Lacosamide improves biochemical, genotoxic, and mitochondrial parameters after PTZ-kindling model in mice.

This study evaluated the effect of lacosamide (LCM) on biochemical and mitochondrial parameters after PTZ kindling in mice. Male mice were treated on alternative days for a period of 11 days with LCM (20, 30, or 40 mg/kg), saline, or diazepam (2 mg/kg), before PTZ administration (50 mg/kg). The hippocampi were collected to evaluate free radicals, the activities of superoxide dismutase (SOD), catalase (CAT), and the mitochondrial complexes I-III, II, and II-III, as well as Bcl-2 and cyclo-oxygenase-2 (COX-2) expressions. Hippocampi, blood, and bone marrow were collected for genotoxic and mutagenic evaluations. LCM 40 mg/kg increased latency and decreased percentage of seizures, only on the 3rd day of observation. The dose of 30 mg/kg only showed positive effects on the percentage of seizures on the 2nd day of observation. LCM decreased free radicals and SOD activity and the dose of 40 mg/kg were able to increase CAT activity. LCM 30 and 40 mg/kg improved the enzymatic mitochondrial activity of the complex I-III and LCM 30 mg/kg improved the activity of the complex II. In the comet assay, the damage induced by PTZ administration was reduced by LCM 20 and 30 mg/kg. The dose of 20 mg/kg increased COX-2 expression while the highest dose used, 40 mg/kg, was able to reduce this expression when compared to the group treated with LCM 20 mg/kg. Although LCM did not produce the antiepileptogenic effect in vivo, it showed the neuroprotective effect against oxidative stress, bioenergetic dysfunction, and DNA damage induced by the repeated PTZ administration.

Neonatal morphine exposure and maternal deprivation alter nociceptive response and central biomarkers' levels throughout the life of rats.

In the present study, we investigated the effect of repeated neonatal morphine exposure and/or maternal deprivation(MD) on the nociceptive response and central biomarkers' BDNF, IL-1ß, and IL-4 levels at postnatal days 16(PND16), 30(PND30), and 60(PND60). At birth, the litters were standardized to contain 8 pups/dam (n = 58). From PND1 to PND10, the pups of the deprived groups were separated daily from their mothers for 3 h and divided into 5 groups: control(C), saline(S), morphine(M), deprived-saline(DS), and deprived-morphine(DM). The pups received subcutaneous injections of saline/morphine (5 µg) in the mid-scapular area between PND8 and PND14. Nociceptive responses were assessed by hot plate(HP) and tail-flick(TFL) tests and biomarker levels by ELISA. Thermal hyperalgesia(HP) was found in all assessments for the M, DS, and DM groups, and a decrease in nociceptive threshold(TFL) was found in the DS group at PND16; M and DM groups at PND30; and M, DS, and DM groups at PND60. There were interactions between treatment/deprivation/timepoint in all central biomarkers' levels. The current study indicates that neonatal exposure to morphine and MD, which occurs in the pediatric ICU, can alter the nociceptive and neuroinflammatory responses.

Gamma-decanolactone attenuates acute and chronic seizures in mice: a possible role of adenosine A1 receptors.

This study aimed to investigate the possible gamma-decanolactone mechanisms of action in the GABAergic and adenosine systems using the aminophylline-induced acute crisis model and the pentylenetetrazole-induced kindling model. In the acute model, male mice received administration of bicuculline (GABAA receptor antagonist), 8-cyclopentyl-1,3-dipropylxanthine (A1 receptor antagonist) or ZM241385 (A2A receptor antagonist), 15 min before the treatment with gamma-decanolactone (300 mg/kg). After a single dose of aminophylline was administered, the animals were observed for 60 min. In the chronic model of seizure, 30 min after the treatment with gamma-decanolactone, mice received pentylenetetrazole once every third day. On the last day of kindling, the animals received the same GABA and adenosine antagonists used in the acute model, 15 min before gamma-decanolactone administration. The protein expression of GABAA α1 receptor and adenosine A1 receptor was detected using western blotting technique in hippocampal samples. The results showed that gamma-decanolactone increased the latency to first seizure and decreased seizure occurrence in the acute and chronic models. The adenosine A2A receptor antagonist and GABAA receptor antagonist were not able to change gamma-decanolactone behavioral seizure induced by aminophylline or pentylenetetrazole. The administration of adenosine A1 receptor antagonist reversed the protective effect of gamma-decanolactone in both models. In addition, gamma-decanolactone promoted an increase in the expression GABAA α1 receptor, in the hippocampus. The results suggest that the neuroprotective effect of gamma-decanolactone observed during the investigation could have a straight connection to its action on A1 adenosine receptors.

Transcranial direct current stimulation (tDCS) affects neuroinflammation parameters and behavioral seizure activity in pentylenetetrazole-induced kindling in rats.

Despite the introduction of new antiepileptic drugs, about 30 % of patients with epilepsy are refractory to drug therapy. Thus, the search for non-pharmacological interventions such as transcranial direct current stimulation (tDCS) may be an alternative, either alone or in combination with low doses of anticonvulsants. This study evaluated the effect of anodal (a-tDCS) and cathodal tDCS (c-tDCS) on seizure behavior and neuroinflammation parameters. Rats were submitted to the kindling model induced by pentylenetetrazole (PTZ) using diazepam (DZP) as anticonvulsant standard. tDCS groups were submitted to 10 sessions of a-tDCS or c-tDCS or SHAM-tDCS. Every 3 days they received saline (SAL), low dose of DZP (alone or in combination with tDCS) or effective dose of DZP 30 min before administration of PTZ, totaling 16 days of protocol. Neither a-tDCS nor c-tDCS reduced the occurrence of clonic forelimb seizures (convulsive motor seizures - stage 3 by the adapted Racine scale we based on). Associated with DZP, c-tDCS (c-tDCS/DZP0.15) increased the latency to first clonic forelimb seizure on the 10th and 16th days. Hippocampal IL-1ß levels were reduced by c-tDCS and c-tDCS/DZP0.15. In contrast, these treatments induced an increase in cortical IL-1ß levels. Hippocampal TNF-α levels were not altered by c-tDCS or a-tDCS, but c-tDCS and c-tDCS/DZP0.15 increased those levels in cerebral cortex. Cortical NGF levels were increased by c-tDCS and c-tDCS/DZP0.15. a-tDCS/DZP0.15 reduced hippocampal BDNF levels and c-tDCS/DZP0.15 increased these levels in cerebral cortex. In conclusion, c-tDCS alone or in combination with a low dose of DZP showed to affect neuroinflammation, improving central neurotrophin levels and decreasing hippocampal IL-1ß levels after PTZ-induced kindling without statistically significant effect on seizure behavior.

Transcranial direct current stimulation combined with exercise modulates the inflammatory profile and hyperalgesic response in rats subjected to a neuropathic pain model: Long-term effects.

BACKGROUND: Behavioral alterations, like mechanical and thermal hyperalgesia, and modulation of biomarkers in the peripheral and central nervous systems (CNS) are markers of chronic pain. Transcranial direct current stimulation (tDCS) with exercise is a promising therapy for pain due to its neuromodulatory capacity. OBJECTIVE: To assess the individual effects of tDCS, exercise, and the two combined on the nociceptive response and BDNF, IL-1ß, and IL-4 levels in the CNS structures of rats in a chronic pain model. METHODS: For 8 consecutive days after the establishment of chronic neuropathic pain by inducing a constriction injury to the sciatic nerve (CCI), the rats received tDCS, exercise, or both treatments combined (20 min/day). The hyperalgesic response was assessed by von Frey and hot plate tests at baseline, 7, and 14 days after CCI surgery and immediately, 24 h, and 7 days after the end of treatment. The BDNF, IL-1ß, and IL-4 levels were assessed in the cerebral cortex, brainstem, and spinal cord by enzyme-linked immunosorbent assay at 48 h and 7 days after the end of treatment. RESULTS: The CCI model triggered marked mechanical and thermal hyperalgesia. However, bimodal tDCS, aerobic exercise, and the two combined relieved nociceptive behavior for up to 7 days following treatment completion. CONCLUSIONS: Bimodal tDCS, aerobic exercise, or both treatments combined promoted analgesic effects for neuropathic pain. Such effects were reflected by cytokine modulation throughout the spinal cord-brainstem-cerebral cortex axis.

Maternal Deprivation and Sex Alter Central Levels of Neurotrophins and Inflammatory Cytokines in Rats Exposed to Palatable Food in Adolescence.

Maternal deprivation (MD) in rodents is used to simulate human-infant early life stress, which leads to neural, hormonal, and behavioral alterations. Palatable food (PF) can reduce the stress response, and individuals use it as a self-applied stress relief method. Thus, the present study aimed to evaluate the effect of the association between MD in the early life (P1-P10) and PF consumption (condensed milk, P21-P44) in the central neuroplasticity (BDNF/NGF levels) and central neuroinflammatory parameters (TNF-α, IL-6, and IL-10 levels) in male and female Wistar rats in the adolescence. In addition, weight-related parameters (weight gain, Lee Index, and relative adipose tissue weight) were evaluated. PF exposure increased relative adipose tissue weight; however, it did not lead to a change in animals' body weight. MD reduced hypothalamic BDNF and NGF levels, and hippocampal TNF-α levels in male and female rats. Animals of both sexes that received PF, exhibited reduced hypothalamic NGF levels. Neuroinflammatory marker evaluations showed that male rats were more susceptible to the interventions than female rats, since MD reduced their cortical IL-10 levels and PF increased their IL-6 levels. Differences in the Lee index, central BDNF, TNF-α, and IL-6levels were observed between sexes. Male animals per se presented greater Lee index. Female rats had higher BDNF and IL-6 levels in the hippocampus and hypothalamus and higher hypothalamic TNF-α levels than those observed in males. In conclusion, there were more noticeable effects of MD than PF on the variables measured in this study. Sex effect was identified as an important factor and influenced most of the neurochemical measures in this study. In this way, we suggest including both female and male animals in researches to improve the quality of translational studies.

Rosmarinic acid improves oxidative stress parameters and mitochondrial respiratory chain activity following 4-aminopyridine and picrotoxin-induced seizure in mice.

Studies have indicated that epilepsy, an important neurological disease, can generate oxidative stress and mitochondrial dysfunction, among other damages to the brain. In this context, the use of antioxidant compounds could provide neuroprotection and help to reduce the damage caused by epileptic seizures and thereby the use of anticonvulsant drugs. Rosmarinic acid (RA) is an ester of caffeic acid and 3,4-dihydroxyphenylactic acid that prevents cell damage caused by free radicals, acting as an antioxidant. It also presents anti-inflammatory, antimutagenic, and antiapoptotic properties. In this work, we used two models of acute seizure, 4-aminopyridine (4-AP) and picrotoxin (PTX)-induced seizures in mice, to investigate the anticonvulsant, antioxidant, and neuroprotective profile of RA. Diazepam and valproic acid, antiepileptic drugs already used in the treatment of epilepsy, were used as positive controls. Although RA could not prevent seizures in the models used in this study, neither enhance the latency time to first seizure at the tested doses, it exhibited an antioxidant and neuroprotective effect. RA (8 and 16 mg/kg) decreased reactive oxygen species production, superoxide dismutase activity, and DNA damage, measured in hippocampus, after seizures induced by PTX and 4-AP. Catalase activity was decreased by RA only after seizures induced by 4-AP. The activity of the mitochondrial complex II was increased by RA in hippocampus samples after both seizure models. The results obtained in this study suggest that RA is able to reduce cell damage generated by the 4-AP and PTX seizures and therefore could represent a potential candidate in reducing pathophysiological processes involved in epilepsy.

Maternal deprivation alters nociceptive response in a gender-dependent manner in rats.

The present study aimed at investigating both the early and long-term effects of maternal deprivation as well as gender on neuromotor reflexes, anxiety behavior and thermal nociceptive responses. A total of 64 Wistar rats pups (32 males, 32 females) were utilized and were deprived of their mother for 3 h/daily, from postnatal day 1 (P1) until P10. Successively, animals were divided into 2 groups: control group (C) - pups no subjected to intervention; and the maternal-deprived group (MD): pups subjected to maternal deprivation. The neuromotor reflexes were evaluated through the righting reflex and negative geotaxis tests; the exploratory behavior by open field test (OFT); the anxiety-like behavior by elevated plus-maze test (EPM); the thermal nociceptive responses byhot plate (HP) and tail-flick (TFL) tests. All the animals subjected to maternal deprivation showed a delayed reflex response at P8 in the negative geotaxis test. In contrast, the OFT at P20 identified an effect of gender on the outer crossings and grooming as well as an interaction between gender and maternal deprivation on latency. Additionally, effect of maternal deprivation in the open and closed arms as well as gender effect in the protected head-dipping (PHD) and non-protected head-dipping (NPHD) were observed at P20 (EPM). In contrast, there were a gender effect on latency and an interaction between gender and maternal deprivation on rearing at P42. Moreover, in nociceptive tests was observed an analgesic effect induced by maternal deprivation; however, in the TFL test, only deprived females showed this effect. Surprisingly, only control animals presented an ontogeny nociceptive effect in the HP testat P21 and P43, which may be related to an increase in the inhibitory nociceptive pathways throughout life. In this way, we suggest maternal deprivation to be able to anticipate the maturation of the inhibitory nociceptive pathway. In conclusion, maternal deprivation induced a delayed reflex response at P8 and altered the anxiety and nociceptive behaviors according to the time after exposure to this stressor, in a gender-specific manner.

Neuropharmacological Profile of Gamma-Decanolactone on Chemically-induced Seizure in Mice.

BACKGROUND: Gamma-Decanolactone (GD) is a monoterpene compound that presents anticonvulsant effect in acute and chronic models of epilepsy and it acts as a noncompetitive glutamate antagonist. OBJECTIVE: This study evaluated the anticonvulsant profile and the possible mechanism of action of GD in seizures induced by isoniazid (INH; 250 mg/kg), picrotoxin (PCT; 5 mg/kg) and 4- aminopyridine (4-AP; 13 mg/kg) in male mice. METHOD: Thirty minutes before the convulsants administration, animals received a single administration of saline, GD (100 or 300 mg/kg) or the positive control diazepam (DZP; 2 mg/kg). The parameters evaluated were the latency to the first seizure and the occurrence of clonic forelimb seizures. The rotarod performance test was used to evaluate the neurotoxicity of GD (300 mg/kg). Also, the DZPinduced sleep test was used. RESULTS: DZP increased the latency to the first seizure on all used models and decreased the percentage of seizures in two of the three behavioral models. GD was able to prolong the latency to the first seizure and decreased the percentage of seizures induced by INH and 4-AP, but not by PCT. It reduced the latency to fall off the rotarod test only at the time of 30 min. In the DZP-induced sleep test, GD shortened the onset and prolonged the time of sleep. CONCLUSION: Our findings suggested that GD reduced the convulsive behavior in the seizure models used here and it could modulate GABA pathways and affect potassium channels directly or indirectly, involving more than one cellular target in the central nervous system.

Gamma-Decanolactone Improves Biochemical Parameters Associated with Pilocarpine-Induced Seizures in Male Mice.

BACKGROUND AND OBJECTIVE: Gamma-decanolactone (GD) is a monoterpene effective against seizures induced by pentylenetetrazole. The mechanism of action of GD is likely to be via glutamate antagonism. GD also inhibits intracellular reactive oxygen species (ROS) generation and the lipopolysaccharide-induced expression of inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-α) in vitro. Considering the neuropharmacological profile of GD studied so far, we investigated the effect of intraperitoneal administration of GD 100 and 300 mg/kg on pilocarpine (PIL)-induced status epilepticus (SE) in mice. METHODS: GD was administered 30 min before PIL. Behavioral (latency to first seizure and the percentage of clonic forelimb seizures), biochemical, and oxidative stress parameters were evaluated. DNA damage in the cerebral cortex of mice was assessed using the comet assay and mutagenic activity of GD was evaluated using Salmonella/microsome assay in TA100, TA98, TA97a, TA102, and TA1535 strains, with and without metabolic activation (S9 mix). RESULTS: The behavioral results showed that only the latency to the first clonic seizure increased in the groups treated with GD 300 mg/kg, but not when the animals received GD 100 mg/kg. Both GD doses were able to increase superoxide dismutase and catalase activities, inducing a decrease in ROS and nitrite production and in DNA damage in the cerebral cortex. GD was not able to induce base pair substitution and frameshift mutations in the absence or in the presence of metabolic activation. CONCLUSION: These findings demonstrate that GD does not improve behavioral parameters in the PIL model, but it was able to protect seizure-related oxidative stress and DNA damage in mice, without inducing gene mutations.

DNA damage and oxidative stress induced by seizures are decreased by anticonvulsant and neuroprotective effects of lobeline, a candidate to treat alcoholism.

The alkaloid lobeline (Lob) has been studied due to its potential use in treatment of drug abuse. This study evaluates the possible anticonvulsant and neuroprotective activities of Lob to obtain new information on its properties that could confirm it as a candidate in the treatment of alcohol addiction. The anticonvulsant effect of Lob was evaluated using a pilocarpine-induced seizure model. In addition, possible neuroprotective effects were investigated measuring DNA damage using the comet assay, assessing free radical levels by dichlorofluorescein diacetate (DCF) oxidation, and measuring the antioxidant potential using the α, α-diphenyl-ß-picrylhydrazyl (DPPH) scavenging assay, besides measuring superoxide dismutase (SOD) and catalase (CAT) enzyme activities in brain tissues. Lobeline increased the latency to the first seizure and decreased the percentage of seizures in a similar way as diazepam, used as control. DNA damage induced by Pil and hydrogen peroxide were decreased in hippocampus and cerebral cortex from mice treated with Lob. The levels of free radicals and CAT activity increased in cortex and hippocampus, respectively, in mice treated with Pil. Lobeline decreased CAT in hippocampus, leading to similar values as in the saline negative control. In conclusion, Lob has anticonvulsant and neuroprotective actions that may be mediated by antioxidant-like mechanisms, indicating its potential as candidate drug in alcoholism therapy.

Rosmarinic Acid Attenuates the Activation of Murine Microglial N9 Cells through the Downregulation of Inflammatory Cytokines and Cleaved Caspase-3.

OBJECTIVE: The present study evaluated the ability of rosmarinic acid (RA) to inhibit microglia activation induced by lipopolysaccharide (LPS) in the N9 murine microglial cell line, and investigated the putative mechanisms involved in this process. METHODS: In all tests, N9 murine microglial cells were pretreated with RA (0.1, 1.0, and 10 µM) for 20 h and exposed to LPS (1 µM/mL) for 4 h. Cell viability was measured by Trypan blue exclusion assay. Flow cytometry was used to detect reactive oxygen species (ROS), quantify cleaved caspase-3, and analyze the mitochondrial electrochemical potential. iNOS, Arg-1, TNF-α, IL-1ß, and IL-6 proteins were analyzed by Western blotting, and their antigens were detected using the chemiluminescence technique. The effect of RA on DNA was evaluated by the Comet assay. RESULTS: RA attenuated the expression of the M1 marker iNOS and the levels of proinflammatory factors, including TNF-α, IL-1ß, and IL-6; it increased the expression of the M2 marker Arg-1, and inhibited, at least in part, ROS generation and loss of mitochondrial outer membrane permeabilization through the inhibition of cleaved caspase-3 activation. RA also inhibited DNA damage, reassuring cell protection. CONCLUSIONS: The results suggested a protective effect of RA through downregulation of inflammatory cytokines and cleaved caspase-3.

Manual acupuncture improves parameters associated with oxidative stress and inflammation in PTZ-induced kindling in mice.

The use of acupuncture in the treatment of central nervous system (CNS) disorders is an age-old 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, superoxide dismutase (SOD), and catalase (CAT) activities, nitrite content, and deoxyribonucleic acid (DNA) damage in cerebral cortex. Mice received PTZ (60mg/kgs.c.) once every three days for 16days, totaling six treatments. MAC was applied at acupoint GV20 daily during the entire experimental protocol. Diazepam (DZP) (2mg/kg) was used as positive control. Also, we evaluated the MAC effect associated with DZP (MAC/DZP) at a low dose (0.15mg/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.

Behavioral and genotoxic evaluation of rosmarinic and caffeic acid in acute seizure models induced by pentylenetetrazole and pilocarpine in mice.

The goal of this study was to investigate the effects of rosmarinic acid (RA) and caffeic acid (CA) in the acute pentylenetetrazole (PTZ) and pilocarpine (PIL) seizure models. We also evaluated the effect of RA and CA on the diazepam (DZP)-induced sleeping time test and its possible neuroprotective effect against the genotoxic damage induced by PTZ and PIL. Mice were treated intraperitoneally (i.p.) with saline, RA (2 or 4 mg/kg), or CA (4 or 8 mg/kg) alone or associated to low-dose DZP. After, mice received a single dose of PTZ (88 mg/kg) or PIL (250 mg/kg) and were monitored for the percentage of seizures and the latency to first seizure (LFS) >3 s. Vigabatrin and DZP were used as positive controls. In the DZP-induced sleeping time test, mice were treated with RA and CA and 30 min after receiving DZP (25 mg/kg, i.p.). The alkaline comet assay was performed after acute seizure tests to evaluate the antigenotoxic profiles of RA and CA. The doses of RA and CA tested alone did not reduce the occurrence of seizures induced by PTZ or PIL. The association of 4 mg/kg RA + low-dose DZP was shown to increase LFS in the PTZ model, compared to the group that received only the DZP. In the DZP-induced sleeping time test, the latency to sleep was reduced by 4 mg/kg RA and 8 mg/kg CA. The PTZ-induced genotoxic damage was not prevented by RA or CA, but the PIL-induced genotoxic damage was decreased by pretreatment with 4 mg/kg RA (in cortex) and 4 mg/kg CA (in hippocampus). In conclusion, RA and CA presented neuroprotective effect against PIL-induced genotoxic damage and reduced the latency to DZP-induced sleep. Of the rosmarinic acid, 4 mg/kg enhanced the DZP effect in the increase of latency to clonic PTZ-induced seizures.

Toxicological evaluation of Pterocaulon polystachyum extract: a medicinal plant with antifungal activity.

Pterocaulon polystachyum DC is a native species to southern Brazil, Paraguay, Uruguay and northeastern Argentina. It is utilized to treat animal problems popularly diagnosed as "mycoses". The antifungal and amebicidal activity of its hexane extract has been previously reported, although there are no studies confirming the safety of this plant for therapeutic purposes to date. Hence, this study investigates the toxic effects of a hexane extract of Pterocaulon polystachyum administered as acute and subacute oral treatments. After acute treatment the extract caused alterations in biochemical parameters, morphological alterations in tissues and was genotoxic, according to the comet assay; neither mortality nor visible signs of lethality were seen in mice. Similarly subacute treatment caused important differences in biochemical parameters and tissues, between control and treated groups. The results also revealed genotoxicity in kidney tissue, though no mutagenicity was detected by the micronucleus test. No animal died during the treatment period.