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Keywords:

  • 3-nitropropionic acid;
  • energy metabolism deficit;
  • excitotoxicity;
  • l-carnitine;
  • neuroprotection;
  • quinolinic acid

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Excitotoxicity and disrupted energy metabolism are major events leading to nerve cell death in neurodegenerative disorders. These cooperative pathways share one common aspect: triggering of oxidative stress by free radical formation. In this work, we evaluated the effects of the antioxidant and energy precursor, levocarnitine (l-CAR), on the oxidative damage and the behavioral, morphological, and neurochemical alterations produced in nerve tissue by the excitotoxin and free radical precursor, quinolinic acid (2,3-pyrindin dicarboxylic acid; QUIN), and the mitochondrial toxin, 3-nitropropionic acid (3-NP). Oxidative damage was assessed by the estimation of reactive oxygen species formation, lipid peroxidation, and mitochondrial dysfunction in synaptosomal fractions. Behavioral, morphological, and neurochemical alterations were evaluated as markers of neurotoxicity in animals systemically administered with l-CAR, chronically injected with 3-NP and/or intrastriatally infused with QUIN. At micromolar concentrations, l-CAR reduced the three markers of oxidative stress stimulated by both toxins alone or in combination. l-CAR also prevented the rotation behavior evoked by QUIN and the hypokinetic pattern induced by 3-NP in rats. Morphological alterations produced by both toxins (increased striatal glial fibrillary acidic protein-immunoreactivity for QUIN and enhanced neuronal damage in different brain regions for 3-NP) were reduced by l-CAR. In addition, l-CAR prevented the synergistic action of 3-NP and QUIN to increase motor asymmetry and depleted striatal GABA levels. Our results suggest that the protective properties of l-CAR in the neurotoxic models tested are mostly mediated by its characteristics as an antioxidant agent.

Abbreviations used
3-NP

3-nitropropionic acid

DCF

dichlorofluorescein

GFAP

glial fibrillary acidic protein

H&E

hematoxylin–eosin

l-CAR

l-carnitine

LP

lipid peroxidation

MDA

malondialdehyde

MTT

thiazolyl blue tetrazolium bromide

NMDAr

NMDA receptors

OPA

o-phtaldialdehyde

PBS

phosphate-buffered saline

QUIN

2,3-pyrindin dicarboxylic acid

ROS

reactive oxygen species

RS

reactive substances

TBA

thiobarbituric acid

Neurodegenerative events constitute a major cause of neurological disorders in humans. Neurodegeneration is the result of different toxic processes which, in a concerted manner, produce severe and specific patterns of brain cell damage (Coyle and Puttfarcken 1993; Santamaría and Jiménez 2005). In particular, human disorders presenting neurodegeneration as a hallmark, such as Huntington’s disease, have been hypothesized to involve depleted energy metabolism and excitotoxicity as triggering factors for deadly cascades (Brouillet et al. 1999; Cowan and Raymond 2006; Nakamura and Animoff 2007). Excitotoxicity is defined as a toxic mechanism characterized by a sustained stimulation of excitatory amino acid receptors (Olney et al. 1971; Schwarcz et al. 1978; Nicholls et al. 2007), whereas disrupted energy metabolism is a mechanism resulting from the altered function of energy supplies, mainly the mitochondria – the ‘mitochondrial hypothesis’– (Beal 2004; Lin and Beal 2006; Nicholls et al. 2007). Despite their obvious differences, these two major mechanisms share common aspects: (i) over-activation of inner pathways leading to cell death; (ii) deficient homeostasis of calcium; and (iii) recruitment of reactive oxygen species (ROS) and further oxidative/nitrosative stress (Beal 2004; Santamaría and Jiménez 2005; Lin and Beal 2006; Nicholls et al. 2007; Sas et al. 2007). Moreover, it is known that specific deficiencies in energy supplies produce indirect excitotoxic events (Zeevalk et al. 1995; Brouillet et al. 1999), while excitotoxicity eventually produces a disrupted energy metabolism (Brouillet et al. 1999; Ribeiro et al. 2006). Indeed, it is accepted that these two mechanisms may be acting in a concerted (synergistic) manner, thus potentiating each other (Albin and Greenamyre 1992; Jacquard et al. 2006; Del Rio et al. 2007).

Quinolinic acid (2,3-pyrindin dicarboxylic acid; QUIN), an endogenous metabolite of l-tryptophan at the kynurenine pathway (Stone 1993), has been proven to be toxic in the CNS, and this feature has been mostly explained by its ability to over-activate NMDA receptors (NMDAr) (Stone 1993). However, the limited excitatory properties of QUIN on NMDAr (weak NMDAr agonist) do not completely explain its neurotoxic potency (De Carvalho et al. 1996; Brown et al. 1998), a question that has served to suggest that additional mechanisms, such as oxidative stress (Rodríguez-Martínez et al. 2000) and disrupted energy metabolism (Ribeiro et al. 2006) may be accounting for its toxicity. In contrast, 3-nitropropionic acid (3-NP), a mycotoxin produced by Arthrinium spp (Alexi et al. 1998), is responsible of neurotoxicity through the irreversible inhibition of succinate dehydrogenase at the mitochondrial respiratory chain in a mechanism directly involving a disrupted energy metabolism, further recruiting excitotoxicity and oxidative stress (Alexi et al. 1998). Both toxins have been largely tested separately to produce toxic paradigms characterized by neuronal damage to basal ganglia, as it occurs in Huntington’s disease (Shear et al. 1998). In this regard, new perspectives on the study of toxic mechanisms for these molecules are under current investigation as their co-administration at subtoxic concentrations might better resemble the physiopathological events occurring in neurodegenerative disorders (Jacquard et al. 2006).

Considering the evidence described above, it can be assumed that agents exhibiting either antioxidant or precursor metabolic properties (or both) may have potential therapeutic value at experimental and/or clinical levels. Levocarnitine (l-CAR) is an interesting candidate to be tested in neurodegenerative models given its reported properties as an antioxidant and as an energy precursor (Gülçin 2006). l-CAR, an l-lysine derivative, seems to play a primary role as a mediator of long chain fatty acid transport into the mitochondria, thus facilitating entrance into β-oxidation cycle and ATP production (Virmani and Binienda 2004; Gülçin 2006; Wang et al. 2007). In neurons, l-CAR can be mediating the transfer of acetyl groups for acetylcholine synthesis (Nalecz and Nalecz 1996), while influencing signal transduction pathways and gene expression (Binienda and Ali 2001). l-CAR is derived from both dietary sources and endogenous biosynthesis, and constitutes an important cofactor for peroxisomal oxidation of long chain fatty acids through carnitine acetyltransferase and carnitine acylcarnitine translocase (Gülçin 2006), thus allowing the formation of acetyl-l-carnitine, which also exhibits antioxidant, metabolic and neuroprotective properties (Picconi et al. 2006). Neutralization of toxic acyl-CoA metabolites in mitochondria (Stumpf et al. 1985), reduction of age-dependent mitochondrial functional decay through restoration of mitochondrial membrane potential and oxygen consumption (Gadaleta et al. 1998; Hagen et al. 2002), and its antiradical activity (Gülçin 2006), are among the positive actions of l-CAR. Therefore, the aim of this work was to investigate whether pharmacologically targeting redox and energy metabolism in nerve tissue through this molecule may result in neuroprotection in paradigms exhibiting different toxic mechanisms. For this purpose, in vitro and in vivo experiments devoted to the characterization of oxidative stress, mitochondrial dysfunction, aberrant behavior, and morphological and neurochemical alterations were performed in isolated brain synaptosomal P2 fractions and brain tissue from rats exposed to 3-NP and/or QUIN. Given that positive effects of l-CAR have been previously reported in the toxic model produced by acute administration of 3-NP to rats, here we tested the effects of l-CAR on 3-NP toxicity under chronic conditions. In addition, since Jacquard et al. (2006) have recently addressed the issue of a synergistic action between these two toxins when co-administered at subtoxic concentrations, we also tested the sensitiveness of this double challenge to the potential protective actions of l-CAR under in vitro and in vivo conditions as a first approach to this new paradigm.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Reagents

3-Nitropropionic acid, QUIN, malondialdehyde (MDA), thiazolyl blue tetrazolium bromide (MTT), mercaptopropionic acid, and GABA were all obtained from Sigma Chemical Co. (St Louis, MO, USA). l-CAR was from Sigma-Tau (Pomezia, Rome, Italy). All other reagents were obtained from other known commercial sources. Deionized water from a Milli-RQ system (Millipore, Bedford, MA, USA), was used for preparation of solutions.

Animals

Male Wistar bred in-house rats (250–300 g) were used throughout the study. For all experimental purposes, animals were housed five-per cage in acrylic box cages and provided with a standard commercial rat chow diet (Laboratory rodent diet 5001; PMI Feeds Inc., Richmond, IN, USA) and water ad libitum. Housing room was maintained under constant conditions of temperature (25 ± 3°C), humidity (50 ± 10%), and lighting (12 h light/dark cycles). A total of 16 rats were used for in vitro experiments (eight brains for measurement of lipid peroxidation (LP) and eight more for MTT reduction assay), whereas 142 rats more were employed for in vivo experiments. All procedures with animals were carried out according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the local guidelines on the ethical use of animals from the Health Ministry of Mexico. During the experiments, all efforts were made to minimize animal suffering.

In vitro experiments

Preparation and incubation of brain crude synaptosomal P2 fractions

Brain synaptosomal P2 fractions represent a suitable preparation for in vitro experiments as they functionally behave as minicells. Crude synaptosomal fractions were obtained from whole rat brains according to a previous report (Pérez-De La Cruz et al. 2005). Briefly, animals were killed by decapitation and their brains rapidly removed and homogenized in 10 volumes (w/v) of 0.32 mol/L sucrose. Homogenates were centrifuged at 1073 g for 10 min and resulting supernatants recentrifuged at 17 172 g for 15 min. Pellets were then gently resuspended in buffer-HEPES solution (0.1 mol/L NaCl, 0.01 mol/L NaH2PO4, 0.05 mol/L NaHCO3, 0.01 mol/L CaCl2, 0.006 mol/L glucose, and 0.01 mol/L HEPES, pH 7.4) and freshly employed for the experiments. Aliquots of 500 μL were incubated at 37°C for 120 min in a shaking water bath, in the presence of QUIN (100 μmol/L), 3-NP (1 mmol/L), combined subtoxic concentrations of these two toxins (166.6 μmol/L 3-NP + 21.03 μmol/L QUIN, meaning one third of CE50 for each toxin, as determined by our own experiments – data not shown), FeSO4 (10 μmol/L) as positive control for ROS formation and LP (Zaleska and Floyd 1985), and/or increasing concentrations (10–1000 μmol/L) of l-CAR. Samples were adjusted to a final volume of 1 mL with HEPES solution. CE50 for both toxins were experimentally determined first by constructing concentration–response curves of lipid peroxidation (a deleterious consequence of ROS formation) as the end-point assay, and then mathematically calculated (Lehmann 1980) and graphically confirmed. Once calculated, these subtoxic concentrations were also employed for another experimental purpose, the estimation of mitochondrial function.

Determination of ROS formation

Reactive oxygen species formation was measured in synaptosomal fractions, according to a previous report (Santamaría et al. 2001). Briefly, synaptosomal P2 fractions isolated from whole brains were diluted in nine volumes of 40 mmol/L Tris plus HEPES buffer and incubated with 5 μmol/L 2′,7′-dichlorofluorescein (DCF) diacetate reagent at 37°C for 120 min in the presence of different treatments. At the end of incubation, fluorescent signals were recorded at excitation and emission wavelengths of 488 and 532 nm, respectively, in a Perkin-Elmer LS55 Luminescence Spectrometer (Perkin-Elmer, Waltham, MA, USA). Results were expressed as nmol of DCF per gram of tissue (wet weight) per milliliter.

Assay of LP

Measurement of LP by thiobarbituric acid (TBA) is an accurate method to characterize oxidative damage to membrane lipids. LP was detected in brain synaptosomal fractions by measuring the formation of TBA-reactive substances (TBA-RS), according to a previous report (Pérez-De La Cruz et al. 2006). Two hundred and fifty microliters aliquots containing the synaptosomal fractions (already exposed to different treatments) were added with 500 μL of the TBA reagent (0.75 g TBA + 15 g trichloroacetic acid + 2.54 mL HCl) and incubated at 94°C for 30 min in a boiling water bath. A pink chromophore was produced in samples in direct relation with the amount of peroxidized products. Samples were then kept on ice for 5 min and centrifuged at 3000 g for 15 min. Optical density from the supernatants was measured in a ThermoSpectronic Genesys 8 Spectrometer (Cole-Parmer, Vernon Hills, IL, USA) at a 532 nm wavelength. Final amounts of TBA-RS – mostly MDA – were calculated by interpolation of values in an MDA standard curve and corrected by the content of protein per sample (Lowry et al. 1951). Results were expressed as nmol of TBA-RS per milligram of protein. Data from six experiments per group were collected.

Estimation of mitochondrial function by MTT reduction

Thiazolyl blue tetrazolium bromide reduction was measured in brain synaptosomal P2 fractions as a partial index of the functional status of the respiratory chain and mitochondrial function, according to a method previously described (Pérez-De La Cruz et al. 2005). Briefly, 600 μL aliquots containing synaptosomal fractions already exposed to different treatments, were added to 8 μL of MTT (5 mg/mL) and incubated at 37°C for 60 min. Samples were then centrifuged at 15 300 g, and pellets were resuspended in 1 mL of isopropanol for 1 h. A second step of centrifugation was performed at 1700 g for 3 min. Quantification of formazan was estimated in supernatants by optical density in a ThermoSpectronic Genesys 8 Spectrometer at a 570 nm wavelength. Results were expressed as the percentage of MTT reduction with respect of control values. Data from six experiments per group were collected.

In vivo experiments

Drug administration scheme

Three different toxic models were performed; two of them currently employed as experimental models of Huntington's disease (HD) (Shear et al. 1998): (i) The first one consisted of a typical excitotoxic lesion to rats through a single infusion of QUIN into the right corpus striatum. (ii) The second model was developed to produce a sustained inhibition of energy metabolism through the repeated administration of 3-NP during a month. For this second model, we choose a long period of exposure of rats to the toxin for two reasons: (a) the chronic infusion of 3-NP to rats mimics in a better fashion the patterns of behavioral changes and neurodegeneration seen in HD (Borlongan et al. 1995); and (b) previous reports in literature (Binienda and Ali 2001; Binienda et al. 2001) have already described the protective properties of l-CAR on the acute markers of toxicity evoked by 3-NP, but until now, whether such positive properties might be lasting for longer periods in rats chronically exposed to 3-NP, still remains untested. (iii) The third model consisted of a sensitization of the excitotoxic lesion produced by a subtoxic dose of QUIN through a previous administration of subtoxic doses of 3-NP to rats (synergistic model). All experiments were performed during the morning (starting every day at 7:00 am).

For the excitotoxic model, four groups of six rats per group were designed: Groups I (Sham) and III (QUIN) received vehicle (saline, i.p.), while Groups II (l-CAR) and IV (l-CAR + QUIN) received l-CAR (100 mg/kg, i.p.) for five consecutive days. Groups I and II also received a single infusion of vehicle into the right striatum 60 min after the last vehicle or l-CAR injection, whereas Groups III and IV received a single 1 μL intrastriatal infusion of QUIN (240 nmol/μL) 60 min after the last infusion of vehicle or l-CAR. Intrastriatal injections were performed at the following coordinates: +0.5 anterior to bregma, −2.6 lateral to bregma, and −4.5 ventral to the dura (Paxinos and Watson 1998). Animals were kept alive during 7 days post-lesion for behavioral and immunohistochemical purposes.

For the sustained (chronic) energy metabolism depletion model, four groups of six rats per group were designed as follows: Groups I (Control) and III (3-NP) received vehicle (saline i.p.) every 2 days for a period of 30 days; Groups II (l-CAR) and IV (l-CAR + 3-NP) received l-CAR (100 mg/kg, i.p.) every 2 days for 30 days. Groups I and II also received a second administration of vehicle 30 min after the first injection, every 2 days for the same period, whereas Groups III and IV received 3-NP (7.5 mg/kg, i.p.) 30 min after the first injection and along the same period. Doses of l-CAR and 3-NP were estimated according to previous reports (Borlongan et al. 1997; Binienda et al. 2004). At the end of the 30 days period of treatment, animals were kept alive four more days for histological purposes.

For the synergistic toxic model, eight groups of seven rats per group were treated as follows: Groups I–IV received a first injection of vehicle (saline i.p.) daily for 10 days, whereas Groups V–VIII received l-CAR (100 mg/kg, i.p.) daily for the same period. Groups I, III, V, and VII also received a second injection of vehicle (saline i.p.) daily for 5 days from the day 6 of the first injection (30 min later), while II, IV, VI, and VIII received 3-NP (3.5 mg/kg, i.p.) as the second injection, under the same conditions. Finally, Groups I, II, V, and VI received an infusion of saline (1 μL) into the right striatum (same coordinates mentioned above) on the day 10 of the first i.p. (2 h after the second injection), and Groups III, IV, VII, and VIII received QUIN (60 nmol/μL) under the same conditions.

Behavioral tests

Behavioral performances were evaluated by two different tests, depending on the experimental model employed:

  • • 
    For those animals intrastriatally lesioned with QUIN, or exposed to the combined toxic actions of 3-NP + QUIN, rotation behavior was quantified according to a previous report (Pérez-De La Cruz et al. 2005). Six days after intrastriatally lesioned, rats from all groups were loaded with apomorphine (1 mg/kg, s.c.) and separated into individual acrylic box cages. Five minutes later, the number of ipsilateral rotations to the lesioned side was recorded every 5 min for 60 min. Each rotation was defined as a complete turn (360°). Results were expressed as the total number of turns in 60 min.
  • • 
    For rats systemically infused with 3-NP, kinetic patterns of motility were recorded in a Versamax Animal Activity Monitor and Analyzer (AccuScan Instruments Inc., Columbus, OH, USA) every 2 days for 1 h, immediately after each toxin administration. Collected criteria from the equipment included, horizontal and vertical activity, total number of movements, and total walked distances. Results were represented as time courses of activity.
Histological examination

Brain tissues were collected and histologically processed according to previous descriptions (Rodríguez et al. 1999). Twenty-four hours after the last 3-NP administration, or 7 days after QUIN injection (individual models), rats from all groups were anesthetized i.p. with 0.3 mL of 3.5% chloral hydrate and perfused transcardially with phosphate-buffered saline (PBS) containing heparin (1/500, v/v), followed by 10% (v/v) formaldehyde solution (4°C). Brains were removed, post-fixed in 10% formalin for 24 h, and immersed in paraffin. Fixed tissues were serially sectioned in an 820 HistoSTAT microtome (American Instrument Exchange, Inc., Haverhill, MA, USA). Striatal, hippocampal, and cortical sections (5–7 μm) were obtained every 100 μm, covering a total distance of 300 μm (for the case of the QUIN lesioned brains, striatal sections were obtained at 100 μm anterior and 100 μm posterior to the needle tract). All sections were stained with hematoxylin–eosin (H&E) to visualize cell bodies.

Immunohistochemical detection of glial fibrillary acidic protein

Immunohistochemical detection of glial fibrillary acidic protein (GFAP) was employed as a morphological marker of reactive gliosis in the individual models. Briefly, brains were fixed in 4%p-formaldehyde for 1 week, embedded in paraffin, and sliced in 7-μm thick sections. All sections were immunohistochemically labeled and deparaffined in a series of xylenes and graded alcohols, and rinsed three times in PBS. After washing, sections were incubated in 1% H2O2 for 10 min to quench endogenous peroxidase activity. Once again, sections were washed in PBS (two times for 5 min), incubated in 10 μmol/L sodium citrate (pH 6.0) in a water bath for 30 min, rinsed three times in PBS buffer, and incubated in 1% bovine serum albumin to block non-specific immunoreactivity. After two consecutive Tris buffer washes, sections were incubated overnight at 4°C in Tris buffer containing the primary monoclonal antibody that recognizes GFAP (1 : 100 dilutions, DAKO; Dakocytomation, Carpinteria, CA, USA). Secondary antibody was swine anti-rabbit conjugated with horseradish peroxidase (DAKO Kit; Dakocytomation), and visualized with diaminobenzidine. Sections were counterstained with hematoxylin, and immunohistochemically stained sections were mounted and cover. Single labeled sections were examined by light microscopy in a Leica DMLS Galen III microscope (Leica Microsystems, Inc., Wetzlar, Germany) with a digital color video camera SSC-DC14 coupled (Sony Electronics, Teanek, NJ, USA).

Quantitative assessment of lesions

For cell counting from histologically prepared sections, the number of neuronal cells, either preserved or damaged, was obtained as an average of 10 randomly selected fields of three sections per brain. The criteria followed to score damaged neurons included pyknotic nuclei, cytoplasmic vacuolation, and neuronal atrophy. The rate of neuronal damage per field was calculated considering the density of lesioned cells versus total density.

For cell counting from immunohistochemically prepared sections, the number of immunoreactive cells for GFAP was scored in 10 randomly selected fields of three sections per brain. Results were expressed as the number of immunoreactive cells per mm2.

Striatal GABA content

The striatal levels of GABA were assessed in tissue samples from those animals belonging to the synergistic toxic model as a current index of neurochemical deficit in this brain region. GABA was measured by HPLC with fluorometric detection, as previously described by us (Pérez-De La Cruz et al. 2005). Seven days after the striatal infusion of QUIN or vehicle (1 day after the motor asymmetry evaluation), rats from the different experimental groups were administered with mercaptopropionic acid (1.2 mmol/kg, i.v.) and killed by decapitation 2 min later. Striatal tissue samples were dissected, homogenized in 15 volumes of methanol–water (85% v/v), and centrifuged (3000 g, 15 min). Supernatants were stored at −70°C until the chromatographic analysis was performed. Pre-column derivatization with an o-phtaldialdehyde (OPA) reagent was performed for the fluorometric detection of OPA-derivatized amino acids in a BAS CC-5/PM-80 chromatographic station (West Lafayette, IN, USA) with a BAS FL-45A fluorometric detector coupled. An OPA-HS Alltech reversed-phase column (Alltech Biotechnology, Lexington, KY, USA) was employed. Results were expressed as micrograms of GABA per gram of wet tissue.

Statistical analysis

All data are presented as mean ± one SEM. Results from cell counting and estimation of the rate of neuronal damage were analyzed by non-parametric anova (Kruskal–Wallis) followed by comparison with Mann–Whitney’s test. All other data were analyzed by one-way anova and post hoc Tukey’s or Bonferroni’s tests, using the software Prism 3.02 (GraphPad, San Diego, CA, USA). Values of p < 0.05 were considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

l-CAR decreases the ROS formation induced by QUIN, 3-NP, and FeSO4

Reactive oxygen species formation was estimated by the 2′,7′-DCF diacetate fluorescence test in brain synaptosomal P2 fractions exposed to QUIN (100 μmol/L), 3-NP (1000 μmol/L), or FeSO4 (10 μmol/L), and treated with increasing concentrations of l-CAR (Table 1). QUIN, 3-NP, and FeSO4 produced enhanced levels of ROS formation when compared with control (136%, 203%, and 383%, respectively). Significant protective effects of 50 μmol/L l-CAR were observed with QUIN-, 3-NP-, and FeSO4-induced ROS formation (−64% of QUIN alone, −51% of 3-NP alone, and −49% of FeSO4 alone). Despite the fact that l-CAR displayed an apparent concentration–response effect when tested against each toxic agent, it only achieved levels comparable with control when tested at 50 μmol/L against QUIN. In consideration of these results, we employed the 50 μmol/L concentration of l-CAR for the next in vitro experiments. l-CAR alone produced no effects on ROS formation at concentrations ranging 10–500 μmol/L, but its effect at higher concentrations seems to be unspecific as at 1000 μmol/L l-CAR increased ROS formation in 58% above the control.

Table 1.   ROS formation (nmol DCF/g tissue/mL) in rat brain synaptosomal P2 fractions in the presence of QUIN (100 μmol/L), 3-NP (1000 μmol/L), FeSO4 (10 μmol/L), and l-CAR (10–1000 μmol/L)
Treatmentl-CAR concentrations [μmol/L]
[0][10][25][50][100][250][500][1000]
  1. Values are mean ± SEM (n = 8 experiments per group). *p < 0.05 and **p < 0.01, differences versus Control; p < 0.05, differences versus QUIN, 3-NP or FeSO4 alone. One-way anova followed by Tukey’s test. QUIN, quinolinic acid; 3-NP, 3-nitropropionic acid; l-CAR, levocarnitine.

l-CAR0.36 ± 0.030.29 ± 0.060.26 ± 0.020.22 ± 0.090.28 ± 0.130.32 ± 0.030.38 ± 0.130.57 ± 0.21
QUIN0.85 ± 0.14*0.78 ± 0.12*0.57 ± 0.020.31 ± 0.090.34 ± 0.110.30 ± 0.07
3-NP1.09 ± 0.17**0.98 ± 0.14*0.76 ± 0.090.53 ± 0.110.45 ± 0.070.42 ± 0.13
FeSO41.74 ± 0.36**1.55 ± 0.05**1.13 ± 0.07*0.89 ± 0.16*,†0.82 ± 0.11*,†0.97 ± 0.04*,†

QUIN-, 3-NP-, and FeSO4-induced lipid peroxidation are reduced by l-CAR

Figure 1 summarizes the effect of l-CAR (50 μmol/L) on lipid peroxidation produced by toxic concentrations of QUIN (100 μmol/L), 3-NP (1000 μmol/L), or FeSO4 (10 μmol/L) in synaptosomal preparations obtained from rat brain. Basal levels of LP were 0.68 nmol MDA/mg protein. The three toxic agents tested separately enhanced the levels of LP when compared with control values (65% for QUIN, 191% for 3-NP, and 381% for FeSO4). In contrast, synaptosomal fractions co-incubated with l-CAR plus the different toxins exhibited peroxidative values significantly lower than those of their respective toxins alone (50% below QUIN alone and 15% below control for l-CAR + QUIN; 57% below 3-NP alone and 26% above control for l-CAR + 3-NP; 67% below FeSO4 alone and 57% above control for l-CAR + FeSO4). Clearly, these results support an antioxidant role of l-CAR in the different toxic models tested.

image

Figure 1.  Effect of levocarnitine (l-CAR) on quinolinate (QUIN)-, 3-nitropropionate (3-NP), and FeSO4-induced lipid peroxidation (formation of TBA-reactive substances) in rat brain synaptosomal P2 fractions. l-CAR (50 μmol/L) was added to synaptosomal fractions in the presence of QUIN (100 μmol/L), 3-NP (1000 μmol/L), or FeSO4 (10 μmol/L). Mean ± SEM of six experiments per group. *p < 0.05 and **p < 0.01, statistically different of control; one-way anova followed by Dunnett’s test.

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The concerted actions of QUIN and 3-NP on lipid peroxidation are reduced by l-CAR

LP was evidenced in synaptosomal P2 fractions by the levels of TBA-RS. Results of these experiments are also shown in Fig. 2. Basal levels of LP were 0.75 nmol MDA/mg protein. Following the issue of a possible synergistic action of depleted energy metabolism and excitotoxicity to produce neurotoxicity (Jacquard et al. 2006), subtoxic concentrations of QUIN (21.03 μmol/L) and 3-NP (166.6 μmol/L) were tested either separately or together, to produce oxidative damage. Both toxins, when tested separately, produced moderate peroxidative effects (28% for QUIN and 48% for 3-NP, above the control). However, when added together to synaptosomal fractions, a potentiated oxidative effect was detected by a 135% increase in LP above the control (83% above QUIN alone and 59% above 3-NP alone). Such an effect provides evidence of a synergistic mechanism produced by these toxins to exacerbate oxidative damage as a triggering factor for their combined toxicity. Interestingly, the addition of increasing concentrations of l-CAR (10–1000 μmol/L) to synaptosomal fractions incubated in the presence of both toxins resulted in a protective concentration–response effect (112 to 39% above the control) that did not reach basal levels, thus suggesting that l-CAR is a protective and antioxidant agent, as it has been demonstrated in several reports dealing with different toxic paradigms (Binienda and Ali 2001; Gülçin 2006; Wang et al. 2007).

image

Figure 2.  Concentration–response curve of levocarnitine (l-CAR) on quinolinate (QUIN) plus 3-nitropropionate (3-NP)-induced lipid peroxidation (formation of TBA-reactive substances) in rat brain synaptosomal P2 fractions. Increasing concentrations of l-CAR (10–1000 μmol/L) were added to synaptosomal fractions in the presence of subtoxic concentrations of QUIN (21.03 μmol/L) plus 3-NP (166.6 μmol/L) – one third of CE50 for both cases. Toxins were also tested separately at the same concentrations. Mean ± SEM of six experiments per group.

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l-CAR ameliorates the synergistic effect of 3-NP plus QUIN on mitochondrial dysfunction

Reduction of MTT was employed as an approach to the reductant capacity of synaptosomes. Data of MTT reduction are shown in Fig. 3, where basal levels were set at 100%. Again, subtoxic concentrations of QUIN (21.03 μmol/L) and 3-NP (166.6 μmol/L) were tested either separately or together. QUIN and 3-NP, when tested separately, produced moderate decays in MTT reduction (21% and 32% below controls, respectively); however, when administered together, the effect of 3-NP plus QUIN on MTT reduction reached 60% depletion, when compared with control values (48% and 38% below QUIN and 3-NP, respectively). Interestingly, the effect evoked by the combination of the two toxins did not reach a level of potentiation. In contrast, the addition of increasing concentrations of l-CAR (10–1000 μmol/L) to synaptosomal fractions incubated under the 3-NP + QUIN condition resulted in a concentration-dependent recovery of mitochondrial MTT reduction capability (60–20% below the control), although again, such a recovery did not achieve control levels.

image

Figure 3.  Concentration–response curve of levocarnitine (l-CAR) on quinolinate (QUIN) plus 3-nitropropionate (3-NP)-induced decreased reduction of MTT in rat brain synaptosomal P2 fractions. Increasing concentrations of l-CAR (10–1000 μmol/L) were added to synaptosomal fractions in the presence of subtoxic concentrations of QUIN (21.03 μmol/L) plus 3-NP (166.6 μmol/L) – one third of CE50 for both cases. Toxins were also tested separately at the same concentrations. Mean ± SEM of six experiments per group.

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The behavioral alterations evoked by QUIN and 3-NP (separately) are prevented by l-CAR

Assessment of behavioral alterations both in the excitotoxic and the energy-deficient models (Fig. 4) included two different markers: the estimation of circling behavior for those animals intrastriatally lesioned with QUIN and the monitoring of motor activity (kinetic patterns, including horizontal and vertical movements, total walked distance, etc.) for rats continuously exposed to 3-NP.

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Figure 4.  Effect of levocarnitine (l-CAR) on motor alterations induced by quinolinate (QUIN) and 3-nitropropionate (3-NP) to rats. In (a) animals received l-CAR (100 mg/kg, i.p.) or vehicle for five consecutive days, and 2 h after the last injection, rats received a single administration of QUIN (1 μL, 240 nmol) or sterile saline into the right striatum. Six days later, animals were administered with apomorphine (1 mg/kg, s.c.), and rotation behavior was evaluated for 60 min. Each bar represents mean ± SEM of six rats per group. *p < 0.001, different of control; one-way anova followed by Tukey’s test. In (b) rats were administered with l-CAR (100 mg/kg, i.p.) or vehicle every 2 days for 30 days. Animals also received an administration of 3-NP (7.5 mg/kg, i.p.) or vehicle 30 min after the first injection, every 2 days for the same period. Three kinetic patterns of motility (horizontal activity, total walked distance, and vertical activity) were recorded every 2 days for 1 h, immediately after each toxin administration. Each point represents mean ± SEM of six rats per group. The small graphics represent the total cumulative activity. *p < 0.05, different of control; one-way anova followed by Tukey’s test.

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Six days after the striatal infusion of QUIN to rats, rotation behavior challenged by a subcutaneous injection of apomorphine was recorded for 60 min (Fig. 4a). Sham animals displayed a moderate average of ipsilateral turns (one of the six rats tested produced six turns per hour, another one exhibited only one turn, and the other four produced no turns). In contrast, all QUIN-treated animals (n = 6) displayed a considerable number of rotations when compared with control (6700%), and this effect was significantly prevented by pre-treatment of rats with l-CAR (14% below the control). The group treated with l-CAR alone produced no significant changes in rotation behavior when compared with control (5%).

Patterns of motor activity evoked by 3-NP in rats are presented in Fig. 4b. In all kinetic markers evaluated (horizontal activity, total walked distance, and vertical activity), 3-NP produced hypoactive patterns when compared with control rats beginning in the first days of evaluation (except for few points where the activity was similar to that of control animals). In general terms, l-CAR treatment significantly prevented the hypokinetic pattern evoked by 3-NP in most of the times and patterns evaluated, while l-CAR alone sometimes increased basal activity. These trends are better observed in the small figures showing total cumulative activities.

l-CAR protected the nerve tissue from morphological alterations induced by QUIN or 3-NP (separately)

In our study, morphological changes in nerve tissue were assessed in both experimental models (Figs 5 and 6) by two methods performed to evidence cell damage: H&E staining and immunohistochemical detection of GFAP.

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Figure 5.  Effect of levocarnitine (l-CAR) on quinolinic acid (QUIN)-induced immunoreactivity against glial fibrillary acidic protein (GFAP) in the rat striatum. Animals received l-CAR (100 mg/kg, i.p.) for five consecutive days, and on day 5, QUIN (240 nmol/μL) was infused into the right striatum. Six days later, coronal brain sections were obtained. Seven micrometer thick coronal sections (all equidistant to the needle tract by 100 μm) were incubated overnight with GFAP antibody, counterstained with hematoxylin–eosin, and observed under light microscopy. In (a) micrographs showing GFAP-immunoreactive cells. Arrows indicate typical GFAP-positive cells (40× magnification). In (b) Counting of GFAP-immunoreactive cells per field. Mean ± SEM of 10 randomly selected fields from three sections per brain (three brains per group were counted). *p < 0.01, different of Sham; +p < 0.05, different of QUIN. Kruskal–Wallis anova followed by Mann–Whitney’s test.

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image

Figure 6.  Effect of levocarnitine (l-CAR) on 3-nitropropionic acid (3-NP)-induced nerve tissue damage in the rat brain. Animals received l-CAR (100 mg/kg, i.p.) every 2 days for 30 days. Thirty minutes after each l-CAR administration, rats received 3-NP (7.5 mg/kg, i.p.) for the same 30 days. Striatal, hippocampal, and cortical coronal sections (7-μm thick) were stained with hematoxylin–eosin, and observed under light microscopy. In (a, b, and c) micrographs show tissue damage from caudate putamen, dentate gyrus, and frontal cortex, respectively. The estimated rate of neuronal damage is also shown. Arrows indicate preserved and damaged neuronal cells (40× magnification). Small squares (right bottom) represent 100× magnifications. Mean ± SEM of 10 randomly selected fields from three sections per brain (three brains per group were counted). *p < 0.01, different of Sham; +p < 0.05, different of QUIN. Kruskal–Wallis anova followed by Mann–Whitney’s test.

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In contrast with a well-preserved appearance of the striatal tissue from Sham animals, the infusion of QUIN resulted in considerable cell loss and atrophy in this region (images not shown), where H&E staining revealed extensive neuronal cell loss along the dorsal striatum, abundant pycnotic nuclei, and destruction of neuropil. Treatment of rats with l-CAR alone produced a normal appearance of striatal tissue which was similar to that of Sham, while administered as a pre-treatment to QUIN-treated animals, prevented all alterations evoked by the toxin (images not shown). Some of these changes were also seen in the immunohistochemical assay (Fig. 5a), where QUIN produced not only an intensive reactivity against GFAP, but also a background showing extensive damaged neuropil and pycnotic nuclei. Normal appearances were seen in Sham and l-CAR + QUIN-treated animals, where a moderate reactive gliosis may correspond to a normal reaction to the mechanical lesion produced by the insertion of the needle. Interestingly, the appearance of the striatal tissue in animals treated with l-CAR alone is even more preserved and less reactive than Sham, suggesting that l-CAR could be preventing even the basal reactive gliosis. These observations were confirmed by quantitative analysis (Fig. 5b) showing that basal positive immunoreactivity against GFAP was significantly enhanced in QUIN-treated animals (140% above Sham), remained almost the control levels in l-CAR + QUIN treated rats (24% above Sham), and decreased even below control levels in the l-CAR group (45% below Sham).

On the other hand, while the continuous administration of 3-NP to rats produced considerable nerve tissue damage – evidenced by cell loss and atrophy – in the three regions analyzed (Fig. 6a, b, and c), pre-treatment of animals with l-CAR produced a significant recovery in the integrity of the nerve tissue. These findings were strengthened by the quantitative assessment of nerve tissue damage, which show l-CAR-induced significant reductions in the rates of neuronal damage produced by 3-NP in the striatum (68% below 3-NP), hippocampus (70% below 3-NP), and frontal cortex (68% below 3-NP). Again, l-CAR alone did not affect the morphology of these regions. Remarkably, in contrast to the rats infused with QUIN, an intense GFAP-immunoreactivity was found in nerve tissue from both 3-NP- and l-CAR + 3-NP-treated animals in the same three regions (images not shown), reflecting a lack of protective effect of l-CAR, at least at this level. Apparently, the glial response evoked by 3-NP as a result of nerve cell damage was so intense that l-CAR was unable to reduce it significantly, although this metabolite was partially able to preserve neuronal integrity at the three regions tested.

Motor asymmetry and depleted striatal GABA levels produced by the synergistic action of 3-NP plus QUIN are prevented by l-CAR

Figure 7 shows data of the effect of l-CAR on circling behavior (behavioral marker of striatal damage) and striatal GABA depletion (neurochemical marker of striatal cell loss) of rats exposed to the synergistic toxic model produced in vivo through the sensitization of the excitotoxic effects of QUIN by the previous treatment of rats with 3-NP. Both toxins were administered at subtoxic doses.

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Figure 7.  Effect of levocarnitine (l-CAR) on motor asymmetry induced by the combined administration of 3-nitropropionate (3-NP) plus quinolinate (QUIN) to rats. Animals received l-CAR (100 mg/kg, i.p.) or vehicle (V1) for 10 consecutive days. From day 6 of l-CAR treatment, rats were injected with 3-NP (3.5 mg/kg, i.p.) or saline (V2) every day for five consecutive days just 30 min after every l-CAR administration. On day 10 of l-CAR treatment, rats also received a single infusion of QUIN (1 μL, 60 nmol) or sterile saline (Sham) into the right striatum just 2 h after the last 3-NP injection. Six days later, animals were administered with apomorphine (1 mg/kg, s.c.), and rotation behavior was evaluated for 60 min. One day later, the striatal tissues from these rats were obtained and prepared for chromatographic analysis of total GABA content. Black bars represent data of behavioral experiments (left ‘Y’ axis), while white bars show results of GABA measurement (right ‘Y ’ axis). Each bar represents mean ± SEM of seven rats per group. a< 0.05 and A< 0.01, different of control; b< 0.05, different of 3-NP + QUIN treatment; one-way anova followed by Tukey’s test.

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Motor asymmetry was slightly, but significantly increased in the group exposed to QUIN alone when compared with Sham-control group, whereas this rotation behavior was exacerbated in the group receiving 3-NP + QUIN (4450% above Sham-control, 185% above QUIN alone). Pre-treatment of rats with l-CAR prevented the ipsilateral turns produced by QUIN alone (90% below QUIN), and significantly reduced this behavior in 3-NP + QUIN-treated animals (74% below 3-NP + QUIN).

GABA levels were significantly decreased only in the 3-NP + QUIN group (40% below Sham-control group), and almost completely preserved by pretreatment of animals with l-CAR (47% above 3-NP + QUIN, 12% below Sham-control). Of note, in both cases (motor asymmetry and GABA levels), a potentiation of the effects of QUIN by the pre-administration for 5 of 3-NP for 5 days, was evident as the magnitude of such effects was much stronger than the mere addition of their individual actions.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Findings of this work describe the protective effects of l-CAR on QUIN- and/or 3-NP-induced neurotoxicity and oxidative damage under in vitro and in vivo conditions.

In vitro findings

Reactive oxygen species formation represents the first key step to initiate oxidative damage. Our data confirm that the toxins tested initiate a cascade of oxidative events in nerve tissue primarily through ROS formation (Santamaría et al. 2001; Jara-Prado et al. 2003). Either through a deficient energy metabolism (Brouillet et al. 1999) or an excitotoxic/pro-oxidant event (Stone 1993; Santamaría and Jiménez 2005), it seems that both 3-NP and QUIN immediately and vigorously recruit the formation of ROS. Our results also demonstrate a concentration-dependent protective action of l-CAR on the ROS formation induced by the toxins, probably through its demonstrated property as a free radical scavenger (Gülçin 2006).

On the other hand, LP constitutes an evidence of oxidative damage to membrane lipids through the action of ROS. The effects of toxic concentrations of QUIN and 3-NP on LP in synaptosomal fractions have been previously reported by us (Jara-Prado et al. 2003; Pérez-De La Cruz et al. 2005, 2006). Our present findings support those reports and suggest that synaptosomal membrane lipids are common targets for the oxidative actions of ROS in the models tested. The peroxidative potency of QUIN in brain synaptosomal fractions was considerable, although its significance appears masked by those higher effects of 3-NP and FeSO4. In the case of 3-NP, recent reports provide enough evidence of its oxidative toxicity by means of different antioxidant strategies (melatonin, dehydroepiandrosterone, transcranial magnetic stimulation, etc.), all employed to reduce its neurotoxic effects (Tunez et al. 2004, 2005, 2006). Our results also support an antioxidant role of l-CAR in the tested models as this metabolite was able to abolish LP.

In regard to the simultaneous exposure of synaptosomal fractions to subtoxic concentrations of QUIN and 3-NP, we found a potentiation in LP that was reduced by l-CAR. Cooperative pathways seem to be responsible for nerve tissue damage, but when testing this hypothesis through the mere use of a single toxin with a predominant mechanism, other processes simply appear as downstream events. In this work, we exposed brain synaptosomal fractions to a simultaneous condition of excitotoxicity, decreased energy metabolism, and oxidative stress as a first approach to the physiopathological conditions occurring during the onset and progress of degenerative events in some neurological disorders. This ‘double challenge’ is indeed not new: Jacquard et al. (2006) have recently addressed this issue, demonstrating that mitochondrial defects amplify NMDAr activation-mediated neurodegeneration through a rise in Ca2+ levels coming from intracellular sources. For this reason, we assume that this paradigm is more representative of human neurodegenerative conditions than isolated models of toxicity, and therefore, it needs to be explored in further studies. In the meantime, these findings suggest that energy depletion, excitotoxicity and oxidative stress simultaneously might account to produce neurotoxicity. Furthermore, the addition of increasing concentrations of l-CAR to synaptosomal fractions incubated under the 3-NP + QUIN condition resulted in a concentration-dependent recovery of mitochondrial MTT reduction, thus suggesting that l-CAR might also be acting at a metabolic level to ameliorate the mitochondrial dysfunction in the model evoked by 3-NP + QUIN.

In vivo findings

In relation to motor alterations, motor asymmetry has been currently employed to evidence extensive functional lesions of the caudate produced after QUIN infusion (Santamaría et al. 2003; Pérez-De La Cruz et al. 2005). The fact that l-CAR prevented such behavioral alterations in the model evoked by QUIN as well as in the combined model evoked by 3-NP + QUIN, is indicative of its broad spectrum of neuroprotective actions under different stressing conditions, as rotation behavior is tightly related with striatal cell loss – which was further confirmed by the morphological examination in the QUIN model – or the neurochemical preservation of GABA in the synergistic model, as described forward. Although other antioxidants have also shown positive effects on QUIN-induced rotation behavior (Santamaría et al. 2003; Pérez-Severiano et al. 2004), l-CAR seems to be a more promising antioxidant and neuroprotective agent as judging by its combined ability to stimulate cell metabolism while reducing oxidative stress.

The fact that 3-NP alone produced hypoactive patterns since the first days of exposure results an interesting and intriguing observation, since this finding is in partial disagreement with previous reports describing altered kinetic patterns for 3-NP (Borlongan et al. 1995, 1997). In those reports, early hyperactivity and late hypoactivity were described when monitoring the time course of motor alterations produced by 3-NP. Thereafter, we described a qualitative hyperkinetic pattern produced by 3-NP (30 mg/kg) in animals just 2 h after the toxin administration (Herrera-Mundo et al. 2006). Although we do not have an explanation for these discrepancies, a possibility could be that, in contrast to these reports, we used a lower dose of 3-NP (7.5 mg/kg) under a different scheme of administration, which in turn might result in a lack of hyperlocomotion during the early stages of 3-NP toxicity. Interestingly, l-CAR alone sometimes turned the animals more active along the different markers evaluated. Nonetheless, this effect should not be compared with those reported for 3-NP (Borlongan et al. 1995, 1997; Herrera-Mundo et al. 2006), as l-CAR-treated animals did not displayed other features of uncontrolled behavior currently produced by 3-NP, such as stereotyped paddling or rigid extension of hindlimbs. In contrast, those animals receiving the l-CAR pre-treatment and infused with 3-NP exhibited horizontal, vertical and ambulatory patterns close to control animals, thereby suggesting an ameliorative effect of l-CAR on 3-NP-induced hypokinesia, that was confirmed when the total cumulative activities were represented. Given the positive effects reported for l-CAR against 3-NP-induced neurotoxicity (Binienda et al. 2001, 2004) and our own findings, it is likely that its neuroprotective properties might achieve a considerable level of nerve tissue preservation, thus allowing major integrative behavioral tasks to be preserved.

The histochemical and morphological alterations produced by QUIN and 3-NP (individual models) in brain tissue showed a tight relation with the motor disturbances evoked by the toxins. The degeneration produced by 3-NP in the striatum, cortex and hippocampus, as well as the intense striatal immunoreactivity against GFAP generated by QUIN, were all significantly attenuated by l-CAR, thereby emphasizing its neuropreventive and neuroprotective properties. In further support of this consideration, l-CAR also exhibited an important degree of prevention against the combined effects of 3-NP and QUIN (synergistic model) on the striatal GABA levels. Striatal GABA depletion has been accepted as a hallmark of QUIN toxicity (Stone 1993), but at subtoxic doses such as the one employed in our synergistic model (60 nmol/μL), its effects are not expected to be significant, unless they may be facilitated by some specific condition, such as a depleted energy metabolism. As GABA depletion is a selective characteristic of toxic models of HD, it is evident that the potentiation of QUIN-induced excitotoxicity by 3-NP not only support the observations of Jacquard et al. (2006) on this paradigm, but also resembles this specific feature of HD, and therefore, l-CAR gains relevance as a neuroprotective agent.

Final remarks

Altogether, the results of this work demonstrate that l-CAR exerts antioxidant and neuroprotective properties in an excitotoxic/pro-oxidant model (QUIN), a deficient metabolic model (3-NP), and the combined actions of both toxins (3-NP + QUIN). l-CAR has been suggested to exert its neuroprotective effects through two major mechanisms: facilitation of long chain fatty acid transport into the mitochondria for β-oxidation cycle (Wang et al. 2007), and scavenging of ROS and antioxidant activity (Gülçin 2006). Of consideration, glucose is likely to be the dominant source of energy in the brain, instead of fatty acids, so whether fatty acid transport is playing a role in the protective properties of l-CAR in these models remains to be investigated. In the meantime, this consideration suggests that the antioxidant properties of l-CAR could be more responsible of neuroprotection.

An additional plausible explanation for the positive actions of l-CAR is that of an antiapoptotic activity proved in a toxic model produced by MPP+, in a process involving the regulation of the Bax and BCL-2 genes expression at the mitochondrial membrane (Wang et al. 2007). These genes are related with stabilization of membrane permeability, thus preserving mitochondrial integrity, suppressing the release of cytochrome c, and inhibiting cell death (Yang et al. 1997). Whether l-CAR is acting by this pathway in the toxic models tested in this work will be subject of further studies. For final consideration, the neuroprotective effects of l-CAR on 3-NP-induced oxidative damage have been suggested to be because of several factors (such as mitochondrial energy enhancement by alternative energy sources like ketone bodies, enhancement of oxidative metabolism by preventing lactate accumulation, and preservation of membrane phospholipids by prevented accumulation of fatty acyl-CoAs), but not to direct interference with succinate dehydrogenase (Binienda et al. 2001). New challenges for neuroprotective agents include their capability to act at different levels against toxic insults. Results of this work suggest that this profile is fulfilled by l-CAR.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This work was supported by CONACyT-México Grant 48370-Q (AS). VP-DLC and MNH-M are scholarship holders from CONACyT-México (Scholarship Grants 200241 and 218229, respectively). DS-A and MNH-M also received a scholarship from Fundación Armstrong-México.

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  3. Materials and methods
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  5. Discussion
  6. Acknowledgements
  7. References
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