3). The stained cells correspond well to the dark cells (as seen in the ultrastructural studies) and are mainly located in the medial area of the SNpc. TUNEL positive nigral cells in the MPTP group show the peculiarity of positive staining in the cytoplasm (Fig. 3).
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Fig. 2. Representative unicryl semithin sections (1 mm) stained with toluidine blue (A, C and E) and TH immunohistochemistry in paraffin sections (5 mm) (B, D and F) of the substantia nigra of C57bl / 6 mice. Control (A, B); MPTP (C, D); melatonin plus MPTP (E, F). Notice that loss of neurons in the substantia nigra (toluidine staining) is correlated with a parallel loss of the TH-positive neurons in the MPTP-treated animals. Prevention by melatonin of the MPTP cytotoxic effect in terms of cell death and TH labeling loss can be also observed. Bars represents 100 mm.
4. Discussion In the present work a chronic experimental model of Parkinson’s disease was used. Daily administration of a low dose (15 mg / kg bw) of MPTP during 35 days results
in the death of 52% of the SNpc neurons with a parallel decrease in the TH immunoreactivity in this brain area. Although some authors reported nigral cell death after short-term treatments in mice [53], the actual death of the cells has not been confirmed as reports base their results on
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Fig. 3. Detection of DNA fragmentation by TUNEL in the substantia nigra. (A) MPTP-treated mice which also received melatonin treatment. Most of the cells are unlabeled (arrowheads) and present the same morphological features as the control (not shown). Bar represents 25 mm. The insert displays some TUNEL positive neurons with the labeling restricted to the nucleus (arrows). Bar represents 10 mm. A representative unicryl semithin section counterstained with toluidine blue is shown. (B) Representative unicryl semithin section of the MPTP-treated mice stained with toluidine blue. Notice that it shows both the dark (arrowheads) and the pale (arrows) neurons. Bar represents 25 mm. (C) MPTP-treated mice. Numerous shrunken neurons with positive staining can be observed (arrowheads). Observe that non-shrunken neurons are unlabeled (arrows). Bar represents 25 mm. Note in the insert that, in contrast to (A), the labeled neurons exhibit cytoplasmic labeling (arrowheads) in addition to nuclear labeling (arrows). Bar represents 10 mm. A representative unicryl semithin section counterstained with toluidine blue is shown.
measuring striatal dopamine or TH-positive immunohistochemistry. Zhang et al. [58] showed that after 5 days of MPTP administration TH reactivity and dopamine decrease in the striatum, but TH-positive neurons do not change in the substantia nigra, indicating that only the striatal terminals are affected and that nigral neurons are not dead. Some authors even report that although some dopaminergic cells suspend TH activity after MPTP injection, they are not actually dead and will eventually recover after some time [25]. Cochiolo et al. [11] found only scarce mitochondrial damage in the cells of the substantia nigra 24 h after single MPTP administration of 20 and 40 mg / kg; the striatum, however, was already affected, with axonal degeneration and microglia reaction. This indicates that MPTP damage probably starts at the level of the axons and the synaptic end buttons, which would therefore be the first part of the cell sensitive to the cessation of energy production from the damaged mitochondria, the first target of MPTP. Lesions of these kind do not ensure that the entire cell will die, a spontaneous or induced recovery being possible. This could explain why Arai et al. [5] found lesions only in the neuronal bodies when brain was studied after 1 month following 5-day treatment with MPTP. After 1 month many of the neurons with axonal changes would be either dead or fully recovered, as previously suggested by Lewandowska et al. [30]. This could therefore be, besides the adaptive plasticity of the non-affected axons suggested by these authors, the reason why authors find dopamine and TH recovery some time after a single or a few injections of the drug. This important finding indicates that several injections of the drug should be given in order to effectively induce nigral cell death to really emulate Parkinson’s disease. It also suggests that reactive TH in the striatum is not the appropriate parameter to conclude that an agent prevents nigral cell death. Additional counting of cells in the SNpc using a basic dye, as hinted by other authors [25,53], would be necessary since the absence of TH is functional data and does not necessarily imply cellular death and or exclude the possibility of regeneration. In the present work most of the remaining neurons, excluding the already dead cells, also presented alterations. Several of these cells were demonstrated to be undergoing a non-necrotic death as evaluated by TUNEL. The socalled dark cells described in the ultrastructural study, correspond well with the TUNEL positive cells. The fact that they show DNA fragmentation but not the typical apoptotic morphology indicates that they are undergoing a non-necrotic cell death that could well represent the morphologically known dark degeneration. Therefore, dark degeneration is a type of cell death that might well either be alternative to necrotic and apoptotic cell death or a subtype of apoptosis. An unexpected finding from this study is the TUNEL staining of part of the cytoplasm in the dark degenerated
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Fig. 5. Ultrastructural aspect of the glial cells and the nerve fibers in the substantia nigra. MPTP plus melatonin (A); MPTP (C–G). (A) Oligodendrocyte showing the same ultrastructural features as seen in the control group (not shown). The Golgi apparatus areas (G) are well defined and the rough endoreticulum displays single short cisternae (arrows). The nucleus is located eccentric with clumped chromatin beneath the nuclear envelope (arrowheads). Bar represents 1 mm. (B) Detail of a damaged oligodendrocyte showing some mitochondria with clear matrix and loss of the cristae (m). Bar represents 0.5 mm. (C) Detail of a damaged oligodendrocyte with Golgi apparatus cisternae (G) fragmented into vesicles. Bar represents 0.5 mm. (D) Damaged oligodendrocyte with clearing of the cytoplasm due to the breaking up of its components. The nucleus (n) is denser and shows bigger clumps of heterochromatin than that in panel A. The perinuclear space is dilated (arrowhead). Inside the myelinated axons empty membrane limited spaces (stars) can be also observed. Bar represents 1 mm. (E) This panel shows a swelling myelinated axon (star) surrounded by non-swelling axons (asterisks). Bar represents 1 mm. (F and G) Myelinated and unmyelinated nerve fibers displaying abnormal dense filamentous material. Bar represents 0.5 mm (F) and 0.25 mm (G).
Fig. 4. Ultrastructural aspect of the neurons of the substantia nigra pars compacta. Melatonin plus MPTP (A, B); MPTP (C–F). (A) Neurons present the same ultrastructural characteristics as those in the control group (not shown). The nucleus (n) is euchromatic with irregular outline. Both the rough endoplasmic reticulum cisternae (r) and the mitochondria (m) are numerous and scattered throughout the perikarion. The Golgi apparatus (G) is well developed. Bar represents 1 mm. (B) Sometimes the Golgi apparatus displays a reticular organization. Bar represents 1 mm. (C) Light damaged neuron showing very few mitochondria (m), some of which present a clear matrix (mx). The nucleus (n) displays an irregular outline. Bar represents 1 mm. (D) Breakdown areas of the plasma membrane (arrows) can be also observed in some of the light neurons (N) and oligodendrocytes (O). Bar represents 1 mm. (E) Dark damaged neuron that displays a very dense perikarion with many clear mitochondria (m) and a well developed rough endoplasmic reticulum (r). These neurons also show a collapsed nucleus with the chromatin very fragmented (arrows). Bar represents 1 mm. The Golgi apparatus cisternae (G) are dilated (insert). Bar represents 1 mm. (F) Damaged neuron that presents moderately dense cytoplasm. Both rough endoplasmic reticulum cisternae (r) and perinuclear space (p) are dilated. The nucleus (n) is very indented. The neuropil presents numerous empty spaces (star). Bar represents 1 mm. The insert show a very thin myelin sheath surrounding one of them. Bar represents 0.1 mm.
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neurons of the substantia nigra (in addition to the nuclear staining). No such staining was present in DNA-ase-treated positive controls, indicating that it is specific for the dark degenerating cells. Mitochondrial DNA (mtDNA) fragmentation after oxidative stress had been previously reported by others [14,22,39] and was found unlabeled by TUNEL [14]. Hayakawa et al. [22], proposed that random double-strand breaks could be formed after free radical attack, followed by rejoining of mtDNA with formation of minicircles. This could be the reason why Cui et al. [14] did not find TUNEL labeling mtDNA fragmentation after 60-min ischemia. MPTP induces direct inhibition of complex I of the mitochondrial respiratory chain [37] producing a large number of free radicals and an energy crisis in the mitochondria. It is possible therefore, that after a chronic (35-day) MPTP treatment the oxidative attack is so aggressive that double strands are too small to form minicircles anymore, mtDNA being totally fragmented and 39OH termini free to be TUNEL-labeled. These TUNEL positive (nucleus and cytoplasm) cells that correspond to the dark degenerated cells would represent cells committed to die. Light cells observed in the optical and ultrastructural studies of our work are not stained by TUNEL. Some of them showing membrane breaking could represent cells with irreversible degeneration, while others, with nonaffected membranes, could represent cells with decreased metabolism waiting for better circumstances to recover. These two types of degenerating cells correspond well with the description of Maxwell [32] who, after ischemiareperfusion (he too found both cell types), considered light cells with intact membranes as cells capable of recovering after the insult. Although signs of oxidative stress were not evaluated in the present work, remaining as a priority for further studies, the fact that dopaminergic cells in the substantia nigra are unequivocally dead and that this death is chronically occurring by non-necrotic cell death confirm the suitability of this model for the study of Parkinson’s disease. Melatonin is a neurohormone classically known to be involved in the regulation of the circadian and seasonal rhythms [10] and to present sleep-inducing properties [17]. Since Tan et al. [54] showed its antioxidant properties, multiple reports have been made analyzing the antioxidant mechanisms of melatonin (for review see Ref. [47]). This antioxidant agent has been shown to protect neurons against neurotoxin damage in several in vitro neuronal systems [31,33,41]. Mayo et al. [33] also reported that melatonin regulated the mRNA for antioxidant enzymes in dopaminergic cells after the addition of the neurotoxin 6-OHDA, indicating that this could be the mechanism involved in the neuroprotective properties of this agent. In vivo studies with melatonin in experimental models ˜ of Parkinson’s disease are however scarce. Acuna-Castroviejo et al. [1] found this agent prevented an increase in
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lipid peroxidation and a decrease in TH immunoreactivity in the striatum after a single dose of MPTP. Results reported by these authors are very relevant, since melatonin was able to avoid the damage caused by this drug in the striatal dopaminergic axons. Ortiz et al. [38] reported, using DNA electrophoresis, apoptosis of midbrain neurons induced by a single dose of MPTP. Melatonin also prevented both cell damage and DNA fragmentation. Finally, melatonin was able to counteract the decrease in striatal TH immunoreactivity and the loss of complex I activity produced in rats after acute 6-OHDA administration [15,27]. The study presented here demonstrates that melatonin prevents nigral dopaminergic cell damage produced by chronic administration of low doses of MPTP in terms of the number of nigral cells present, their functionality as measured by TH immunohistochemistry and protein concentration (Western blot), and their healthy appearance as seen by TUNEL and ultra-structural morphological features. It is shown, for the first time, that melatonin clearly prevents nigral dopaminergic cell death induced by a chronic treatment used as an experimental model of Parkinson’s disease. In both the acute studies [1,38] and the present work, melatonin was given before and during the administration of the neurotoxin, indicating that melatonin may prevent neurotoxin damage. Nevertheless, the present results are not able to discern if this antioxidant may improve an already established disease, and further studies are still necessary in order to clarify this point. Elucidation of the mechanisms of how melatonin protects against MPTP has not been undertaken in this study. MPTP toxicity is based on direct inhibition of complex I of the electron transport chain in the mitochondria by its metabolite MPP1. Inhibition of the said complex has also been reported in the substantia nigra of patients suffering Parkinson’s disease. This inhibition causes energy depletion and increases free radical concentration in the mitochondria. The antioxidant effects of melatonin and its protective effects against the uncoupling of the electron transport chain of several toxins in the ˜ mitochondria have been well summarized by Acuna-Castroviejo et al. [2]. All these data altogether push us to undertake further studies based on this hypothesis. Levels of the antioxidant melatonin tend to decrease with age in contrast to the increased incidence of neurodegenerative diseases. Aging and neurodegenerative diseases have been proposed as a consequence of the imbalance (physiological or toxin-induced) between oxidant production by the organism and its antioxidant defense system. Other constituents of this antioxidant system have not been found to decrease with age, melatonin being the only one matching this age-related pattern. This points to an increase in free radicals which are endogenously or exogenously produced. The protective effect of melatonin demonstrated in abundant cell culture experiments together with the in vivo protection against the acutely 6-OHDA and
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MPTP-induced cell damage and the chronically-induced damage by MPTP reported here, make melatonin a plausible candidate in the prevention of the appearance of these diseases and give a clue to its use as a treatment to avoid disease progression.
[12] [13]
[14]
Acknowledgements This work was funded by grants from the FEDER funding (European Union) and CICYT (Spain) to C.R. (1FD97-0009) and from CICYT to C.R. (SAF00-0010). R.M.S. was supported by a postdoctoral fellowship from the IUOPA. F.H. was supported by a BEFI fellowship from the Program of Biomedical Research of the Health Institute ˜ Carlos III (FIS). The authors wish to thank Fernando Janez and Carlos Villa from the Service of Electron Microscopy of the University of Oviedo for their technical assistance during the electron microscope study. The contribution from ASTURPHARMA S.A. is also acknowledged.
[15]
[16]
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