Acute restraint stress reduces hippocampal oxidative damage and behavior in rats: Effect of S-allyl cysteine Ana Laura Col´ın-Gonz´alez, Hugo Becerr´ıl, Bianca Rub´ı Flores-Reyes, Ismael Torres, Enrique Pinz´on, Daniel Santamar´ıa-Del Angel, Isaac T´unez, Iris Serratos, Jos´e Pedraza-Chaverr´ı, Abel Santamar´ıa PII: DOI: Reference:
S0024-3205(15)00332-X doi: 10.1016/j.lfs.2015.06.009 LFS 14416
To appear in:
Life Sciences
Received date: Revised date: Accepted date:
8 April 2015 14 May 2015 12 June 2015
Please cite this article as: Col´ın-Gonz´ alez Ana Laura, Becerr´ıl Hugo, Flores-Reyes Bianca Rub´ı, Torres Ismael, Pinz´on Enrique, Santamar´ıa-Del Angel Daniel, T´ unez Isaac, Serratos Iris, Pedraza-Chaverr´ı Jos´e, Santamar´ıa Abel, Acute restraint stress reduces hippocampal oxidative damage and behavior in rats: Effect of S-allyl cysteine, Life Sciences (2015), doi: 10.1016/j.lfs.2015.06.009
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Acute restraint stress reduces hippocampal oxidative damage
PT
and behavior in rats: effect of S-allyl cysteine
RI
Running head: Restraint stress and S-allyl cysteine
SC
No. of pages with figures: 36. No. of pages without figures: 29.
NU
Ana Laura Colín-González1, Hugo Becerríl1,†, Bianca Rubí Flores-Reyes1,†,#, Ismael Torres2, Enrique Pinzón2, Daniel Santamaría-Del Angel3, Isaac Túnez4,5, Iris Serratos6, José
MA
Pedraza-Chaverrí7, Abel Santamaría1,*
1
AC CE P
TE
D
Laboratorio de Aminoácidos Excitadores, Instituto Nacional de Neurología y Neurocirugía, Mexico City, Mexico. 2 Unidad de Bioterio, Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico City, Mexico. 3 Laboratorio de Neuroquímica, Instituto Nacional de Pediatría, Mexico City, Mexico 4 Departmento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), Hospital Universitario Reina Sofia/Universidad de Córdoba, Cordoba, Spain. 5 Red Temática de Investigación Cooperativa en Envejecimiento y Fragilidad (RETICEF). 6 Departamento de Química, Universidad Autónoma Metropolitana-Iztapalapa, Mexico City, Mexico. 7 Departamento de Biología, Facultad de Química, Universidad Nacional Autónoma de México, Mexico City, Mexico. † #
These two authors equally contributed to this work. Programa Delfín, Mexico.
*Corresponding author. Laboratorio de Aminoácidos Excitadores, Instituto Nacional de Neurología y Neurocirugía, Insurgentes Sur No. 3877, México D.F. 14269, México. E-mail address: [email protected]
1
ACCEPTED MANUSCRIPT Abstract Aims: This simple study was designed to investigate whether acute restraint stress can
PT
generate changes in behavioral tests and hippocampal endpoints of oxidative stress in rats,
RI
and if the antioxidant S-allyl cysteine (SAC) can prevent these alterations. Materials and methods: We evaluated motor activity, forced swimming and anxiety behavior, as well as
SC
the hippocampal levels of lipid peroxidation and the activities of glutathione-related
NU
enzymes in animals submitted to mild immobilization. The effect of SAC (100 mg⁄ kg, i.p.), given to rats every day 30 minutes before starting the immobilization session, was also
MA
investigated. Immobilization (restraint) stress was induced for a period of 6 h per day for five consecutive days. Key findings: Our results indicate that, under the tested conditions,
D
acute restraint stimulates compensatory behavioral tasks (motor activity, anxiety and forced
TE
swimming) to counteract the stressing conditions prevailing, and selectively increased the
AC CE P
levels of lipid peroxidation and the enzyme activities of glutathione-S-transferase (GST) and glutathione peroxidase (GPx) in the hippocampus also as adaptive responses. SAC exhibited preventive effects in the stressed group as it improved behavior, reduced lipid peroxidation and prevented the increase of GST and GPx activities, suggesting that this antioxidant blunted primary pro-oxidative stimuli induced by restraint stress. Significance: Findings of this work also confirm that the use of antioxidants such as SAC can provide effective protection against the acute oxidative damage associated with anxiety produced by stressing conditions.
Key words: Oxidative stress; Antioxidant defense; Restraint stress; S-Allyl cysteine.
2
ACCEPTED MANUSCRIPT Introduction By definition, stress refers as to any given condition affecting the integrity of biological
PT
systems. Since acute stress is more related with the expression of adaptive responses, it is
RI
characterized by early compensatory responses oriented to restore homeostatic conditions [44] and it provides relevant information on the origin and nature of ongoing harmful
SC
events in the nervous system. Stress is often associated with psychiatric (depression,
NU
anxiety, and panic) and neurodegenerative disorders (Alzheimer’s disease, Parkinson’s disease, etc.). Oxidative stress, a condition involving a mishandled excessive formation of
MA
reactive oxygen and nitrogen species (ROS/RNS), produces damage of different molecules, organs and tissues, and it has been documented in studies using different models of stress in
D
animals [16,24,25,41,43].
TE
Animal immobilization through different experimental protocols has been employed as a
AC CE P
stress-inducing model consisting of deprivation of spontaneous movements [16]. Stress induced by immobilization produces both psychological and physical alterations, triggering oxidative damage in different brain regions, including the hippocampus, brain cortex and striatum [2,25,28,29,40,49]. Acute immobilization diminishes the activity of antioxidant enzymes -including superoxide dismutase (SOD)- and the levels of reduced glutathione (GSH) [2,41,49]. Consequently, different antioxidants can ameliorate the deleterious events generated by pro-oxidant conditions, therefore supporting their use as therapeutic tools to counteract oxidative damage occurring during stress episodes [10]. We have reported that complete restraint stress induced to rats for 24 h not only resembles the changes in oxidative damage previously reported by others through an increased striatal lipid peroxidation and decreased SOD activity, but also that this stress can be prevented by the 3
ACCEPTED MANUSCRIPT antioxidant L-carnitine (L-CAR) and the anti-anxiolytic drug diazepam [32,33]. Therefore, the search for antioxidants with better and wider neuroprotective profiles is pursued.
PT
S-allylcysteine (SAC) is the most abundant organosulfur molecule found in aged garlic
RI
extracts (Fig. 1). SAC exhibits antioxidant properties, including its capacity to scavenge superoxide radical (O2˙-) [23,30,31] and to inhibit hydrogen peroxide (H2O2) formation
SC
[7,21,30]. Through these properties, SAC has been shown to exert neuroprotective effects
NU
in different neurotoxic paradigms, including the reduction of the amyloid-beta peptideinduced oxidative damage, learning deficits [39], and apoptosis [37], while it exerted
MA
neurotrophic actions on cultured rat hippocampal neurons [36]. SAC also reduced neurotoxicity and oxidative stress during excitotoxic events [38], as well as during
D
depletion of energy metabolism [20]. New clues on the protective actions of SAC have
TE
been recently revealed through the neurotoxic model induced by 1-methyl-4-phenyl-
AC CE P
1,2,3,6-tetrahydropyridine (MPTP) in the rat nervous system. Accordingly, SAC was not only able to reduce oxidative stress [13], but it also exerted modulatory actions on the antioxidant transcription factor Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), thus providing an integral antioxidant response through phase 2 enzymes [14]. The present study is aimed to characterize the effects of SAC on different restraint stressinduced behavioral alterations and endpoints of oxidative activity in the rat hippocampus. We chose the hippocampus because it is known that stress causes the release of corticosteroids in this region, which can modify cognitive aspects of behavioral performance for hours [22].
4
ACCEPTED MANUSCRIPT Materials and methods Reagents
PT
SAC was synthesized according to previous reports [13,14]. In brief, L-cysteine reacts
RI
with allyl bromide and the product was purified by recrystallization from ethanol-water. The final product was compared for its identification with its corresponding standard by
SC
melting point. The chemical structure of this compound is shown in Fig. 1. All other
NU
reagents were obtained from known commercial sources. Animals
MA
All procedures with animals were strictly carried out according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the local guidelines on the
D
ethical use of animals from the Ministry of Health, Mexico. A total of 40 male Wistar rats
TE
(260–280 g) were used throughout the study. Animals were obtained from the vivarium of
AC CE P
the Faculty of Medicine from the Universidad Nacional Autónoma de México. Rats were kept in polycarbonate cages in the same room where the immobilization protocol was performed. Before the immobilization experiments began, animals were kept in groups of five per cage with free access to food (Laboratory rodent diet 5001; PMI Feeds Inc., Richmond, IN, USA) and water, and under controlled environmental conditions (constant room temperature (25 ± 3˚C), humidity (50 ± 10%) and light⁄darkness cycles (12:12 h)). Restraint stress protocol and drug administration Animals were randomly assigned to one of four different experimental groups (ten animals per group). In the stress group, immobilization was applied daily for a period of 6 h for 5 consecutive days using an individual rodent restraint device made of plexiglass fenestrate (Model 544-RR, Flat Bottom Restrainer 3.25” X 8”; Plas Labs Inc., Lansing MN, 5
ACCEPTED MANUSCRIPT USA). This device allowed the full rodent immobilization, during which, animals were completely deprived of food and water. Figure 2A shows a photograph of the employed
PT
device. Control and treatment groups with no stress were isolated, but not subjected to
RI
immobilization. For this case, each rat was kept in a small individual cage (30 x 30 x 20 cm) during 6 h for 5 consecutive days. SAC (100 mg/kg, i.p., prepared in distilled water)
SC
was administered 30 min before isolation (control condition) or immobilization. The dose
NU
of SAC used in this study was the same reported to be effective in an excitotoxic model produced by 3-nitropropionic acid in rats [13]. Every stress session was carried out from
MA
8:00 am to 2:00 pm to avoid any effects due to changes in circadian rhythms. At the end of the immobilization period (5 days), animals were subjected to the behavioral protocols and
D
further sacrificed by decapitation, their brains were collected and their hippocampi were
TE
immediately dissected out on ice. Tissue homogenates were obtained and used to estimate
AC CE P
different markers of oxidative activity (lipid peroxidation, antioxidant enzymes activity, and protein content). Behavioral tests
1) Motor activity
Motor activity was estimated in a Versamax Animal Activity Monitor and Analyzer open field device (AccuScan Instruments, Inc, Columbus, Ohio; Figure 2B) for 20 min after the last immobilization event and before removing the animal brains for biochemical analysis, as previously reported [13]. Animals from all groups were deposited in the open plate of the device and observed for 15 min. The collected criteria from the equipment included horizontal and vertical activity, as well as total distance walked. Results were expressed as individual recordings collected along the whole test. 6
ACCEPTED MANUSCRIPT 2) Elevated plus maze The elevated plus maze was used in this study to evaluate a possible anxiety-related
PT
behavior in animals from the different groups, following previous specifications provided
RI
for this test [47]. Briefly, 60 min after submitted to motor activity evaluation and 120 min after challenged in the forced swimming test, rats were deposited in the plus maze device
SC
(Figure 2D) for 5 min and parameters like the time spent and entries made on the open and
NU
closed arms were recorded as an index of the preference of the animals for secure and protected areas (closed arms), or for natural instinctive exploration (open arms). The maze
MA
was constructed upon technical specifications (dimensions and shape) previously established [47].
D
3) Forced swimming
TE
The forced swimming test is a key behavioral challenge to explore depressive-like
AC CE P
behavior. The test was carried out 60 min for each animal after the motor activity assessment, and was performed according to specifications provided by Slattery and Cryan [45] with a 15 min pre-test. Animals were placed into a plastic cylinder (Figure 2C) full of water (23-25°C) and forced to swim for a maximum time of 10 min. During such period, passive (immobility time) and active (struggling and swimming times) behaviors were recorded and analyzed. Lipid peroxidation assay The formation of thiobarbituric acid-reactive substances (TBA-RS) was used as an index to determine the levels of lipid peroxidation [13]. Fifty µl aliquots of the tissue homogenates were added to 100 µl of the TBA reagent (0.75 g of TBA + 15 g of trichloroacetic acid + 2.54 mL of HCl) and incubated at 100°C for 20 min. Samples were 7
ACCEPTED MANUSCRIPT then kept on ice for 5 min and centrifuged at 3,000 x g for 15 min. The optical density of supernatants was measured in a Thermo Spectronic Genesys 8 Spectrometer at 532 nm.
PT
Results were calculated as nmoles of malondialdehyde (MDA) per mg protein and further
RI
expressed as percent of TBA-RS formation vs. control. Antioxidant enzymes activity
SC
Hippocampal samples were homogenized in 500 µl of lysis buffer pH 7.4 (containing 10
NU
mM Tris-HCl, 15 mM NaCl, 0.25mM sucrose and proteases inhibitors), and centrifuged at 13,000 x g for 20 min. The supernatants were used to determine glutathione-S-transferase
MA
(GST), glutathione peroxidase (GPx), glutathione reductase (GR), and glucose 6-phosphate dehydrogenase activities (G6PD). The protein content in all samples was measured by
AC CE P
Assessment of GST activity
TE
activities per mg of protein.
D
Lowry’s method [27] in separate samples of homogenized tissue to correct the enzyme
The method described by Habig et al. [18] was used to evaluate GST activity, where 1chloro-2,4-di-nitrobenzene was employed as substrate. Optical density was detected at 340 nm. The results were expressed as enzyme units needed to transform 1 mol of substrate per minute per milligram of protein and further expressed as percent vs. control. Assessment of GPx activity For GPx activity detection, the reaction mixture consisted of 50 mM potassium phosphate pH 7.0, 1 mM EDTA, 1 mM NaN3, 0.2 mM NADPH, 1 U/ml of glutathione reductase, and 1 mM GSH, according to our previous report [6]. Briefly, 45 µl of homogenates were added to 225 ml of mixture and allowed to incubate for 5 min at room temperature before the initiation of the reaction by the addition of 30 µl of 0.25 mM H2O2. The optical density 8
ACCEPTED MANUSCRIPT was recorded at 340 nm for 5 min and the activity was calculated from the slope of the concentration curve as moles of NADPH oxidized per min. GPx activity was expressed as
PT
U/mg protein and further expressed as percent vs. control.
RI
Assessment of GR activity
GR activity was assayed by a method previously described [5]. The reaction mixture
SC
consisted of 100 mM phosphate buffer (pH 7.6), 1 mM EDTA, 1 mM oxidized glutathione
NU
and 1 mM NADPH. Forty five µl of tissue homogenates was added to 255 µl of reaction mixture. The enzyme activity was quantitated by measuring the disappearance of NADPH
MA
each 15 second during 2 min at 340 nm. Final results were expressed as nmol of oxidized NADPH/min/mg protein, using a molar extinction coefficient of 6.22 x 103 M-1cm-1 and
TE
Assessment of G6PDH activity
D
further expressed as percent vs. control.
AC CE P
G6PDH activity was examined by a method previously described [17]. The reaction mixture consisted of 55 mM Tris-HCl (pH 7.8) plus 3.3 mM MgCl2, 100 mM D-glucose-6phosphate, and 6 mM β-NADP+. Ten µl of tissue homogenates was added to 290 µl of reaction mixture, in a total volume of 0.3 ml. The enzyme activity was quantitated by measuring the formation of NADPH at 340 nm. Final results were expressed as U/mg protein using a molar extinction coefficient of 9.6 x 103 M-1cm-1 and further expressed as percent vs. control. Statistical analysis All results were expressed as mean values ± S.E.M. Data were analyzed by one-way ANOVA followed by a Tukey’s test for multiple comparisons, using the software Prism 4
9
ACCEPTED MANUSCRIPT (GraphPad, San Diego, CA, USA). Values of P
S0024-3205(15)00332-X doi: 10.1016/j.lfs.2015.06.009 LFS 14416
To appear in:
Life Sciences
Received date: Revised date: Accepted date:
8 April 2015 14 May 2015 12 June 2015
Please cite this article as: Col´ın-Gonz´ alez Ana Laura, Becerr´ıl Hugo, Flores-Reyes Bianca Rub´ı, Torres Ismael, Pinz´on Enrique, Santamar´ıa-Del Angel Daniel, T´ unez Isaac, Serratos Iris, Pedraza-Chaverr´ı Jos´e, Santamar´ıa Abel, Acute restraint stress reduces hippocampal oxidative damage and behavior in rats: Effect of S-allyl cysteine, Life Sciences (2015), doi: 10.1016/j.lfs.2015.06.009
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Acute restraint stress reduces hippocampal oxidative damage
PT
and behavior in rats: effect of S-allyl cysteine
RI
Running head: Restraint stress and S-allyl cysteine
SC
No. of pages with figures: 36. No. of pages without figures: 29.
NU
Ana Laura Colín-González1, Hugo Becerríl1,†, Bianca Rubí Flores-Reyes1,†,#, Ismael Torres2, Enrique Pinzón2, Daniel Santamaría-Del Angel3, Isaac Túnez4,5, Iris Serratos6, José
MA
Pedraza-Chaverrí7, Abel Santamaría1,*
1
AC CE P
TE
D
Laboratorio de Aminoácidos Excitadores, Instituto Nacional de Neurología y Neurocirugía, Mexico City, Mexico. 2 Unidad de Bioterio, Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico City, Mexico. 3 Laboratorio de Neuroquímica, Instituto Nacional de Pediatría, Mexico City, Mexico 4 Departmento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), Hospital Universitario Reina Sofia/Universidad de Córdoba, Cordoba, Spain. 5 Red Temática de Investigación Cooperativa en Envejecimiento y Fragilidad (RETICEF). 6 Departamento de Química, Universidad Autónoma Metropolitana-Iztapalapa, Mexico City, Mexico. 7 Departamento de Biología, Facultad de Química, Universidad Nacional Autónoma de México, Mexico City, Mexico. † #
These two authors equally contributed to this work. Programa Delfín, Mexico.
*Corresponding author. Laboratorio de Aminoácidos Excitadores, Instituto Nacional de Neurología y Neurocirugía, Insurgentes Sur No. 3877, México D.F. 14269, México. E-mail address: [email protected]
1
ACCEPTED MANUSCRIPT Abstract Aims: This simple study was designed to investigate whether acute restraint stress can
PT
generate changes in behavioral tests and hippocampal endpoints of oxidative stress in rats,
RI
and if the antioxidant S-allyl cysteine (SAC) can prevent these alterations. Materials and methods: We evaluated motor activity, forced swimming and anxiety behavior, as well as
SC
the hippocampal levels of lipid peroxidation and the activities of glutathione-related
NU
enzymes in animals submitted to mild immobilization. The effect of SAC (100 mg⁄ kg, i.p.), given to rats every day 30 minutes before starting the immobilization session, was also
MA
investigated. Immobilization (restraint) stress was induced for a period of 6 h per day for five consecutive days. Key findings: Our results indicate that, under the tested conditions,
D
acute restraint stimulates compensatory behavioral tasks (motor activity, anxiety and forced
TE
swimming) to counteract the stressing conditions prevailing, and selectively increased the
AC CE P
levels of lipid peroxidation and the enzyme activities of glutathione-S-transferase (GST) and glutathione peroxidase (GPx) in the hippocampus also as adaptive responses. SAC exhibited preventive effects in the stressed group as it improved behavior, reduced lipid peroxidation and prevented the increase of GST and GPx activities, suggesting that this antioxidant blunted primary pro-oxidative stimuli induced by restraint stress. Significance: Findings of this work also confirm that the use of antioxidants such as SAC can provide effective protection against the acute oxidative damage associated with anxiety produced by stressing conditions.
Key words: Oxidative stress; Antioxidant defense; Restraint stress; S-Allyl cysteine.
2
ACCEPTED MANUSCRIPT Introduction By definition, stress refers as to any given condition affecting the integrity of biological
PT
systems. Since acute stress is more related with the expression of adaptive responses, it is
RI
characterized by early compensatory responses oriented to restore homeostatic conditions [44] and it provides relevant information on the origin and nature of ongoing harmful
SC
events in the nervous system. Stress is often associated with psychiatric (depression,
NU
anxiety, and panic) and neurodegenerative disorders (Alzheimer’s disease, Parkinson’s disease, etc.). Oxidative stress, a condition involving a mishandled excessive formation of
MA
reactive oxygen and nitrogen species (ROS/RNS), produces damage of different molecules, organs and tissues, and it has been documented in studies using different models of stress in
D
animals [16,24,25,41,43].
TE
Animal immobilization through different experimental protocols has been employed as a
AC CE P
stress-inducing model consisting of deprivation of spontaneous movements [16]. Stress induced by immobilization produces both psychological and physical alterations, triggering oxidative damage in different brain regions, including the hippocampus, brain cortex and striatum [2,25,28,29,40,49]. Acute immobilization diminishes the activity of antioxidant enzymes -including superoxide dismutase (SOD)- and the levels of reduced glutathione (GSH) [2,41,49]. Consequently, different antioxidants can ameliorate the deleterious events generated by pro-oxidant conditions, therefore supporting their use as therapeutic tools to counteract oxidative damage occurring during stress episodes [10]. We have reported that complete restraint stress induced to rats for 24 h not only resembles the changes in oxidative damage previously reported by others through an increased striatal lipid peroxidation and decreased SOD activity, but also that this stress can be prevented by the 3
ACCEPTED MANUSCRIPT antioxidant L-carnitine (L-CAR) and the anti-anxiolytic drug diazepam [32,33]. Therefore, the search for antioxidants with better and wider neuroprotective profiles is pursued.
PT
S-allylcysteine (SAC) is the most abundant organosulfur molecule found in aged garlic
RI
extracts (Fig. 1). SAC exhibits antioxidant properties, including its capacity to scavenge superoxide radical (O2˙-) [23,30,31] and to inhibit hydrogen peroxide (H2O2) formation
SC
[7,21,30]. Through these properties, SAC has been shown to exert neuroprotective effects
NU
in different neurotoxic paradigms, including the reduction of the amyloid-beta peptideinduced oxidative damage, learning deficits [39], and apoptosis [37], while it exerted
MA
neurotrophic actions on cultured rat hippocampal neurons [36]. SAC also reduced neurotoxicity and oxidative stress during excitotoxic events [38], as well as during
D
depletion of energy metabolism [20]. New clues on the protective actions of SAC have
TE
been recently revealed through the neurotoxic model induced by 1-methyl-4-phenyl-
AC CE P
1,2,3,6-tetrahydropyridine (MPTP) in the rat nervous system. Accordingly, SAC was not only able to reduce oxidative stress [13], but it also exerted modulatory actions on the antioxidant transcription factor Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), thus providing an integral antioxidant response through phase 2 enzymes [14]. The present study is aimed to characterize the effects of SAC on different restraint stressinduced behavioral alterations and endpoints of oxidative activity in the rat hippocampus. We chose the hippocampus because it is known that stress causes the release of corticosteroids in this region, which can modify cognitive aspects of behavioral performance for hours [22].
4
ACCEPTED MANUSCRIPT Materials and methods Reagents
PT
SAC was synthesized according to previous reports [13,14]. In brief, L-cysteine reacts
RI
with allyl bromide and the product was purified by recrystallization from ethanol-water. The final product was compared for its identification with its corresponding standard by
SC
melting point. The chemical structure of this compound is shown in Fig. 1. All other
NU
reagents were obtained from known commercial sources. Animals
MA
All procedures with animals were strictly carried out according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the local guidelines on the
D
ethical use of animals from the Ministry of Health, Mexico. A total of 40 male Wistar rats
TE
(260–280 g) were used throughout the study. Animals were obtained from the vivarium of
AC CE P
the Faculty of Medicine from the Universidad Nacional Autónoma de México. Rats were kept in polycarbonate cages in the same room where the immobilization protocol was performed. Before the immobilization experiments began, animals were kept in groups of five per cage with free access to food (Laboratory rodent diet 5001; PMI Feeds Inc., Richmond, IN, USA) and water, and under controlled environmental conditions (constant room temperature (25 ± 3˚C), humidity (50 ± 10%) and light⁄darkness cycles (12:12 h)). Restraint stress protocol and drug administration Animals were randomly assigned to one of four different experimental groups (ten animals per group). In the stress group, immobilization was applied daily for a period of 6 h for 5 consecutive days using an individual rodent restraint device made of plexiglass fenestrate (Model 544-RR, Flat Bottom Restrainer 3.25” X 8”; Plas Labs Inc., Lansing MN, 5
ACCEPTED MANUSCRIPT USA). This device allowed the full rodent immobilization, during which, animals were completely deprived of food and water. Figure 2A shows a photograph of the employed
PT
device. Control and treatment groups with no stress were isolated, but not subjected to
RI
immobilization. For this case, each rat was kept in a small individual cage (30 x 30 x 20 cm) during 6 h for 5 consecutive days. SAC (100 mg/kg, i.p., prepared in distilled water)
SC
was administered 30 min before isolation (control condition) or immobilization. The dose
NU
of SAC used in this study was the same reported to be effective in an excitotoxic model produced by 3-nitropropionic acid in rats [13]. Every stress session was carried out from
MA
8:00 am to 2:00 pm to avoid any effects due to changes in circadian rhythms. At the end of the immobilization period (5 days), animals were subjected to the behavioral protocols and
D
further sacrificed by decapitation, their brains were collected and their hippocampi were
TE
immediately dissected out on ice. Tissue homogenates were obtained and used to estimate
AC CE P
different markers of oxidative activity (lipid peroxidation, antioxidant enzymes activity, and protein content). Behavioral tests
1) Motor activity
Motor activity was estimated in a Versamax Animal Activity Monitor and Analyzer open field device (AccuScan Instruments, Inc, Columbus, Ohio; Figure 2B) for 20 min after the last immobilization event and before removing the animal brains for biochemical analysis, as previously reported [13]. Animals from all groups were deposited in the open plate of the device and observed for 15 min. The collected criteria from the equipment included horizontal and vertical activity, as well as total distance walked. Results were expressed as individual recordings collected along the whole test. 6
ACCEPTED MANUSCRIPT 2) Elevated plus maze The elevated plus maze was used in this study to evaluate a possible anxiety-related
PT
behavior in animals from the different groups, following previous specifications provided
RI
for this test [47]. Briefly, 60 min after submitted to motor activity evaluation and 120 min after challenged in the forced swimming test, rats were deposited in the plus maze device
SC
(Figure 2D) for 5 min and parameters like the time spent and entries made on the open and
NU
closed arms were recorded as an index of the preference of the animals for secure and protected areas (closed arms), or for natural instinctive exploration (open arms). The maze
MA
was constructed upon technical specifications (dimensions and shape) previously established [47].
D
3) Forced swimming
TE
The forced swimming test is a key behavioral challenge to explore depressive-like
AC CE P
behavior. The test was carried out 60 min for each animal after the motor activity assessment, and was performed according to specifications provided by Slattery and Cryan [45] with a 15 min pre-test. Animals were placed into a plastic cylinder (Figure 2C) full of water (23-25°C) and forced to swim for a maximum time of 10 min. During such period, passive (immobility time) and active (struggling and swimming times) behaviors were recorded and analyzed. Lipid peroxidation assay The formation of thiobarbituric acid-reactive substances (TBA-RS) was used as an index to determine the levels of lipid peroxidation [13]. Fifty µl aliquots of the tissue homogenates were added to 100 µl of the TBA reagent (0.75 g of TBA + 15 g of trichloroacetic acid + 2.54 mL of HCl) and incubated at 100°C for 20 min. Samples were 7
ACCEPTED MANUSCRIPT then kept on ice for 5 min and centrifuged at 3,000 x g for 15 min. The optical density of supernatants was measured in a Thermo Spectronic Genesys 8 Spectrometer at 532 nm.
PT
Results were calculated as nmoles of malondialdehyde (MDA) per mg protein and further
RI
expressed as percent of TBA-RS formation vs. control. Antioxidant enzymes activity
SC
Hippocampal samples were homogenized in 500 µl of lysis buffer pH 7.4 (containing 10
NU
mM Tris-HCl, 15 mM NaCl, 0.25mM sucrose and proteases inhibitors), and centrifuged at 13,000 x g for 20 min. The supernatants were used to determine glutathione-S-transferase
MA
(GST), glutathione peroxidase (GPx), glutathione reductase (GR), and glucose 6-phosphate dehydrogenase activities (G6PD). The protein content in all samples was measured by
AC CE P
Assessment of GST activity
TE
activities per mg of protein.
D
Lowry’s method [27] in separate samples of homogenized tissue to correct the enzyme
The method described by Habig et al. [18] was used to evaluate GST activity, where 1chloro-2,4-di-nitrobenzene was employed as substrate. Optical density was detected at 340 nm. The results were expressed as enzyme units needed to transform 1 mol of substrate per minute per milligram of protein and further expressed as percent vs. control. Assessment of GPx activity For GPx activity detection, the reaction mixture consisted of 50 mM potassium phosphate pH 7.0, 1 mM EDTA, 1 mM NaN3, 0.2 mM NADPH, 1 U/ml of glutathione reductase, and 1 mM GSH, according to our previous report [6]. Briefly, 45 µl of homogenates were added to 225 ml of mixture and allowed to incubate for 5 min at room temperature before the initiation of the reaction by the addition of 30 µl of 0.25 mM H2O2. The optical density 8
ACCEPTED MANUSCRIPT was recorded at 340 nm for 5 min and the activity was calculated from the slope of the concentration curve as moles of NADPH oxidized per min. GPx activity was expressed as
PT
U/mg protein and further expressed as percent vs. control.
RI
Assessment of GR activity
GR activity was assayed by a method previously described [5]. The reaction mixture
SC
consisted of 100 mM phosphate buffer (pH 7.6), 1 mM EDTA, 1 mM oxidized glutathione
NU
and 1 mM NADPH. Forty five µl of tissue homogenates was added to 255 µl of reaction mixture. The enzyme activity was quantitated by measuring the disappearance of NADPH
MA
each 15 second during 2 min at 340 nm. Final results were expressed as nmol of oxidized NADPH/min/mg protein, using a molar extinction coefficient of 6.22 x 103 M-1cm-1 and
TE
Assessment of G6PDH activity
D
further expressed as percent vs. control.
AC CE P
G6PDH activity was examined by a method previously described [17]. The reaction mixture consisted of 55 mM Tris-HCl (pH 7.8) plus 3.3 mM MgCl2, 100 mM D-glucose-6phosphate, and 6 mM β-NADP+. Ten µl of tissue homogenates was added to 290 µl of reaction mixture, in a total volume of 0.3 ml. The enzyme activity was quantitated by measuring the formation of NADPH at 340 nm. Final results were expressed as U/mg protein using a molar extinction coefficient of 9.6 x 103 M-1cm-1 and further expressed as percent vs. control. Statistical analysis All results were expressed as mean values ± S.E.M. Data were analyzed by one-way ANOVA followed by a Tukey’s test for multiple comparisons, using the software Prism 4
9
ACCEPTED MANUSCRIPT (GraphPad, San Diego, CA, USA). Values of P
