Cell Biochem Biophys DOI 10.1007/s12013-014-0048-8
ORIGINAL PAPER
Edaravone Protects Neurons in the Rat Substantia Nigra Against 6-Hydroxydopamine-Induced Oxidative Stress Damage Xiqi Liu • Rushing Shao • Meng Li Guofeng Yang
•
Ó Springer Science+Business Media New York 2014
Abstract To investigate the mechanism of the neuroprotective effect of edaravone in substantia nigra (SN) of the 6-OHDA-induced rat model of Parkinson’s disease. Animal model of Parkinson’s disease was induced in male Sprague– Dawley rats by injecting 6-OHDA into the left medial forebrain bundle. Subsequently, rats were intraperitoneally injected with 0.3, 1, or 3 mg/kg of edaravone for 14 days or with 3 mg/kg edaravone for 14 days followed by 14 days of no treatment. We evaluated the effect of edaravone on the rotational and normal behavior of the rats, and on the number of tyrosine hydroxylase (TH)-positive cells, the amount of Nissl bodies, and the levels of glutathione (GSH), and malondialdehyde (MDA) in the SN. Edaravone treatment at 3 mg/kg significantly reduced apomorphineinduced rotational behavior (P \ 0.01), improved the spontaneous behavior, prevented the decrease in the levels of TH-positive cells, Nissl bodies and GSH, and inhibited the increase in the levels of MDA (P \ 0.05) in SN of rats with 6-OHDA-induced PD. Edaravone exerted a long-term neuroprotective effects in 6-OHDA-induced PD animal model by attenuating changes in the levels of GSH and MDA in SN, caused by oxidative stress. Edaravone prevented 6-OHDA-induced behavioral changes and de-pigmentation of SN that results from the loss of dopaminergic neurons.
X. Liu R. Shao M. Li Department of Neurology, The Central Hospital of Cangzhou City, Cangzhou 061000, Hebei, China G. Yang (&) Department of Neurology, The Second Hospital of Hebei Medical University, Shijiazhuang 050000, China e-mail: [email protected]; [email protected]
Keywords Parkinson’s disease Edaravone 6-OHDA Glutathione Malondialdehyde
Introduction Parkinson’s disease (PD) is a progressive neurodegenerative disorder, that is characterized by cumulative neuronal loss. It is characterized by a number of motor function abnormalities, such as bradykinesia, tremors, muscular rigidity, and postural instability, mainly caused by the progressive loss of dopaminergic neurons located within a pigmented region of the brain called the substantia nigra (SN) [1]. A number of pathogenic factors have been implicated in causing PD including oxidative stress, mitochondrial dysfunction, inflammation, excitotoxicity, and signals that mediate apoptosis [2]. The brain is especially susceptible to oxidative damage because of its high levels of polyunsaturated fatty acids and relatively low antioxidant activity [3]. Previous reports show that the significant oxidative damage to lipids, proteins, and DNA observed in postmortem PD striatum and within the SN [4–10], correlated with decreased levels of glutathione (GSH), one of the body’s most important antioxidants and a biomarker of oxidative stress [11, 12], and increased levels of malondialdehyde (MDA), a marker of lipid peroxidation [5]. Since existing medical care for PD primarily focuses on symptom management, and oxidative stress plays a pivotal role in the degeneration of dopaminergic neurons of the SN, compounds with antioxidant capacities may be efficient in stopping or slowing down the progression of PD [13, 14]. Edaravone (3-methyl-1-phenyl-2-pyrazolin-5one) is a potent hydroxy radical scavenger and antioxidant [15] that is able to suppress the production of nitric oxide
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and reactive oxygen species by activated microglia [16]. Recent studies showed that edaravone has in vitro and in vivo anti-apoptotic, anti-oxidative, and anti-inflammatory neuroprotective effects in animal model of PD with 6-hydroxydopamine (6-OHDA)-induced selective dopaminergic neuronal loss in the SN [17]. The present study sought to determine the effect of edaravone on GSH and MDA levels in the SN and corpus striatum and its short-term and long-term neuroprotective effect in a rat model of 6-OHDA-induced PD.
Methods Experimental Animals Seventy-two healthy adult male Sprague–Dawley rats, weighing 250–300 g, were provided by the Laboratory Animal Center, Hebei Medical University, China. All experiments using animals were performed in accordance with protocols approved by the Medical Ethics Committee of Hebei Medical University. Animals were divided into three groups: normal control (n = 6), normal saline control (n = 6), and edaravone group (n = 24). Animals in the edaravone group were further divided into subgroups based on the edaravone concentration used and the length of treatment: 0.3 mg/kg for 14 days (n = 6), 1 mg/kg for 14 days (n = 6), 3 mg/kg for 14 days (n = 6), and 3 mg/kg for 28 days (n = 6).
Shanghai, China) was slowly inserted via a cranial hole of approximately 2 mm in diameter until it reached the target site. 4 lL of 6-OHDA (2 lg/lL) containing 0.02 % ascorbic acid was slowly injected at a speed of 9 ng/s; the micro-syringe needle was left in place for 10 min and then withdrawn at a speed of 1 mm/min. The sham-operated group received 4 lL of normal saline containing 0.02 % ascorbic acid. Rats in the normal control group did not receive any treatment. After the surgery, the cranial hole was plugged with gelatin sponge, and the wound was treated with penicillin, sterilized, and sutured. Rats were then returned to cages. Rats in the edaravone groups were intraperitoneally administered 1 ml saline 30 min after the surgery, and 0.3, 1, or 3 mg/kg edaravone (once a day) for a total of 14 days. Alternatively, rats were administered 3 mg/kg edaravone for 14 days and raised for additional 14 days after stopping edaravone treatment. All the behavioral and histological changes were evaluated at 14 and 27 days post-surgery. Rotational Behavior Test At 14 and 28 days after surgery, the model group rats were intraperitoneally administered 0.5 mg/kg apomorphine to induce rotational behavior (i.e. circling) to the right (healthy) side. Rotation recording started 5 min after apomorphine administration for a total of 30 min. Only rats which exhibited over 210 rotations over a period of 30 min were considered for further analysis as a successfully established animal model.
Reagents and Antibodies Histological Staining 6-OHDA, ascorbic acid, apomorphine, anti-tyrosine hydroxylase antibody, sodium pentobarbital, and diaminobenzidine were purchased from Sigma (Sigma, St. Louis, MO, USA). Edaravone was supplied by Simcere (Simcere Pharmaceutical Group, Nanjing, China). Rat model of Parkinson Disease Rats were anesthetized by intraperitoneal injection of sodium pentobarbital (40 mg/kg body weight). After the disappearance of posterior limb rebound reflex and corneal reflex, animal head was fixed in a stereotaxic instrument (MICRO4; World Precision Instruments, Inc., Sarasota, FL, USA), hair was removed and skin disinfected with iodine tincture and alcohol. An approximately 2 cm length midline incision was made to fully expose bregma. Threedimensional coordinates of the left medial forebrain bundle were determined as described by Paxinos and Watson [18]: 2.8 mm posterior to bregma, 2.0 mm left lateral to the midline, and 8.2 mm inferior to the dura mater. A microsyringe needle (Guangzheng Medical Instrument Co., Ltd.,
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After behavioral tests, rats were deeply anesthetized, rapidly perfused with 150–200 mL of normal saline (37 °C), followed by perfusion with 4 % formaldehyde (4 °C; 200 mL rapid perfusion and 300 mL slow perfusion). The harvested brain was treated with 4 % formaldehyde for 4 h at 4 °C, and subsequently placed in 20 and 30 % glucose-containing phosphate-buffered solutions at 4 °C. The corpus striatum and midbrain tissues were cut into 20 lm coronal sections using freezing microtome (Leica CM 1900, Bensheim, Germany). After rinsing, sections were stained by toluidine blue to visualized Nissl bodies. Tyrosine hydroxylase (TH)poisitive cells in the SN were visualized by Streptavidin– biotin (SABC) complex immunohistochemistry method using anti-tyrosine hydroxylase antibody and SABC kit (Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) according to manufacturer’s instructions. Images were acquired using inverted microscope (BX50WI, Olympus, Japan) at 9400 magnification. Ten coronal sections of the SN were selected from each group, a number of Nissl bodies and TH-poisitive cells in the compact part of the
Cell Biochem Biophys Table 1 Effect of edaravone on the apomorphine-induced rotation behavior in rats with 6-OHDA-induced PD (times/30 min) ðx sÞ
Table 2 Effect of edaravone on the number of Nissl bodies ðx sÞ in the substantia nigra of PD rat model (number/high-fold field of view)
Group
n
xs
Group
n
Left
Right
Control
6
.0000 ± .0000
Control
10
152.1900 ± 6.9125
155.7933 ± 5.1050
NS
6
241.0000 ± 15.9374*
NS
10
34.2300 ± 4.1543*m
ED-0.3 mg14 days
6
237.8333 ± 12.9061*
ED-0.3 mg
10
34.5300 ± 3.8993*m
149.3300 ± 6.3505
ED-1mg14 days
6
234.8333 ± 11.0348*
ED-1 mg
10
37.5100 ± 2.5740*m
150.3800 ± 9.8178
ED-3 mg 14 days
6
215.1667 ± 8.3766 * **
63.0200 ± 6.3099* **m
153.8867 ± 3.9327
6
219.0000 ± 8.0000* **
ED-3 mg 14 days
10
ED-3 mg 28 days
ED-3 mg 28 days
10
63.6333 ± 4.6784* **m
154.7333 ± 4.0393
PD in rats was induced by 6-OHDA injection into the medial forebrain bundle. Control group untreated animals, NS animals injected with 4 lL of normal saline, ED animals treated with edaravone * P \ 0.01 versus control; ** P \ 0.05 versus NS, ED-0.3 mg and ED-1 mg groups
148.3633 ± 7.3868
Control group untreated animals, NS animals injected with 4 lL of normal saline, ED animals treated with edaravone * P \ 0.05 versus control; ** P \ 0.05 vs NS,ED-0.3-mg and ED1 mg groups mP \ 0.05 versus right
SN in three non-overlapping fields per section were counted, and mean values were calculated. GSH and MDA Tissue Content Measurement 24 h after administration of the last edaravone dose, animals were decapitated, the brains were quickly harvested, and the corpus striatum and SN were dissociated on ice. Blood was removed with normal saline at 4 °C, and the corpus striatum and SN were blotted dry and snap-frozen at -80 °C. Collected brain tissues were weighed, chopped into small blocks with eye scissors, and disrupted using glass homogenizer. GSH content in the bilateral corpus striata and the compact part of the SN was detected by a Total Glutathione Quantification Kit (Dojindo Laboratories, Kumamoto, Japan), following the kits’ instructions. MDA levels were analyzed using TBARS Assay Kit (Cayman Chemical, Ann Arbor, MI, USA).
animals exhibited notably increased rotational behavior (circling to the healthy side; [210 times/min) as compared to untreated control group (P \ 0.01). Intraperitoneal injections of edaravone at the highest concentration (3 mg/kg) caused significant decrease in the number of observed rotations comparing to saline-treated animals (NS group), and rats treated for 14 days with lower concentrations of edaravone (ED group) (0.3 and 1 mg/kg; P \ 0.05). The decrease in the amount of apomorphineinduced rotations observed in rats treated with edaravone at 3 mg/kg for 14 days coincided with the increase in the ad libitum creeping behavior. This behavior was not observed in the NS group and in animals, treated with lower doses of edaravone (data not shown) (Table 1). Effect of Edaravone on the Rat Corpus Striatum and the Substantia Nigra
Statistical Analysis All data are expressed as mean ± SD and were statistically analyzed using SPSS 13.0 software. Independent sample t test was used for comparison between two groups, and analysis of variance was performed for comparison among multiple groups. A level of P \ 0.05 was considered statistically significant.
Results Effect of Edaravone on Apomorphine-Induced Rotational Behavior We first evaluated the effect of edaravone on the apomorphine-induced rotational behavior in rats with 6-OHDA medial forebrain bundle lesions. Apomorphine-treated
We next assessed the effect of edaravone on the nerve cells in the left corpus striatum and the compact part of the SN of PD rats. We observed markedly lower number of Nissl bodies in the left compact part of the SN than in the right side in both NS and ED groups, comparing to the untreated control group (P \ 0.05, Table 2). The number of Nissl bodies in the left compact part of the SN of the rats in the NS and ED groups was significantly lower than that in the normal control group (P \ 0.05, Table 2; Fig. 1a). Animals treated with the high dose of edaravone (3 mg/kg) exhibited over 1.7-fold increase in the number of Nissl bodies in the left compact part of the SN, comparing to NS group, or rats, treated with lower doses (0.3 and 1 mg/kg) of the drug (P \ 0.05). There was no significant difference in the number of Nissl bodies between edaravone 3 mg/kg 14 day group and edaravone 3 mg/kg 28 day group (Table 2; Fig. 1a).
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Cell Biochem Biophys Fig. 1 Effect of edaravone on the nerve cells in the left corpus striatum and the compact part of the substantia nigra. Rats with 6-OHDA-induced PD were treated with increased doses of edaravone as described in Materials and Methods, and a number of Nissl bodies (a) and TH-positive cells (b) in coronal sections of the substantia nigra were visualized by toluidine blue staining and SABC immunohistochemistry, respectively, at 9400 magnification
Table 3 Effect of edaravone on the amount of TH-positive cells in the substantia nigra of PD rat model (piece/HP) ðx sÞ Group
n
Left 39.9500 ± 4.0498
Control
10
NS
10
3.8700 ± 1.1576*m
Right 40.1000 ± 2.4000 37.4500 ± 2.7444
ED-0.3 mg
10
4.0000 ± .9615*m
37.7000 ± 2.4815
ED-1 mg
10
3.9100 ± .8386*m
38.0600 ± 2.1157
ED-3 mg 14 days
10
11.8000 ± 2.6617* **m
38.5300 ± 3.0919
ED-3 mg 28 days
10
11.4500 ± 2.6875* **m
38.5400 ± 3.0446
Control group untreated animals, NS animals injected with 4 lL of normal saline, ED animals treated with edaravone * P \ 0.05 versus control; ** P \ 0.05 versus NS, ED-0.3 mg and ED-1 mg groups; mP \ 0.05 versus right
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NS- and edaravone-treated animals had significantly lower levels of TH-positive cells in the compact part of the SN, comparing to the untreated control group (Table 3; P \ 0.05), with consistently higher number of TH-positive cells in the left compact part of the SN comparing to the right side (P \ 0.05). Administering higher dose of edaravone (3 mg/kg) increased the number of TH-positive cells in the left compact part over 2.5-fold comparing to NS or ED group treated with lower concentrations of edaravone (1.3 and 1 mg/kg; P \ 0.05). However, there was no significant difference in TH-positive cell numbers in tissues harvested immediately after treatment with 3 mg/kg edaravone for 14 days or tissues obtained from animals raised for additional 14 days after the last edaravone administration (ED 28 days group) (Table 3; Fig. 1b).
Cell Biochem Biophys Table 4 Effect of edaravone on the levels of GSH in the left substantia nigra and striatum of PD rat model (mg/g prot) ðx sÞ Group
n Substantia nigra
Striatum
Control
6 7.1800 ± .5391
7.5667 ± .5064
NS
6 4.1700 ± .4074*
2.8350 ± .8099*
ED-0.3 mg
6 4.2867 ± .4040*
2.6833 ± .8923*
ED-1 mg
6 4.1400 ± .4213*
3.2467 ± 1.0979*
ED-3 mg 14 days
6 5.1367 ± .7225* **
4.1933 ± 3.7339* ***
ED-3 mg 28 days
6 5.1867 ± .6866* **
4.4267 ± .9781* ***
Control group untreated animals, NS animals injected with 4 lL of normal saline, ED animals treated with edaravone *P \ 0.05 versus control; **P \ 0.05 versus NS,ED-0.3 mg and ED1 mg groups; ***P \ 0.05 versus and ED-0.3 mg groups
Table 5 Effect of edaravone on the concentration of MDA in the left SN and striatum of PD rat model induced by 6-OHDA (nmol/mgprot) ðx sÞ Group
n
Substantia nigra
Striatum
Control
6
4.8225 ± .6699
4.2458 ± .8447
NS
6
10.7662 ± 1.6463*
11.5387 ± 1.4051*
ED-0.3 mg
6
10.9785 ± 1.2321*
12.1502 ± 1.2436*
ED-1 mg
6
10.6545 ± 1.2132*
10.8913 ± 1.2456*
ED-3 mg 14 days
6
7.5605 ± 1.8058* **
6.8862 ± 2.0480* **
ED-3 mg 28 days
6
7.5652 ± .6909* **
7.2552 ± .8585* **
Control group untreated animals, NS animals injected with 4 lL of normal saline, ED animals treated with edaravone * P \ 0.05 versus control; ** P \ 0.05 versus NS, ED-0.3 mg and ED-1 mg groups
Effects of Edaravone on the GSH and MDA Levels in the Substantia Nigra and Corpus Striatum Previous studies show that GSH, that participates in the regulation of cellular oxidation, is decreased in the SN and putamen of PD patients [19–23]. We measured the levels of GSH and MDA in the SN and striatum of the erdaravonetreated PD rats. Animals, in the ED group treated with the highest dose of edaravone (3 mg/kg) exhibited significantly higher GSH levels in the SN, comparing to the NS, ED 0.3 mg/kg, and ED 1 mg/kg groups (P \ 0.05). GSH levels in the corpus striatum of the ED 3 mg/kg group were significantly higher than in the NS and ED 0.3 mg/kg groups (P \ 0.05). However, there was no significant difference in GSH levels between animals in ED-3 mg 14 days and ED-3 mg 28 days groups (Table 4). High dose of edaravone (3 mg/kg) led to significantly reduced MDA levels in the SN and corpus striatum comparing to NS, ED 0.3 mg/kg, and ED 1 mg/kg groups
(P \ 0.05). No difference in MDA levels was detected between ED-3 mg 14 days and ED-3 mg 28 days groups (Table 5).
Discussion The pharmacologic treatment of Parkinson disease can be divided into neuroprotective and symptomatic therapy. Current approach to treating for PD is primarily symptomatic and based on a dopamine replacement strategy using the dopamine precursor L-dopa. This symptomatic treatment cannot fundamentally prevent PD or stop disease progression. Moreover, chronic treatment with L-dopa is associated with the development of complications such as motor fluctuation, dyskinesia, and dementia. In the recent years, a substantial amount of studies focused on developing neuroprotective therapy for PD that potentially can slow, stop, or reverse disease progression and at the same time prevent the development of cognitive impairment. Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) is a novel potent free radical scavenger that was recently shown to reduce brain damage caused by ischemia–reperfusion injury, traumatic brain injury, and stroke [24–26]. Edaravone has lipophilic groups, and exhibits good cell membrane permeability and ability to pass through blood– brain barrier. Recent studies show that edaravone protects brain nerve cells by reducing reactive oxygen species, inhibiting apoptosis, blocking nonenzymatic peroxidation and lipoxygenase activity, and preventing vascular endothelial cell injury [27–32]. Wu et al. [33] detected high levels of dopamine transporter in the bilateral corpus striata, cerebral cortex, and cerebella of a rat model of PD induced by 6-OHDA using a gamma counter. Their report showed that intraperitoneal injection of high dose of edaravone can significantly inhibit the reduction in dopamine transporter levels in the injured side compared to the normal control group (P \ 0.05), indicating that edaravone can strengthen tissue’s anti-oxidative capacity. Our results show that all rats with 6-OHDA-induced PD exhibited significantly increased rotational behavior (circling to the healthy side, [210 times/min) after apomorphine administration, comparing to untreated animals. Intraperitoneal injection of high doses of edaravone was able to significantly reduce circling and also improved spontaneous behavior, as indicated by diminished ad libitum creeping toward the injured site in animals, treated with 3 mg/kg edaravone after 14 days of treatment with the drug. To our knowledge, this is the first report of the effect of edaravone on spontaneous behavior in rat model of PD. We may speculate that edaravone-induced inhibition of free radical damage to dopaminergic neurons prevents
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further loss of dopaminergic neurons, and thereby slows down the progression of PD, and improving the behavior of rats with 6-OHDA-induced disease. Edaravone can improve methamphetamine hydrochloride-induced dopaminergic neuron degeneration and increase dopaminergic neuron survival, but it does not influence the activation of microglia. This suggests that edaravone exerts its protective effect by inhibiting the formation of free radicals, the downstream product of activated microglia, rather than inhibiting the activation of microglia [34–37]. Studies show that intravenous administration of edaravone at 250 and 100 mg/kg in the early stage of PD progression can inhibit the loss of dopaminergic neurons in the SN, suggesting that edaravone’s effect against free radical damage is associated with its anti-apoptotic effect [38–41]. Our results showed that high doses of edaravone (3 mg/kg), but not lower doses (0.3 and 1 mg/kg) prevented the decrease in the amount of Nissl bodies and TH-positive cells in the SN of rats with 6-OHDA-induced injury. These findings demonstrate that edaravone exhibits protective effects on nerve cells in the SN in a dose-dependent manner, probably by inhibiting the formation of free radicals. GSH is a ubiquitous thiol tripeptide that alone or in concert with enzymes reduces superoxide radicals, hydroxyl radicals, and peroxynitrites inside the cells. GSH is also involved in DNA synthesis and repair, protein synthesis, cellular immunity, and enzymatic reaction. Results obtained from an autopsy show that the SN and putamen of PD patients are highly sensitive to free radical damage and has 30–60 %-reduced levels of glutathione disulfide, accompanied by an increase in iron levels. No decrease in the GSH levels was detected in any other regions of the brain of PD patients or in the brain of patients with other neurodegenerative diseases [19–23, 42, 43]. Our results show that edaravone at a higher dose of 3 mg/kg, but not at low concentration of 0.3 and 1 mg/kg, significantly increased the level of GSH in the SN and corpus striatum of rats with 6-OHDA-induced PD. These findings suggest that edaravone exerts neuroprotective effect on rat SN injured by 6-OHDA that is associated with the increase in GSH level. MDA is one of the markers of lipid peroxidation that has been found to be significantly increased in the SN of PD patients, compared to other brain regions and control tissue [44]. Our results demonstrate that edaravone at 3 mg/kg significantly inhibited the increase in the levels of MDA, induced by 6-OHDA, while 0.3 and 1 mg/kg edaravone did not have an effect on MDA levels. These findings suggest that edaravone shows a dose-dependent neuroprotective effect on PD rats that, in agreement with previous studies, is likely associated with edaravone-induced inhibition of free radical activity [45–47]. In conclusion, we investigated the long-term protective effect of edaravone on dopaminergic neurons by observing
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the changes in rat behavior, tissue morphology, and neurobiochemistry at two time points: immediately following edaravone treatment, and 14 days after the last administration of the drug. The neuroprotective effect of high doses of edaravone on SN and corpus striatum with 6-OHDA-induced injury was maintained for 14 days after administration of the last edaravone injection, and was similar to its immediate therapeutic effect. To our knowledge, this is the first report showing that edaravone has a long-term protective effect in the case of neuronal injury. We demonstrate that the possible mechanism of this protective effect involves edaravone-promoted changes in GSH and MDA levels in the SN and corpus striatum of a rat model of 6-OHDA-induced PD.
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29. Shinohara, Y., & Inoue, S. (2013). Cost-effectiveness analysis of the neuroprotective agent edaravone for noncardioembolic cerebral infarction. Journal of Stroke & Cerebrovascular Diseases, 22(5), 668–674. 30. Takabatake, Y., Uno, E., & Wakamatsu, K. (2003). The clinical effect of combination therapy with edaravone and sodium ozagrel for acute cerebral infarction. No To Shinkei, 55(7), 589–593. 31. Chan, W. S., Durairajan, S. S., & Lu, J. H. (2009). Neuroprotective effects of Astragaloside IV in 6-hydroxydopamine-treated primary nigral cell culture. Neurochemistry International, 55(6), 414–422. 32. Chaturvedi, R. K., Shukla, S., Seth, K., et al. (2006). Neuroprotective and neurorescue effect of black tea extract in 6-hydroxydopamine-lesioned rat model of Parkinson’s disease. Neurobiology of Disease, 22(2), 421–434. 33. Wu, Guan, Bao, Shi Yao, Luo, Weifeng, et al. (2006). According to the adr of Parkinson’s disease rat dopamine transporter protection study[J]. Journal of clinical neurology, 12(4), 296–298. 34. Anno, M., Mizuno, T., Kato, H., et al. (2005). The radical scavenger edaravone prevents oxidative neurotoxicity induced by peroxynitrite and activated microglia[J]. Neuropharmacology, 48, 283–290. 35. Yuan, W. J., Yasuhara, T., Shingo, T., et al. (2008). Neuroprotective effects of edaravone administration on 6-OHDA-treated dopaminergic neurons[J]. BMC Neuroscience, 9, 75. 36. Vernon, A. C., Zbarsky, V., Datla, K. P., et al. (2007). Subtype selective antagonism of substantia nigra pars compacta Group I metabotropic glutamate receptors protects the nigrostriatal system against 6-hydroxydopamine toxicity in vivo. Journal of Neurochemistry, 103(3), 1075–1091. 37. Va¨a¨na¨nen, A. J., Rauhala, P., Tuominen, R. K., et al. (2006). KDI tripeptide of gamma1 laminin protects rat dopaminergic neurons from 6-OHDA induced toxicity. Journal of Neuroscience Research, 84(3), 655–665. 38. Plaitakis, A., & Shashidharan, P. (2000). Glutamate transport and metabolism in dopaminergic neurons of substantia nigra: Implications for the pathogenesis of Parkinson’s disease. Journal of Neurology, 247(2), 1125–1135. 39. Nakamura, K., & Wiatr, T. (2000). Preferential resistance of dopaminergic neurons to the toxicity of glutathione depletion is independent of cellular glutathione peroxidase and is mediated by tetrahydrobiopterin[J]. Journal of Neurochemistry, 74(6), 2305–2314. 40. Nakamura, K., Wang, W., & Kang, U. J. (1997). The role of glutathione in dopaminergic neuronal survival. Journal of Neurochemistry, 69(5), 1850–1858. 41. Gramsbergen, J. B., Sandberg, M., Møller Dall, A., Kornblit, B., & Zimmer, J. (2002). Glutathione depletion in nigrostriatal slice cultures: GABA loss, dopamine resistance and protection by the tetrahydrobiopterin precursor sepiapterin. Brain Research, 935(1–2), 47–58. 42. Rojas, P., Serrano-Garcı´a, N., & Mares-Sa´mano, J. J. (2008). EGb761 protects against nigrostriatal dopaminergic neurotoxicity 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinsonism in mice: Role of oxidative stress. The European Journal of Neuroscience, 28(1), 41–50. 43. Rojas, P., Serrano-Garcı´a, N., & Medina-Campos, O. N. (2011). S-Allylcysteine, a garlic compound, protects against oxidative stress in 1-methyl-4-phenylpyridinium-induced parkinsonism in mice. The Journal of Nutritional Biochemistry, 22(10), 937–944. 44. Ross, B. M., Moszczynska, A., Erlich, J., & Kish, S. J. (1998). Low activity of key phospholipid catabolic and anabolic enzymes in human substantia nigra: Possible implications for Parkinson’s disease. Neuroscience, 83(3), 791–798.
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ORIGINAL PAPER
Edaravone Protects Neurons in the Rat Substantia Nigra Against 6-Hydroxydopamine-Induced Oxidative Stress Damage Xiqi Liu • Rushing Shao • Meng Li Guofeng Yang
•
Ó Springer Science+Business Media New York 2014
Abstract To investigate the mechanism of the neuroprotective effect of edaravone in substantia nigra (SN) of the 6-OHDA-induced rat model of Parkinson’s disease. Animal model of Parkinson’s disease was induced in male Sprague– Dawley rats by injecting 6-OHDA into the left medial forebrain bundle. Subsequently, rats were intraperitoneally injected with 0.3, 1, or 3 mg/kg of edaravone for 14 days or with 3 mg/kg edaravone for 14 days followed by 14 days of no treatment. We evaluated the effect of edaravone on the rotational and normal behavior of the rats, and on the number of tyrosine hydroxylase (TH)-positive cells, the amount of Nissl bodies, and the levels of glutathione (GSH), and malondialdehyde (MDA) in the SN. Edaravone treatment at 3 mg/kg significantly reduced apomorphineinduced rotational behavior (P \ 0.01), improved the spontaneous behavior, prevented the decrease in the levels of TH-positive cells, Nissl bodies and GSH, and inhibited the increase in the levels of MDA (P \ 0.05) in SN of rats with 6-OHDA-induced PD. Edaravone exerted a long-term neuroprotective effects in 6-OHDA-induced PD animal model by attenuating changes in the levels of GSH and MDA in SN, caused by oxidative stress. Edaravone prevented 6-OHDA-induced behavioral changes and de-pigmentation of SN that results from the loss of dopaminergic neurons.
X. Liu R. Shao M. Li Department of Neurology, The Central Hospital of Cangzhou City, Cangzhou 061000, Hebei, China G. Yang (&) Department of Neurology, The Second Hospital of Hebei Medical University, Shijiazhuang 050000, China e-mail: [email protected]; [email protected]
Keywords Parkinson’s disease Edaravone 6-OHDA Glutathione Malondialdehyde
Introduction Parkinson’s disease (PD) is a progressive neurodegenerative disorder, that is characterized by cumulative neuronal loss. It is characterized by a number of motor function abnormalities, such as bradykinesia, tremors, muscular rigidity, and postural instability, mainly caused by the progressive loss of dopaminergic neurons located within a pigmented region of the brain called the substantia nigra (SN) [1]. A number of pathogenic factors have been implicated in causing PD including oxidative stress, mitochondrial dysfunction, inflammation, excitotoxicity, and signals that mediate apoptosis [2]. The brain is especially susceptible to oxidative damage because of its high levels of polyunsaturated fatty acids and relatively low antioxidant activity [3]. Previous reports show that the significant oxidative damage to lipids, proteins, and DNA observed in postmortem PD striatum and within the SN [4–10], correlated with decreased levels of glutathione (GSH), one of the body’s most important antioxidants and a biomarker of oxidative stress [11, 12], and increased levels of malondialdehyde (MDA), a marker of lipid peroxidation [5]. Since existing medical care for PD primarily focuses on symptom management, and oxidative stress plays a pivotal role in the degeneration of dopaminergic neurons of the SN, compounds with antioxidant capacities may be efficient in stopping or slowing down the progression of PD [13, 14]. Edaravone (3-methyl-1-phenyl-2-pyrazolin-5one) is a potent hydroxy radical scavenger and antioxidant [15] that is able to suppress the production of nitric oxide
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and reactive oxygen species by activated microglia [16]. Recent studies showed that edaravone has in vitro and in vivo anti-apoptotic, anti-oxidative, and anti-inflammatory neuroprotective effects in animal model of PD with 6-hydroxydopamine (6-OHDA)-induced selective dopaminergic neuronal loss in the SN [17]. The present study sought to determine the effect of edaravone on GSH and MDA levels in the SN and corpus striatum and its short-term and long-term neuroprotective effect in a rat model of 6-OHDA-induced PD.
Methods Experimental Animals Seventy-two healthy adult male Sprague–Dawley rats, weighing 250–300 g, were provided by the Laboratory Animal Center, Hebei Medical University, China. All experiments using animals were performed in accordance with protocols approved by the Medical Ethics Committee of Hebei Medical University. Animals were divided into three groups: normal control (n = 6), normal saline control (n = 6), and edaravone group (n = 24). Animals in the edaravone group were further divided into subgroups based on the edaravone concentration used and the length of treatment: 0.3 mg/kg for 14 days (n = 6), 1 mg/kg for 14 days (n = 6), 3 mg/kg for 14 days (n = 6), and 3 mg/kg for 28 days (n = 6).
Shanghai, China) was slowly inserted via a cranial hole of approximately 2 mm in diameter until it reached the target site. 4 lL of 6-OHDA (2 lg/lL) containing 0.02 % ascorbic acid was slowly injected at a speed of 9 ng/s; the micro-syringe needle was left in place for 10 min and then withdrawn at a speed of 1 mm/min. The sham-operated group received 4 lL of normal saline containing 0.02 % ascorbic acid. Rats in the normal control group did not receive any treatment. After the surgery, the cranial hole was plugged with gelatin sponge, and the wound was treated with penicillin, sterilized, and sutured. Rats were then returned to cages. Rats in the edaravone groups were intraperitoneally administered 1 ml saline 30 min after the surgery, and 0.3, 1, or 3 mg/kg edaravone (once a day) for a total of 14 days. Alternatively, rats were administered 3 mg/kg edaravone for 14 days and raised for additional 14 days after stopping edaravone treatment. All the behavioral and histological changes were evaluated at 14 and 27 days post-surgery. Rotational Behavior Test At 14 and 28 days after surgery, the model group rats were intraperitoneally administered 0.5 mg/kg apomorphine to induce rotational behavior (i.e. circling) to the right (healthy) side. Rotation recording started 5 min after apomorphine administration for a total of 30 min. Only rats which exhibited over 210 rotations over a period of 30 min were considered for further analysis as a successfully established animal model.
Reagents and Antibodies Histological Staining 6-OHDA, ascorbic acid, apomorphine, anti-tyrosine hydroxylase antibody, sodium pentobarbital, and diaminobenzidine were purchased from Sigma (Sigma, St. Louis, MO, USA). Edaravone was supplied by Simcere (Simcere Pharmaceutical Group, Nanjing, China). Rat model of Parkinson Disease Rats were anesthetized by intraperitoneal injection of sodium pentobarbital (40 mg/kg body weight). After the disappearance of posterior limb rebound reflex and corneal reflex, animal head was fixed in a stereotaxic instrument (MICRO4; World Precision Instruments, Inc., Sarasota, FL, USA), hair was removed and skin disinfected with iodine tincture and alcohol. An approximately 2 cm length midline incision was made to fully expose bregma. Threedimensional coordinates of the left medial forebrain bundle were determined as described by Paxinos and Watson [18]: 2.8 mm posterior to bregma, 2.0 mm left lateral to the midline, and 8.2 mm inferior to the dura mater. A microsyringe needle (Guangzheng Medical Instrument Co., Ltd.,
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After behavioral tests, rats were deeply anesthetized, rapidly perfused with 150–200 mL of normal saline (37 °C), followed by perfusion with 4 % formaldehyde (4 °C; 200 mL rapid perfusion and 300 mL slow perfusion). The harvested brain was treated with 4 % formaldehyde for 4 h at 4 °C, and subsequently placed in 20 and 30 % glucose-containing phosphate-buffered solutions at 4 °C. The corpus striatum and midbrain tissues were cut into 20 lm coronal sections using freezing microtome (Leica CM 1900, Bensheim, Germany). After rinsing, sections were stained by toluidine blue to visualized Nissl bodies. Tyrosine hydroxylase (TH)poisitive cells in the SN were visualized by Streptavidin– biotin (SABC) complex immunohistochemistry method using anti-tyrosine hydroxylase antibody and SABC kit (Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) according to manufacturer’s instructions. Images were acquired using inverted microscope (BX50WI, Olympus, Japan) at 9400 magnification. Ten coronal sections of the SN were selected from each group, a number of Nissl bodies and TH-poisitive cells in the compact part of the
Cell Biochem Biophys Table 1 Effect of edaravone on the apomorphine-induced rotation behavior in rats with 6-OHDA-induced PD (times/30 min) ðx sÞ
Table 2 Effect of edaravone on the number of Nissl bodies ðx sÞ in the substantia nigra of PD rat model (number/high-fold field of view)
Group
n
xs
Group
n
Left
Right
Control
6
.0000 ± .0000
Control
10
152.1900 ± 6.9125
155.7933 ± 5.1050
NS
6
241.0000 ± 15.9374*
NS
10
34.2300 ± 4.1543*m
ED-0.3 mg14 days
6
237.8333 ± 12.9061*
ED-0.3 mg
10
34.5300 ± 3.8993*m
149.3300 ± 6.3505
ED-1mg14 days
6
234.8333 ± 11.0348*
ED-1 mg
10
37.5100 ± 2.5740*m
150.3800 ± 9.8178
ED-3 mg 14 days
6
215.1667 ± 8.3766 * **
63.0200 ± 6.3099* **m
153.8867 ± 3.9327
6
219.0000 ± 8.0000* **
ED-3 mg 14 days
10
ED-3 mg 28 days
ED-3 mg 28 days
10
63.6333 ± 4.6784* **m
154.7333 ± 4.0393
PD in rats was induced by 6-OHDA injection into the medial forebrain bundle. Control group untreated animals, NS animals injected with 4 lL of normal saline, ED animals treated with edaravone * P \ 0.01 versus control; ** P \ 0.05 versus NS, ED-0.3 mg and ED-1 mg groups
148.3633 ± 7.3868
Control group untreated animals, NS animals injected with 4 lL of normal saline, ED animals treated with edaravone * P \ 0.05 versus control; ** P \ 0.05 vs NS,ED-0.3-mg and ED1 mg groups mP \ 0.05 versus right
SN in three non-overlapping fields per section were counted, and mean values were calculated. GSH and MDA Tissue Content Measurement 24 h after administration of the last edaravone dose, animals were decapitated, the brains were quickly harvested, and the corpus striatum and SN were dissociated on ice. Blood was removed with normal saline at 4 °C, and the corpus striatum and SN were blotted dry and snap-frozen at -80 °C. Collected brain tissues were weighed, chopped into small blocks with eye scissors, and disrupted using glass homogenizer. GSH content in the bilateral corpus striata and the compact part of the SN was detected by a Total Glutathione Quantification Kit (Dojindo Laboratories, Kumamoto, Japan), following the kits’ instructions. MDA levels were analyzed using TBARS Assay Kit (Cayman Chemical, Ann Arbor, MI, USA).
animals exhibited notably increased rotational behavior (circling to the healthy side; [210 times/min) as compared to untreated control group (P \ 0.01). Intraperitoneal injections of edaravone at the highest concentration (3 mg/kg) caused significant decrease in the number of observed rotations comparing to saline-treated animals (NS group), and rats treated for 14 days with lower concentrations of edaravone (ED group) (0.3 and 1 mg/kg; P \ 0.05). The decrease in the amount of apomorphineinduced rotations observed in rats treated with edaravone at 3 mg/kg for 14 days coincided with the increase in the ad libitum creeping behavior. This behavior was not observed in the NS group and in animals, treated with lower doses of edaravone (data not shown) (Table 1). Effect of Edaravone on the Rat Corpus Striatum and the Substantia Nigra
Statistical Analysis All data are expressed as mean ± SD and were statistically analyzed using SPSS 13.0 software. Independent sample t test was used for comparison between two groups, and analysis of variance was performed for comparison among multiple groups. A level of P \ 0.05 was considered statistically significant.
Results Effect of Edaravone on Apomorphine-Induced Rotational Behavior We first evaluated the effect of edaravone on the apomorphine-induced rotational behavior in rats with 6-OHDA medial forebrain bundle lesions. Apomorphine-treated
We next assessed the effect of edaravone on the nerve cells in the left corpus striatum and the compact part of the SN of PD rats. We observed markedly lower number of Nissl bodies in the left compact part of the SN than in the right side in both NS and ED groups, comparing to the untreated control group (P \ 0.05, Table 2). The number of Nissl bodies in the left compact part of the SN of the rats in the NS and ED groups was significantly lower than that in the normal control group (P \ 0.05, Table 2; Fig. 1a). Animals treated with the high dose of edaravone (3 mg/kg) exhibited over 1.7-fold increase in the number of Nissl bodies in the left compact part of the SN, comparing to NS group, or rats, treated with lower doses (0.3 and 1 mg/kg) of the drug (P \ 0.05). There was no significant difference in the number of Nissl bodies between edaravone 3 mg/kg 14 day group and edaravone 3 mg/kg 28 day group (Table 2; Fig. 1a).
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Cell Biochem Biophys Fig. 1 Effect of edaravone on the nerve cells in the left corpus striatum and the compact part of the substantia nigra. Rats with 6-OHDA-induced PD were treated with increased doses of edaravone as described in Materials and Methods, and a number of Nissl bodies (a) and TH-positive cells (b) in coronal sections of the substantia nigra were visualized by toluidine blue staining and SABC immunohistochemistry, respectively, at 9400 magnification
Table 3 Effect of edaravone on the amount of TH-positive cells in the substantia nigra of PD rat model (piece/HP) ðx sÞ Group
n
Left 39.9500 ± 4.0498
Control
10
NS
10
3.8700 ± 1.1576*m
Right 40.1000 ± 2.4000 37.4500 ± 2.7444
ED-0.3 mg
10
4.0000 ± .9615*m
37.7000 ± 2.4815
ED-1 mg
10
3.9100 ± .8386*m
38.0600 ± 2.1157
ED-3 mg 14 days
10
11.8000 ± 2.6617* **m
38.5300 ± 3.0919
ED-3 mg 28 days
10
11.4500 ± 2.6875* **m
38.5400 ± 3.0446
Control group untreated animals, NS animals injected with 4 lL of normal saline, ED animals treated with edaravone * P \ 0.05 versus control; ** P \ 0.05 versus NS, ED-0.3 mg and ED-1 mg groups; mP \ 0.05 versus right
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NS- and edaravone-treated animals had significantly lower levels of TH-positive cells in the compact part of the SN, comparing to the untreated control group (Table 3; P \ 0.05), with consistently higher number of TH-positive cells in the left compact part of the SN comparing to the right side (P \ 0.05). Administering higher dose of edaravone (3 mg/kg) increased the number of TH-positive cells in the left compact part over 2.5-fold comparing to NS or ED group treated with lower concentrations of edaravone (1.3 and 1 mg/kg; P \ 0.05). However, there was no significant difference in TH-positive cell numbers in tissues harvested immediately after treatment with 3 mg/kg edaravone for 14 days or tissues obtained from animals raised for additional 14 days after the last edaravone administration (ED 28 days group) (Table 3; Fig. 1b).
Cell Biochem Biophys Table 4 Effect of edaravone on the levels of GSH in the left substantia nigra and striatum of PD rat model (mg/g prot) ðx sÞ Group
n Substantia nigra
Striatum
Control
6 7.1800 ± .5391
7.5667 ± .5064
NS
6 4.1700 ± .4074*
2.8350 ± .8099*
ED-0.3 mg
6 4.2867 ± .4040*
2.6833 ± .8923*
ED-1 mg
6 4.1400 ± .4213*
3.2467 ± 1.0979*
ED-3 mg 14 days
6 5.1367 ± .7225* **
4.1933 ± 3.7339* ***
ED-3 mg 28 days
6 5.1867 ± .6866* **
4.4267 ± .9781* ***
Control group untreated animals, NS animals injected with 4 lL of normal saline, ED animals treated with edaravone *P \ 0.05 versus control; **P \ 0.05 versus NS,ED-0.3 mg and ED1 mg groups; ***P \ 0.05 versus and ED-0.3 mg groups
Table 5 Effect of edaravone on the concentration of MDA in the left SN and striatum of PD rat model induced by 6-OHDA (nmol/mgprot) ðx sÞ Group
n
Substantia nigra
Striatum
Control
6
4.8225 ± .6699
4.2458 ± .8447
NS
6
10.7662 ± 1.6463*
11.5387 ± 1.4051*
ED-0.3 mg
6
10.9785 ± 1.2321*
12.1502 ± 1.2436*
ED-1 mg
6
10.6545 ± 1.2132*
10.8913 ± 1.2456*
ED-3 mg 14 days
6
7.5605 ± 1.8058* **
6.8862 ± 2.0480* **
ED-3 mg 28 days
6
7.5652 ± .6909* **
7.2552 ± .8585* **
Control group untreated animals, NS animals injected with 4 lL of normal saline, ED animals treated with edaravone * P \ 0.05 versus control; ** P \ 0.05 versus NS, ED-0.3 mg and ED-1 mg groups
Effects of Edaravone on the GSH and MDA Levels in the Substantia Nigra and Corpus Striatum Previous studies show that GSH, that participates in the regulation of cellular oxidation, is decreased in the SN and putamen of PD patients [19–23]. We measured the levels of GSH and MDA in the SN and striatum of the erdaravonetreated PD rats. Animals, in the ED group treated with the highest dose of edaravone (3 mg/kg) exhibited significantly higher GSH levels in the SN, comparing to the NS, ED 0.3 mg/kg, and ED 1 mg/kg groups (P \ 0.05). GSH levels in the corpus striatum of the ED 3 mg/kg group were significantly higher than in the NS and ED 0.3 mg/kg groups (P \ 0.05). However, there was no significant difference in GSH levels between animals in ED-3 mg 14 days and ED-3 mg 28 days groups (Table 4). High dose of edaravone (3 mg/kg) led to significantly reduced MDA levels in the SN and corpus striatum comparing to NS, ED 0.3 mg/kg, and ED 1 mg/kg groups
(P \ 0.05). No difference in MDA levels was detected between ED-3 mg 14 days and ED-3 mg 28 days groups (Table 5).
Discussion The pharmacologic treatment of Parkinson disease can be divided into neuroprotective and symptomatic therapy. Current approach to treating for PD is primarily symptomatic and based on a dopamine replacement strategy using the dopamine precursor L-dopa. This symptomatic treatment cannot fundamentally prevent PD or stop disease progression. Moreover, chronic treatment with L-dopa is associated with the development of complications such as motor fluctuation, dyskinesia, and dementia. In the recent years, a substantial amount of studies focused on developing neuroprotective therapy for PD that potentially can slow, stop, or reverse disease progression and at the same time prevent the development of cognitive impairment. Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) is a novel potent free radical scavenger that was recently shown to reduce brain damage caused by ischemia–reperfusion injury, traumatic brain injury, and stroke [24–26]. Edaravone has lipophilic groups, and exhibits good cell membrane permeability and ability to pass through blood– brain barrier. Recent studies show that edaravone protects brain nerve cells by reducing reactive oxygen species, inhibiting apoptosis, blocking nonenzymatic peroxidation and lipoxygenase activity, and preventing vascular endothelial cell injury [27–32]. Wu et al. [33] detected high levels of dopamine transporter in the bilateral corpus striata, cerebral cortex, and cerebella of a rat model of PD induced by 6-OHDA using a gamma counter. Their report showed that intraperitoneal injection of high dose of edaravone can significantly inhibit the reduction in dopamine transporter levels in the injured side compared to the normal control group (P \ 0.05), indicating that edaravone can strengthen tissue’s anti-oxidative capacity. Our results show that all rats with 6-OHDA-induced PD exhibited significantly increased rotational behavior (circling to the healthy side, [210 times/min) after apomorphine administration, comparing to untreated animals. Intraperitoneal injection of high doses of edaravone was able to significantly reduce circling and also improved spontaneous behavior, as indicated by diminished ad libitum creeping toward the injured site in animals, treated with 3 mg/kg edaravone after 14 days of treatment with the drug. To our knowledge, this is the first report of the effect of edaravone on spontaneous behavior in rat model of PD. We may speculate that edaravone-induced inhibition of free radical damage to dopaminergic neurons prevents
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further loss of dopaminergic neurons, and thereby slows down the progression of PD, and improving the behavior of rats with 6-OHDA-induced disease. Edaravone can improve methamphetamine hydrochloride-induced dopaminergic neuron degeneration and increase dopaminergic neuron survival, but it does not influence the activation of microglia. This suggests that edaravone exerts its protective effect by inhibiting the formation of free radicals, the downstream product of activated microglia, rather than inhibiting the activation of microglia [34–37]. Studies show that intravenous administration of edaravone at 250 and 100 mg/kg in the early stage of PD progression can inhibit the loss of dopaminergic neurons in the SN, suggesting that edaravone’s effect against free radical damage is associated with its anti-apoptotic effect [38–41]. Our results showed that high doses of edaravone (3 mg/kg), but not lower doses (0.3 and 1 mg/kg) prevented the decrease in the amount of Nissl bodies and TH-positive cells in the SN of rats with 6-OHDA-induced injury. These findings demonstrate that edaravone exhibits protective effects on nerve cells in the SN in a dose-dependent manner, probably by inhibiting the formation of free radicals. GSH is a ubiquitous thiol tripeptide that alone or in concert with enzymes reduces superoxide radicals, hydroxyl radicals, and peroxynitrites inside the cells. GSH is also involved in DNA synthesis and repair, protein synthesis, cellular immunity, and enzymatic reaction. Results obtained from an autopsy show that the SN and putamen of PD patients are highly sensitive to free radical damage and has 30–60 %-reduced levels of glutathione disulfide, accompanied by an increase in iron levels. No decrease in the GSH levels was detected in any other regions of the brain of PD patients or in the brain of patients with other neurodegenerative diseases [19–23, 42, 43]. Our results show that edaravone at a higher dose of 3 mg/kg, but not at low concentration of 0.3 and 1 mg/kg, significantly increased the level of GSH in the SN and corpus striatum of rats with 6-OHDA-induced PD. These findings suggest that edaravone exerts neuroprotective effect on rat SN injured by 6-OHDA that is associated with the increase in GSH level. MDA is one of the markers of lipid peroxidation that has been found to be significantly increased in the SN of PD patients, compared to other brain regions and control tissue [44]. Our results demonstrate that edaravone at 3 mg/kg significantly inhibited the increase in the levels of MDA, induced by 6-OHDA, while 0.3 and 1 mg/kg edaravone did not have an effect on MDA levels. These findings suggest that edaravone shows a dose-dependent neuroprotective effect on PD rats that, in agreement with previous studies, is likely associated with edaravone-induced inhibition of free radical activity [45–47]. In conclusion, we investigated the long-term protective effect of edaravone on dopaminergic neurons by observing
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the changes in rat behavior, tissue morphology, and neurobiochemistry at two time points: immediately following edaravone treatment, and 14 days after the last administration of the drug. The neuroprotective effect of high doses of edaravone on SN and corpus striatum with 6-OHDA-induced injury was maintained for 14 days after administration of the last edaravone injection, and was similar to its immediate therapeutic effect. To our knowledge, this is the first report showing that edaravone has a long-term protective effect in the case of neuronal injury. We demonstrate that the possible mechanism of this protective effect involves edaravone-promoted changes in GSH and MDA levels in the SN and corpus striatum of a rat model of 6-OHDA-induced PD.
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