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Keywords:

  • cell loss;
  • free radicals;
  • Huntington's disease;
  • 3-nitropropionic acid;
  • succinate dehydrogenase;
  • transcranial magnetic stimulation

Abstract

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Conclusion
  7. References

An investigation was conducted on the effect of transcranial magnetic field stimulation (TMS) on the free radical production and neuronal cell loss produced by 3-nitropropionic acid in rats. The effects of 3-nitropropionic acid were evaluated by examining the following changes in: the quantity of hydroperoxides and total radical-trapping antioxidant potential (TRAP), lipid peroxidation products, protein carbonyl groups, reduced glutathione (GSH) content, glutathione peroxidase (GSH-Px), catalase and succinate dehydrogenase (SDH) activities; total nitrite and cell death [morphological changes, quantification of neuronal loss and lactate dehydrogenase (LDH) levels]. Our results reveal that 3-nitropropionic acid induces oxidative and nitrosative stress in the striatum, prompts cell loss and also shows that TMS prevents the harmful effects induced by the acid. In conclusion, the results show the ability of TMS to modify neuronal response to 3-nitropropionic acid.

Abbreviations used
DMSO

dimethyl sulfoxide

GSH

reduced glutathione

GSH-Px

glutathione peroxidase

4–HDA

4-hydroxyalkenals

i.p.

intraperitoneally

LDH

lactate dehydrogenase

MDA

malondialdehyde

NO

nitric oxide

NOx

total nitrite (nitrite and nitrate)

ROS

reactive oxygen species

SDH

succinate dehydrogenase

TMS

transcranial magnetic field stimulation

TRAP

total radical-trapping antioxidant potential

Transcranial magnetic stimulation (TMS) of the human brain was originally developed as a diagnostic tool to investigate neurological disorders, particularly the physiological functioning of motor pathways (Rossini and Rossi 1998). Likewise, a possible effect of TMS on mood was first reported in 1987 (Bickford et al. 1987). On the other hand, the in vitro studies showed that magnetic stimulation analogous to TMS increased the overall viability of mouse monoclonal hippocampal HT22 cells and they had a neuroprotective effect against oxidative stressors such as β-amyloid and glutamate (Post et al. 1999). In addition, different studies have demonstrated that an extremely low frequency magnetic field (60 Hz, 0.7 mT) induces cell differentiation and increases neurofilaments and neurite growth (Feria-Velasco et al. 1998; Verdugo-Díaz et al. 1998; Arias-Carrión et al. 2004). Additionally, evidence exists indicating that TMS modifies vulnerability to neuronal insults (Fujiki et al. 2003) and that it improves Parkinsonian and depressive symptoms (Sandyk 1999; Fitzgerald et al. 2002; Calvo-Merino and Haggard 2004; Di Lazzaro et al. 2004; Fregni et al. 2004; Hajak et al. 2004; Lefaucheur et al. 2004; Miniussi et al. 2005). Moreover, a recent study showed that repetitive TMS induces ascorbate free radical and ataxia severity declined, and cerebellar hemispheric blood flow increased in spinocerebellar degenerations. Thus, the spinocerebellar degeneration patients showed negative correlations between ataxia severity and cerebellar hemispheric blood flow or superoxide scavenging activity (Ihara et al. 2005). Furthermore, Brusa et al. (2005) have found that repetitive TMS of 1 Hz induced a significant reduction of abnormal involuntary movements in Huntington's disease patients (Brusa et al. 2005).

Huntington's disease is a progressive neurodegenerative disorder associated with severe degeneration of basal ganglia neurones, especially the intrinsic neurones of the striatum (Martin and Gusella 1986). One toxin that is used to induce changes in animals similar to that seen in Huntington's disease is 3-nitropropionic acid (Beal et al. 1993; Beal 1998). 3-Nitropropionic acid is reported to interrupt mitochondrial electron transport. This mycotoxin is a selective inhibitor of succinate dehydrogenase (SDH, EC 1.3.99.1) (Alston et al. 1977; Coles et al. 1979) and homogeneously inhibits SDH in the rat brain (Gould et al. 1985; Alexi et al. 1998; Brouillet et al. 1998), an enzyme located in the mitochondrial inner membrane and responsible for the oxidation of succinate to fumarate. This phenomenon induces a reduction in ATP production and gives rise to free radicals, both nitric oxide and reactive oxygen species (Schulz et al. 1995; Tabrizi et al. 2000; Pérez-Severino et al. 2002). Oxidative stress and excitotoxicity are two conditions leading to cell death, by both necrosis and apoptosis (Pang and Geddes 1997), which are thought to be important in several neurodegenerative diseases; they are relevant to the striatal cell loss seen in Huntington's disease and are gaining prominence for 3-nitropropionic lesions (La Fontaine et al. 2002; Vis et al. 2004). Additionally, other pathogenic mechanisms of 3-nitropropionic acid include the activation of NMDA receptors, perturbed calcium homeostasis, caspase activation, mitochondrial permeability transition induction and mitochondrial calcium overload (Lee and Chang 2004).

The main object of this study is to evaluate the effects of TMS (60 Hz and 0.7 mT) on cell loss and degeneration, as well as oxidative damage in the 3-nitropropionic acid model of Huntington's disease in Wistar rats (Borlongan et al. 1995; Kodsi and Swerdlow 1997).

Experimental procedures

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Conclusion
  7. References

Chemical reagents and administered products

3-Nitropropionic acid, dimethyl sulfoxide (DMSO) and other reagents were purchased from Sigma (St Louis, MO, USA).

Animals

All animal welfare and procedures were in accordance with the European Communities Council Directive of 24 November 1986 (86/609/ECC) and the RD 223/1988, and were approved by the University of Cordoba's Bioethics Committee, Spain. Two-month-old male Wistar rats weighing between 190 and 210 g at the beginning of the study were purchased from Charles River (Barcelona, Spain). They were kept under controlled conditions of temperature (20–23°C), illumination (12-h light/12-h dark cycle, lights on at 08:00 h) and were provided with food (Purina®, Barcelona, Spain) and water ad libitum.

3-Nitropropionic acid (in DMSO 0.1%) was administered intraperitoneally (i.p.) at a dose of 20 mg/kg body weight for 4 days consecutively, whereas TMS was applied, either on its own or combined with 3-nitropropionic acid. In some animals the treatment with TMS began 4 days before and continued for 4 days after the first injection of 3-nitropropionic acid (TMS plus 3-nitropropionic acid), whereas in another group, TMS was applied for 4 days after the last injection of 3-nitropropionic acid (3-nitropropionic acid plus TMS). Different experiments were performed to attain the programmed objective: (i) experiment 1 was designed to study the effect of TMS on total radical-trapping antioxidant potential (TRAP) and hydroperoxides formation in the 3-nitropropionic acid model (n = 8 animals per group); (ii) experiment 2 was designed to quantify the effect of TMS on oxidative stress and changes in SDH activity induced by 3-nitropropionic acid in the striatal synaptosomes (n = 8 animals per group); (iii) experiment 3 was performed to determine the histological injury triggered by 3-nitropropionic acid in the striatum and the effect of TMS administration (n = 4 animals per group); (iv) and experiment 4 analysed lactate dehydrogenase (LDH) levels in plasma and striatum, and nitric oxide concentration in striatum (n = 8 animals per group). In all the experiments the rats were divided as follows: (i) control; (ii) TMS; (iii) mock, which involved placing the animals in the same TMS apparatus (plastic cylindrical cage) for the same period of time, but without any actual stimulation (no oscillatory magnetic field); (iv) 3-nitropropionic acid; (v) 3-nitropropionic acid plus mock; (vi) TMS plus 3-nitropropionic acid; and (vii) 3-nitropropionic acid plus TMS.

Transcranial magnetic field stimulation

Animals were placed in plastic cylindrical cages designed to keep them immobile while receiving magnetic stimulation to their heads. Each coil consisted of 1000 turns of enamelled copper wire (7 cm diameter) contained in plastic boxes (10.5 × 10.5 × 3.5 cm). A pair of Helmholtz coils (selected to minimize electric field effects) generated the fields (Dhan 1000(tm); Magnetoterapia S.A. de C.V., Mexico DF, Mexico). The stimulation consisted of an oscillatory magnetic field in the form of a sinusoidal wave with a frequency of 60 Hz and amplitude of 0.7 mT applied for 2 h in the morning and 2 h in the afternoon. Animals showed no signs of discomfort (i.e. agitation) when exposed to TMS. The daily exposure time was selected on the basis of previous reports demonstrating in vivo neuronal differentiation of chromaffin cells (Drucker-Colín et al. 1994; Feria-Velasco et al. 1998). The 2-hour exposure to TMS induced a maximum of 0.5–1°C temperature increase in the chamber. The two coils were located dorsally and ventrally to the head. The distance between each coil and the midpoint of the head was approximately 6 cm. TMS was administered for a period of 8 days, beginning 4 days before and continuing for 4 days after the first injection of 3-nitropropionic acid for the TMS + 3-nitropropionic acid group; whereas for the 3-nitropropionic + TMS acid group, the TMS began after the last injection of 3-nitropropionic acid and continued for 4 days. The length of in vivo exposure was determined on the basis of previous studies from our group that showed the protective effect of some substances on 3-nitropropionic acid induced oxidative stress when they were administered over 8 days (Montilla et al. 2004; Túnez et al. 2004a,b, 2005).

TRAP measurement

Antioxidants fall during prevention the initiation of oxidation (hydroperoxides reduction) or the peroxy radicals formation (lipid peroxidation of polyunsaturated fatty acids blocked). TRAP evaluated the activity of natural antioxidants. Thus, this parameter was determined in whole-brain homogenates by measuring the luminal chemiluminiscence intensity induced by 2,2′-azo-bis-2-amidinopropane hydrochloride (ABAP) according to the method of (Číz̆ováet al. 2004), with some modifications as previously described (Uotila et al. 1994). TRAP values were calculated as nanomoles of Trolox per milligram of protein (nmol Trolox/mg protein). The luminescence was measured using the luminescence function of a Multi-detector (Synergy™ HT Multi-Detection Microplate Readers, Bio-Tek, Vermont, USA) with emission at 690 nm.

Hydroperoxides measurement

Hydroperoxides are prominent early products of peroxidation. Nascent hydroperoxides can have several possible fates such as iron-catalysed one-electron reduction to chain-initiating free radicals, which exacerbate peroxidative damage. Thus, increased levels of hydroperoxides are frequently associated with the oxidative mechanisms involved in pathological conditions. Hydroperoxides formation was used to evaluate reactive oxygen species (ROS). The formation of peroxides was detected in whole brain using an oxidant-sensing fluorescent probe, 2′,7′-dichlorofluorescin diacetate (DCFH-DA), which, in the presence of H2O2, is oxidized to DCF (Cathcart et al. 1983). The total fluorescence was measured using a fluorophotometer (model 930; Zuzi, Shanghai, China) with excitation and emission at 485 nm and 530 nm, respectively. The values were expressed as nanomoles of reactive oxygen species per milligram of protein (nmol ROS/mg protein).

Striatal synaptosomes

The brain was removed and striatum (caudate/putamen) was isolated and suspended in 2 mL ice-cold isolation buffer (0.32 m sucrose, 20 mm HEPES, 4 µg/mL leupeptin, 4 µg/mL pepstatin, 5 µg/mL aprotinin, 20 µg/mL type II-S soybean trypsin inhibitor, 0.2 mm phenylmethylsulphonyl fluoride, 2 mm EGTA, 2 mm EDTA, pH 7.2). The homogenate was centrifuged at 450 g for 10 min at 4°C, and the supernatant was transferred to a new tube. The remaining pellet was resuspended in 1.5 mL homogenization buffer and centrifuged at 450 g for 10 min at 4°C. The supernatant fractions were combined and centrifuged at 20 000 g for 10 min at 4°C, and the resulting crude synaptosomal pellet was resuspended in 2 mL de Locke's buffer (154 mm NaCl, 5.6 mm KCl, 2.3 mm CaCl2, 1.0 mm MgCl2, 3.6 mm NaHCO3, 5 mm glucose, 5 mm HEPES, at a pH 7.2; Springere et al. 1997).

Assay of SDH activity

SDH is selectively inhibited by 3-nitropropionic acid. This phenomenon triggers a drop in energy production and it leads to oxidative stress. The evaluation of its activity is important to confirm the effect induced by 3-nitropropionic acid administration, and it enables the verification of the neuroprotective effect of experimental treatment. This assay was performed on each of the Percoll/Metrizamide gradient interfaces. Each assay reaction contained the following solutions: 650 µL phosphate buffer solution (containing 0.3 m d-mannitol and 5.0 mm magnesium chloride, pH 7.03), 125 µL of 0.04 m sodium azide, 125 µL of 0.50 mm dichloroindophenol, 125 µL of 0.2 m succinate and 400 µL of a gradient interface. The gradient interface was added last to initiate the reaction. These reactions were allowed to proceed at 25 ± 2°C and the discoloration caused by the reduction of dichloroindophenol was monitored over a 40-min period at 600 nm (Strack et al. 2001). The absorbance was evaluated in a spectrophotometer (UV-1603; Shimadzu, Kyoto, Japan). The values are presented in units per milligram of protein (U/mg protein).

Assay of lipid peroxidation products

Measurement of malondialdehyde (MDA) + 4-hydroxyalkenals (4–HDA) has been used an indicator of lipid peroxidation. Lipid peroxidation is a mechanism of cellular injury. The levels of lipid peroxidation products were determined using reagents purchased from Oxis International (LPO-586 kit; Oxis International, Portland, OR, USA). The level of lipid peroxidation is expressed as nanomoles of MDA + 4–HDA per milligram of protein (nmol/mg protein), and absorbance was evaluated in a spectrophotometer (UV-1603; Shimadzu) at a wavelength of 586 nm.

Assay of protein carbonyl groups

The protein carbonylation has been used as an indicator of protein oxidative damage. The content of protein carbonyls was evaluated using the method of Levine et al. (1990). Samples (500 µg) were incubated with 500 µL of a 10-mm solution of 2,4-dinitrophenylhydrazine in 2 n HCl for 60 min. Subsequently, the proteins were precipitated from the solutions with the use of 500 µL of 20% trichloroacetate. Then the proteins were washed three times with a solution of ethanol and ethylacetate (1 : 1 v/v) and dissolved in 1 mL of 6 m guanidine (containing 20 mm of phosphate buffer, pH 2.3, in trifluoroacetic acid) at 37°C. The carbonyls were evaluated in a spectrophotometer (UV-1603; Shimadzu) at wavelength 360 nm. The results are presented in nanomoles per milligram of protein (nmol/mg protein).

Assay of GSH content

GSH plays a central role as a co-enzyme in different enzymes such as GSH-Px and glutathione-S-transferase (GST). Additionally, this tripeptide comprises an important part of the anti-oxidant system. The determination of the levels of GSH were carried out using reagents purchased from Oxis International, i.e. the GSH-420 kit. The content of GSH is expressed as GSH nanomoles per milligram of protein (nmol/mg protein), and the absorbance was evaluated in a spectrophotometer (UV-1603; Shimadzu) at wavelength 420 nm.

Glutathione peroxidase (GSH–Px) activity assay

Glutathione peroxidase (GSH–Px) (E.C. 1.11.1.9) is an enzyme that acts as a catalyst in the oxidation of glutathione in the presence of hydrogen peroxide (H2O2) to yield oxidized glutathione and water (H2O). Its activity was evaluated by the Flohé and Gunzler method (1984). The tissues were homogenized in ice-cold buffer (0.1 m KH2PO4/K2HPO4, pH 7.0, plus 29.2 mg EDTA in 100 mL of distilled water and 10.0 mg digitonin in 100 mL of distilled water, final volume, 2000 mL) to produce a homogenate. The homogenates were then centrifuged at 10 000 g for 10 min at 4°C. The glutathione peroxidase assay is based on the oxidation of NADPH to NAD+, catalysed by a limiting concentration of glutathione reductase, with maximum absorbance at 340 nm. The content of GSH–Px is expressed as units per milligram of protein (U/mg protein), and absorbance was evaluated in a spectrophotometer (UV-1603; Shimadzu).

Assay of catalase activity

Catalase (E.C. 1.11.1.6) is an enzyme present in many animal cells. This enzyme acts as a catalyst in the dismutation of H2O2 to oxygen (O2) and H2O. It was assayed following the method of Aebi (1984), by the rate of decomposition of H2O2 at 240 nm. H2O2 (10 mm) was used as reagent, with the rate of dismutation of H2O2 to water and O2 being proportional to the concentration of catalase, and with maximum absorbance at 240 nm. The absorbance was evaluated in a spectrophotometer (UV-1603; Shimadzu). The results are presented in units per milligram of protein (U/mg protein).

Tissue processing and histological analysis

The whole brains were rapidly removed and fixed by immersion in 10% buffered formaldehyde. Subsequently they were embedded in paraffin wax, cut into 8-μm thick sections and stained with 0.025% cresyl violet (Nissl-stained). Sections were examined under bright-field illumination on a Leitz Orthoplan microscope (Herramientas Leitz S.L., Barcelona, Spain).

We analyse three different rostro-caudal levels (anterior, middle and posterior) spaced apart by 400 μm. In each level four sections of 8 μm thickness were obtained. To examine the cell density, the neurones were counted bilaterally on at least two sections at each level at a × 400 magnification by an investigator who was blind for the treatment of the animals. We used a counting frame (area) of 0.071 mm2, which was randomly placed at five different zones and the counts were averaged. A hand-held counter was used to count both healthy and pyknotic cells. The estimates of total neuronal number were based on counting nuclei. In addition it is important to clarify that the phrase ‘number of neurones’ shall signify neuronal density (healthy and pyknotic cells) in this study.

LDH release

LDH catalyses the interconversion of lactate and pyruvate. The amount of this enzyme may be used as a marker of tissue breakdown. LDH in the striatal homogenate and plasma were assayed using a kit purchased from Sigma, i.e. 340-LD. The activity of LDH is expressed as units per milligram of protein (U/mg protein) and units per litre (U/L), respectively; and absorbance was evaluated in a spectrophotometer (UV-1603; Shimadzu) at wavelength 340 nm.

Assay of total nitrite values as a marker of nitric oxide levels

Nitric oxide (NO) is a free gas produced endogenously by a variety of mammalian cells. This molecule induces vasodilatation; it inhibits platelet aggregation and adhesion to the vascular endothelium and elevates the intracellular level of cyclic GMP.

Total nitrite (nitrite and nitrate; NOx) was used as a marker of NO levels and assayed following the Griess method (Ricart-Janéet al. 2002) in homogenates of striatum. This assay uses the determination of nitrite as an indicator of NO production in biological samples. Nitric oxide is transformed in nitrate and nitrite. Because a colourimetric reagent (the Greiss reagent) exists for the determination of nitrite, it is common practice to use either enzymatic or chemical reduction to convert all nitrates to nitrite in a sample and measure total nitrite as an indicator of NO production. Nitrate was reduced to nitrite by incubating a sample aliquot (150 µL) for 15 min at 37°C in the presence of 0.1 U/mL nitrate reductase, 50 µm NADPH and 5 µm flavin-adenine dinucleotide in a final volume of 160 µL. When nitrate reduction is complete, total nitrite is then determined spectophotometrically by using the Griess reaction. Griess reagent is composed of a mixture of sulfanilamide 2% (w/v) and N-(1-naphthyl) ethylene-diamine 0.2% (w/v). The reaction was monitored at 540 nm. The absorbance was evaluated in a spectrophotometer (UV-1603; Shimadzu). The values are presented in micromoles per milligram of protein (μmol/mg protein).

Protein estimation

The protein concentration was determined by the Lowry method (Lowry et al. 1951) in a spectrophotometer (UV-1603; Shimadzu), using bovine serum albumin as a standard.

Statistical analysis

Statistical analysis of the data was accomplished by means of the Spss® statistical software package (SPSS Ibérica, Madrid, Spain). The Shapiro–Wilk test did not show a significant departure from normality in the distribution of variance values. To evaluate variations in data, a one-way analysis of variance (one-way anova) was corrected with the Bonferroni test. The level of statistical significance was set at p < 0.05. All results are expressed as mean ± SD.

Results

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Conclusion
  7. References

We did not find a statistically significant difference between the control, vehicle (DMSO 0.1%), mock (animals were placed in plastic cylindrical cages but did not receive TMS) or TMS alone groups, for the parameters analysed.

Changes in free radicals production markers and SDH activity in rats treated with 3-nitropropionic acid and untreated rats: effects of TMS

3-Nitropropionic acid produced a significant increase of protein carbonyl groups and lipid peroxidation product levels in the striatal nucleus synaptosomes (protein carbonylation, 1.33 ± 0.18 nmol/mg protein in the control group vs. 5.67 ± 0.51 nmol/mg protein in the 3-nitropropionic group, p < 0.001; lipid peroxidation products, 6.14 ± 0.65 nmol/mg protein in the control group vs. 25.23 ± 2.87 nmol/mg protein in the 3-nitropropionic group, p < 0.001; Fig. 1). Similarly, 3-nitropropionic acid triggered a reduction in GSH content (1.02 ± 0.13 nmol/mg protein in the control group vs. 0.20 ± 0.02 nmol/mg protein in the 3-nitropropionic group; p < 0.001; Fig. 2) and catalase and GSH–Px activities (catalase activity, 0.27 ± 0.03 U/mg protein in the control group vs. 0.01 ± 0.51 U/mg protein in the 3-nitropropionic group, p < 0.001; GSH–Px activity, 1.11 ± 0.12 U/mg protein in the control group vs. 0.83 ± 0.08 U/mg protein in the 3-nitropropionic group, p < 0.001; Fig. 2). Moreover, 3-nitropropionic acid produced a significant decrease in SDH activity in the striatum (40.63 ± 2.84 U/mg protein in the control group vs. 12.47 ± 0.90 U/mg protein in the 3-nitropropionic group; p < 0.001; Fig. 3). These effects were prevented and reversed by the administration of TMS (Figs 1–3).

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Figure 1. Changes in the levels of lipid peroxidation products (nanomoles of MDA + 4–HDA per milligram of protein; nmol/mg protein) and protein carbonylated levels (nanomoles per milligram of protein; nmol/mg protein) in the striatal synaptosomes of rats treated with 3-nitropropionic acid either alone or in combination with TMS. Values are means ± SD, n = 8 animals per group (ap < 0.001 vs. control; dp < 0.001 vs. 3-nitropropionic acid; hp < 0.01 vs. TMS + 3-nitropropionic acid). anova between groups: lipid peroxides, d.f. = 6, F = 435.281, p < 0.0001; protein carbonylation, d.f. = 6, F = 185.533, p < 0.0001.

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image

Figure 2. Changes in the GSH content (nanomoles per milligram of protein; nmol/mg protein) and GSH–Px (units per milligram of protein; U/mg protein) and catalase (units per milligram of protein; U/mg protein) activities in the striatal synaptosomes of rats treated with 3-nitropropionic acid either alone or in combination with TMS. Values are means ± SD, n = 8 animals per group (ap < 0.001 vs. control; bp < 0.01 vs. control; dp < 0.001 vs. 3-nitropropionic acid; gp < 0.001 vs. TMS + 3-nitropropionic acid; hp < 0.01 vs. TMS + 3-nitropropionic acid). anova between groups: GSH content, d.f. = 6, F = 208.765, p < 0.0001; GSH–Px activity, d.f. = 6, F = 10.036, p < 0.0001; catalase, d.f. = 6, F = 45.368, p < 0.0001.

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Figure 3. Changes in the succinate dehydrogenase activity in striatal synaptosomes. Values are means ± SD, n = 8 animals per group (ap < 0.001 vs. control; dp < 0.001 vs. 3-nitropropionic acid; gp < 0.001 vs. TMS + 3-nitropropionic acid). anova between groups: d.f. = 6, F = 752.140, p < 0.0001.

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This acid enhanced levels of hydroperoxides in homogenates throughout the entire brain (p < 0.001; Table 1), as well as NOx in striatal homogenates (1.09 ± 0.08 µmol/mg protein in the control group vs. 6.55 ± 0.43 µmol/mg protein in the 3-nitropropionic group; p < 0.001; Fig. 4). Additionally, 3-nitropropionic acid decreased TRAP levels (p < 0.001; Table 1).

Table 1.  TMS effects on changes in the hydroperoxide levels and TRAP content induced by 3-nitropropionic acid on the whole brain
 Hydroperoxides nmol ROS/mg proteinTRAP nmol Trolox/mg protein
  1. Values are means ± SEM, n = 8 animals per group. anova between groups: hydroperoxides, d.f. = 6, F = 9.672, p < 0.0001; TRAP, d.f. = 6, F = 41.621, p < 0.0001. ap < 0.001 vs. control; dp < 0.001 vs. 3-nitropropionic acid; fp < 0.05 vs. 3-nitropropionic acid.

Control85.19 ± 0.938.92 ± 2.91
TMS84.07 ± 2.987.79 ± 1.12
Mock83.78 ± 3.888.00 ± 2.75
3-Nitropropionic acid90.55 ± 1.04a3.94 ± 0.40a
3-Nitropropionic acid + mock89.73 ± 1.974.01 ± 0.53
TMS + 3-nitropropionic acid86.03 ± 0.90d5.93 ± 0.44d
3-Nitropropionic acid + TMS84.94 ± 3.84f6.40 ± 1.07d
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Figure 4. Effects of 3-nitropropionic acid and TMS on total nitrites (NOx) levels in striatal homogenates. Values are means ± SD, n = 8 animals per group (ap < 0.001 vs. control; dp < 0.001 vs. 3-nitropropionic acid; gp < 0.001 vs. TMS + 3-nitropropionic acid). anova between groups: d.f. = 6, F = 407.910, p < 0.0001.

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Cytoprotection by TMS against cell loss induced by 3-nitropropionic acid in rats

At low magnification a decrease in neuronal density was evident in the 3-nitropropionic acid group compared with the other groups (Fig. 5). Healthy neurones stained with cresyl violet stain were robust in shape and had a pale and spherical or slightly oval nucleus and a single large nucleolus (Fig. 6). The cytoplasm of the neurones could also be seen clearly. In the other groups, pyknotic neurones were observed, although their number varied depending on the group (Fig. 6).

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Figure 5. Low-magnification views of coronal sections stained with cresyl-fast violet of four groups. Note the low neuronal density in panel (b). (a) Control group; (b) 3-nitropropionic group; (c) TMS + 3-nitropropionic group; (d) 3-nitropropionic acid + TMS group. Scale bars: 250 µm.

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Figure 6. Sections stained with cresyl-fast-violet. The arrows indicate normal (thick arrow) and pyknotic (thin arrow) neurones. Panels (a) and (b) show the control group (the insert shows higher magnification of the neuronal cells with their nucleoli). Panels (c) and (d) show the 3-nitropropionic group (the insert shows higher magnification of degenerating neurones showing cytoplasmic vacuolation and pyknotic nuclei). Panels (e) and (f) show the TMS + 3-nitropropionic group. Panels (g) and (h) show the 3-nitropropionic + TMS group. Scale bar = 40 μm in (a), (c), (e) and (g). Scale bar = 25 μm in (b), (d), (f) and (h); scale bars in the inserts = 10 μm.

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Neuronal degeneration may present a continuum of neurodegenerative morphologies. In this study, we found pyknotic neurones (Fig. 6) that were stained darkly, no nucleus or nucleolus was visible, and the cells were shrunken and sickle- or raisin-shaped. Other neurones showed a smaller nucleus with condensed chromatin and swollen cytoplasm. Additionally, neuropil vacuolization and reactive gliosis were observed in 3-nitropropionic acid (Fig. 6).

Cell loss was determined by neuronal density in the striatum (Figs 6 and 7). 3-Nitropropionic acid decreased the amount of neurones in the striatum by 41.33% (Figs 6 and 7) compared with the control group (76.45 ± 2.36 total number of cells per area in the control group vs. 44.85 ± 6.71 total number of cells per area in the 3-nitropropionic group; p < 0.001). The administration of TMS prevented or reversed the cell loss induced by 3-nitropropionic acid (62.10 ± 7.68 total number of cells per area in the TMS + 3-nitropropionic acid group and 56.05 ± 8.55 total number of cells in the 3-nitropropionic acid + TMS vs. 44.85 ± 6.71 total number of cells per area in the 3-nitropropionic group; p < 0.001; Figs 6 and 7). Additionally, the ratio of healthy cells to pyknotic cells (HC : PC) was lower in the 3-nitropropionic group than the control group (HC : PC, 0.47 in the 3-nitropropionic group vs. 190.25 in the control group). When the rats were treated with TMS, the proportion between cells was greater in the TMS + 3-nitorpropionic acid and 3-nitropropionic acid + TMS groups than in the 3-nitropropionic group (HC : PC, 3.24 in the TMS + 3-nitropropionic group and 3.05 in the 3-nitropropionic acid + TMS group vs. 0.47 in the 3-nitropropionic acid group; Fig. 7).

image

Figure 7. Effects of TMS on neuronal cell loss and degeneration in the striatum of rats either treated or not with 3-nitropropionic acid. Values are means ± SD of the number of cells present in five striatal areas of four brains, n = 4 animals per group (ap < 0.001 vs. control; bp < 0.001 vs. 3-nitropropionic acid). anova between groups: total neurones/area, d.f. = 4, F = 53.551, p < 0.0001; normal neurones/area, d.f. = 4, F = 153.878, p < 0.0001; degenerated neurones/area, d.f. = 4, F = 119.845, p < 0.0001.

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The LDH levels were used as necrosis markers (Table 2). 3-Nitropropionic acid administration significantly enhanced LDH content in rats (p < 0.001; Fig. 5 and Table 2). This effect was either partially prevented or reversed by the administration of TMS (p < 0.001; Fig. 6, Table 2).

Table 2.  TMS effects on changes in the LDH activity induced by 3-nitropropionic acid on the striatum
 Plasma LDH U/LStriatal LDH U/mg protein
  1. Values are means ± SEM, n = 8 animals per group. anova between groups: brain LDH, d.f. = 6, F = 5.023, p < 0.0001; plasma LDH, d.f. = 6, F = 42.553, p < 0.0001. ap < 0.001 vs. control; dp < 0.001 vs. 3-nitropropionic acid.

Control50.88 ± 1.055.12 ± 0.38
TMS50.72 ± 2.954.98 ± 0.15
Mock51.59 ± 1.074.93 ± 0.27
3-Nitropropionic acid62.85 ± 0.96a7.87 ± 0.72a
3-Nitropropionic acid + mock63.01 ± 1.027.79 ± 0.92
TMS + 3-nitropropionic acid47.57 ± 1.95d4.80 ± 0.46d
3-Nitropropionic acid + TMS48.48 ± 0.56d5.37 ± 0.28d

Discussion

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Conclusion
  7. References

The most important finding of the present study is that the TMS (60 Hz and 0.7 mT) can prevent and reverse the process of neuronal degeneration and oxidative stress triggered by 3-nitropropionic acid administration. Although there is evidence indicating that TMS has a protective effect in neuropsychiatry disorders (Sandyk 1999; Fujiki et al. 2003; Arias-Carrión et al. 2004; Calvo-Merino et al. 2004; Di Lazzaro et al. 2004; Fregni et al. 2004; Hajak et al. 2004; Lefaucheur et al. 2004; Miniussi et al. 2005), we must acknowledge that the scientific evidence regarding this point is controversial. Thus, the results change according to the experimental model used, target organ, animal age and pattern intensity, time and type of exposure (Post et al. 1999; Arafa et al. 2003; Koyama et al. 2004; Lai and Singh 2004; Lee et al. 2004).

Effects of TMS on free radical production and reduction of SDH activity induced by 3-nitropropionic acid

It is well known that free radicals and other reactive species are involved in various physiological and pathological conditions, namely, the ageing process and Alzheimer's, Parkinson's and Huntington's disease, etc. (Marlatt et al. 2004).

Our results show that greater oxidative stress occurred when the male Wistar rats were treated with 3-nitropropionic acid, which is in line with the findings of other authors who have revealed the significant oxidative damage of 3-nitropropionic acid (Binienda 2003; Montilla et al. 2004; Rosenstock et al. 2004; Túnez et al. 2004a,b, 2005; Mandavilli et al. 2005). Additionally, we reveal a reduction in MDA + 4–HDA levels, protein carbonylation content and hydroperoxide production, together with increased GSH content, TRAP values and antioxidative enzyme activities prompted by TMS. These data demonstrate the protective effect of TMS, preventing or reversing oxidative stress prompted by 3-nitropropionic acid, and supporting the important role played by oxygen radicals in neurodegenerative diseases, cell damage, loss and survival, and cell differentiation and proliferation (Garcia et al. 2001; Prolla and Mattson 2001; Lee et al. 2004).

3-Nitropropionic acid triggered an increase in NOx levels in striatal homogenates. These results concur with other reports, which found that the neurotoxin also increased levels of NO in the transgenic mouse model of Huntington's disease, and that it induces NOx (Pérez-Severino et al. 2002). TMS, on the other hand, prevented and reversed the effects of 3-nitropropionic acid on NOx values. Vardimon et al. (1999) suggest a connection between oxidative stress and the impact on transcription factors, and also that differentiation itself induces proteins that quench reactive oxygen species. In addition, Pérez-Severino et al. (2002) and Sun et al. (2005) have shown that reductions in the NO levels by the inhibition of nitric oxide synthase (NOS) are correlated with increases in neurogenesis.

Previous reports have shown that NO is a diffusible intercellular messenger with multiple functions in the cardiovascular, immunological and nervous systems (Moncada et al. 1991). In the brain, NO has an anti-proliferative action (Estrada et al. 1997), mitochondrial disruption (Cassina and Radi 1996) and striatal oxidative damage (Pérez-Severino et al. 2002). These effects may be caused by: (i) prolonged action of poly-(ADP-ribose)-polymerase (PARP; Zhang et al. 1994); (ii) disruption of calcium homeostasis (Brorson et al. 1997); and (iii) inhibition of neuronal energy production (Brorson et al. 1999). Although it is not intrinsically unstable, NO avidly combines with the superoxide anion (O2) to form peroxynitrite (ONOO), which is a highly reactive free radical, and has been shown to mediate much of the neurotoxicity of NO (Bolaños et al. 1995). In addition, other research projects demonstrate that peroxynitrite causes significant reductions in the activities of NADH dehydrogenase (complex I) and succinate dehydrogenase (complex II) (Vatassery et al. 2004). These data are in line with our results, which showed that 3-nitropropionic acid reduced SDH activity in the brain. Moreover, TMS administration blocked the effects caused by 3-nitropropionic acid on SDH activity. This effect is confirmed by other authors who found that magnetic fields enhance the activity of SDH in liver tissue and lymphocytes (Gorczynska et al. 1986; Pop et al. 1989; Gorczynska and Wegrzynowicz 1991; Temur'iants and Shekhotkin 1995). Collectively, the data indicate that: (i) TMS might regulate SDH activity by its effect on the oxidative balance, a phenomenon that could be a result of the protective effect of TMS on cell loss and damage induced by 3-nitropropionic acid; and (ii) mitochondria and free radicals play an important role in Huntington's disease and in our experimental model. Additionally, this effect might be an important finding given that SDH activity is consistently found to be lower in the striatum of patients suffering from Huntington's disease (Gu et al. 1996; Tabrizi et al. 1999).

TMS effects on brain cell loss and neuronal degeneration induced by 3-nitropropionic acid

We have found that 3-nitropropionic acid prompted a decrease in neuronal density and morphological changes characteristic of neuronal degeneration and a reduction in cell intensity, as well as increases of LDH content in the brain and plasma. The reduction in the cell intensity is indicative of cell loss through neuronal degeneration that triggers the death of neurones. In addition, the values obtained for LDH and the morphological changes seem to indicate that cell death occurs through necrosis in our experimental model. These data are in line with studies that suggest that 3-nitropropionic acid enhances vulnerability and cell death in the brain through mitochondrial dysfunction (Vis et al. 2002), in addition to leading to decreased ATP synthesis and an increased production of free radicals able to induce cell death (McCracken et al. 2001). Additionally, Vis et al. (2004) have found increases of LDH activity in the culture medium in response to increasing concentrations of 3-nitropropionic acid exposure, as well as dose-dependent neuronal loss induced by this mycotoxin, and an increasing number of Nissl-stained cells with either nuclear condensation or fragmentation, pointing to overall cell death and predominantly necrotic cell death. Those data concur with our own findings in this study. Furthermore, in our experimental model, the administration of TMS prevented and reversed the cell loss and degeneration induced by 3-nitropropionic acid administration returning the neuronal density to a state of normality.

Conclusion

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Conclusion
  7. References

Our findings suggested that: (i) TMS decreases oxidative and nitrosative stress and (ii) TMS reduces cell loss. In short, TMS reveals two possible actions: (i) it either protects against the effect of 3-nitropropionic acid or (ii) it avoids the 3-nitropropionic acid effect.

These results indicate the neuroprotective effect of TMS because they show the following:

  • (i)
    anti-oxidative action characterized by stimulation, at least, of the activity of anti-oxidative enzymatic systems, an increase in GSH content, decrease in lipid peroxidation products and protein carbonylation and a reduction of NO levels;
  • (ii)
    an increase in SDH activity;
  • (iii)
    a reduction of cellular loss and damage.

Viewed collectively, it seems to indicate that the effects observed on the quantified biomarkers of oxidative stress, as well as the effect on SDH activity, could be partly the result of the maintenance of cellular integrity induced by TMS. Additionally, these events would explain the differences observed between administration of TMS either before or after the lesion. Thus, decreased cellular loss would be accompanied by a lesser degree of oxidative stress and increased SDH activity. Therefore, it is probable that the observed differences can be due to the degree existing damage at the moment for beginning the treatment with TMS (the beginning of the TMS administration in TMS + 3-nitropropionic acid group agrees with the non-existance of cell damage, whereas the beginning of treatment with TMS in 3-nitropropionic acid + TMS group happens with the maximum degree of cell damage).

These findings demonstrate the beneficial effect of TMS and its possible interest for therapeutic strategies, particularly in the development of a neuroprotective therapy, as it can be used as an exogenous agent to increase the survival of neuronal cells. This would slow down, reduce and improve the cell loss that occurs in neurodegenerative diseases, especially Huntington's disease, thus helping clinical improvement. However, further research is required to evaluate the mechanisms involved in the properties of TMS, as well as its possible therapeutic potential.

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