Combination of phenobarbital with phenytoin and pregabalin produces synergy in the mouse tonic-clonic seizure model: An isobolographic analysis.

AIMS: Despite many antiepileptic drugs (AEDs) are available to treat epilepsy, there is still about 30% of epilepsy patients inadequately treated with these AEDs. For these patients, polytherapy with two or three AEDs to fully control their seizure attacks is recommended. Unfortunately, polytherapy is always associated with drug interactions, whose nature may be beneficial, neutral or unfavorable. To determine a type of interaction for the combination of three AEDs (i.e., phenobarbital [PB], phenytoin [PHT] and pregabalin [PGB]) at the fixed-ratio of 1:1:1, we used a model of tonic-clonic seizures in male albino Swiss mice. MATERIALS AND METHOD: Tonic-clonic seizures in mice were evoked by a current (sine-wave, 25 mA, 500 V, 0.2 s stimulus duration) delivered via auricular electrodes. The anticonvulsant effects of the three-drug combination (PB, PHT and PGB) in terms of suppression of tonic-clonic seizures in mice were assessed with type I isobolographic analysis. Potential acute side effects for the mixture of PB, PHT and PGB along with total brain concentrations of the AEDs were determined to confirm pharmacodynamic nature of observed interaction. RESULTS: The three-drug combination of PB, PHT and PGB (at the fixed-ratio of 1:1:1) exerted synergistic interaction (at P < 0.01) in the mouse model of tonic-clonic seizures. The combination of PB, PHT and PGB did not produce any side effects in experimental animals, when measuring long-term memory, muscular strength and motor coordination. The measurement of total brain concentrations of PB, PHT and PGB was conducted to confirm that none of the three AEDs significantly influenced total brain concentrations (pharmacokinetic profiles) of the other co-administered AEDs in mice. CONCLUSIONS: The synergistic pharmacodynamic interaction for the combination of PB, PHT and PGB observed in this preclinical study can be translated into clinical settings and this favorable AED combination is worthy of being recommended to some patients with refractory epilepsy.

Chronic unpredictable stress-induced reduction in the hippocampal brain-derived neurotrophic factor (BDNF) gene expression is antagonized by zinc treatment.

Preclinical data indicate the antidepressant activity of zinc and the involvement of the brain-derived neurotrophic factor (BDNF) in this mechanism. The present study investigates the effect of chronic (16 days) combined treatment with zinc (15 mg/kg zinc hydroaspartate) and imipramine (5 mg/kg) in chronic unpredictable stress (CUS) on the BDNF mRNA level in the rat brain. Moreover, serum zinc concentrations were also assessed. CUS induced a significant reduction in the BDNF mRNA level in the hippocampus by 21% but had no effect in the frontal cortex. Repeated treatment with zinc induced a significant increase in the BDNF mRNA level in the hippocampus in the unstressed animals by 12% and as in the chronically stressed animals by 14%, compared to the appropriate controls. Imipramine treatment did not affect this factor. However, combined treatment of zinc and imipramine induced a 12% elevation of the BDNF mRNA level in the stressed but not in the unstressed rats. CUS induced a 19% reduction in the serum zinc concentration, whereas combined treatment of zinc and imipramine reduced this concentration by 24% in the unstressed and increased it (by 20%) in the stressed animals. These results indicate that: 1) CUS induces a reduction in the BDNF gene expression with a concomitant diminution of serum zinc concentration and 2) the CUS-induced reduction in the BDNF gene expression is antagonized by chronic treatment with zinc.

Different pattern of changes in calcium binding proteins immunoreactivity in the medial prefrontal cortex of rats exposed to stress models of depression.

Reductions in the number and size of neurons in the medial prefrontal cortex (mPFC) have been documented in many post-mortem studies of depressed patients and animals exposed to stress. Here, we examined the effect of chronic unpredictable stress (CUS) and chronic mild stress (CMS) on specific populations of neurons in the rat mPFC. Antibodies directed against parvalbumin (PV), calbindin D-28K (CB) and active caspase-3 have been used to quantify the numerical density of PV-immunoreactive (PV-ir), CB-ir and active caspase-3-ir cells, and to measure the relative optical density of neuropil. CUS decreased the density of CB-ir neurons and the optical density of CB-ir neuropil. In turn, CMS increased the densities of both CB-ir neurons and neuropil, while PV-ir neurons and PV-ir neuropil were not changed. The frequency distribution of neuronal surface areas was significantly different only for PV-ir neurons, and only between the control and CUS group. CMS reduced the density of active caspase-3-ir cells while CUS did not. We concluded that the mPFC reveals a different pattern of changes in neurons containing calcium binding proteins and active caspase-3 immunoreactivity in response to CUS and CMS.

Alterations in hippocampal calcium-binding neurons induced by stress models of depression: a preliminary assessment.

In this study, the neuropathological changes induced by chronic unpredictable stress (CUS) and chronic mild stress (CMS) in calbindin D-28K (CB) and parvalbumin (PV) immunoreactive neurons in the rat hippocampus were demonstrated. We used immunohistochemical techniques to quantify the numerical density and morphological changes of PV immunoreactive and CB immunoreactive neurons in the dentate gyrus (DG) and the CA1 and CA3 regions of the hippocampus. We also assessed cell proliferation (Ki-67) and apoptotic processes (active caspase-3) in the DG. We found a significant decrease (16.6% for CUS and 13.3% for CMS) in the numerical density of granule cells (GC), alterations in the CB immunoreactive cells of the GC in the DG and an impairment of mossy fiber CB immunolabelling in the CA3. These changes were not accompanied by a decrease in Ki-67 labeling or the level of caspase-3 in the DG. These data indicate a stress-induced reduction of calcium binding neuron parameters, which may be related to the behavioral paradigms exhibited in these models.

Influence of zinc supplementation on imipramine effect in a chronic unpredictable stress (CUS) model in rats.

Zinc is an endogenous modulator of neuronal activity and may play an important role in the pathogenesis of depression. Recent studies have shown that zinc exhibits antidepressant-like activity in some models of depression in rodents. Our previous studies have shown that the footshock-induced fighting behavior was reduced in the rats subjected to chronic unpredictable stress (CUS). This test is used as the new experimental model of depression. Various antidepressant drugs given repeatedly prevented this kind of behavioral depression. The aim of the present study was to evaluate the effect of prolonged treatment with zinc hydroaspartate and to examine if zinc supplementation could modulate the imipramine effect in CUS model of behavioral depression in rats. The experiments were carried out on male Wistar rats. Chronic stress (persisting for 16 days) was induced by the modified method described by Katz et al. Zinc hydroaspartate at the dose of 30 mg/kg/day or 15 mg/kg/day and imipramine at the dose of 5 mg/kg/day were administered once daily for 14 days. Imipramine was given (ip) 1 h before every stress session and zinc hydroaspartate (ip) l h before the antidepressant. The footshock-induced fighting behavior test was performed 48 h after the last session of the chronic stress. It was demonstrated that in chronically stressed rats the number of fighting attacks was significantly reduced (by about 75%). Zinc hydroaspartate at the dose of 30 mg/kg/day, given alone, prevented the deficit in fighting behavior in chronically stressed rats. Neither imipramine at the dose of 5 mg/kg/day nor zinc hydroaspartate (15 mg/kg/day) administered alone changed the intensity of fighting behavior in chronically stressed rats. However, when imipramine was given at the same dose in the rats pretreated with zinc hydroaspartate (15 mg/kg/day) the deficit of fighting behavior was not observed. The present results indicate that zinc similarly to antidepressants protects the rats against the CUS-induced behavioral depression. Moreover, our findings suggest that zinc supplementation could potentiate the antidepressant effect of imipramine.

Effect of NMDA receptor antagonists on behavioral impairment induced by chronic treatment with dexamethasone.

Severe and prolonged stress but also long-term treatment with glucocorticoids (GCs) have been described to cause brain damage (especially hippocampal and striatal neurons) in humans as well as in animals. GCs potentiate stress or ischemia-induced accumulation of excitatory amino acids (EAA) in the extracellular space of the hippocampus. It was shown that EAA play a major role in various neurologic disorders with cognitive dysfunction. Many authors suggested the neuroprotective effect of N-methyl-D-aspartate (NMDA) glutamate receptor antagonists in some acute or chronic neurodegenerative diseases. On the other hand, many NMDAreceptor antagonists produce highly undesirable side-effects at the doses within their putative therapeutic range. The aim of the present study was to evaluate the behavioral effects (memory performance, motor coordination, lethality and body weight) of MK-801 or memantine (MEMAN, non-competitive NMDAreceptor antagonists) (at the doses of 25 and 50 microg/kg/day or 2.5 and 5.0 mg/kg/day, respectively) on neurotoxicity induced by dexamethasone (DEX) administered chronically at the doses of 40 or 80 mg/kg/day in mice. It was shown that prolonged treatment (for 10 days) with DEX at the dose of 80 mg/kg/day (but not at 40 mg/kg/day) significantly decreased the retention time in the memory task in mice and impaired the motor coordination in "chimney" test. Neither MK-801 nor MEMAN (at the both doses used) were able to counteract the behavioral impairment induced by DEX administration. Moreover, the potentiation of the body weight reduction and lethality induced by DEX were noted in mice co-treated with MK-801 or MEMAN. The above findings suggest that MK-801 or MEMAN at the doses used have no neuroprotective effect. On the contrary, both NMDA receptor antagonists potentiate the toxicity of DEX given chronically.

Effect of chronic treatment with dexamethasone on brain dopamine receptors in mice.

Glucocorticoids are expressed in the central nervous system. Radioligand binding studies have shown their presence in the neurons of the limbic system, a structure involved in mood control and subtle regulation of hypothalamic-pituitary-adrenal (HPA) axis. Structures of the limbic system are also rich in dopaminergic innervation. It has been hypothesized that glucocorticoids may be important in causing and perpetuating depression. Our previous study has demonstrated that dexamethasone decreases the locomotor activity of mice and counteracts the hyperactivity induced by agonists of dopamine receptors. The aim of the present study was to find the possible mechanism responsible for these behavioral effects of dexamethasone. So we sought to examine the influence of chronic dexamethasone treatment on selective radioligand binding to dopamine D(1) ([(3)H]SCH 23390) and D(2) ([(3)H]spiperone) receptors in the brain of mice. The male Albino Swiss mice received dexamethasone (4, 8 or 16 mg/kg/day) for 14 days. The striatum and limbic system structures were isolated and the binding procedure was performed 3.5 or 48 h after the last injection. It was shown that 3.5 h after the last dose of dexamethasone (4 mg/kg/day), specific D(2) receptor binding was statistically significantly increased (by 64%) in the limbic system. On the contrary, the tendency to the reduction of specific D(2) receptor binding was observed in the striatum. Dexamethasone treatment did not influence the specific binding to D(1) receptors in any structure of the brain.

Antidepressants in chronic unpredictable mild stress (CUMS)-induced deficit of fighting behavior.

Chronic unpredictable stress (CUS) is one of the behavioral models resembling in some respects (loss of normal aggresiveness) human depression. In the present study, consistent with the ethical principles for scientific experiments on animals, we have decided to modify the CUS procedure. In this new modified model named chronic unpredictable mild stress (CUMS), we have introduced mild stressor (14 h period of 45 degrees cage tilt) instead of one severe stressor (20 s exposure to electric footshock). The purpose of the present study was to determine whether this new procedure CUMS, similarly to CUS, affected the footshock-induced fighting behavior. We have also investigated the effect of antidepressant drugs with different pharmacological profiles (imipramine, mianserin, fluoxetine, moclobemide, tianeptine) and anxiolytic drug (oxazepam) on fighting behavior in rats submitted to CUMS. It was found that in rats subjected to CUMS procedure the number of fighting attacks was significantly reduced (by about 80%). Prolonged treatment (once daily, for 14 days) with imipramine (10 mg/kg/day), tianeptine (12.5 mg/kg/day), mianserin (10 mg/kg/day), moclobemide (50 mg/kg/day), fluoxetine (10 mg/kg/day), but not oxazepam (5 mg/kg/day) prevented the deficit in fighting behavior in rats subjected to CUMS. In conclusion, the results of the present study indicate that CUMS, similarly to CUS procedure, induced behavioral deficit in rats which was normalized by antidepressants with a different pharmacological profile.

Histological examination of the kidney after experimental administration of MK-801 and dexamethasone.

The aim of the research was histological assessment of the influence of MK-801 (NMDA receptor antagonist) and dexamethasone on the kidney. The experiment was carried out on adult Albino-Swiss mouse males. MK-801 was administered in the dose of 0.3 mg/kg/24 h for 8 days, dexamethasone--in the toxic dose of 120 mg/kg/24 h. Kidney slices stained with hematoxylin and eosin and with PAS method were examined with light microscope. The performed experiments revealed that MK-801 causes morphological changes in the shape of slight narrowing of the urinary spaces in renal corpuscles and narrowing of the lumen of the proximal convoluted tubules and dexamethasone administered in toxic doses causes dilatation of these spaces with kidney's hyperemia. MK-801 intensifies morphological changes of the kidney induced by toxic doses of dexamethasone.

Effect of imipramine on brain D-1 and 5-HT-2A receptors in a chronic unpredictable stress model in rats.

Chronic unpredictable stress (CUS) model of depression is one of the well validated animal models of depression. In this paper, we report the results of investigations into dopaminergic D-1 and serotonergic 5-HT-2A receptors in the brain of rats subjected to CUS procedure and treated chronically with imipramine. We have examined the dopaminergic D-1 ([3H-SCH 23390) in the limbic area and serotonergic 5-HT-2A ([3H-ketanserin) receptors in the cerebral cortex by a saturation radioligand binding method in rats subjected to CUS paradigm, imipramine, both CUS and imipramine and control animals. CUS procedure resulted in a significant 36% increase in the D-1 receptor density in the limbic system, which was attenuated by chronic imipramine treatment. Also a 21% increase in the density of 5-HT-2A receptors in the cerebral cortex induced by CUS was reduced by chronic imipramine treatment. The present data indicate that the increases in the density of brain D-1 and 5-HT-2A receptors of rats subjected to CUS, which are "normalized" by imipramine, might be involved in the pathophysiology of "animal depression" (and, thus, in pathophysiology of human depression) and in the mechanism of antidepressant therapy.

Repeated treatment with selective serotonin reuptake inhibitors but not anxiolytics prevents the stress-induced deficit of fighting behavior.

Several animal models of "depression" have been examined. One of them is chronic unpredictable stress (CUS)-induced deficit of fighting behavior in rats. In the present study, we compared the effects of two antidepressants (fluoxetine or fluvoxamine) and three anxiolytics (buspirone, lorazepam or oxazepam) on the electric footshock-induced fighting behavior in the pairs of male Wistar rats exposed to CUS procedure (16-day application of various unpredictable stressors). It was found that, in chronically stressed rats, the number of fighting attacks was significantly reduced (by about 70%). Prolonged (for 14 days) treatment of rats with fluoxetine or fluvoxamine (both at the dose of 10 mg/kg/day) counteracted the deficit of aggression induced by the chronic stress. On the contrary, the anxiolytics: lorazepam (0.5 mg/kg/day), oxazepam (5 mg/kg/day) or buspirone (0.2 mg/kg/day) administered for 14 days, did not modify the deficit of fighting induced by CUS procedure. It must be underlined that prolonged treatment with all used drugs did not change the intensity of fighting in normal (unstressed) rats. In conclusion, prolonged treatment with antidepressant drugs prevents the CUS-induced deficit of fighting behavior, whereas no beneficial effect of anxiolytic agents was found.