Aleksandra Rašić-Marković

The activity of erythrocyte and brain Na+/K+ and Mg2+-ATPases in rats subjected to acute homocysteine and homocysteine thiolactone administration

Hyperhomocysteinemia is associated with various pathologies including cardiovascular disease, stroke, and cognitive dysfunctions. Systemic administration of homocysteine can trigger seizures in animals, and patients with homocystinuria suffer from epileptic seizures. Available data suggest that homocysteine can be harmful to human cells because of its metabolic conversion to homocysteine thiolactone, a reactive thioester. A number of reports have demonstrated a reduction of Na+/K+-ATPase activity in cerebral ischemia, epilepsy and neurodegeneration possibly associated with excitotoxic mechanisms. The aim of this study was to examine the in vivo effects of d,l-homocysteine and d,l-homocysteine thiolactone on Na+/K+- and Mg2+-ATPase activities in erythrocyte (RBC), brain cortex, hippocampus, and brain stem of adult male rats. Our results demonstrate a moderate inhibition of rat hippocampal Na+/K+-ATPase activity by d,l-homocysteine, which however expressed no effect on the activity of this enzyme in the cortex and brain stem. In contrast,d,l-homocysteine thiolactone strongly inhibited Na+/K+-ATPase activity in cortex, hippocampus and brain stem of rats. RBC Na+/K+-ATPase and Mg2+-ATPase activities were not affected by d,l-homocysteine, while d,l-homocysteine thiolactone inhibited only Na+/K+-ATPase activity. This study results show that homocysteine thiolactone significantly inhibits Na+/K+-ATPase activity in the cortex, hippocampus, and brain stem, which may contribute at least in part to the understanding of excitotoxic and convulsive properties of this substance.

Correlation between electrocorticographic and motor phenomena in lindane-induced experimental epilepsy in rats.

We report a study on the relation between open-field behavior and electroencephalographic (EEG) changes during lindane-induced seizures in 2-month-old adult male Wistar rats. For chronic EEG recordings and power spectra analysis, 3 electrodes were implanted into the skull. Three groups of animals, (i) saline-injected control (n = 6), (ii) DMSO-treated (n = 6), and (iii) lindane intraperitoneally administered: L(4) (4 mg/kg, n = 10), L(6) (6 mg/kg, n = 11), and L(8) (8 mg/kg, n = 11), were observed for 30 min for the occurrence of convulsive behavior. It was assessed by incidence of motor seizures, and seizure severity grade was determined by a descriptive rating scale (0, no response; 1, head nodding, lower jaw twitching; 2, myoclonic body jerks, bilateral forelimb clonus with full rearing; 3, progression to generalized clonic convulsions followed by tonic extension of fore and hind limbs and tail; 4, status epilepticus). EEG signal and spectral analyses were suitable to describe the dynamics of complex behavioral responses. Incidence and severity of epileptic manifestations, recorded as high voltage spike-wave complexes, polyspikes, sleep-like patterns in EEG, and power spectra changes, were greater in lindane-treated groups in a dose-dependent manner compared with control or DMSO-treated groups. Our results suggest good correlation between lindane-induced epileptiform activity and behavioral changes.

The role of nitric oxide in convulsions induced by lindane in rats.

Lindane is an organochloride pesticide and scabicide. It evokes convulsions mainly trough the blockage of GABA(A) receptors. Nitric oxide (NO), gaseous neurotransmitter, has contradictor role in epileptogenesis due to opposite effects of L-arginine, precursor of NO syntheses (NOS), and L-NAME (NOS inhibitor) observed in different epilepsy models. The aim of the current study was to determine the effects of NO on the behavioral and EEG characteristics of lindane-induced epilepsy in male Wistar albino rats. The administration of L-arginine (600, 800 and 1000 mg/kg, i.p.) in dose-dependent manner significantly increased convulsion incidence and severity and shortened latency time to first convulsion elicited by lower lindane dose (4 mg/kg, i.p.). On the contrary, pretreatment with L-NAME (500, 700 and 900 mg/kg, i.p.) decreased convulsion incidence and severity and prolonged latency time to convulsion following injection with a convulsive dose of lindane (8 mg/kg, i.p.). EEG analyses showed increase of number and duration of ictal periods in EEG of rats receiving l-arginine prior to lindane and decrease of this number in rats pretreated with L-NAME. These results support the conclusion that NO plays a role of endogenous convulsant in rat model of lindane seizures.

Finasteride improves motor, EEG, and cellular changes in rat brain in thioacetamide-induced hepatic encephalopathy

Neurosteroids are involved in the pathogenesis of hepatic encephalopathy (HE). This study evaluated the effects of finasteride, inhibitor of neurosteroid synthesis, on motor, EEG, and cellular changes in rat brain in thioacetamide-induced HE. Male Wistar rats were divided into the following groups: 1) control; 2) thioacetamide-treated group, TAA (300 mg·kg(-1)·day(-1)); 3) finasteride-treated group, FIN (50 mg·kg(-1)·day(-1)); and 4) group treated with FIN and TAA (FIN + TAA). Daily doses of TAA and FIN were administered in three subsequent days intraperitoneally, and in the FIN + TAA group FIN was administered 2 h before every dose of TAA. Motor and reflex activity was determined at 0, 2, 4, 6, and 24 h, whereas EEG activity was registered about 24 h after treatment. The expressions of neuronal (NeuN), astrocytic [glial fibrilary acidic protein (GFAP)], microglial (Iba1), and oligodendrocyte (myelin oligodendrocyte glycoprotein) marker were determined 24 h after treatment. While TAA decreased all tests, FIN pretreatment (FIN + TAA) significantly improved equilibrium, placement test, auditory startle, head shake reflex, motor activity, and exploratory behavior vs. the TAA group. Vital reflexes (withdrawal, grasping, righting and corneal reflex) together with mean EEG voltage were significantly higher (P < 0.01) in the FIN + TAA vs. the TAA group. Hippocampal NeuN expression was significantly lower in TAA vs. control (P < 0.05). Cortical Iba1 expression was significantly higher in experimental groups vs. control (P < 0.05), whereas hippocampal GFAP expression was increased in TAA and decreased in the FIN + TAA group vs. control (P < 0.05). Finasteride improves motor and EEG changes in TAA-induced HE and completely prevents the development of hepatic coma.

Exercise Decreases Susceptibility to Homocysteine Seizures: the Role of Oxidative Stress

The aim of the study was to examine the effects of chronic exercise training on seizures induced by homocysteine thiolactone (HCT) in adult rats. Rats were assigned to: sedentary control; exercise control; sedentary+HCT; exercise+HCT group. Animals in the exercise groups ran 30 min daily on a treadmill for 30 consecutive days (belt speed 20 m/min), while sedentary rats spent the same time on the treadmill (speed 0 m/min). On the 31st day, the HCT groups received HCT (8.0 mmol/kg), while the control groups received vehicle. Afterwards, convulsive behavior and EEG activity were registered. Lipid peroxidation, superoxide dismutase (SOD) and catalase (CAT) activity were ascertained in the rat hippocampus. No signs of seizures were registered in sedentary and exercise control rats. Seizure latency was increased, while number of seizure episodes and spike-and-wave discharges (SWD) in EEG were decreased in the exercise+HCT compared to the sedentary+HCT group. Seizure incidence, the severity thereof and duration of SWDs were not significantly different between these groups. Exercise partly prevented increase of lipid peroxidation and decrease of the SOD and CAT activity after HCT administration. These results indicate beneficial effects of exercise in model of HCT-induced seizures in rats, what could be, at least in part, a consequence of improved antioxidant enzymes activity.

Paradoxical sleep deprivation potentiates epilepsy induced by homocysteine thiolactone in adult rats.

There is an intriguing and still poorly understood relationship between sleep deprivation and epilepsy. It has recently been shown that paradoxical sleep deprivation decreases levels of homocysteine, an amino acid involved together with its thiolactone metabolite in epileptogenesis. The aim of the present study was to investigate the effects of paradoxical sleep deprivation on homocysteine thiolactone (H)-induced seizures in rats, a model of generalized seizures. Selective deprivation of paradoxical sleep in adult male Wistar rats was achieved by the platform method. Animals with implanted electrodes for electroencephalogram (EEG) registration were assigned to appropriate experimental conditions (dry cage for control, large platform for stress control and small platform for paradoxical sleep deprivation) and 72 h later were intraperitoneally treated with either H (5.5 mmol/kg) or saline (0.9% NaCl). This study showed that paradoxical sleep deprivation increased the incidence and number of H-induced seizure episodes, shortened latency time to seizures and led to significant rates of lethality after H administration, but without effect on the seizure severity. Paradoxical sleep deprivation increased the number and duration of spikes-and-wave discharges, while decreased latency to its appearance in EEG. Judging by the behavioral and EEG findings, it could be concluded that paradoxical sleep deprivation can provoke the expression of factors that can potentiate H-induced seizures in adult rats.

Different Sensitivity of Various Brain Structures to Thioacetamide-Induced Lipid Peroxidation

Thioacetamide (TAA) exerts hepatotoxic, neurotoxic and carcinogenic effects. The aim of our study was to investigate the effects of TAA on lipid peroxidation and catalase activity in various rat brain regions. Male Wistar rats were divided into following groups: 1. control, saline-treated; 2. thioacetamide-treated groups, TAA300 (300 mg/kg), TAA600 (600 mg/kg) and TAA900 (900 mg/kg). Daily dose of TAA (300 mg/kg) was administered intraperitoneally once (TAA300), twice (TAA600) and three times (TAA900) in consecutive days. Brain samples were collected 24 h after the last dose of TAA and malondialdehyde (MDA) level and catalase activity were determined in cortex, brainstem and hippocampus. MDA level was significantly increased while catalase activity was significantly lower in all brain regions in TAA900 group in comparison with control group. In TAA600 MDA level was increased in the brainstem and cortex when compared to control (p < 0.01). The same dose of TAA 600 mg/kg induced a significant decline in catalase activity in the brainstem and cortex and an increase in its activity in the hippocampus when compared to control (p < 0.01). In TAA300 an increase in MDA level was evident only in the brainstem. Catalase activity was significantly higher in the cortex and hippocampus in TAA300 group in comparison with control (p < 0.01). Based on these results, it may be concluded that various rat brain regions have different sensitivity to TAA-induced lipid peroxidation with hippocampus being less sensitive than cerebral cortex and brainstem.

Influence of NR2B-Selective NMDA Antagonist on Lindane-Induced Seizures in Rats

Ifenprodil is a novel NMDA receptor antagonist that selectively inhibits receptors containing the NR2B subunit. Lindane, a widely used pesticide and scabicide, evokes seizures mainly through the blockade of the γ-aminobutyric acid type A receptor complex. The aim of this study was to determine the effects of ifenprodil on the behavioral and electroencephalographic (EEG) manifestations of seizures in lindane-treated rats. Adult male Wistar rats with three electrodes implanted into the skull were treated intraperitoneally (i.p.) with lindane 8 mg/kg and observed for seizure behavior and EEG during the next 30 min. Seizure behavior was assessed by incidence, severity (determined by a descriptive rating scale ranging from 0 to 4) and duration of latency. Increasing doses of ifenprodil (5, 10, 20 mg/kg, i.p.) or vehicle were injected 30 min prior to lindane administration. Ifenprodil decreased the incidence and severity of lindane seizures and prolonged the latency to seizures in a dose-dependent manner. 20 mg/kg of ifenprodil significantly de- creased the incidence (p < 0.05) and severity (p < 0.05) of seizures when compared to the vehicle treatment. Latency to seizures was significantly prolonged by 10 and 20 mg/kg of ifenprodil. The estimated ED50 value of ifenprodil was 15.53 (5.48–15.20) mg/kg. The lindane-induced bursts of spiking activity in EEG were not completely suppressed by the applied doses of ifenprodil. These results indicate that ifenprodil alleviates behavioral seizures and modifies EEG characteristics of lindane seizures in rats, thus showing the involvement of NMDA receptors containing the NR2B subunit in the mechanisms of lindane convulsions.

Multidisciplinary approach to nitric oxide signaling: Focus on the gastrointestinal and the central nervous system.

Nitric oxide (NO), with close historical ties to cardiovascular physiology, is an important endogenous mediator, the most potent natural vasorelaxant known, involved in many biological functions . NO acts as an intra/intercellular signalling molecule and is a mediator in almost all organ systems . NO is known to occur in many cells types, including vascular endothelial cells, neurons, and epithelial cells . The transmitter of nonadrenergic, noncholinergic (NANC) inhibitory neurons has been the subject of hundreds of investigations over the past 3 decades. Recent evidence suggests that NO may serve as a NANC inhibitory signaling molecule in the gastrointestinal (GI) tract. NO serves as the primary enteric inhibitory neurotransmitter in GI muscles, and nitrergic neurons regulate gut tone, phasic contractile amplitude and frequency, and inhibitory reflexes . In the central nervous system (CNS) NO is involved in some major processes such as memory through longterm potentiation (LTP) and learning . This gasotransmitter also contributes to a pathogenesis of epilepsy. The role of NO in the generation of epilepsy is contradictory since there is evidence of its proconvulsive and anticonvulsive effects . In this review, we will discuss about the possible role of NO as neurotransmitter in the GI and CNS, with focus on the contribution of NO-mediated signaling pathways in the GI motility and CNS excitability. Physiological functions of nitric oxide

Homocysteine thiolactone-induced seizures in adult rats are aggravated by inhibition of inducible nitric oxide synthase

Homocysteine and its metabolites (homocysteine thiolactone (HT)) induce seizures via different but still not well-known mechanisms. The role of nitric oxide (NO) in epileptogenesis is highly contradictory and depends on, among other factors, the source of NO production. The aim of the present study was to examine the effects of aminoguanidine, selective inhibitor of inducible NO synthase (iNOS), on HT-induced seizures. Aminoguanidine (50, 75, and 100 mg/kg, intraperitoneally (i.p.)) was injected to rats 30 min prior to inducing HT (5.5 mmol/kg, i.p.). Seizure behavior was assessed by seizure incidence, latency time to first seizure onset, number of seizure episodes, and their severity during observational period of 90 min. Number and duration of spike and wave discharges (SWDs) were determined in electroencephalogram (EEG). Seizure latency time was significantly shortened, while seizure incidence, number, and duration of HT-induced SWD in EEG significantly increased in rats receiving aminoguanidine 100 mg/kg before subconvulsive dose of HT. Aminoguanidine in a dose-dependent manner also significantly increased the number of seizure episodes induced by HT and their severity. It could be concluded that iNOS inhibitor (aminoguanidine) markedly aggravates behavioral and EEG manifestations of HT-induced seizures in rats, showing functional involvement of iNOS in homocysteine convulsive mechanisms.