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Manual acupuncture improves parameters associated with oxidative stress and inflammation in PTZ-induced kindling in mice
Accepted Manuscript Title: Manual acupuncture improves parameters associated with oxidative stress and inflammation in PTZ-induced kindling in mice Authors: Alexsandro Lu´ıs Frantz, Gabriela Gregory Regner, Pricila Pfluger, ¨ Vanessa Rodrigues Coelho, Lucas Lima da Silva, Cassiana Macagnan Viau, Marcele Silva de Souza, Juliana Bondan da Silva, Jaqueline Nascimento Picada, Jenifer Saffi, Patr´ıcia Pereira PII: DOI: Reference:
S0304-3940(17)30786-3 http://dx.doi.org/10.1016/j.neulet.2017.09.044 NSL 33122
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
28-4-2017 21-9-2017 22-9-2017
Please cite this article as: Alexsandro Lu´ıs Frantz, Gabriela Gregory Regner, Pricila Pfluger, ¨ Vanessa Rodrigues Coelho, Lucas Lima da Silva, Cassiana Macagnan Viau, Marcele Silva de Souza, Juliana Bondan da Silva, Jaqueline Nascimento Picada, Jenifer Saffi, Patr´ıcia Pereira, Manual acupuncture improves parameters associated with oxidative stress and inflammation in PTZ-induced kindling in mice, Neuroscience Lettershttp://dx.doi.org/10.1016/j.neulet.2017.09.044 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Manual acupuncture improves parameters associated with oxidative stress and inflammation in PTZ-induced kindling in mice Alexsandro Luís Frantz1, Gabriela Gregory Regner1, Pricila Pflüger1, Vanessa Rodrigues Coelho1, Lucas Lima da Silva1, Cassiana Macagnan Viau1, Marcele Silva de Souza2, Juliana Bondan da Silva3, Jaqueline Nascimento Picada3, Jenifer Saffi2, Patrícia Pereira1 * 1
Laboratory of Neuropharmacology and Preclinical Toxicology, Federal University of Rio
Grande do Sul, Porto Alegre, RS, Brazil 2
Department of Basic Health Science, Laboratory of Genetic Toxicology, UFCSPA, Porto
Alegre, RS, Brazil 3
Laboratory of Genetic Toxicology, Lutheran University of Brazil, Canoas, RS, Brazil,
*Corresponding author: Dr. Patrícia Pereira 1
Laboratory
of
Neuropharmacology
and
Preclinical
Toxicology,
Department
of
Pharmacology, Institute of Basic Health Sciences, Federal University of Rio Grande do Sul, Sarmento Leite 500/305, Porto Alegre, RS, CEP: 90.050-170, Brazil Tel/Fax: +55 51 33083121; e-mail address: [email protected]
Highlights MAC/SAL/PTZ and MAC/DZP0.15/PTZ groups decreased the formation of ROS MAC/SAL/PTZ and MAC/DZP0.15/PTZ groups decreased release of nitric oxide MAC/SAL/PTZ and MAC/DZP0.15/PTZ increased SOD and CAT activity MAC/SAL/PTZ and MAC/DZP0.15/PTZ reduced the expression of TNF-α Acupuncture had a protective effect against neuroinflammation and DNA damage
Abstract The use of acupuncture in the treatment of central nervous system (CNS) disorders is an ageold practice. Although only a few studies have proved its efficacy, evidence has indicated the use of acupuncture to treat different types of seizures. Therefore, the present study aimed to evaluate the effect of manual acupuncture (MAC) using the chemical kindling model. The role of MAC in oxidative stress and inflammation after pentylenetetrazole (PTZ)-induced kindling was investigated by measuring reactive oxygen species (ROS) production,
[1]
superoxide dismutase (SOD), and catalase (CAT) activities, nitrite content, and deoxyribonucleic acid (DNA) damage in cerebral cortex. Mice received PTZ (60 mg/kg s.c.) once every three days for 16 days, totaling six treatments. MAC was applied at acupoint GV20 daily during the entire experimental protocol. Diazepam (DZP) (2 mg/kg) was used as positive control. Also, we evaluated the MAC effect associated with DZP (MAC/DZP) at a low dose (0.15 mg/kg). The results demonstrated that MAC or MAC/DZP were not able to reduce significantly seizure occurrence or to increase the latency to the first seizure during treatment. MAC/DZP promoted a difference in the first latency to seizure only on the third day. PTZ-induced kindling caused significant neuronal injury, oxidative stress, increased DNA damage, nitric oxide production, and expression of the pro-inflammatory Tumor Necrosis Factor-α (TNF-α). These effects were reversed by treatment with MAC or MAC/DZP. These results indicated that the stimulation of acupoint GV20 by MAC showed no potential antiepileptogenic effect in the model used, although it greatly promoted neuronal protection, which may result from antioxidant and anti-inflammatory effects observed here.
Keywords:
DNA
damage,
Kindling,
Manual
acupuncture,
Oxidative
stress,
Pentylenetetrazole, TNF-α.
Introduction Epilepsy is a chronic neurological disorder characterized by recurrent seizures, and it is estimated to affect approximately 50 million people worldwide. Globally, close to 2.4 million people are diagnosed with epilepsy each year [1]. Epilepsy has detrimental effects on patient health and quality of life, placing great psychological and economic burdens on the family members [2]. Despite the increasing number of new antiepileptic drugs (AEDs) with better pharmacokinetic, safety, and tolerability profiles introduced over the past 20 years, such advancement has not had a substantial effect on seizure remission for patients with drugresistant epilepsy [3, 4, 5, 6]. Nowadays, complementary and alternative medicine such as acupuncture (AC) has sparked considerable interest concerning the treatment of several diseases, including epilepsy [7]. Despite the widespread use of AC for pain management, explaining the relationship between AC’s clinical effects and its neurobiological mechanisms is still a challenge to modern science [8, 9, 10]. Several studies have shown that AC modulates the endogenous analgesic pathways by activating the opioid, serotonergic, and noradrenergic systems [11, 12]. Besides, different AC modalities are being investigating in experimental models in rodents [13]. These studies [2]
have shown that AC improves neuronal function, reduces ROS generation, increases superoxide dismutase (SOD) activity, decreases malondialdehyde (MDA) levels, reduces expression of inflammatory cytokines including interleukin-1𝛽 (IL-1𝛽), interleukin-6 (IL-6), and tumor necrosis factor-𝛼 (TNF-𝛼), and improves parameters associated with epilepsy as the suppression of pilocarpine-induced epileptiform EEGs [6, 13]. Oxidative stress in the brain is caused by the imbalance between the generation and detoxification of reactive oxygen species (ROS) and nitrogen species (RNS), which damage brain cells and play important roles in brain inflammation, aging, and degeneration [14]. Excess free radical production is associated with injuries to cell structures, including lipid peroxidation, DNA damage, and enzyme inactivation. The central nervous system (CNS) becomes highly sensitive due to its high oxygen consumption and the low antioxidant defense activities, resulting in increased susceptibility to oxidative stress associated with several CNS disorders including epilepsy [15]. Thus, the goal of this study was to determine the effect of manual acupuncture (MAC) on seizures using a pentylenetetrazole (PTZ)-induced kindling model in mice. In addition, the modulation of oxidative stress parameters, nitric oxide (NO) production, and DNA damage by MAC was evaluated. The association of MAC and diazepam (DZP), a therapeutically useful anticonvulsant, was also investigated in this study.
Material and Methods Animals Seventy-two male CF-1 mice (2-3 months of age, 30-40 g) obtained from the breading colony of the Federal University of Rio Grande do Sul (UFRGS) were used in this work. Animals were housed in plastic cages (5 per cage), with water and food ad libitum, under a 12 h light/dark cycle (lights on at 7:00 AM), and at a constant temperature of 23 ± 2°C. All experimental procedures were carried out in accordance to the national and international legislation (Guidelines of Brazilian Council of Animal Experimentation – CONCEA – and EU Directive 2010/63/EU for animal experiments), with the approval of the Committee on the Ethical Use of Animals of UFRGS (authorization number 28514). All protocols were designed aiming to reduce the number of animals used to a minimum, as well as to minimize their suffering. Mice were assigned randomly into eight groups.
Pharmacological agents
[3]
PTZ was purchased from Sigma-Aldrich Co. (St. Louis, MO, U.S.A.). DZP (Compaz®) was purchased from Cristália Produtos Químicos e Farmacêuticos Ltda and was used as the reference anticonvulsant drug for comparison (positive control). PTZ and DZP were dissolved in saline solution/NaCl 0.9% (SAL) and administrated subcutaneously (s.c.) and intraperitoneally (i.p.) respectively at a volume of 10 mL/kg body weight. Primary antibodies (anti-TNF-α [Sc52746] and anti-β-actin [Sc69879]), and secondary antibodies were obtained from Santa Cruz Biotechnology (California, USA).
Experimental protocol and MAC treatment PTZ-induced kindling model was carried out as described by De Oliveira et al. [16] with minor modifications. The severity of seizures was observed by the adapted Racine scale (0, no convulsive behavior; 1, myoclonic jerks; 2, bilateral forelimb clonus lasting less than 3 sec; 3, bilateral forelimb clonus lasting more than 3 sec; 4, tonic-clonic seizure; 5, death). The PTZ dose used was 60 mg/kg s.c., obtained through a pilot test. This convulsant agent was administered every three days, totaling six treatments (16 days). Mice were allocated into eight groups that received MAC or not. Animals given the MAC treatment were divided in three groups and received only SAL (MAC/SAL/SAL), SAL and PTZ (MAC/SAL/PTZ) or DZP 0.15 mg/kg and PTZ (MAC/DZP0.15/PTZ). Animals that did not receive MAC were divided in four groups and received only SAL (SAL/SAL), SAL and PTZ (SAL/PTZ), DZP 2 mg/kg and PTZ (DZP2/PTZ) or DZP 0.15 mg/kg and PTZ (DZP0.15/PTZ). One group was called Sham/SAL/PTZ. A schematic diagram demonstrating the experimental design is show in the Figure 1. Animals treated with MAC received one application daily during a 16-day experimental protocol before the drug treatment. AC needles (Dong Bang Acupuncture Inc., Republic of Korea) were inserted to a depth of 3 mm at acupoint GV20 (Baihui) [17], located at the midmost point of the parietal bone, and turned at a rate of two spins per second for 15 s, for 10 min. Right after MAC treatment, animals were given intraperitoneal injections of DZP (0.15 mg/kg) or SAL, 30 min prior PTZ administration. The “nonacu” was also used to control the non-specific effects of AC stimulation (sham group). This point was set on the gluteus muscle, approximately 3 mm away from the lateral side of the tail, and was simulated concurrently with the application on the acupoint. Injections were given between 7 and 11 a.m. Immediately after PTZ injection, mice were placed individually in acrylic observation chambers for 30 min and behavioral seizure was observed. The occurrence of bilateral [4]
forelimb clonus as long as or lasting longer than 3 s (stage 3 in the adapted Racine scale) and the latency to the first seizure were used to compare the groups. DZP, a gamma-aminobutyric acid A receptors (GABAAR) agonist, was used in this study as a positive control (2 mg/kg). Also, a low DZP dose (0.15 mg/kg) was used to assess its association with MAC in the PTZ-induced kindling. DZP doses were determined according to pilot tests (dose-response).
Tissue sample On the last day of treatment, 30 min after PTZ administration, the animals were killed by decapitation and their brains were dissected. Frozen cortex from each treated mouse was homogenized in ice-cold phosphate buffer (KCl 140 mmol/L, phosphate (20 mmol/L, pH 7.4) and centrifuged at 12,000 rpm for 10 min. The supernatant was used for biochemical measurements. We used three samples, each one from each different animal, for all biochemical measurements. Protein concentration was measured according to the method described by Lowry et al. [18] using serum bovine albumin as standard. Samples of peripheral blood, cortex, and hippocampus were collected for the genotoxicity assay and bone marrow extracted from both femurs was used in the micronucleus test. We used five samples, each one from each different animal, for the genotoxicity assay and the micronucleus test.
Free radical levels assay To investigate the regulatory effects of MAC on the generation of intracellular ROS, the samples were examined by monitoring the changes in fluorescence intensity of the probe, DCFH-DA. Homogenates were overlaid with 100 μL of 25 μM dichlorofluorescein diacetate (DCFDA) and were placed back in the incubator for additional 30 min at 37°C. At the end of the incubation period, plates were removed, and fluorescence of the homogenates was measured on a SpectraMax M2e Microplate Reader (Molecular Devices, MDS Analytical Technologies, Sunnyvale, California). The excitation/emission wavelengths for DCFDA were 480/520 nm. Values of relative fluorescence (RFU) were expressed as RFU mg−1 protein [19].
Superoxide dismutase activity (SOD) SOD catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide (H2O2). Because of this, it is an important antioxidant defense in most cells exposed to oxygen. SOD activity was evaluated by quantifying the inhibition of superoxide-dependent autoxidation of epinephrine, observing the absorbance of the samples at 480 nm [20]. Briefly, 170 µL of a [5]
mixture containing 50 mM glycine buffer pH 10.2 and 10 mM catalase was added to 20 µL of homogenate. After that, 10 µL of epinephrine were added and absorbance was immediately recorded every 30 s for 12 min at 480 nm in a SpectraMax M2e Microplate Reader (Molecular Devices, MDS Analytical Technologies, Sunnyvale, California). The inhibition of epinephrine autoxidation occurs in the presence of SOD, whose activity can be then indirectly assayed spectrophotometrically. One SOD unit is defined as the amount of SOD necessary to inhibit 50% of epinephrine autoxidation and the specific activity is reported as SOD Units/mg protein.
Catalase activity (CAT) CAT is an intracellular enzyme, found in most organisms, which breaks down H2O2 and is an important defense of the cells. CAT activity was assayed according to the method described by Chance and Machley [21], based on the disappearance of H2O2 at 240 nm. Briefly, 10 µL of homogenate was added to 180 µL of 20 mM potassium phosphate buffer pH 7.2. Subsequently, 10 µL of 5 mM H2O2 was added and absorbance was immediately recorded every 30 s for 10 min, using a SpectraMax M2e Microplate Reader (Molecular Devices, MDS Analytical Technologies, Sunnyvale, California). One CAT unit was defined as one µmol of hydrogen peroxide consumed per minute and the specific activity was calculated as CAT units/mg protein.
Alkaline comet assay The alkaline comet assay was used to detect recent DNA damage induced by the treatments, such as single- and double strand breaks, alkali-labile sites, and DNA–DNA and DNA– protein cross-links. The assay was performed as described in Tice et al. [22] with minor modifications [23]. Briefly, the total cortex and hippocampus were dissected from 5 animals per group and placed in 0.5 mL cold phosphate-buffered saline (PBS) and finely minced in order to obtain a cellular suspension. Blood samples were placed in 15 mL anticoagulant (heparin sodium 25,000 IU- Liquemine). These suspension cells from cortex, hippocampus, and peripheral blood were embedded in 95 μL 0.75% low melting point agarose (Gibco BRL). The mixture (cell/agarose) was spread on a fully frosted microscope slide coated with a layer of 300 μL normal melting agarose (1%) (Gibco BRL). After solidification, slides were placed in lysis buffer (2.5 M NaCl, 100 mM EDTA, and 10 mM Tris, freshly added 1% Triton X-100 (Sigma) and 10% DMSO, pH 10.0) for 48 h at 4°C. Subsequently, the slides were incubated in freshly made alkaline buffer (300 mM NaOH and 1 mM EDTA, pH > 13) [6]
for 20 min at 4 °C. An electric current of 300 mA and a voltage of 25 V (0.90 V/cm) was applied for 15 min to induce DNA electrophoresis. The slides were then neutralized (0.4 M Tris, pH 7.5), fixed, and stained with silver. Cells were analyzed in an optical microscopic based on the shape of 100 randomly selected cells (50 cells from each of two replicate slides) from each tissue per animal. The extent of DNA damage was evaluated classifying comets into five categories based on the length of migration and/or the perceived relative proportion of the DNA in the tail to the size of head (nucleus): 0 representing undamaged cells (comets with no tail) and 1–4 representing increasing relative tail intensities and smaller head size. Two parameters, damage index (DI) and damage frequency (DF), were used to evaluate DNA damage. The DI of a group could range from 0 (completely undamaged = 100 cells x 0) to 400 (maximum damage = 100 cells x 4). DF (%) was calculated for each sample based on the number of cells with tail versus those without tail [24].
Micronucleus test The micronucleus test was performed to measure possible increase in the genomic instability by clastogenicity (chromosome breakage) and aneugenicity (chromosome lagging due to dysfunction of the mitotic apparatus) induced by the treatments. This test was performed according to the US Environmental Protection Agency Gene-Tox Program [25]. Bone marrow extracted of both femurs from 5 animals per group was suspended in fetal calf serum and smeared on clean glass slides. Slides were air-dried, fixed in methanol, stained in 10% Giemsa and coded for a blind analysis. Cyclophosphamide at 50 mg/kg (a single dose i.p) was used as positive control and the animals of this group were euthanized 24 h after the injection. To avoid false negative results and to obtain a measure of toxicity on bone marrow, the polychromatic erythrocyte: normocromatic erythrocyte (PCE:NCE) ratio was scored in 1,000 cells. The incidence of a micronucleus (MN) was observed in 2,000 PCEs for each animal.
Measurement of Nitric oxide production The production of nitric oxide (NO) was estimated by measuring the amount of nitrite, a stable metabolite of NO [26]. Briefly, 100 µL of homogenate was mixed with 100 µL of the Griess reagent (1% sulfanilamide; 0.1% naphthylethylenediamine; 2.5% H3PO4) at environment temperature. After 20 min, absorbance was measured at 540 nm using SpectraMax M2e Microplate Reader (Molecular Devices, MDS Analytical Technologies, Sunnyvale, California). A nitrite calibration curve was used to convert absorbance to nitrite concentration in µM. [7]
TNF-Alpha Expression Frozen cortex from each treated mouse was homogenized in ice-cold buffer (Tris/acetate 20 mM, pH 7.5, sucrose, EDTA 1 mM, EGTA 1 mM, Triton X-100 1%, ortovanadate 1 mM, sodium glycerophosphate 1 mM, sodium fluoride 5 mM, sodium pyrophosphate 1 mM, βmercaptoethanol 5 mM, bezamidine 1 mM, PMSF 35 μg/mL, leupeptine 5 μg/mL). The samples were homogenized and incubated on ice for 20 min before being centrifuged at 12,000 g for 15 min. The supernatants were assayed for protein concentration. For the western-blot analysis, a 30-μg sample of total proteins was separated on SDSpolyacrylamide gel 15% and transferred to a polyvinylidene difluoride membrane. The membranes were blocked by incubation in TBS buffer containing Tween-20 0.1%, and dried milk 5% for 1 h at room temperature, and then washed with TBS buffer containing Tween-20 0.1%. Next, membranes were blotted overnight at 4°C with the primary antibodies (1/1,000) mentioned above. The blots were washed three times with TBS 0.1% Tween and developed with peroxidase-linked secondary antibodies (1/3,000). All blots were developed by ECL Western Blotting Detection Kit Reagent and detected using a ChemiDoc (Bio-Rad) imaging system [27].
Data analysis The results of PTZ-induced kindling, occurrence of clonic seizures, and latency time to its onset were analyzed using Fisher's exact probability test and the generalized estimating equations (GEE) followed by the post hoc Bonferroni test, respectively. All experiments were independently repeated at least three times, with triplicate samples for each treatment. Results are expressed as means ± standard deviation (SD). Data were analyzed by one-way analysis of variance (ANOVA), and means were compared using the Tukey test, and the P value of ≤ 0.05 considered as statistically significant. Data were analyzed using GraphPad Prism v.5 program (Intuitive Software for Science, San Diego, CA, U.S.A.) and Statistical Package for Social Sciences (SPSS, Chicago, USA) v.18.0.
Results This work aimed to evaluate the effect of MAC or its association with DZP on PTZ-induced kindling (MAC/DZP/PTZ). After the last treatment, 100% of the animals in the SAL/PTZ group (negative control) showed seizure behavior lasting at least 3 s. These results confirm
[8]
that the subconvulsive PTZ dose used in this study (60 mg/kg) was able to induce a “kindling state” in mice after six treatments (Figure 2A). The development of PTZ-kindling was only prevented by pretreatment with DZP 2 mg/kg (P ≤ 0.001). MAC was not able to change the convulsive behavior of the animals (occurrence of seizure or latency to the first bilateral forelimb clonus lasting more than 3 sec; Figure 2A and B). The group MAC/DZP0.15/PTZ showed a significant increase in the latency to first seizure only on the third and fourth days of treatment (Figure 2B), although it is possible to observe a behavior that differed from that of the negative control both in the percentage of seizure and latency in all the days of treatment. There is evidence that higher ROS levels may be produced in PTZ-induced seizures. Therefore, we monitored the changes in fluorescence intensity of DCFDA to assess intracellular ROS induced by PTZ seizure. The results indicated the suppressive effect of MAC/SAL/PTZ and MAC/DZP0.15/PTZ on ROS production, showing a significant difference from the control group SAL/PTZ (P ≤ 0.05; Figure 3). DZP 2 mg/kg was able to decrease ROS production significantly (DZP2/PTZ). On the other hand, DZP0.15/PTZ given alone did not change the level of DCF. In order to investigate whether the antioxidant properties of MAC were mediated by an increase in antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT) activities were
measured
(Figures
4
and
5).
The
treatment
with
MAC/SAL/PTZ
and
MAC/DZP0.15/PTZ increased SOD and CAT activities after PTZ-induced kindling in cerebral cortex when compared to the SAL/PTZ group (P ≤ 0.05). Both SOD and CAT activities were higher in the DZP2/PTZ, compared to negative control. DZP0.15/PTZ was not able to induce any change. As shown in Figure 6, MAC/SAL and MAC/DZP0.15/PTZ inhibited NO production (P < 0.05). NO production was lower in the group treated with DZP2/PTZ, while DZP0.15/PTZ was not able to produce change. The levels of TNF-α were increased by PTZ, as shown in Figure 7A-B (SAL/PTZ). The present results show that MAC significantly inhibited the pro-inflammatory factor (P ≤ 0.05), as well as MAC/DZP0.15/PTZ (P ≤ 0.01), suggesting its capacity to change this inflammatory parameter alone or in association with a benzodiazepine drug. The alkaline comet assay detects DNA strand breaks and alkali-labile sites. Our results showed that PTZ-induced seizures promoted significant DNA damage measured in cerebral cortex, hippocampus and peripheral blood (Table 1). Damage Index (DI) and Damage Frequency (DF) values for the MAC/SAL/SAL group were not statistically different from [9]
those exhibited by the SAL/SAL group. Similarly, Sham/SAL/PTZ and SAL/PTZ groups did not differ from each other in DI and DF. Thus, the results were compared to the SAL/SAL and SAL/PTZ groups. In hippocampus, all groups treated with PTZ showed increased DI in comparison to the SAL/SAL group, except for the MAC/DZP0.15/PTZ group, suggesting a trend of MAC in association with DZP to protect DNA against PTZ injuries in hippocampus (Table 1). When groups that received PTZ were compared to the SAL/PTZ group, no statistical difference was observed. In cerebral cortex, the DZP0.15/PTZ and DZP2/PTZ groups showed lower DI in comparison to SAL/PTZ, indicating the protective effect of DZP. All groups showed a significant difference in relation to the SAL/SAL group. In peripheral blood, there was an increase in the DI and DF values only for the SAL/PTZ and Sham/SAL/PTZ groups in comparison to the SAL/SAL group. The PTZ groups treated with MAC, DZP (0.15 and 2 mg/kg) or the association MAC/DZP0.15/PTZ were not able to decrease DNA damage significantly, in comparison to SAL/PTZ (P > 0.05; Table 1). There was no statistical difference in the ratio of PCE/NCE and micronucleus frequency among the groups. Only the cyclophosphamide positive control group showed increased micronucleus frequency in comparison to the SAL/SAL group (P ≤ 0.001; Table 2).
Discussion MAC’s ability to modulate seizures in a PTZ-induced kindling model was evaluated based on the beneficial effects of AC on oxidative stress and inflammatory parameters, as shown in a few previous studies [13, 28, 29]. Although MAC alone did not produce a less convulsive behavior, it was able to reduce the ROS production, increase SOD and CAT activities, and improve the inflammatory parameter measured after the PTZ-induced kindling. Likewise, studies have provided evidence that AC reduces oxidative stress and free radical formation in Parkinson’s disease in rodent models [30] and reverses the overproduction of ROS in vascular dementia model in rats, even suggesting an antioxidant property of AC [31]. Contributing to this evidence, researchers have shown that AC increased the activities of catalase, superoxide dismutase, and glutathione peroxidase in the hippocampus of alcoholic rats [32], also increasing the serum level of SOD in ischemic hypoxic neonate rats with cerebral palsy [33]. This is the first attempt to evaluate the protective effect of MAC on the PTZ-induced kindling model associated with oxidative stress and neuroinflammation parameters.
[10]
Recent studies have shown that inflammatory cytokine expressions were distinctively induced under various damaging conditions [34]. Contributing to neuronal cell death through a sustained inflammatory response, microglial cells are specialized macrophages with a role in the immune system, whereas astrocytes, the most numerous glial cells, induce inflammatory mediators such as interleukin-1β (IL-1 β) and tumor necrosis factor-α (TNF-α) when activated [35]. Here, PTZ administration increased cortical pro-inflammatory TNF-α level, known to participate in the induction and maintenance of epilepsy, as revealed using other epileptic models. Studies have demonstrated that the overexpression of TNF-α, among other inflammatory cytokines in mouse brains, is linked to spontaneous seizures, underscoring its crucial role in epilepsy. Hence, high levels of brain TNF-α in PTZ-kindled mice may be related to a pro-epileptogenic potential, possibly lowering the threshold of seizure induction and thus setting the basis for the onset of epilepsy [36]. In this scenario, our results showed that
MAC
(MAC/SAL/PTZ)
and
MAC
associated
with
a
low
DZP
dose
(MAC/DZP0.15/PTZ) significantly inhibited this pro-inflammatory factor. This result corroborates the findings of a previous study that showed that MAC applied in rats submitted to spinal cord injury was able to reduce expression of inflammatory cytokines, including IL1B, IL-6 and TNF-α [13]. Persistent seizures increase ROS, causing damage in macromolecules, including DNA [37, 38]. In this study, the increased DNA damage in brain tissues of the SAL/PTZ group might be associated with imbalance of the redox state caused by the seizures. In cerebral cortex, the treatment with DZP2/PTZ, which inhibited the seizures, decreased PTZ-induced DNA damage. Although MAC improved the antioxidant status, it was not able to decrease DNA damage in brain tissues significantly. The MAC/DZP0.15/PTZ treatment tended to decrease DNA damage in hippocampus, suggesting a protective effect from the association between MAC and DZP at low doses. Furthermore, there was an increase in DNA damage in peripheral blood in two kindling groups, SAL/PTZ and Sham/SAL/PTZ, in comparison to the SAL/SAL group, suggesting a systemic injury induced by PTZ. In order to evaluate if this DNA damage could lead to mutation, the micronucleus test was performed. As shown in Table 2, PTZ was not able to increase the micronucleus frequency in bone marrow. Concerning micronucleus frequency, DZP and MAC likewise did not induce significant increase which could represent risks to genomic stability. The main effect of most antiepileptic agents is to enhance the response to GABAA by facilitating the opening of gamma-aminobutyric acid (GABA)-activated chloride channels. [11]
Benzodiazepines increase the frequency of channel opening events, rising chloride ion conductance and inhibiting the action potential [39]. However, the clinical uses of benzodiazepines are limited by their side effects such as psychomotor impairment, sedation, myorelaxation, ataxia, amnesia, potentiation of other central depressant drugs, and dependence liability [40]. So, an alternative which can improve their efficacy is needed, decreasing undesirable effects and thus reducing tolerance and dependence. Unlike DZP, whose anticonvulsant mechanisms are well known, several studies have proposed different mechanisms to explain the effects of AC, including clinical evaluations that have demonstrated that AC produces positive effects on several types of epilepsy. Such beneficial effects include the absence of seizure as well of febrile convulsion, generalized clonic-tonic seizure, and even status epilepticus (SE). The overall therapeutic benefits include the improvement of electroencephalogram (e.g. the reduction of spike wave and desynchronization), of epileptic symptoms (e.g. decrease of the seizure frequency and shortness of attack episodes), of severity of SE, of functional recovery, and of life quality [41, 42, 43, 44]. Animal studies have demonstrated the antiepileptic effects of AC in varied experimental models. Chen et al. [45] observed that electroacupuncture (EA) produces nearly 27% reduction of PTZ-induced epileptiform neuronal activities of ventroposterior lateral thalamus of rats. EA has an anti-epileptic effect, which is related to its role in downregulating the increased hippocampal Ca2+ level in PTZ-kindled epilepsy in rats [46]. AC intervention has a protective effect on pyramidal cells of hippocampal CA 1 and CA 3 regions in epileptic rats, which is associated with the normal function of intracellular PI(3)K/Akt signaling pathway [17]. EA, when applied at head acupoints (e.g. GV20), prevents atrophy of some limbic structures and improves cognitive deficits in pilocarpine-epileptic rats. This effect has been shown to depend on the serotonergic system [47]. Apart from that, AC facilitates the release of neuropeptides, modulating gene expression in neuronal cells in the CNS, eliciting profound physiological effects and activating self-healing mechanisms. AC also enhances neurogenesis in several diseases through concomitant stimulation at ST36 and GV20, which improves hippocampus-related neuropathologies such as epilepsy and ischemia, inducing cell proliferation and neuroblast differentiation [48]. Here, the pretreatment with the combination of MAC and a low dose of DZP decreased the occurrence of seizures, increasing the latency to the first seizure, but not significantly. Yet, such result is still important if we consider the use of a lower dose of DZP, which could reduce the intensity of sedation or immobility. Combined treatment of MAC with DZP also [12]
decreases ROS levels and NO production, in addition to increasing SOD and CAT activities, tending to decrease DNA damage in brain tissue. DZP has distinct actions as positive allosteric modulator of GABAA receptors, although the mechanism underlying MAC effects should be elucidated. This combination mitigates neuroinflammation following PTZ-induced kindling model, as demonstrated by inhibition of TNF-α expression, maybe without the adverse side effects associated with high dose benzodiazepines. Consequently, a combination therapy including MAC and a benzodiazepine may become a novel therapeutic strategy in the attenuation of neurological symptoms in epileptogenesis. Conclusions Our findings reveal that the stimulation of acupoint GV20 by MAC did not decrease seizures induced by PTZ in the kindling model, but MAC alone or MAC in association with a low DZP dose demonstrated neuroprotective effects, improving brain antioxidant defenses, decreasing the expression of the pro-inflammatory TNF-α factor, and tending to decrease DNA damage in hippocampus after PTZ-induced kindling. Thus, MAC should be more thoroughly investigated in further studies focused on its potential use in epilepsy and other disorders associated with the CNS, or as a possible adjuvant in conventional pharmacotherapy.
Author’s contributions CMV, JS, and PP contributed to the experimental designs. ALF, GGR, PF, VRC, LLS, MSS, JBS, and CMV collected and analyzed the experimental data. JNP and PP were responsible for manuscript writing. All authors read and approved the final manuscript.
Ethics approval and consent to participate Not applicable.
Funding This work was supported by CNPq (grant N. 307064/2013-1) and UFRGS (N. 28514).
Availability of data and material Not applicable.
[13]
Competing interests The authors declare that they have no competing interests.
Consent for publication We accept the conditions of submission and the BioMed Central Copyright and License Agreement.
Acknowledgments We thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Federal University of Rio Grande do Sul (UFRGS) for supporting the development of this study.
[14]
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Figure legends
Figure 1. A schematic diagram illustrating the chronological events of the experiments.
[21]
Figure 2. The effect of MAC on PTZ-induced kindling in mice. (A) Percentage of animals presenting seizures (bilateral forelimb clonus lasting more than 3 sec) in the SAL/PTZ, DZP2/PTZ, DZP0.15/PTZ, MAC/SAL/PTZ, MAC/DZP0.15/PTZ, Sham/SAL/PTZ groups during repeated PTZ administration (60 mg/kg, s.c.). N = 9 animals per group (**P ≤ 0.01, and ***P ≤ 0.001; Fisher’s Exact Test, compared to the SAL/PTZ group). (B) Latency to the 1st bilateral forelimb clonus lasting more than 3 sec in mice submitted to PTZ-kindling. N = 9 animals per group (**P ≤ 0.01, and ***P ≤ 0.001, compared to the SAL/PTZ group). The comparisons between the six groups, according to the number of treatments, were analyzed by using the Generalized Estimation Equation (GEE). Bonferroni multiple comparisons were used to complete the evaluation Statistical calculations.
[22]
Figure 3. The effect of MAC on oxidative stress promoted by PTZ-induced kindling model in the cerebral cortex of mice. DCF was used to determine ROS generation in cells homogenates. Increasingly oxidizing extracellular conditions demonstrated a rise in DCF fluorescence, and are indicative of higher intracellular ROS levels. Values represent the mean ± SD of N = 3 animals per group. *P ≤ 0.05, ***P ≤ 0.001 compared to the SAL/PTZ group; #P ≤ 0.001 compared to the SAL/SAL group.
[23]
Figure 4. The effect of MAC on the activity of the antioxidant enzyme Superoxide dismutase (SOD) in the mice cerebral cortex. Values represent the mean ± SD of N = 3 animals per group. *P ≤ 0.05, ***P ≤ 0.001 compared to the SAL/PTZ group; #P ≤ 0.05 compared to the SAL/SAL group.
[24]
Figure 5. The effect of MAC on the activity of the antioxidant enzyme catalase in the mice cerebral cortex. Values represent the mean ± SD of N = 3 animals per group. *P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001 compared to the SAL/PTZ group; #P ≤ 0.01 compared to the SAL/SAL group.
Figure 6. The effect of MAC on NO production promoted by PTZ-induced kindling model in the cerebral cortex of mice. Values represent the mean ± SD of N = 3 animals per group. *P ≤ 0.05, **P ≤ 0.01 compared to the SAL/PTZ group; #P ≤ 0.05, compared to the SAL/SAL group.
[25]
Figure 7. The effect of MAC on the expression of TNF-α protein in the cerebral cortex of mice. The TNF-α protein was examined by western blot analysis. Values represent the mean ± SD of N = 3 animals per group. *P ≤ 0.05, **P ≤ 0.01 compared to the SAL/PTZ group; #P ≤ 0.01 compared to the SAl/SAL group.
[26]
Table 1. Effect of manual AC on DNA damage promoted by pentylenetetrazole (PTZ)-induced kindling model in mice. Hippocampus
Cortex
Peripheral blood
Group
DI a
DF b
DI a
DF b
DI a
DF b
SAL/SAL
66.6 ±10.8
66.0 ± 10.6
74.8 ± 13.6
73.8 ± 12.9
44.6 ± 6.9
44.6 ± 6.9
MAC SAL/SAL
69.2 ± 16.1
68.0 ± 15.7
50.4 ± 5.8
50.4 ± 5.8
42.4 ± 10.0
42.4 ± 10.0
Sal/PTZ
269.3 ± 46.0 ### 98.3 ± 1.2 ### 213.0 ± 40.8 ###
97.0 ± 2.8 ## 85.5 ± 18.9 # 78.5 ± 12.0 ##
Sham SAL/PTZ
237.3 ± 11.1 ### 98.7 ± 2.3 ### 196.0 ± 26.0 ###
96.0 ± 2.9 #
83.5 ± 11.8 # 78.5 ± 4.4 ##
MAC SAL/PTZ
194.5 ± 21.3 #
100.0 ± 0 ###
170.0 ± 17.9 ###
82.3 ±14.1
56.4 ± 8.6
55.4 ± 9.0
DZP 2.0 mg/kg/PTZ
183.2 ± 86.7 #
89.6 ± 9.6 ##
140.2 ± 39.6 #** 84.8 ± 10.4
79.4 ± 27.8
67.0 ± 15.2
DZP 0.15 mg/kg/PTZ
235.3 ± 82.7 ### 98.8 ± 1.5 ### 155.2 ± 33.1 ## * 89.4 ± 6.5
59.2 ± 25.1
54.0 ± 18.7
51.8 ± 16.0
51.8 ± 16.0
MAC DZP 0.15 mg/kg/PTZ 151.6 ± 39.2
95.8 ± 4.0 ### 186.5 ± 13.8 ###
93.0 ±8.9
N = 5 animals per group. aDI can range from 0 (completely undamaged, 100 cells × 0) to 400 (with maximum damage, 100 × 4). bDF was calculated based on number of cells with tail versus those with no tail. *P ≤ 0.05; **P ≤ 0.01 in comparison to SAL/PTZ group (ANOVA, Tukey test); #P ≤ 0.05;
##
P ≤ 0.01;
###
P ≤ 0.001 in comparison to SAL/SAL group (ANOVA,
Tukey
test).
[27]
Table 2. Mutagenic activity in bone marrow after PTZ-induced kindling model in mice. MNPCE ª in 2,000 PCE
Ratio PCE/NCE b
Mean ± SD
Mean ± SD
SAL/SAL
6.0 ± 1.2
1.8± 0.1
MAC Sal/Sal
8.0 ± 1.9
1.7± 0.2
SAL/PTZ
9.5 ± 1.0
1.5 ± 0.3
Sham SAL/PTZ
10.8 ± 2.6
1.5 ± 0.1
MAC SAL/PTZ
8.2 ± 2.7
1.6 ± 0.2
DZP 2.0 mg/kg/PTZ
8.3 ± 3.3
1.5± 0.5
DZP 0.15 mg/kg/PTZ
9.2 ± 2.3
1.4± 0.1
MAC DZP 0.15 mg/kg/PTZ
7.6 ± 1.8
1.5± 0.3
Cyclophosphamidec
29.2 ± 5.9 ###
1.3 ± 0.1
Group
a
MNPCE: micronucleus in polychromatic erythrocytes.
b c
Ratio PCE/NCE: ratio polychromatic erythrocytes/normochromatic erytrocytes.
Positive control (50 mg/kg single dose, i.p.).
N = 5 animals per group. ###
P ≤0.001: significant difference in comparison with the saline/saline group (ANOVA,
Tukey test).
[28]
S0304-3940(17)30786-3 http://dx.doi.org/10.1016/j.neulet.2017.09.044 NSL 33122
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
28-4-2017 21-9-2017 22-9-2017
Please cite this article as: Alexsandro Lu´ıs Frantz, Gabriela Gregory Regner, Pricila Pfluger, ¨ Vanessa Rodrigues Coelho, Lucas Lima da Silva, Cassiana Macagnan Viau, Marcele Silva de Souza, Juliana Bondan da Silva, Jaqueline Nascimento Picada, Jenifer Saffi, Patr´ıcia Pereira, Manual acupuncture improves parameters associated with oxidative stress and inflammation in PTZ-induced kindling in mice, Neuroscience Lettershttp://dx.doi.org/10.1016/j.neulet.2017.09.044 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Manual acupuncture improves parameters associated with oxidative stress and inflammation in PTZ-induced kindling in mice Alexsandro Luís Frantz1, Gabriela Gregory Regner1, Pricila Pflüger1, Vanessa Rodrigues Coelho1, Lucas Lima da Silva1, Cassiana Macagnan Viau1, Marcele Silva de Souza2, Juliana Bondan da Silva3, Jaqueline Nascimento Picada3, Jenifer Saffi2, Patrícia Pereira1 * 1
Laboratory of Neuropharmacology and Preclinical Toxicology, Federal University of Rio
Grande do Sul, Porto Alegre, RS, Brazil 2
Department of Basic Health Science, Laboratory of Genetic Toxicology, UFCSPA, Porto
Alegre, RS, Brazil 3
Laboratory of Genetic Toxicology, Lutheran University of Brazil, Canoas, RS, Brazil,
*Corresponding author: Dr. Patrícia Pereira 1
Laboratory
of
Neuropharmacology
and
Preclinical
Toxicology,
Department
of
Pharmacology, Institute of Basic Health Sciences, Federal University of Rio Grande do Sul, Sarmento Leite 500/305, Porto Alegre, RS, CEP: 90.050-170, Brazil Tel/Fax: +55 51 33083121; e-mail address: [email protected]
Highlights MAC/SAL/PTZ and MAC/DZP0.15/PTZ groups decreased the formation of ROS MAC/SAL/PTZ and MAC/DZP0.15/PTZ groups decreased release of nitric oxide MAC/SAL/PTZ and MAC/DZP0.15/PTZ increased SOD and CAT activity MAC/SAL/PTZ and MAC/DZP0.15/PTZ reduced the expression of TNF-α Acupuncture had a protective effect against neuroinflammation and DNA damage
Abstract The use of acupuncture in the treatment of central nervous system (CNS) disorders is an ageold practice. Although only a few studies have proved its efficacy, evidence has indicated the use of acupuncture to treat different types of seizures. Therefore, the present study aimed to evaluate the effect of manual acupuncture (MAC) using the chemical kindling model. The role of MAC in oxidative stress and inflammation after pentylenetetrazole (PTZ)-induced kindling was investigated by measuring reactive oxygen species (ROS) production,
[1]
superoxide dismutase (SOD), and catalase (CAT) activities, nitrite content, and deoxyribonucleic acid (DNA) damage in cerebral cortex. Mice received PTZ (60 mg/kg s.c.) once every three days for 16 days, totaling six treatments. MAC was applied at acupoint GV20 daily during the entire experimental protocol. Diazepam (DZP) (2 mg/kg) was used as positive control. Also, we evaluated the MAC effect associated with DZP (MAC/DZP) at a low dose (0.15 mg/kg). The results demonstrated that MAC or MAC/DZP were not able to reduce significantly seizure occurrence or to increase the latency to the first seizure during treatment. MAC/DZP promoted a difference in the first latency to seizure only on the third day. PTZ-induced kindling caused significant neuronal injury, oxidative stress, increased DNA damage, nitric oxide production, and expression of the pro-inflammatory Tumor Necrosis Factor-α (TNF-α). These effects were reversed by treatment with MAC or MAC/DZP. These results indicated that the stimulation of acupoint GV20 by MAC showed no potential antiepileptogenic effect in the model used, although it greatly promoted neuronal protection, which may result from antioxidant and anti-inflammatory effects observed here.
Keywords:
DNA
damage,
Kindling,
Manual
acupuncture,
Oxidative
stress,
Pentylenetetrazole, TNF-α.
Introduction Epilepsy is a chronic neurological disorder characterized by recurrent seizures, and it is estimated to affect approximately 50 million people worldwide. Globally, close to 2.4 million people are diagnosed with epilepsy each year [1]. Epilepsy has detrimental effects on patient health and quality of life, placing great psychological and economic burdens on the family members [2]. Despite the increasing number of new antiepileptic drugs (AEDs) with better pharmacokinetic, safety, and tolerability profiles introduced over the past 20 years, such advancement has not had a substantial effect on seizure remission for patients with drugresistant epilepsy [3, 4, 5, 6]. Nowadays, complementary and alternative medicine such as acupuncture (AC) has sparked considerable interest concerning the treatment of several diseases, including epilepsy [7]. Despite the widespread use of AC for pain management, explaining the relationship between AC’s clinical effects and its neurobiological mechanisms is still a challenge to modern science [8, 9, 10]. Several studies have shown that AC modulates the endogenous analgesic pathways by activating the opioid, serotonergic, and noradrenergic systems [11, 12]. Besides, different AC modalities are being investigating in experimental models in rodents [13]. These studies [2]
have shown that AC improves neuronal function, reduces ROS generation, increases superoxide dismutase (SOD) activity, decreases malondialdehyde (MDA) levels, reduces expression of inflammatory cytokines including interleukin-1𝛽 (IL-1𝛽), interleukin-6 (IL-6), and tumor necrosis factor-𝛼 (TNF-𝛼), and improves parameters associated with epilepsy as the suppression of pilocarpine-induced epileptiform EEGs [6, 13]. Oxidative stress in the brain is caused by the imbalance between the generation and detoxification of reactive oxygen species (ROS) and nitrogen species (RNS), which damage brain cells and play important roles in brain inflammation, aging, and degeneration [14]. Excess free radical production is associated with injuries to cell structures, including lipid peroxidation, DNA damage, and enzyme inactivation. The central nervous system (CNS) becomes highly sensitive due to its high oxygen consumption and the low antioxidant defense activities, resulting in increased susceptibility to oxidative stress associated with several CNS disorders including epilepsy [15]. Thus, the goal of this study was to determine the effect of manual acupuncture (MAC) on seizures using a pentylenetetrazole (PTZ)-induced kindling model in mice. In addition, the modulation of oxidative stress parameters, nitric oxide (NO) production, and DNA damage by MAC was evaluated. The association of MAC and diazepam (DZP), a therapeutically useful anticonvulsant, was also investigated in this study.
Material and Methods Animals Seventy-two male CF-1 mice (2-3 months of age, 30-40 g) obtained from the breading colony of the Federal University of Rio Grande do Sul (UFRGS) were used in this work. Animals were housed in plastic cages (5 per cage), with water and food ad libitum, under a 12 h light/dark cycle (lights on at 7:00 AM), and at a constant temperature of 23 ± 2°C. All experimental procedures were carried out in accordance to the national and international legislation (Guidelines of Brazilian Council of Animal Experimentation – CONCEA – and EU Directive 2010/63/EU for animal experiments), with the approval of the Committee on the Ethical Use of Animals of UFRGS (authorization number 28514). All protocols were designed aiming to reduce the number of animals used to a minimum, as well as to minimize their suffering. Mice were assigned randomly into eight groups.
Pharmacological agents
[3]
PTZ was purchased from Sigma-Aldrich Co. (St. Louis, MO, U.S.A.). DZP (Compaz®) was purchased from Cristália Produtos Químicos e Farmacêuticos Ltda and was used as the reference anticonvulsant drug for comparison (positive control). PTZ and DZP were dissolved in saline solution/NaCl 0.9% (SAL) and administrated subcutaneously (s.c.) and intraperitoneally (i.p.) respectively at a volume of 10 mL/kg body weight. Primary antibodies (anti-TNF-α [Sc52746] and anti-β-actin [Sc69879]), and secondary antibodies were obtained from Santa Cruz Biotechnology (California, USA).
Experimental protocol and MAC treatment PTZ-induced kindling model was carried out as described by De Oliveira et al. [16] with minor modifications. The severity of seizures was observed by the adapted Racine scale (0, no convulsive behavior; 1, myoclonic jerks; 2, bilateral forelimb clonus lasting less than 3 sec; 3, bilateral forelimb clonus lasting more than 3 sec; 4, tonic-clonic seizure; 5, death). The PTZ dose used was 60 mg/kg s.c., obtained through a pilot test. This convulsant agent was administered every three days, totaling six treatments (16 days). Mice were allocated into eight groups that received MAC or not. Animals given the MAC treatment were divided in three groups and received only SAL (MAC/SAL/SAL), SAL and PTZ (MAC/SAL/PTZ) or DZP 0.15 mg/kg and PTZ (MAC/DZP0.15/PTZ). Animals that did not receive MAC were divided in four groups and received only SAL (SAL/SAL), SAL and PTZ (SAL/PTZ), DZP 2 mg/kg and PTZ (DZP2/PTZ) or DZP 0.15 mg/kg and PTZ (DZP0.15/PTZ). One group was called Sham/SAL/PTZ. A schematic diagram demonstrating the experimental design is show in the Figure 1. Animals treated with MAC received one application daily during a 16-day experimental protocol before the drug treatment. AC needles (Dong Bang Acupuncture Inc., Republic of Korea) were inserted to a depth of 3 mm at acupoint GV20 (Baihui) [17], located at the midmost point of the parietal bone, and turned at a rate of two spins per second for 15 s, for 10 min. Right after MAC treatment, animals were given intraperitoneal injections of DZP (0.15 mg/kg) or SAL, 30 min prior PTZ administration. The “nonacu” was also used to control the non-specific effects of AC stimulation (sham group). This point was set on the gluteus muscle, approximately 3 mm away from the lateral side of the tail, and was simulated concurrently with the application on the acupoint. Injections were given between 7 and 11 a.m. Immediately after PTZ injection, mice were placed individually in acrylic observation chambers for 30 min and behavioral seizure was observed. The occurrence of bilateral [4]
forelimb clonus as long as or lasting longer than 3 s (stage 3 in the adapted Racine scale) and the latency to the first seizure were used to compare the groups. DZP, a gamma-aminobutyric acid A receptors (GABAAR) agonist, was used in this study as a positive control (2 mg/kg). Also, a low DZP dose (0.15 mg/kg) was used to assess its association with MAC in the PTZ-induced kindling. DZP doses were determined according to pilot tests (dose-response).
Tissue sample On the last day of treatment, 30 min after PTZ administration, the animals were killed by decapitation and their brains were dissected. Frozen cortex from each treated mouse was homogenized in ice-cold phosphate buffer (KCl 140 mmol/L, phosphate (20 mmol/L, pH 7.4) and centrifuged at 12,000 rpm for 10 min. The supernatant was used for biochemical measurements. We used three samples, each one from each different animal, for all biochemical measurements. Protein concentration was measured according to the method described by Lowry et al. [18] using serum bovine albumin as standard. Samples of peripheral blood, cortex, and hippocampus were collected for the genotoxicity assay and bone marrow extracted from both femurs was used in the micronucleus test. We used five samples, each one from each different animal, for the genotoxicity assay and the micronucleus test.
Free radical levels assay To investigate the regulatory effects of MAC on the generation of intracellular ROS, the samples were examined by monitoring the changes in fluorescence intensity of the probe, DCFH-DA. Homogenates were overlaid with 100 μL of 25 μM dichlorofluorescein diacetate (DCFDA) and were placed back in the incubator for additional 30 min at 37°C. At the end of the incubation period, plates were removed, and fluorescence of the homogenates was measured on a SpectraMax M2e Microplate Reader (Molecular Devices, MDS Analytical Technologies, Sunnyvale, California). The excitation/emission wavelengths for DCFDA were 480/520 nm. Values of relative fluorescence (RFU) were expressed as RFU mg−1 protein [19].
Superoxide dismutase activity (SOD) SOD catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide (H2O2). Because of this, it is an important antioxidant defense in most cells exposed to oxygen. SOD activity was evaluated by quantifying the inhibition of superoxide-dependent autoxidation of epinephrine, observing the absorbance of the samples at 480 nm [20]. Briefly, 170 µL of a [5]
mixture containing 50 mM glycine buffer pH 10.2 and 10 mM catalase was added to 20 µL of homogenate. After that, 10 µL of epinephrine were added and absorbance was immediately recorded every 30 s for 12 min at 480 nm in a SpectraMax M2e Microplate Reader (Molecular Devices, MDS Analytical Technologies, Sunnyvale, California). The inhibition of epinephrine autoxidation occurs in the presence of SOD, whose activity can be then indirectly assayed spectrophotometrically. One SOD unit is defined as the amount of SOD necessary to inhibit 50% of epinephrine autoxidation and the specific activity is reported as SOD Units/mg protein.
Catalase activity (CAT) CAT is an intracellular enzyme, found in most organisms, which breaks down H2O2 and is an important defense of the cells. CAT activity was assayed according to the method described by Chance and Machley [21], based on the disappearance of H2O2 at 240 nm. Briefly, 10 µL of homogenate was added to 180 µL of 20 mM potassium phosphate buffer pH 7.2. Subsequently, 10 µL of 5 mM H2O2 was added and absorbance was immediately recorded every 30 s for 10 min, using a SpectraMax M2e Microplate Reader (Molecular Devices, MDS Analytical Technologies, Sunnyvale, California). One CAT unit was defined as one µmol of hydrogen peroxide consumed per minute and the specific activity was calculated as CAT units/mg protein.
Alkaline comet assay The alkaline comet assay was used to detect recent DNA damage induced by the treatments, such as single- and double strand breaks, alkali-labile sites, and DNA–DNA and DNA– protein cross-links. The assay was performed as described in Tice et al. [22] with minor modifications [23]. Briefly, the total cortex and hippocampus were dissected from 5 animals per group and placed in 0.5 mL cold phosphate-buffered saline (PBS) and finely minced in order to obtain a cellular suspension. Blood samples were placed in 15 mL anticoagulant (heparin sodium 25,000 IU- Liquemine). These suspension cells from cortex, hippocampus, and peripheral blood were embedded in 95 μL 0.75% low melting point agarose (Gibco BRL). The mixture (cell/agarose) was spread on a fully frosted microscope slide coated with a layer of 300 μL normal melting agarose (1%) (Gibco BRL). After solidification, slides were placed in lysis buffer (2.5 M NaCl, 100 mM EDTA, and 10 mM Tris, freshly added 1% Triton X-100 (Sigma) and 10% DMSO, pH 10.0) for 48 h at 4°C. Subsequently, the slides were incubated in freshly made alkaline buffer (300 mM NaOH and 1 mM EDTA, pH > 13) [6]
for 20 min at 4 °C. An electric current of 300 mA and a voltage of 25 V (0.90 V/cm) was applied for 15 min to induce DNA electrophoresis. The slides were then neutralized (0.4 M Tris, pH 7.5), fixed, and stained with silver. Cells were analyzed in an optical microscopic based on the shape of 100 randomly selected cells (50 cells from each of two replicate slides) from each tissue per animal. The extent of DNA damage was evaluated classifying comets into five categories based on the length of migration and/or the perceived relative proportion of the DNA in the tail to the size of head (nucleus): 0 representing undamaged cells (comets with no tail) and 1–4 representing increasing relative tail intensities and smaller head size. Two parameters, damage index (DI) and damage frequency (DF), were used to evaluate DNA damage. The DI of a group could range from 0 (completely undamaged = 100 cells x 0) to 400 (maximum damage = 100 cells x 4). DF (%) was calculated for each sample based on the number of cells with tail versus those without tail [24].
Micronucleus test The micronucleus test was performed to measure possible increase in the genomic instability by clastogenicity (chromosome breakage) and aneugenicity (chromosome lagging due to dysfunction of the mitotic apparatus) induced by the treatments. This test was performed according to the US Environmental Protection Agency Gene-Tox Program [25]. Bone marrow extracted of both femurs from 5 animals per group was suspended in fetal calf serum and smeared on clean glass slides. Slides were air-dried, fixed in methanol, stained in 10% Giemsa and coded for a blind analysis. Cyclophosphamide at 50 mg/kg (a single dose i.p) was used as positive control and the animals of this group were euthanized 24 h after the injection. To avoid false negative results and to obtain a measure of toxicity on bone marrow, the polychromatic erythrocyte: normocromatic erythrocyte (PCE:NCE) ratio was scored in 1,000 cells. The incidence of a micronucleus (MN) was observed in 2,000 PCEs for each animal.
Measurement of Nitric oxide production The production of nitric oxide (NO) was estimated by measuring the amount of nitrite, a stable metabolite of NO [26]. Briefly, 100 µL of homogenate was mixed with 100 µL of the Griess reagent (1% sulfanilamide; 0.1% naphthylethylenediamine; 2.5% H3PO4) at environment temperature. After 20 min, absorbance was measured at 540 nm using SpectraMax M2e Microplate Reader (Molecular Devices, MDS Analytical Technologies, Sunnyvale, California). A nitrite calibration curve was used to convert absorbance to nitrite concentration in µM. [7]
TNF-Alpha Expression Frozen cortex from each treated mouse was homogenized in ice-cold buffer (Tris/acetate 20 mM, pH 7.5, sucrose, EDTA 1 mM, EGTA 1 mM, Triton X-100 1%, ortovanadate 1 mM, sodium glycerophosphate 1 mM, sodium fluoride 5 mM, sodium pyrophosphate 1 mM, βmercaptoethanol 5 mM, bezamidine 1 mM, PMSF 35 μg/mL, leupeptine 5 μg/mL). The samples were homogenized and incubated on ice for 20 min before being centrifuged at 12,000 g for 15 min. The supernatants were assayed for protein concentration. For the western-blot analysis, a 30-μg sample of total proteins was separated on SDSpolyacrylamide gel 15% and transferred to a polyvinylidene difluoride membrane. The membranes were blocked by incubation in TBS buffer containing Tween-20 0.1%, and dried milk 5% for 1 h at room temperature, and then washed with TBS buffer containing Tween-20 0.1%. Next, membranes were blotted overnight at 4°C with the primary antibodies (1/1,000) mentioned above. The blots were washed three times with TBS 0.1% Tween and developed with peroxidase-linked secondary antibodies (1/3,000). All blots were developed by ECL Western Blotting Detection Kit Reagent and detected using a ChemiDoc (Bio-Rad) imaging system [27].
Data analysis The results of PTZ-induced kindling, occurrence of clonic seizures, and latency time to its onset were analyzed using Fisher's exact probability test and the generalized estimating equations (GEE) followed by the post hoc Bonferroni test, respectively. All experiments were independently repeated at least three times, with triplicate samples for each treatment. Results are expressed as means ± standard deviation (SD). Data were analyzed by one-way analysis of variance (ANOVA), and means were compared using the Tukey test, and the P value of ≤ 0.05 considered as statistically significant. Data were analyzed using GraphPad Prism v.5 program (Intuitive Software for Science, San Diego, CA, U.S.A.) and Statistical Package for Social Sciences (SPSS, Chicago, USA) v.18.0.
Results This work aimed to evaluate the effect of MAC or its association with DZP on PTZ-induced kindling (MAC/DZP/PTZ). After the last treatment, 100% of the animals in the SAL/PTZ group (negative control) showed seizure behavior lasting at least 3 s. These results confirm
[8]
that the subconvulsive PTZ dose used in this study (60 mg/kg) was able to induce a “kindling state” in mice after six treatments (Figure 2A). The development of PTZ-kindling was only prevented by pretreatment with DZP 2 mg/kg (P ≤ 0.001). MAC was not able to change the convulsive behavior of the animals (occurrence of seizure or latency to the first bilateral forelimb clonus lasting more than 3 sec; Figure 2A and B). The group MAC/DZP0.15/PTZ showed a significant increase in the latency to first seizure only on the third and fourth days of treatment (Figure 2B), although it is possible to observe a behavior that differed from that of the negative control both in the percentage of seizure and latency in all the days of treatment. There is evidence that higher ROS levels may be produced in PTZ-induced seizures. Therefore, we monitored the changes in fluorescence intensity of DCFDA to assess intracellular ROS induced by PTZ seizure. The results indicated the suppressive effect of MAC/SAL/PTZ and MAC/DZP0.15/PTZ on ROS production, showing a significant difference from the control group SAL/PTZ (P ≤ 0.05; Figure 3). DZP 2 mg/kg was able to decrease ROS production significantly (DZP2/PTZ). On the other hand, DZP0.15/PTZ given alone did not change the level of DCF. In order to investigate whether the antioxidant properties of MAC were mediated by an increase in antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT) activities were
measured
(Figures
4
and
5).
The
treatment
with
MAC/SAL/PTZ
and
MAC/DZP0.15/PTZ increased SOD and CAT activities after PTZ-induced kindling in cerebral cortex when compared to the SAL/PTZ group (P ≤ 0.05). Both SOD and CAT activities were higher in the DZP2/PTZ, compared to negative control. DZP0.15/PTZ was not able to induce any change. As shown in Figure 6, MAC/SAL and MAC/DZP0.15/PTZ inhibited NO production (P < 0.05). NO production was lower in the group treated with DZP2/PTZ, while DZP0.15/PTZ was not able to produce change. The levels of TNF-α were increased by PTZ, as shown in Figure 7A-B (SAL/PTZ). The present results show that MAC significantly inhibited the pro-inflammatory factor (P ≤ 0.05), as well as MAC/DZP0.15/PTZ (P ≤ 0.01), suggesting its capacity to change this inflammatory parameter alone or in association with a benzodiazepine drug. The alkaline comet assay detects DNA strand breaks and alkali-labile sites. Our results showed that PTZ-induced seizures promoted significant DNA damage measured in cerebral cortex, hippocampus and peripheral blood (Table 1). Damage Index (DI) and Damage Frequency (DF) values for the MAC/SAL/SAL group were not statistically different from [9]
those exhibited by the SAL/SAL group. Similarly, Sham/SAL/PTZ and SAL/PTZ groups did not differ from each other in DI and DF. Thus, the results were compared to the SAL/SAL and SAL/PTZ groups. In hippocampus, all groups treated with PTZ showed increased DI in comparison to the SAL/SAL group, except for the MAC/DZP0.15/PTZ group, suggesting a trend of MAC in association with DZP to protect DNA against PTZ injuries in hippocampus (Table 1). When groups that received PTZ were compared to the SAL/PTZ group, no statistical difference was observed. In cerebral cortex, the DZP0.15/PTZ and DZP2/PTZ groups showed lower DI in comparison to SAL/PTZ, indicating the protective effect of DZP. All groups showed a significant difference in relation to the SAL/SAL group. In peripheral blood, there was an increase in the DI and DF values only for the SAL/PTZ and Sham/SAL/PTZ groups in comparison to the SAL/SAL group. The PTZ groups treated with MAC, DZP (0.15 and 2 mg/kg) or the association MAC/DZP0.15/PTZ were not able to decrease DNA damage significantly, in comparison to SAL/PTZ (P > 0.05; Table 1). There was no statistical difference in the ratio of PCE/NCE and micronucleus frequency among the groups. Only the cyclophosphamide positive control group showed increased micronucleus frequency in comparison to the SAL/SAL group (P ≤ 0.001; Table 2).
Discussion MAC’s ability to modulate seizures in a PTZ-induced kindling model was evaluated based on the beneficial effects of AC on oxidative stress and inflammatory parameters, as shown in a few previous studies [13, 28, 29]. Although MAC alone did not produce a less convulsive behavior, it was able to reduce the ROS production, increase SOD and CAT activities, and improve the inflammatory parameter measured after the PTZ-induced kindling. Likewise, studies have provided evidence that AC reduces oxidative stress and free radical formation in Parkinson’s disease in rodent models [30] and reverses the overproduction of ROS in vascular dementia model in rats, even suggesting an antioxidant property of AC [31]. Contributing to this evidence, researchers have shown that AC increased the activities of catalase, superoxide dismutase, and glutathione peroxidase in the hippocampus of alcoholic rats [32], also increasing the serum level of SOD in ischemic hypoxic neonate rats with cerebral palsy [33]. This is the first attempt to evaluate the protective effect of MAC on the PTZ-induced kindling model associated with oxidative stress and neuroinflammation parameters.
[10]
Recent studies have shown that inflammatory cytokine expressions were distinctively induced under various damaging conditions [34]. Contributing to neuronal cell death through a sustained inflammatory response, microglial cells are specialized macrophages with a role in the immune system, whereas astrocytes, the most numerous glial cells, induce inflammatory mediators such as interleukin-1β (IL-1 β) and tumor necrosis factor-α (TNF-α) when activated [35]. Here, PTZ administration increased cortical pro-inflammatory TNF-α level, known to participate in the induction and maintenance of epilepsy, as revealed using other epileptic models. Studies have demonstrated that the overexpression of TNF-α, among other inflammatory cytokines in mouse brains, is linked to spontaneous seizures, underscoring its crucial role in epilepsy. Hence, high levels of brain TNF-α in PTZ-kindled mice may be related to a pro-epileptogenic potential, possibly lowering the threshold of seizure induction and thus setting the basis for the onset of epilepsy [36]. In this scenario, our results showed that
MAC
(MAC/SAL/PTZ)
and
MAC
associated
with
a
low
DZP
dose
(MAC/DZP0.15/PTZ) significantly inhibited this pro-inflammatory factor. This result corroborates the findings of a previous study that showed that MAC applied in rats submitted to spinal cord injury was able to reduce expression of inflammatory cytokines, including IL1B, IL-6 and TNF-α [13]. Persistent seizures increase ROS, causing damage in macromolecules, including DNA [37, 38]. In this study, the increased DNA damage in brain tissues of the SAL/PTZ group might be associated with imbalance of the redox state caused by the seizures. In cerebral cortex, the treatment with DZP2/PTZ, which inhibited the seizures, decreased PTZ-induced DNA damage. Although MAC improved the antioxidant status, it was not able to decrease DNA damage in brain tissues significantly. The MAC/DZP0.15/PTZ treatment tended to decrease DNA damage in hippocampus, suggesting a protective effect from the association between MAC and DZP at low doses. Furthermore, there was an increase in DNA damage in peripheral blood in two kindling groups, SAL/PTZ and Sham/SAL/PTZ, in comparison to the SAL/SAL group, suggesting a systemic injury induced by PTZ. In order to evaluate if this DNA damage could lead to mutation, the micronucleus test was performed. As shown in Table 2, PTZ was not able to increase the micronucleus frequency in bone marrow. Concerning micronucleus frequency, DZP and MAC likewise did not induce significant increase which could represent risks to genomic stability. The main effect of most antiepileptic agents is to enhance the response to GABAA by facilitating the opening of gamma-aminobutyric acid (GABA)-activated chloride channels. [11]
Benzodiazepines increase the frequency of channel opening events, rising chloride ion conductance and inhibiting the action potential [39]. However, the clinical uses of benzodiazepines are limited by their side effects such as psychomotor impairment, sedation, myorelaxation, ataxia, amnesia, potentiation of other central depressant drugs, and dependence liability [40]. So, an alternative which can improve their efficacy is needed, decreasing undesirable effects and thus reducing tolerance and dependence. Unlike DZP, whose anticonvulsant mechanisms are well known, several studies have proposed different mechanisms to explain the effects of AC, including clinical evaluations that have demonstrated that AC produces positive effects on several types of epilepsy. Such beneficial effects include the absence of seizure as well of febrile convulsion, generalized clonic-tonic seizure, and even status epilepticus (SE). The overall therapeutic benefits include the improvement of electroencephalogram (e.g. the reduction of spike wave and desynchronization), of epileptic symptoms (e.g. decrease of the seizure frequency and shortness of attack episodes), of severity of SE, of functional recovery, and of life quality [41, 42, 43, 44]. Animal studies have demonstrated the antiepileptic effects of AC in varied experimental models. Chen et al. [45] observed that electroacupuncture (EA) produces nearly 27% reduction of PTZ-induced epileptiform neuronal activities of ventroposterior lateral thalamus of rats. EA has an anti-epileptic effect, which is related to its role in downregulating the increased hippocampal Ca2+ level in PTZ-kindled epilepsy in rats [46]. AC intervention has a protective effect on pyramidal cells of hippocampal CA 1 and CA 3 regions in epileptic rats, which is associated with the normal function of intracellular PI(3)K/Akt signaling pathway [17]. EA, when applied at head acupoints (e.g. GV20), prevents atrophy of some limbic structures and improves cognitive deficits in pilocarpine-epileptic rats. This effect has been shown to depend on the serotonergic system [47]. Apart from that, AC facilitates the release of neuropeptides, modulating gene expression in neuronal cells in the CNS, eliciting profound physiological effects and activating self-healing mechanisms. AC also enhances neurogenesis in several diseases through concomitant stimulation at ST36 and GV20, which improves hippocampus-related neuropathologies such as epilepsy and ischemia, inducing cell proliferation and neuroblast differentiation [48]. Here, the pretreatment with the combination of MAC and a low dose of DZP decreased the occurrence of seizures, increasing the latency to the first seizure, but not significantly. Yet, such result is still important if we consider the use of a lower dose of DZP, which could reduce the intensity of sedation or immobility. Combined treatment of MAC with DZP also [12]
decreases ROS levels and NO production, in addition to increasing SOD and CAT activities, tending to decrease DNA damage in brain tissue. DZP has distinct actions as positive allosteric modulator of GABAA receptors, although the mechanism underlying MAC effects should be elucidated. This combination mitigates neuroinflammation following PTZ-induced kindling model, as demonstrated by inhibition of TNF-α expression, maybe without the adverse side effects associated with high dose benzodiazepines. Consequently, a combination therapy including MAC and a benzodiazepine may become a novel therapeutic strategy in the attenuation of neurological symptoms in epileptogenesis. Conclusions Our findings reveal that the stimulation of acupoint GV20 by MAC did not decrease seizures induced by PTZ in the kindling model, but MAC alone or MAC in association with a low DZP dose demonstrated neuroprotective effects, improving brain antioxidant defenses, decreasing the expression of the pro-inflammatory TNF-α factor, and tending to decrease DNA damage in hippocampus after PTZ-induced kindling. Thus, MAC should be more thoroughly investigated in further studies focused on its potential use in epilepsy and other disorders associated with the CNS, or as a possible adjuvant in conventional pharmacotherapy.
Author’s contributions CMV, JS, and PP contributed to the experimental designs. ALF, GGR, PF, VRC, LLS, MSS, JBS, and CMV collected and analyzed the experimental data. JNP and PP were responsible for manuscript writing. All authors read and approved the final manuscript.
Ethics approval and consent to participate Not applicable.
Funding This work was supported by CNPq (grant N. 307064/2013-1) and UFRGS (N. 28514).
Availability of data and material Not applicable.
[13]
Competing interests The authors declare that they have no competing interests.
Consent for publication We accept the conditions of submission and the BioMed Central Copyright and License Agreement.
Acknowledgments We thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Federal University of Rio Grande do Sul (UFRGS) for supporting the development of this study.
[14]
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[20]
Figure legends
Figure 1. A schematic diagram illustrating the chronological events of the experiments.
[21]
Figure 2. The effect of MAC on PTZ-induced kindling in mice. (A) Percentage of animals presenting seizures (bilateral forelimb clonus lasting more than 3 sec) in the SAL/PTZ, DZP2/PTZ, DZP0.15/PTZ, MAC/SAL/PTZ, MAC/DZP0.15/PTZ, Sham/SAL/PTZ groups during repeated PTZ administration (60 mg/kg, s.c.). N = 9 animals per group (**P ≤ 0.01, and ***P ≤ 0.001; Fisher’s Exact Test, compared to the SAL/PTZ group). (B) Latency to the 1st bilateral forelimb clonus lasting more than 3 sec in mice submitted to PTZ-kindling. N = 9 animals per group (**P ≤ 0.01, and ***P ≤ 0.001, compared to the SAL/PTZ group). The comparisons between the six groups, according to the number of treatments, were analyzed by using the Generalized Estimation Equation (GEE). Bonferroni multiple comparisons were used to complete the evaluation Statistical calculations.
[22]
Figure 3. The effect of MAC on oxidative stress promoted by PTZ-induced kindling model in the cerebral cortex of mice. DCF was used to determine ROS generation in cells homogenates. Increasingly oxidizing extracellular conditions demonstrated a rise in DCF fluorescence, and are indicative of higher intracellular ROS levels. Values represent the mean ± SD of N = 3 animals per group. *P ≤ 0.05, ***P ≤ 0.001 compared to the SAL/PTZ group; #P ≤ 0.001 compared to the SAL/SAL group.
[23]
Figure 4. The effect of MAC on the activity of the antioxidant enzyme Superoxide dismutase (SOD) in the mice cerebral cortex. Values represent the mean ± SD of N = 3 animals per group. *P ≤ 0.05, ***P ≤ 0.001 compared to the SAL/PTZ group; #P ≤ 0.05 compared to the SAL/SAL group.
[24]
Figure 5. The effect of MAC on the activity of the antioxidant enzyme catalase in the mice cerebral cortex. Values represent the mean ± SD of N = 3 animals per group. *P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001 compared to the SAL/PTZ group; #P ≤ 0.01 compared to the SAL/SAL group.
Figure 6. The effect of MAC on NO production promoted by PTZ-induced kindling model in the cerebral cortex of mice. Values represent the mean ± SD of N = 3 animals per group. *P ≤ 0.05, **P ≤ 0.01 compared to the SAL/PTZ group; #P ≤ 0.05, compared to the SAL/SAL group.
[25]
Figure 7. The effect of MAC on the expression of TNF-α protein in the cerebral cortex of mice. The TNF-α protein was examined by western blot analysis. Values represent the mean ± SD of N = 3 animals per group. *P ≤ 0.05, **P ≤ 0.01 compared to the SAL/PTZ group; #P ≤ 0.01 compared to the SAl/SAL group.
[26]
Table 1. Effect of manual AC on DNA damage promoted by pentylenetetrazole (PTZ)-induced kindling model in mice. Hippocampus
Cortex
Peripheral blood
Group
DI a
DF b
DI a
DF b
DI a
DF b
SAL/SAL
66.6 ±10.8
66.0 ± 10.6
74.8 ± 13.6
73.8 ± 12.9
44.6 ± 6.9
44.6 ± 6.9
MAC SAL/SAL
69.2 ± 16.1
68.0 ± 15.7
50.4 ± 5.8
50.4 ± 5.8
42.4 ± 10.0
42.4 ± 10.0
Sal/PTZ
269.3 ± 46.0 ### 98.3 ± 1.2 ### 213.0 ± 40.8 ###
97.0 ± 2.8 ## 85.5 ± 18.9 # 78.5 ± 12.0 ##
Sham SAL/PTZ
237.3 ± 11.1 ### 98.7 ± 2.3 ### 196.0 ± 26.0 ###
96.0 ± 2.9 #
83.5 ± 11.8 # 78.5 ± 4.4 ##
MAC SAL/PTZ
194.5 ± 21.3 #
100.0 ± 0 ###
170.0 ± 17.9 ###
82.3 ±14.1
56.4 ± 8.6
55.4 ± 9.0
DZP 2.0 mg/kg/PTZ
183.2 ± 86.7 #
89.6 ± 9.6 ##
140.2 ± 39.6 #** 84.8 ± 10.4
79.4 ± 27.8
67.0 ± 15.2
DZP 0.15 mg/kg/PTZ
235.3 ± 82.7 ### 98.8 ± 1.5 ### 155.2 ± 33.1 ## * 89.4 ± 6.5
59.2 ± 25.1
54.0 ± 18.7
51.8 ± 16.0
51.8 ± 16.0
MAC DZP 0.15 mg/kg/PTZ 151.6 ± 39.2
95.8 ± 4.0 ### 186.5 ± 13.8 ###
93.0 ±8.9
N = 5 animals per group. aDI can range from 0 (completely undamaged, 100 cells × 0) to 400 (with maximum damage, 100 × 4). bDF was calculated based on number of cells with tail versus those with no tail. *P ≤ 0.05; **P ≤ 0.01 in comparison to SAL/PTZ group (ANOVA, Tukey test); #P ≤ 0.05;
##
P ≤ 0.01;
###
P ≤ 0.001 in comparison to SAL/SAL group (ANOVA,
Tukey
test).
[27]
Table 2. Mutagenic activity in bone marrow after PTZ-induced kindling model in mice. MNPCE ª in 2,000 PCE
Ratio PCE/NCE b
Mean ± SD
Mean ± SD
SAL/SAL
6.0 ± 1.2
1.8± 0.1
MAC Sal/Sal
8.0 ± 1.9
1.7± 0.2
SAL/PTZ
9.5 ± 1.0
1.5 ± 0.3
Sham SAL/PTZ
10.8 ± 2.6
1.5 ± 0.1
MAC SAL/PTZ
8.2 ± 2.7
1.6 ± 0.2
DZP 2.0 mg/kg/PTZ
8.3 ± 3.3
1.5± 0.5
DZP 0.15 mg/kg/PTZ
9.2 ± 2.3
1.4± 0.1
MAC DZP 0.15 mg/kg/PTZ
7.6 ± 1.8
1.5± 0.3
Cyclophosphamidec
29.2 ± 5.9 ###
1.3 ± 0.1
Group
a
MNPCE: micronucleus in polychromatic erythrocytes.
b c
Ratio PCE/NCE: ratio polychromatic erythrocytes/normochromatic erytrocytes.
Positive control (50 mg/kg single dose, i.p.).
N = 5 animals per group. ###
P ≤0.001: significant difference in comparison with the saline/saline group (ANOVA,
Tukey test).
[28]