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Research report
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Sensory-motor performance after acute glutathione depletion by l-buthionine sulfoximine injection into substantia nigra pars compacta
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Mei-Li Díaz-Hung a,∗ , Lisette Blanco Lezcano a , Nancy Pavón Fuentes a , ˜ a , Eduardo Orta Soto a , Rilda León Martínez a , Bárbara Estupinan b Klaudia Martínez Cordovez , Isabel Fernández Jiménez a a b
International Center for Neurological Restoration, Havana, Cuba Faculty of Biology, University of Havana, Cuba
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h i g h l i g h t s • • • •
Transient glutathione depletion leads to sensory-motor impairment in rats. Sensory-motor impairment is accompanied TH-positive cell loss in the SNpc. TH-positive cell loss was not related to oxidative damage. Glutathione depletion is accompanied by antioxidant response in SNpc.
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Article history: Received 22 March 2014 Received in revised form 26 May 2014 Accepted 30 May 2014 Available online xxx
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Keywords: Glutathione Antioxidant enzymes Malondialdehyde Astrocytes Dopaminergic cell Sensory-motor function
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1. Introduction
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Glutathione is the major antioxidant in the living cells. Its deficit has been linked to neurodegenerative disorders as Parkinson’s disease but its role in the etiology of nigral degeneration and sensory-motor performance has been poorly explored. To evaluate the effect of glutathione depletion on nigro-striatal oxidative metabolism and sensory-motor performance in rats, l-buthionine sulfoximine (15 mM) or saline solution was injected into substantia nigra pars compacta (SNpc). Then, oxidative metabolism was studied 24 h and 7 days later in SNpc and corpus striatum (CS). Tyrosine hydroxylase and GFAP immunohistochemistry assays were carried out at 7 days. In addition, animals were evaluated in open field, adhesive removal, staircase and traverse beam tests. Glutathione depletion induced compensatory response in catalase activity and glial response in the in SNpc and no oxidative damage was observed. However, a loss in dopaminergic cells was found. At the same time, animals with glutathione depletion have shown poor performance in behavioral tests except for staircase test. These results suggest that glutathione depletion can be related to sensory-motor dysfunction. © 2014 Published by Elsevier B.V.
The hallmark of Parkinson’s disease (PD) neuropathology is the cell loss in substantia nigra pars compacta (SNpc), directly affecting the dopaminergic (DA) nigro-striatal pathway [7] which is
Abbreviations: PD, Parkinson’s disease; SNpc, substantia nigra pars compacta; DA, dopamine; ROS, reactive oxygen species; GSH, glutathione; CAT, catalase; GPx, glutathione peroxidase; GRD, glutathione reductase; MDA, malondialdehyde; BSO, l-buthionine sulfoximine; CS, corpus striatum. Q3 ∗ Corresponding author at: Immunochemistry department, International Center for Neurological Restoration, Ave 25 No 15805% 158 y 160, Cubanacan, Playa, Havana, CP 11300 Cuba. Tel.: +53 7 271 5353; fax: +53 7 2736028. E-mail addresses: [email protected], [email protected] (M.-L. Díaz-Hung).
responsible for the motor symptoms. The etiology of PD remains unknown although oxidative stress and mitochondrial dysfunction appear to play a crucial role in the demise of DA neurons [35]. Oxidative stress is a complex and dynamic situation characterized by an imbalance between the reactive oxygen species (ROS) production and the availability of antioxidants [24]. Defense system against ROS encompasses enzymes such as superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase as well as glutathione (GSH), urate and vitamin C [9]. These systems prevent the generation of free radicals and inactivate them once they have been formed [29]. GSH is required for many cellular processes. Particularly, it plays an important role in the maintenance and regulation of the cell thiol-redox status. In addition, it has been related with gene expression, cell proliferation, differentiation and apoptosis. Alterations
http://dx.doi.org/10.1016/j.bbr.2014.05.066 0166-4328/© 2014 Published by Elsevier B.V.
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in GSH homeostasis have been implicated in the etiology and/or progression of a number of human diseases [5,24]. A decrease in GSH levels has been described as one of the earliest biochemical alteration in PD. Since then, many research studies have addressed the role of GSH homeostasis in PD progression [27]. The infusion of l-buthionine sulfoximine (BSO), an inhibitor of ␥glutamyl cysteine synthetase, did not kill DA cells [54]. However, further studies have shown that maintaining glutathione levels is critical for protecting DA neurons in SNpc from neurodegeneration [15,28] suggesting that the effect may be quite complex and could involve both, oxidative stress-induced macromolecular damage and disruption of redox signaling [5]. Previous studies have suggested that GSH is important in the brain function and cognition [12,19,22] but sensory-motor function has been poorly explored. Taking into consideration the evidences supporting the theory that oxidative damage proceeds through self-perpetuating mechanisms that last much longer than the initial triggering event [26] we hypothesized that an acute GSH depletion by BSO can trigger the neurodegenerative mechanisms on SNpc which affects the sensory-motor function in rats.
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2. Materials and methods
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2.1. Animals
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Male, Sprague-Dawley rats weighing 250–300 g (CENPALAB, Mayabeque, Cuba), were housed five per cage under a temperature of 22–24 ◦ C, with a relative humidity of 60 ± 5% and a photoperiod of 12 h. Bedding was changed twice per week, and water and food were provided ad libitum. All experimental procedures complied with the ethical principles for animal research established by Clark et al. [17] and the Canadian Council for Animal Care [41,42]. 2.2. Surgical procedure
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Rats were randomly assigned to the experimental groups, anesthetized with chloral hydrate (420 mg/kg body weight, intraperitoneal) and placed in a rodent stereotactic surgery device. An incision was made to expose the cranial Bregma point, from which coordinates (mm) corresponding to the SNpc were set: AP −4.9; ML +1.7 and DV −8.1 [45]. Oxidative stress was induced by injecting 4 L of BSO (15 mM in physiological saline solution) at a rate of 1 L/min, by means of a 10 L Hamilton syringe. The group with the vehicle injury underwent the same surgical procedure, but was administered physiological saline solution instead of BSO. An additional control group was prepared with completely untreated animals.
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2.3. Quantification of GSH
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was recorded. The concentration values were calculated from a GSH standard curve. 2.4. Behavioral test 2.4.1. Open field Rats comprising untreated, vehicle and BSO groups were evaluated in a single session of 5 min 7 days after surgery. Test was started after placing the rat at the center of open field chamber (100 × 100 × 100 cm) and recorded automatically by SMART software (version 2.0, Copyright Panlab, Spain, 2001). Distance traveled, resting time (time in which the rat moves to a speed below 2.5 cm/s) and vertical activity (number of animal’ rises on his hindlimbs) were calculated. 2.4.2. Bilateral tactile stimulation Rats were isolated in the test box and allowed to habituate. Then, square patches of adhesive material (of 1 cm2 approximately) were placed in the plantar surface of both forepaws as stimulus. Once placed the adhesive material, the rats were returned to the test box. It was registered the time that they delayed in retiring the small patches. The test was carried out 7 days after the administration of BSO [47,50]. 2.4.3. Staircase test It was used boxes (28 cm long × 6.6 cm wide × 6.8 cm height) made of transparent acrylic and consisted in: (1) an entrance opening at the base of the posterior end; (2) a central platform 4.7 cm high × 2.9 cm wide and (3) a mobile staircase of six steps high. Two food pellets were placed in a deep indentation made on the surface of each step and in the inferior level (floor of the box). Three days before and during the test, the rats were maintained under restricted feeding (10–12 g per animal). In each trial the animals were placed inside the experimental set during 15 min; and the number of eaten pellets counted [37]. 2.4.4. Traverse beam The rats were place in the half point of a beam (60 cm long) connecting two platforms (60 cm height above the support surface) and it was allowed to go freely to anyone of them. In this test the motor task difficulty was increased gradually using beams of rectangular or circular traverse section placed as follow: rectangular large (2.5 cm wide), circular large (2.5 cm diameter), rectangular small (1 cm wide) and circular small (1 cm diameter). Rats were evaluated two consecutive days (two trials per day in each beam) during 60 s with 10 min inter-trial. The distance traveled and permanence time in the beam was registered automatically by SMART software (version 2.0, Copyright Panlab, Spain, 2001). In addition, we take into consideration the task success as the arrival to the platform. 2.5. Morphological studies
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Twenty four hours or 7 days after BSO or vehicle injections, groups of rats were deeply anesthetized (7% chloral hydrate, 6 mL/kg, i.p.) and decapitated. Their brains were extracted and rinsed in cold saline. Then, the SNpc and corpus striatum (CS) were ipsilaterally dissected. The tissues were frozen in liquid nitrogen, weighed, and stored at −70 ◦ C until the analysis of GSH. Tissue samples were homogenized in a glass-Teflon potter containing 5% 5-sulfosalicylic acid (1:15, w/v) and the protein-free supernatant was isolated by centrifugation at 8160 × g during 10 min. Total GSH was quantified by Tietze’s recycling assay as described by Azbill et al. [53]. Aliquots of the supernatants were incubated for 25 min at 37 ◦ C in a medium containing 0.21 mM NADPH, 0.6 mM DTNB, 6.3 mM EDTA, and 143 mM sodium phosphate pH 7.5. Following the addition of 0.5 U GRD, the absorbance increase at 412 nm due to the formation of 5-thio-nitrobenzoate
Rats were anesthetized with chloral hydrate (420 mg/kg bodyweight, intraperitoneal) and perfused transcardially with phosphate-buffered saline (PBS) followed by 4% formaldehyde. The brains were removed and postfixed in 4% formaldehyde for 24 h and immersed in solutions of sucrose in PBS 7%, 15% and 30% (24 h each one). The midbrain from −4.8 to −6.3 anterior to Bregma was cut on a freezing microtome (Leitz, Wetzlar, Germany) and the coronal sections (20 m) were mounted serially on four gelatin coated slides with 10 sections and 45 m intervals between the adjacent sections. One each fourth slides was stained with cresyl violet and examined with light microscopy or used for immunohistochemistry. For the last purpose, sections were washed with PBS during 1 h and then were dipped in blocking solution of
Please cite this article in press as: Díaz-Hung M-L, et al. Sensory-motor performance after acute glutathione depletion by l-buthionine sulfoximine injection into substantia nigra pars compacta. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.05.066
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Table 1
Q4 Mean ± SEM of the GSH content (nmol/mg of protein). Statistical analysis (ANOVA and Tukey test) showed significant differences between BSO and controls (vehicle and untreated) groups. Substantia nigra pars compacta
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Corpus striatum
Untreated
Vehicle
BSO
Untreated
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BSO
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10.14 ± 0.46 8.88 ± 1.56
3.98 ± 0.62*** 9.37 ± 0.55
23.09 ± 2.10
21.39 ± 2.13 18.7 ± 2.24
11.23 ± 1.48*** 14.53 ± 1.47*
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0.3% H2 O2 in methanol for 20 min in order to quench endogenous peroxidase. The washings were given three times in PBS for 10 min each. Sections were stained with an antibody against glial fibrillar acid protein (GFAP) (1:250) and tyrosine hidroxilase (TH) overnight at 4 ◦ C. Immunoreactivity was imaged with rhodamine and fluorescein-conjugated secondary antibody, respectively. A representative sample (three rats per group) was utilized to semi-count the TH-positive cells using Image J software at a magnification of 20× in each section and percent of density of TH-positive cells calculated.
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2.6. Oxidative stress markers
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2.6.1. Assay for GPx and GRD activities Tissue samples were homogenized in a glass-Teflon potter containing 50 mM Tris/0.1 mM EDTA, pH 7.60 (1/30, w/v) and the cytosolic fraction was isolated by centrifugation at 1500 × g during 30 min at 4 ◦ C. Aliquots of the supernatant were used to measure total protein content [8] and GSH-related enzyme activities by spectrophotometric methods. GPx activity was determined by quantifying the rate of oxidation of GSH by cumene hydroperoxide (total GPX) as catalyzed by the GPx present in the sample as previously described [25]. Briefly, a reaction mixture (final vol. 0.6 mL) containing 0.1 mL sample cytosol, 1.25 mM GSH, 0.187 mM NADPH, 0.3 UmL−1 GRD, 1 mM EDTA disodium salt, and 1 mM NaN3 dissolved in 0.1 M potassium phosphate pH 7.00, was incubated at 25 ◦ C for 5 min. Then, 1 mM cumene hydroperoxide was added and the change in absorbance at 340 nm was recorded. GRD activity was determined by the rate of consumption of NADPH by the GSH regenerating reaction [13]. 2.6.2. Assay for CAT activity Tissues samples were homogenized in 1 M Tris/0.25 M sucrose buffer (pH 7.4) at a tissue/buffer volume ratio of 1/5. The homogenates were centrifuged at 14,000 rpm for 15 min. CAT activity determinations were performed by spectrophotometry, following the decomposition of H2 O2 as described by Aebi [1]. The assay used phosphate buffer at 0.06 M, pH 7.4, a 60 mM solution of H2 O2 in phosphate buffer as the substrate and 21 L of homogenate until final volume 0.6 mL and measuring the absorbance at 240 nm every 2 s for 20 s. A unit of enzyme activity was defined as the amount required to transform 1 mmol of H2 O2 in 1 min at 37 ◦ C.
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2.6.3. Quantification of malondialdehyde The homogeneous sample was obtained using conditions similar to those employed for the CAT assay. Two-hundred microliters of the sample were vortexed with 400 L of a 0.67% thiobarbituric acid solution in 0.2 M HCl, then incubated for 15 min in a water bath at 100 ◦ C and centrifuged at 2448 × g for 10 min at room temperature. The resulting supernatant was used for measuring absorbance at 535 nm. The concentration values were calculated from a MDA standard curve [11].
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2.7. Data processing and statistical analysis
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The values are expressed as mean ± SEM. Normal distribution and homogeneity of variance of the data were tested by the
Kolmogorov–Smirnov and Levene tests, respectively. All variables were analyzed with a one-way ANOVA followed by Tukey’s test, a p value lower than 0.05 was considered significant.
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3. Results
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3.1. Glutathione content
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Single injection of BSO into SNpc produced a significant loss of GSH in both, SNpc (F(2, 23) = 23.2; p < 0.001) and CS (F(2, 23) = 10.21; p < 0.001) at 24 h. The level of GSH decreased by 60% in SNpc and 47% respect the control groups. In the SNpc, the GSH level was totally recovery following 7 days (F(2, 24) = 1.39; p > 0.05); however in the CS it remains lower (F(2, 21) = 4.4; p < 0.05) (Table 1). 3.2. Behavioral test 3.2.1. Open field In BSO rats the distance traveled showed a significant decrease in comparison with vehicle and untreated animals (F(2, 42) = 6.31; p < 0.01). Those animals diminished the exploratory activity and their resting time was increased (F(2, 42) = 8.27; p < 0.001) comparing to vehicle and untreated groups. In addition, vertical activity was significantly diminished (F(2, 41) = 4.87; p < 0.05) (Fig. 1A–C). 3.2.2. Bilateral tactile stimulation Subjects of BSO group increased significantly their latency to remove the tactile stimulus in the left limbs regarding to untreated and vehicle groups (F(2, 36) = 14.48; p < 0.01). No significant differences were observed in the right limbs (F(2, 36) = 2.07; p > 0.05) (Fig. 2). 3.2.3. Staircase test In the staircase test no significant differences were observed in the number of food pellet retrieved among the experimental groups for both forelimbs (F(2, 60) = 0.26; p > 0.05) (Fig. 3). 3.2.4. Traverse beam No differences among groups were found in the success frequency in the beam test although there was a general tendency for all animals to fall in the most difficulty beams. For this reason, just the rectangular large beam data and animals with successful performance were analyzed for quantitative variables. This analysis revealed that BSO rats traveled less distance in the beam regarding the untreated and vehicle animals (F(2, 22) = 7.05; p < 0.01) (Fig. 4) but no differences were found in permanence time (F(2, 22) = 2.84; p > 0.05) (data no showed). 3.3. Morphological studies The staining with cresyl violet showed a loss in neuronal cells as well as prevalence of glial cells in the BSO rats regard to the rest. Atrophy of neural cells was also observed in this group (Fig. 5A). At the same time, it was appreciated a decrease of TH-positive cells, which was significant (F(2, 6) = 9.81; p < 0.05) in BSO animals comparing to untreated and vehicles groups (Fig. 5B). In addition, it
Please cite this article in press as: Díaz-Hung M-L, et al. Sensory-motor performance after acute glutathione depletion by l-buthionine sulfoximine injection into substantia nigra pars compacta. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.05.066
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Fig. 1. Mean ± SEM of untreated, vehicle and BSO groups activities in open field test. (A) Resting time, (B) distance traveled and (C) vertical activity.
Many works has been reported reduced levels of GSH in the SNpc of PD postmortem brain tissue [2] which can be a causative insult for neuronal degeneration. In fact, it has been demonstrated that survival of nigral dopaminergic neurons crucially depends on a tight regulation of their glutathione levels [28]. Most of the studies in vivo have established long-term models of GSH depletion in which it is not surprising to find the deficits in sensory-motor performance due to GSH depletion; however it has been poorly explored. In the present study, we injected BSO directly in the SNpc to evaluate the Fig. 2. Latencies of untreated, vehicle and BSO groups in the bilateral tactile stimulation test for both, left and right limbs. Mean ± SEM are shown.
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was found an increase in the GFAP-positive cells and in the size of astrocytes in BSO rats.
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3.4. Oxidative stress markers
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No significant changes were found neither in GPx nor GRD enzymatic activities for both, SNpc and CS. CAT activity showed a significant increasing in the SNpc of BSO group at 24 h regarding untreated and vehicle groups (F(2, 18) = 6.66; p < 0.01) without significant changes in CS. MDA content was increased in the CS of BSO group comparing with vehicle at 7 days after BSO administration (F(2, 25) = 4.13; p < 0.05) (Fig. 6).
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4. Discussion
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BSO is a selective inhibitor of ␥-glutamyl cysteine synthetase commonly used to study the relationship between GSH and cellular death [14]. GSH levels can be compromised by inherited or acquired defects in the enzymes, transporter, signaling molecules, or transcription factors that are involved in its homeostasis. GSH deficiency accompanies an increased susceptibility to oxidative stress [29].
Fig. 3. Mean ± SEM of food pellet retrieved in the staircase test by both, left and right forelimbs.
Fig. 4. Traverse beam test performance. (A) Typical path traveled in the bar from the beginning (B) until the end (E) points. Note the direct path toward the platform in BSO animals comparing to untreated and vehicle which showed a tendency to walk and explore the beam. (B) Distance traveled in the beam showed as mean ± SEM for untreated, vehicle and BSO groups.
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Fig. 5. Morphological changes in the SNpc. (A) Representative images show the neuronal loss observed with cresyl violet stain, the increased of GFAP positive cells and the TH and decreased GFAP.
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transient GSH depletion effect on motor and sensory modalities, and we report here for the first time that a transient GSH depletion event leads to sensor and motor impairment. In the open field test, rats treated with BSO showed a low horizontal and vertical activity indicating locomotor impairment. In rodents, locomotion is one of the most important components of exploration, a prominent activity of the rat’ spontaneous activity. Locomotor abnormalities are associated with several human disease such as Parkinson’s disease and Huntington’s disease and are also displayed by animal models [6] as was shown in mice treated with paraquat and MPTP [46]. In addition, resting time in open field has been correlated positively with nigral degeneration in rats after proteosomal inhibition [40]. In the bilateral tactile stimulation rats of BSO group displayed a high latency to remove the patches in the left forelimb (contralateral to BSO administration) regarding vehicle and control groups. Although no differences between both forelimbs (left and right) were observed indicating motor asymmetry, this result suggests a tiny sensorial dysfunction. This test is particularly responsive to subtle losses of dopamine [34]. In fact, mild sensory-motor changes can occur when cell loss was as low as 35% in 6-OHDA rats [56]. The phase in which the animal fails to contact the stimulus on the affected side, is transient in rats and the animals respond to contralateral stimulus once the stimulus on the good side is removed [49]. The paradigm followed in the staircase test did not show significant changes between groups contrary to results observed in 6-OHDA rats although this toxin causes the selective and almost completes destruction of DA neurons in the SNpc or nigro-striatal pathway [51]. Probably the circuits involved in fine motor forepaw function were not affected by GSH depletion. Moreover, it is necessary to take into consideration the training before BSO injection. Basal ganglia are critical not only for the initiation and maintenance of movements, but also for the learning and maintenance of procedural memories [55]. It has been found that DA signaling is necessary for normal motor skill learning and synaptic plasticity in
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primary motor cortex but not for the execution of a learned task or for synaptic transmission [36]. We use the traverse beam in this study to examine the balance and motor coordination skills in the animals treated with BSO. Previous reports have showed that mice treated with MPTP spend more time crossing the bar [44]. Moreover, postural and balance impairment were seen with as little as 40% cell loss in 6-OHDA rats [56] in similar task. Unexpectedly and contrary to the studies cited above, in our study all animals have showed the same success frequency but the distance traveled in the bar diminished in BSO rats. The last is in concordance with the observation that untreated and vehicle rats had a tendency to explore the bar, which was recorded in the path toward the platform (Fig. 4B). BSO group diminished the exploratory activity which could be related to an adaptative strategy to compensate a motor impairment, similar result was also observed in open field test where motivational, attentional, or learning processes are also involved [34]. The behavioral changes related above are in accordance with a loss in DA cells and glial activation in the SNpc of BSO rats. Previously, it was observed the same cellular alteration in cell culture treated with BSO [21]. Astrocytes activation could be one response to cellular insult due to GSH depletion in our study. Astroglia serve as a metabolic support for neurons, play a major role in uptake and/or degradation of neurotransmitters and regulate information processing and synaptic connectivity in the brain [43]. Astrocytes protect neurons from ROS toxicity and they are particularly important in detoxify H2 O2 because they provided cysteine precursor to the neurons improving neuronal GSH synthesis [23]. In addition, astrocytes express the transcription factor Nrf2. Under oxidant stress Nrf-2 translocate into the nucleus and interact with antioxidant responsive elements as those involved in GSH biosynthesis [18]. Nrf2-mediated GSH biosynthesis and release from astrocytes protects neurons from oxidative stress [30]. However, repeated activation of astrocytes may leads to an overproduction of ROS and other neurotoxic mediators as IL6 and arachidonic acid, which exacerbate the neuronal damage [31,33,57]. This study shows a GSH depletion of 60% in the SNpc and 47% in the CS after 24 h BSO injection. Seven days later, GSH content was similar to untreated and vehicle groups in the SNpc but in CS it was a tendency to remains decreased. Taken together, these results show the transient character of the injury but also a lower capability to detoxify ROS in both structures. Increasing in ROS generation leads to cellular damage but also generate a compensatory response by antioxidant enzymes such as superoxide dismutase, CAT and GPx. Similar to previous reports [48] we do not found changes in GPx activity. It has been suggested that high substrate concentration could exceed the antioxidant capability of this enzyme [38] which is limited by its instability and low availability [32]. Moreover, absence of changes in GPx activity might benefit the cellular sulfhydryl homeostasis by reducing the consumption of GSH in the GPX reaction [19]. In concordance with a low consumption of GSH in the GPx reaction, no changes in GRD activity were observed. CAT activity was increased in the SNpc of BSO rats at 24 h and it was reestablished at 7 days but in the CS it remains invariable. Although it has been suggested that GPx has a more important role in the Central Nervous System, CAT responds specifically to high H2 O2 . The increase in CAT activity in the SNpc is consistent with the deep decrease of GSH content in this structure. In addition, it has been reported that CAT activity is higher than GPx in this nucleus [4]. In the CS, the decrease in GSH content did not seem to be enough to generate the H2 O2 required to activate CAT. However, it is necessary to underline that low GSH levels can generate oxidative stress, peroxides accumulation and in turn, cellular damage. Under GSH depletion, H2 O2 endogenously produced by monoamine oxidase (MAO) enhances arachidonic acid release
Please cite this article in press as: Díaz-Hung M-L, et al. Sensory-motor performance after acute glutathione depletion by l-buthionine sulfoximine injection into substantia nigra pars compacta. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.05.066
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Fig. 6. Oxidative stress markers in SNpc and CS of untreated, vehicle and BSO groups. It has shown the mean ± SEM. (A), (C), (E) and (G) SNpc. (B), (D), (F) and (H) CS.
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through cellular phospholipase A2 activation in membranes and furthermore, might be converted to hydroxyl (OH− ) radicals by metals such as iron and copper. Both, arachidonic acid and OH− , promote lipid hydroperoxides formation [31] increasing aldehydes content such as MDA [10]. Unexpectedly, no accumulation of MDA content was observed in the SNpc and it was slightly increased in CS at 7 days. This effect is in relationship with CAT activity in both structures. Although in PD patient MDA has been found increase in both, blood and SNpc [20,39,58], this finding suggests that glutathione depletion could lead to cellular death by an oxidative damage-independent mechanism. Previously, it was shown dopamine depletions in the striatum and SNpc after intrastriatal 6-OHDA administration in rats without evidences indicating an increased amount of oxidative stress in SNpc although high levels of 4-hydroxynonenal and protein carbonyl were found I the CS of this animals
[52]. In the present study we found MDA accumulation and low GSH content in CS, suggesting an oxidative unbalance in this structure. It is known that glutathione depletion impairs synaptic plasticity in rat [3]. This fact and the decrease of the TH positive cells could be the mechanism involved in the basal ganglia dysfunction the sensory-motor-related impairment described above. Further studies are necessary to verify this hypothesis. Role of GSH in cell apoptosis remains unclear and clarity is likely to be further complicated by cell type specificity and nature of proapoptotic stimuli. It was observed that GSH/GSSG imbalance can precede loss of mitochondrial integrity, cytochrome c translocation to cytosol and caspase-3 activation and subsequent recovery of cellular GSH/GSSG could not influence the apoptotic endpoint [16]. In agreement with this idea, it has been suggested that neurodegenerative process could begin even if mitochondrial function is recover [44].
Please cite this article in press as: Díaz-Hung M-L, et al. Sensory-motor performance after acute glutathione depletion by l-buthionine sulfoximine injection into substantia nigra pars compacta. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.05.066
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Oxidative damage is not necessary for the transient GSH depletion-induced neuronal death and sensory-motor deficit. This effect could be related to disruption in the redox signaling generating a nigro-striatal dysfunction because the GSH decrease generates a compensatory response observed by an increasing in CAT activity and glial response in SNpc. In addition, absence of antioxidant compensatory mechanism in CS could affect the synaptic plasticity in this structure by accumulation of ROS contributing to the basal ganglia dysfunction. Further studies in redox signaling and synaptic plasticity are required to clarify the mechanisms underlying the GSH deficits.
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Acknowledgements
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A novel dicyclodextrinyl diselenide compound with glutathione peroxidase activity. FEBS J 2007;274:3846–54. [33] McGeer PL, McGeer EG. Glial reactions in Parkinson’s disease. Mov Disord 2008;23:474–83. [34] Meredith GE, Kang UJ. Behavioral models of Parkinson’s disease in rodents: a new look at an old problem. Mov Disord 2006;21:1595–606. [35] Michel PP, Ruberg M, Hirsch E. Dopaminergic neurons reduced to silence by oxidative stress: an early step in the death cascade in Parkinson’s disease? Sci STKE 2006;2006:e19. [36] Molina-Luna K, Pekanovic A, Rohrich S, Hertler B, Schubring-Giese M, RioultPedotti MS, et al. Dopamine in motor cortex is necessary for skill learning and synaptic plasticity. PLoS ONE 2009;4:e7082. [37] Montoya CP, Campbell-Hope LJ, Pemberton KD, Dunnett SB. The “staircase test”: a measure of independent forelimb reaching and grasping abilities in rats. J Neurosci Methods 1991;36:219–28. [38] Ng CF, Schafer FQ, Buettner GR, Rodgers VG. The rate of cellular hydrogen peroxide removal shows dependency on GSH: mathematical insight into in vivo H2O2 and GPx concentrations. Free Radic Res 2007;41:1201–11. [39] Nikam S, Nikam P, Ahaley SK, Sontakke AV. Oxidative stress in Parkinson’s disease. Indian J Clin Biochem 2009;24:98–101. [40] Niu C, Mei J, Pan Q, Fu X. Nigral degeneration with inclusion body formation and behavioral changes in rats after proteasomal inhibition. Stereotact Funct Neurosurg 2009;87:69–81. [41] Olferd E, Cross B, Mc Willian D, Mc Willian A. Guidelines for the use of animals in Psychology. In: Canadian Council on Animal Care (CCAC). Bradda Printing Services Inc.: Ottawa; 1997. p. 155–62. [42] Olferd E, Cross B, Mc Willian D, Mc Willian A. Guidelines for the use of animal in Neuroscience research. In: Canadian Council on Care (CCAC). Ottawa: Bradda Printing Services Inc.; 1997. p. 163–5. [43] Parpura V, Heneka MT, Montana V, Oliet SH, Schousboe A, Haydon PG, et al. Glial cells in (patho)physiology. J Neurochem 2012;121:4–27. [44] Patki G, Che Y, Lau YS. Mitochondrial dysfunction in the striatum of aged chronic mouse model of Parkinson’s disease. Front Aging Neurosci 2009;1:3, http://dx.doi.org/10.3389/neuro.24.003.2009. [45] Paxinos G, Watson C. The rat brain in stereotaxic coordenates. Second Academic Press; 1986. [46] Ren JP, Zhao YW, Sun XJ. Toxic influence of chronic oral administration of paraquat on nigrostriatal dopaminergic neurons in C57BL/6 mice. Chin Med J (Engl) 2009;122:2366–71. [47] Rose FD, Davey MJ, Love S, Dell PA. Environmental enrichment and recovery from contralateral sensory neglect in rats with large unilateral neocortical lesions. Behav Brain Res 1987;24:195–202. [48] Rougemont M, Do KQ, Castagne V. New model of glutathione deficit during development: effect on lipid peroxidation in the rat brain. J Neurosci Res 2002;70:774–83. [49] Schallert T. Behavioral test for preclinical intervention assessment. NeuroRx 2006;3:497–504. [50] Schallert T, Whishaw IQ. 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Contents lists available at ScienceDirect
Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr
Research report
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Sensory-motor performance after acute glutathione depletion by l-buthionine sulfoximine injection into substantia nigra pars compacta
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Mei-Li Díaz-Hung a,∗ , Lisette Blanco Lezcano a , Nancy Pavón Fuentes a , ˜ a , Eduardo Orta Soto a , Rilda León Martínez a , Bárbara Estupinan b Klaudia Martínez Cordovez , Isabel Fernández Jiménez a a b
International Center for Neurological Restoration, Havana, Cuba Faculty of Biology, University of Havana, Cuba
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h i g h l i g h t s • • • •
Transient glutathione depletion leads to sensory-motor impairment in rats. Sensory-motor impairment is accompanied TH-positive cell loss in the SNpc. TH-positive cell loss was not related to oxidative damage. Glutathione depletion is accompanied by antioxidant response in SNpc.
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Article history: Received 22 March 2014 Received in revised form 26 May 2014 Accepted 30 May 2014 Available online xxx
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Keywords: Glutathione Antioxidant enzymes Malondialdehyde Astrocytes Dopaminergic cell Sensory-motor function
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1. Introduction
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Glutathione is the major antioxidant in the living cells. Its deficit has been linked to neurodegenerative disorders as Parkinson’s disease but its role in the etiology of nigral degeneration and sensory-motor performance has been poorly explored. To evaluate the effect of glutathione depletion on nigro-striatal oxidative metabolism and sensory-motor performance in rats, l-buthionine sulfoximine (15 mM) or saline solution was injected into substantia nigra pars compacta (SNpc). Then, oxidative metabolism was studied 24 h and 7 days later in SNpc and corpus striatum (CS). Tyrosine hydroxylase and GFAP immunohistochemistry assays were carried out at 7 days. In addition, animals were evaluated in open field, adhesive removal, staircase and traverse beam tests. Glutathione depletion induced compensatory response in catalase activity and glial response in the in SNpc and no oxidative damage was observed. However, a loss in dopaminergic cells was found. At the same time, animals with glutathione depletion have shown poor performance in behavioral tests except for staircase test. These results suggest that glutathione depletion can be related to sensory-motor dysfunction. © 2014 Published by Elsevier B.V.
The hallmark of Parkinson’s disease (PD) neuropathology is the cell loss in substantia nigra pars compacta (SNpc), directly affecting the dopaminergic (DA) nigro-striatal pathway [7] which is
Abbreviations: PD, Parkinson’s disease; SNpc, substantia nigra pars compacta; DA, dopamine; ROS, reactive oxygen species; GSH, glutathione; CAT, catalase; GPx, glutathione peroxidase; GRD, glutathione reductase; MDA, malondialdehyde; BSO, l-buthionine sulfoximine; CS, corpus striatum. Q3 ∗ Corresponding author at: Immunochemistry department, International Center for Neurological Restoration, Ave 25 No 15805% 158 y 160, Cubanacan, Playa, Havana, CP 11300 Cuba. Tel.: +53 7 271 5353; fax: +53 7 2736028. E-mail addresses: [email protected], [email protected] (M.-L. Díaz-Hung).
responsible for the motor symptoms. The etiology of PD remains unknown although oxidative stress and mitochondrial dysfunction appear to play a crucial role in the demise of DA neurons [35]. Oxidative stress is a complex and dynamic situation characterized by an imbalance between the reactive oxygen species (ROS) production and the availability of antioxidants [24]. Defense system against ROS encompasses enzymes such as superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase as well as glutathione (GSH), urate and vitamin C [9]. These systems prevent the generation of free radicals and inactivate them once they have been formed [29]. GSH is required for many cellular processes. Particularly, it plays an important role in the maintenance and regulation of the cell thiol-redox status. In addition, it has been related with gene expression, cell proliferation, differentiation and apoptosis. Alterations
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in GSH homeostasis have been implicated in the etiology and/or progression of a number of human diseases [5,24]. A decrease in GSH levels has been described as one of the earliest biochemical alteration in PD. Since then, many research studies have addressed the role of GSH homeostasis in PD progression [27]. The infusion of l-buthionine sulfoximine (BSO), an inhibitor of ␥glutamyl cysteine synthetase, did not kill DA cells [54]. However, further studies have shown that maintaining glutathione levels is critical for protecting DA neurons in SNpc from neurodegeneration [15,28] suggesting that the effect may be quite complex and could involve both, oxidative stress-induced macromolecular damage and disruption of redox signaling [5]. Previous studies have suggested that GSH is important in the brain function and cognition [12,19,22] but sensory-motor function has been poorly explored. Taking into consideration the evidences supporting the theory that oxidative damage proceeds through self-perpetuating mechanisms that last much longer than the initial triggering event [26] we hypothesized that an acute GSH depletion by BSO can trigger the neurodegenerative mechanisms on SNpc which affects the sensory-motor function in rats.
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2. Materials and methods
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2.1. Animals
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Male, Sprague-Dawley rats weighing 250–300 g (CENPALAB, Mayabeque, Cuba), were housed five per cage under a temperature of 22–24 ◦ C, with a relative humidity of 60 ± 5% and a photoperiod of 12 h. Bedding was changed twice per week, and water and food were provided ad libitum. All experimental procedures complied with the ethical principles for animal research established by Clark et al. [17] and the Canadian Council for Animal Care [41,42]. 2.2. Surgical procedure
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Rats were randomly assigned to the experimental groups, anesthetized with chloral hydrate (420 mg/kg body weight, intraperitoneal) and placed in a rodent stereotactic surgery device. An incision was made to expose the cranial Bregma point, from which coordinates (mm) corresponding to the SNpc were set: AP −4.9; ML +1.7 and DV −8.1 [45]. Oxidative stress was induced by injecting 4 L of BSO (15 mM in physiological saline solution) at a rate of 1 L/min, by means of a 10 L Hamilton syringe. The group with the vehicle injury underwent the same surgical procedure, but was administered physiological saline solution instead of BSO. An additional control group was prepared with completely untreated animals.
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2.3. Quantification of GSH
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was recorded. The concentration values were calculated from a GSH standard curve. 2.4. Behavioral test 2.4.1. Open field Rats comprising untreated, vehicle and BSO groups were evaluated in a single session of 5 min 7 days after surgery. Test was started after placing the rat at the center of open field chamber (100 × 100 × 100 cm) and recorded automatically by SMART software (version 2.0, Copyright Panlab, Spain, 2001). Distance traveled, resting time (time in which the rat moves to a speed below 2.5 cm/s) and vertical activity (number of animal’ rises on his hindlimbs) were calculated. 2.4.2. Bilateral tactile stimulation Rats were isolated in the test box and allowed to habituate. Then, square patches of adhesive material (of 1 cm2 approximately) were placed in the plantar surface of both forepaws as stimulus. Once placed the adhesive material, the rats were returned to the test box. It was registered the time that they delayed in retiring the small patches. The test was carried out 7 days after the administration of BSO [47,50]. 2.4.3. Staircase test It was used boxes (28 cm long × 6.6 cm wide × 6.8 cm height) made of transparent acrylic and consisted in: (1) an entrance opening at the base of the posterior end; (2) a central platform 4.7 cm high × 2.9 cm wide and (3) a mobile staircase of six steps high. Two food pellets were placed in a deep indentation made on the surface of each step and in the inferior level (floor of the box). Three days before and during the test, the rats were maintained under restricted feeding (10–12 g per animal). In each trial the animals were placed inside the experimental set during 15 min; and the number of eaten pellets counted [37]. 2.4.4. Traverse beam The rats were place in the half point of a beam (60 cm long) connecting two platforms (60 cm height above the support surface) and it was allowed to go freely to anyone of them. In this test the motor task difficulty was increased gradually using beams of rectangular or circular traverse section placed as follow: rectangular large (2.5 cm wide), circular large (2.5 cm diameter), rectangular small (1 cm wide) and circular small (1 cm diameter). Rats were evaluated two consecutive days (two trials per day in each beam) during 60 s with 10 min inter-trial. The distance traveled and permanence time in the beam was registered automatically by SMART software (version 2.0, Copyright Panlab, Spain, 2001). In addition, we take into consideration the task success as the arrival to the platform. 2.5. Morphological studies
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Twenty four hours or 7 days after BSO or vehicle injections, groups of rats were deeply anesthetized (7% chloral hydrate, 6 mL/kg, i.p.) and decapitated. Their brains were extracted and rinsed in cold saline. Then, the SNpc and corpus striatum (CS) were ipsilaterally dissected. The tissues were frozen in liquid nitrogen, weighed, and stored at −70 ◦ C until the analysis of GSH. Tissue samples were homogenized in a glass-Teflon potter containing 5% 5-sulfosalicylic acid (1:15, w/v) and the protein-free supernatant was isolated by centrifugation at 8160 × g during 10 min. Total GSH was quantified by Tietze’s recycling assay as described by Azbill et al. [53]. Aliquots of the supernatants were incubated for 25 min at 37 ◦ C in a medium containing 0.21 mM NADPH, 0.6 mM DTNB, 6.3 mM EDTA, and 143 mM sodium phosphate pH 7.5. Following the addition of 0.5 U GRD, the absorbance increase at 412 nm due to the formation of 5-thio-nitrobenzoate
Rats were anesthetized with chloral hydrate (420 mg/kg bodyweight, intraperitoneal) and perfused transcardially with phosphate-buffered saline (PBS) followed by 4% formaldehyde. The brains were removed and postfixed in 4% formaldehyde for 24 h and immersed in solutions of sucrose in PBS 7%, 15% and 30% (24 h each one). The midbrain from −4.8 to −6.3 anterior to Bregma was cut on a freezing microtome (Leitz, Wetzlar, Germany) and the coronal sections (20 m) were mounted serially on four gelatin coated slides with 10 sections and 45 m intervals between the adjacent sections. One each fourth slides was stained with cresyl violet and examined with light microscopy or used for immunohistochemistry. For the last purpose, sections were washed with PBS during 1 h and then were dipped in blocking solution of
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Table 1
Q4 Mean ± SEM of the GSH content (nmol/mg of protein). Statistical analysis (ANOVA and Tukey test) showed significant differences between BSO and controls (vehicle and untreated) groups. Substantia nigra pars compacta
24 h 7 days
Corpus striatum
Untreated
Vehicle
BSO
Untreated
Vehicle
BSO
9.94 ± 0.94
10.14 ± 0.46 8.88 ± 1.56
3.98 ± 0.62*** 9.37 ± 0.55
23.09 ± 2.10
21.39 ± 2.13 18.7 ± 2.24
11.23 ± 1.48*** 14.53 ± 1.47*
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0.3% H2 O2 in methanol for 20 min in order to quench endogenous peroxidase. The washings were given three times in PBS for 10 min each. Sections were stained with an antibody against glial fibrillar acid protein (GFAP) (1:250) and tyrosine hidroxilase (TH) overnight at 4 ◦ C. Immunoreactivity was imaged with rhodamine and fluorescein-conjugated secondary antibody, respectively. A representative sample (three rats per group) was utilized to semi-count the TH-positive cells using Image J software at a magnification of 20× in each section and percent of density of TH-positive cells calculated.
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2.6. Oxidative stress markers
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2.6.1. Assay for GPx and GRD activities Tissue samples were homogenized in a glass-Teflon potter containing 50 mM Tris/0.1 mM EDTA, pH 7.60 (1/30, w/v) and the cytosolic fraction was isolated by centrifugation at 1500 × g during 30 min at 4 ◦ C. Aliquots of the supernatant were used to measure total protein content [8] and GSH-related enzyme activities by spectrophotometric methods. GPx activity was determined by quantifying the rate of oxidation of GSH by cumene hydroperoxide (total GPX) as catalyzed by the GPx present in the sample as previously described [25]. Briefly, a reaction mixture (final vol. 0.6 mL) containing 0.1 mL sample cytosol, 1.25 mM GSH, 0.187 mM NADPH, 0.3 UmL−1 GRD, 1 mM EDTA disodium salt, and 1 mM NaN3 dissolved in 0.1 M potassium phosphate pH 7.00, was incubated at 25 ◦ C for 5 min. Then, 1 mM cumene hydroperoxide was added and the change in absorbance at 340 nm was recorded. GRD activity was determined by the rate of consumption of NADPH by the GSH regenerating reaction [13]. 2.6.2. Assay for CAT activity Tissues samples were homogenized in 1 M Tris/0.25 M sucrose buffer (pH 7.4) at a tissue/buffer volume ratio of 1/5. The homogenates were centrifuged at 14,000 rpm for 15 min. CAT activity determinations were performed by spectrophotometry, following the decomposition of H2 O2 as described by Aebi [1]. The assay used phosphate buffer at 0.06 M, pH 7.4, a 60 mM solution of H2 O2 in phosphate buffer as the substrate and 21 L of homogenate until final volume 0.6 mL and measuring the absorbance at 240 nm every 2 s for 20 s. A unit of enzyme activity was defined as the amount required to transform 1 mmol of H2 O2 in 1 min at 37 ◦ C.
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2.6.3. Quantification of malondialdehyde The homogeneous sample was obtained using conditions similar to those employed for the CAT assay. Two-hundred microliters of the sample were vortexed with 400 L of a 0.67% thiobarbituric acid solution in 0.2 M HCl, then incubated for 15 min in a water bath at 100 ◦ C and centrifuged at 2448 × g for 10 min at room temperature. The resulting supernatant was used for measuring absorbance at 535 nm. The concentration values were calculated from a MDA standard curve [11].
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2.7. Data processing and statistical analysis
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The values are expressed as mean ± SEM. Normal distribution and homogeneity of variance of the data were tested by the
Kolmogorov–Smirnov and Levene tests, respectively. All variables were analyzed with a one-way ANOVA followed by Tukey’s test, a p value lower than 0.05 was considered significant.
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3. Results
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3.1. Glutathione content
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Single injection of BSO into SNpc produced a significant loss of GSH in both, SNpc (F(2, 23) = 23.2; p < 0.001) and CS (F(2, 23) = 10.21; p < 0.001) at 24 h. The level of GSH decreased by 60% in SNpc and 47% respect the control groups. In the SNpc, the GSH level was totally recovery following 7 days (F(2, 24) = 1.39; p > 0.05); however in the CS it remains lower (F(2, 21) = 4.4; p < 0.05) (Table 1). 3.2. Behavioral test 3.2.1. Open field In BSO rats the distance traveled showed a significant decrease in comparison with vehicle and untreated animals (F(2, 42) = 6.31; p < 0.01). Those animals diminished the exploratory activity and their resting time was increased (F(2, 42) = 8.27; p < 0.001) comparing to vehicle and untreated groups. In addition, vertical activity was significantly diminished (F(2, 41) = 4.87; p < 0.05) (Fig. 1A–C). 3.2.2. Bilateral tactile stimulation Subjects of BSO group increased significantly their latency to remove the tactile stimulus in the left limbs regarding to untreated and vehicle groups (F(2, 36) = 14.48; p < 0.01). No significant differences were observed in the right limbs (F(2, 36) = 2.07; p > 0.05) (Fig. 2). 3.2.3. Staircase test In the staircase test no significant differences were observed in the number of food pellet retrieved among the experimental groups for both forelimbs (F(2, 60) = 0.26; p > 0.05) (Fig. 3). 3.2.4. Traverse beam No differences among groups were found in the success frequency in the beam test although there was a general tendency for all animals to fall in the most difficulty beams. For this reason, just the rectangular large beam data and animals with successful performance were analyzed for quantitative variables. This analysis revealed that BSO rats traveled less distance in the beam regarding the untreated and vehicle animals (F(2, 22) = 7.05; p < 0.01) (Fig. 4) but no differences were found in permanence time (F(2, 22) = 2.84; p > 0.05) (data no showed). 3.3. Morphological studies The staining with cresyl violet showed a loss in neuronal cells as well as prevalence of glial cells in the BSO rats regard to the rest. Atrophy of neural cells was also observed in this group (Fig. 5A). At the same time, it was appreciated a decrease of TH-positive cells, which was significant (F(2, 6) = 9.81; p < 0.05) in BSO animals comparing to untreated and vehicles groups (Fig. 5B). In addition, it
Please cite this article in press as: Díaz-Hung M-L, et al. Sensory-motor performance after acute glutathione depletion by l-buthionine sulfoximine injection into substantia nigra pars compacta. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.05.066
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Fig. 1. Mean ± SEM of untreated, vehicle and BSO groups activities in open field test. (A) Resting time, (B) distance traveled and (C) vertical activity.
Many works has been reported reduced levels of GSH in the SNpc of PD postmortem brain tissue [2] which can be a causative insult for neuronal degeneration. In fact, it has been demonstrated that survival of nigral dopaminergic neurons crucially depends on a tight regulation of their glutathione levels [28]. Most of the studies in vivo have established long-term models of GSH depletion in which it is not surprising to find the deficits in sensory-motor performance due to GSH depletion; however it has been poorly explored. In the present study, we injected BSO directly in the SNpc to evaluate the Fig. 2. Latencies of untreated, vehicle and BSO groups in the bilateral tactile stimulation test for both, left and right limbs. Mean ± SEM are shown.
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was found an increase in the GFAP-positive cells and in the size of astrocytes in BSO rats.
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No significant changes were found neither in GPx nor GRD enzymatic activities for both, SNpc and CS. CAT activity showed a significant increasing in the SNpc of BSO group at 24 h regarding untreated and vehicle groups (F(2, 18) = 6.66; p < 0.01) without significant changes in CS. MDA content was increased in the CS of BSO group comparing with vehicle at 7 days after BSO administration (F(2, 25) = 4.13; p < 0.05) (Fig. 6).
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BSO is a selective inhibitor of ␥-glutamyl cysteine synthetase commonly used to study the relationship between GSH and cellular death [14]. GSH levels can be compromised by inherited or acquired defects in the enzymes, transporter, signaling molecules, or transcription factors that are involved in its homeostasis. GSH deficiency accompanies an increased susceptibility to oxidative stress [29].
Fig. 3. Mean ± SEM of food pellet retrieved in the staircase test by both, left and right forelimbs.
Fig. 4. Traverse beam test performance. (A) Typical path traveled in the bar from the beginning (B) until the end (E) points. Note the direct path toward the platform in BSO animals comparing to untreated and vehicle which showed a tendency to walk and explore the beam. (B) Distance traveled in the beam showed as mean ± SEM for untreated, vehicle and BSO groups.
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Fig. 5. Morphological changes in the SNpc. (A) Representative images show the neuronal loss observed with cresyl violet stain, the increased of GFAP positive cells and the TH and decreased GFAP.
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transient GSH depletion effect on motor and sensory modalities, and we report here for the first time that a transient GSH depletion event leads to sensor and motor impairment. In the open field test, rats treated with BSO showed a low horizontal and vertical activity indicating locomotor impairment. In rodents, locomotion is one of the most important components of exploration, a prominent activity of the rat’ spontaneous activity. Locomotor abnormalities are associated with several human disease such as Parkinson’s disease and Huntington’s disease and are also displayed by animal models [6] as was shown in mice treated with paraquat and MPTP [46]. In addition, resting time in open field has been correlated positively with nigral degeneration in rats after proteosomal inhibition [40]. In the bilateral tactile stimulation rats of BSO group displayed a high latency to remove the patches in the left forelimb (contralateral to BSO administration) regarding vehicle and control groups. Although no differences between both forelimbs (left and right) were observed indicating motor asymmetry, this result suggests a tiny sensorial dysfunction. This test is particularly responsive to subtle losses of dopamine [34]. In fact, mild sensory-motor changes can occur when cell loss was as low as 35% in 6-OHDA rats [56]. The phase in which the animal fails to contact the stimulus on the affected side, is transient in rats and the animals respond to contralateral stimulus once the stimulus on the good side is removed [49]. The paradigm followed in the staircase test did not show significant changes between groups contrary to results observed in 6-OHDA rats although this toxin causes the selective and almost completes destruction of DA neurons in the SNpc or nigro-striatal pathway [51]. Probably the circuits involved in fine motor forepaw function were not affected by GSH depletion. Moreover, it is necessary to take into consideration the training before BSO injection. Basal ganglia are critical not only for the initiation and maintenance of movements, but also for the learning and maintenance of procedural memories [55]. It has been found that DA signaling is necessary for normal motor skill learning and synaptic plasticity in
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primary motor cortex but not for the execution of a learned task or for synaptic transmission [36]. We use the traverse beam in this study to examine the balance and motor coordination skills in the animals treated with BSO. Previous reports have showed that mice treated with MPTP spend more time crossing the bar [44]. Moreover, postural and balance impairment were seen with as little as 40% cell loss in 6-OHDA rats [56] in similar task. Unexpectedly and contrary to the studies cited above, in our study all animals have showed the same success frequency but the distance traveled in the bar diminished in BSO rats. The last is in concordance with the observation that untreated and vehicle rats had a tendency to explore the bar, which was recorded in the path toward the platform (Fig. 4B). BSO group diminished the exploratory activity which could be related to an adaptative strategy to compensate a motor impairment, similar result was also observed in open field test where motivational, attentional, or learning processes are also involved [34]. The behavioral changes related above are in accordance with a loss in DA cells and glial activation in the SNpc of BSO rats. Previously, it was observed the same cellular alteration in cell culture treated with BSO [21]. Astrocytes activation could be one response to cellular insult due to GSH depletion in our study. Astroglia serve as a metabolic support for neurons, play a major role in uptake and/or degradation of neurotransmitters and regulate information processing and synaptic connectivity in the brain [43]. Astrocytes protect neurons from ROS toxicity and they are particularly important in detoxify H2 O2 because they provided cysteine precursor to the neurons improving neuronal GSH synthesis [23]. In addition, astrocytes express the transcription factor Nrf2. Under oxidant stress Nrf-2 translocate into the nucleus and interact with antioxidant responsive elements as those involved in GSH biosynthesis [18]. Nrf2-mediated GSH biosynthesis and release from astrocytes protects neurons from oxidative stress [30]. However, repeated activation of astrocytes may leads to an overproduction of ROS and other neurotoxic mediators as IL6 and arachidonic acid, which exacerbate the neuronal damage [31,33,57]. This study shows a GSH depletion of 60% in the SNpc and 47% in the CS after 24 h BSO injection. Seven days later, GSH content was similar to untreated and vehicle groups in the SNpc but in CS it was a tendency to remains decreased. Taken together, these results show the transient character of the injury but also a lower capability to detoxify ROS in both structures. Increasing in ROS generation leads to cellular damage but also generate a compensatory response by antioxidant enzymes such as superoxide dismutase, CAT and GPx. Similar to previous reports [48] we do not found changes in GPx activity. It has been suggested that high substrate concentration could exceed the antioxidant capability of this enzyme [38] which is limited by its instability and low availability [32]. Moreover, absence of changes in GPx activity might benefit the cellular sulfhydryl homeostasis by reducing the consumption of GSH in the GPX reaction [19]. In concordance with a low consumption of GSH in the GPx reaction, no changes in GRD activity were observed. CAT activity was increased in the SNpc of BSO rats at 24 h and it was reestablished at 7 days but in the CS it remains invariable. Although it has been suggested that GPx has a more important role in the Central Nervous System, CAT responds specifically to high H2 O2 . The increase in CAT activity in the SNpc is consistent with the deep decrease of GSH content in this structure. In addition, it has been reported that CAT activity is higher than GPx in this nucleus [4]. In the CS, the decrease in GSH content did not seem to be enough to generate the H2 O2 required to activate CAT. However, it is necessary to underline that low GSH levels can generate oxidative stress, peroxides accumulation and in turn, cellular damage. Under GSH depletion, H2 O2 endogenously produced by monoamine oxidase (MAO) enhances arachidonic acid release
Please cite this article in press as: Díaz-Hung M-L, et al. Sensory-motor performance after acute glutathione depletion by l-buthionine sulfoximine injection into substantia nigra pars compacta. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.05.066
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Fig. 6. Oxidative stress markers in SNpc and CS of untreated, vehicle and BSO groups. It has shown the mean ± SEM. (A), (C), (E) and (G) SNpc. (B), (D), (F) and (H) CS.
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through cellular phospholipase A2 activation in membranes and furthermore, might be converted to hydroxyl (OH− ) radicals by metals such as iron and copper. Both, arachidonic acid and OH− , promote lipid hydroperoxides formation [31] increasing aldehydes content such as MDA [10]. Unexpectedly, no accumulation of MDA content was observed in the SNpc and it was slightly increased in CS at 7 days. This effect is in relationship with CAT activity in both structures. Although in PD patient MDA has been found increase in both, blood and SNpc [20,39,58], this finding suggests that glutathione depletion could lead to cellular death by an oxidative damage-independent mechanism. Previously, it was shown dopamine depletions in the striatum and SNpc after intrastriatal 6-OHDA administration in rats without evidences indicating an increased amount of oxidative stress in SNpc although high levels of 4-hydroxynonenal and protein carbonyl were found I the CS of this animals
[52]. In the present study we found MDA accumulation and low GSH content in CS, suggesting an oxidative unbalance in this structure. It is known that glutathione depletion impairs synaptic plasticity in rat [3]. This fact and the decrease of the TH positive cells could be the mechanism involved in the basal ganglia dysfunction the sensory-motor-related impairment described above. Further studies are necessary to verify this hypothesis. Role of GSH in cell apoptosis remains unclear and clarity is likely to be further complicated by cell type specificity and nature of proapoptotic stimuli. It was observed that GSH/GSSG imbalance can precede loss of mitochondrial integrity, cytochrome c translocation to cytosol and caspase-3 activation and subsequent recovery of cellular GSH/GSSG could not influence the apoptotic endpoint [16]. In agreement with this idea, it has been suggested that neurodegenerative process could begin even if mitochondrial function is recover [44].
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Oxidative damage is not necessary for the transient GSH depletion-induced neuronal death and sensory-motor deficit. This effect could be related to disruption in the redox signaling generating a nigro-striatal dysfunction because the GSH decrease generates a compensatory response observed by an increasing in CAT activity and glial response in SNpc. In addition, absence of antioxidant compensatory mechanism in CS could affect the synaptic plasticity in this structure by accumulation of ROS contributing to the basal ganglia dysfunction. Further studies in redox signaling and synaptic plasticity are required to clarify the mechanisms underlying the GSH deficits.
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Acknowledgements
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