Excess of miRNA-378a-5p perturbs mitotic fidelity and correlates with breast cancer tumourigenesis in vivo.

BACKGROUND: Optimal expression and proper function of key mitotic proteins facilitate control and repair processes that aim to prevent loss or gain of chromosomes, a hallmark of cancer. Altered expression of small regulatory microRNAs is associated with tumourigenesis and metastasis but the impact on mitotic signalling has remained unclear. METHODS: Cell-based high-throughput screen identified miR-378a-5p as a mitosis perturbing microRNA. Transient transfections, immunofluorescence, western blotting, time-lapse microscopy, FISH and reporter assays were used to characterise the mitotic anomalies by excess miR-378a-5p. Analysis of microRNA profiles in breast tumours was performed. RESULTS: Overexpression of miR-378a-5p induced numerical chromosome changes in cells and abrogated taxol-induced mitotic block via premature inactivation of the spindle assembly checkpoint. Moreover, excess miR-378a-5p triggered receptor tyrosine kinase-MAP kinase pathway signalling, and was associated with suppression of Aurora B kinase. In breast cancer in vivo, we found that high miR-378a-5p levels correlate with the most aggressive, poorly differentiated forms of cancer. INTERPRETATION: Downregulation of Aurora B by excess miR-378a-5p can explain the observed microtubule drug resistance and increased chromosomal imbalance in the microRNA-overexpressing cells. The results suggest that breast tumours may deploy high miR-378a-5p levels to gain growth advantage and antagonise taxane therapy.

Best use of the recommended IFCC reference method, material and values in HbA1C analyses.

The results of Finnish HbA(1C) surveys (Labquality Ltd.) during the past 10 years have undergone continuous improvement with smaller overall coefficients of variation for the HbA(1C) mean values of all methods (from 7.5 to 5.4% for normal and from 8.9 to 4.7% for diabetic samples). Most of the HbA(1C) methods are certified for traceability to the Diabetes Control and Complication Trial (DCCT) designated comparison method, which originally was a high-performance liquid chromatography (HPLC) method (Bio-Rex 70, Bio-Rad) but is no longer in routine use. It was therefore important that the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) had prepared both reference preparations and method for the determination of HbA(1C). However, the very demanding reference method is not realistic for use in clinical laboratories. According to the present study, the mean HbA(1C) values of the Labquality Ltd. showed significant correlations to the HbA(1C) values of The European Reference Laboratory for Glycohemoglobin (r = 0.999) and to the values using the IFCC method (r = 0.999). The reference values of the IFCC method (mainly those of the manufacturer) range from 2.85 to 3.81%, being significantly lower than the present DCCT values (4.0-6.1%). Since it may take some time before consumers are ready to accept the new IFCC reference values for general use, we propose that the IFCC reference materials and method should be used for calibration of the present methods to the well-known DCCT levels.

Effect of lamotrigine treatment on status epilepticus-induced neuronal damage and memory impairment in rat.

Status epilepticus causes neuronal damage that is associated with cognitive impairment. The present study examined whether a novel antiepileptic drug, lamotrigine (LTG), alleviates status epilepticus-induced temporal lobe damage and memory impairment, and compared its efficacy with carbamazepine. Status epilepticus was induced by electric stimulation of the perforant pathway (PP) in rats. Treatment with LTG (12.5 mg/kg, twice a day) was started either 3 days before (preLTG group) or 1 h after (postLTG group) a 60 min PP stimulation. Treatment with carbamazepine (CBZ; 30 mg/kg, twice a day) was started 3 days before (CBZ group) a 60 min PP stimulation. All treatments were continued for 2 weeks. Thereafter, the severity of seizures, seizure-induced neuronal damage, quantitative electroencephalogram (EEG), and memory impairment were compared between vehicle-treated unstimulated and stimulated controls, LTG-treated rats, and CBZ-pretreated rats. Both in the preLTG and postLTG groups, damage to hilar somatostatin-immunoreactive neurons, hippocampal CA3b and CA3a pyramidal cells, and the piriform cortex was mild and did not differ from that in unstimulated controls. Furthermore, CA3c damage in the preLTG group did not differ from that in unstimulated controls. Vehicle-treated stimulated controls and CBZ-pretreated rats, however, had significant damage in the hilus, CA3 subregions, and piriform cortex compared with unstimulated controls (P<0.05 for the stimulated side, contralateral side, or both). Treatment with LTG or CBZ had no effect on the number or duration of behavioral seizures during PP stimulation. They did not affect the baseline EEG or status epilepticus-induced slowing of the EEG. Also, the status epilepticus-induced spatial memory impairment in the Morris water-maze was not attenuated by treatment with LTG or CBZ. Our data demonstrate that treatment with LTG has a mild neuroprotective effect on status epilepticus-induced neuronal damage in rats even when administered after the beginning of status epilepticus.

Chronic elevation of brain GABA levels beginning two days after status epilepticus does not prevent epileptogenesis in rats.

Vigabatrin (VGB) treatment is neuroprotective in various models of status epilepticus (SE) and delays the development of kindling via mechanisms that are assumed to relate to the elevation of GABA levels in the brain. Here, we tested the hypothesis that a chronic elevation of brain GABA levels obtained by VGB treatment prevents the development of spontaneous seizures (i.e. epilepsy) following SE in rats. Self-sustained SE (SSSE) was induced by stimulating the lateral nucleus of the amygdala. Two days later, chronic VGB (75 mg/kg/day) or saline treatment was started via subcutaneous osmotic minipumps. The development of spontaneous seizures was monitored once a week (24 h at a time) using video-EEG recording. Rats were perfused for histology either at the end of the 10-week drug treatment, or later at the end of an 8-week drug-free follow-up period. Before perfusion for histology, spatial learning and memory perform was tested in the Morris water-maze. Spontaneous seizures were observed in 55% (6/11) of the saline-treated and 73% (8/11) of the VGB-treated rats during the 10-week treatment period. Seizure frequency, severity, and duration were similar in VGB-treated rats and controls during and after the drug-treatment period. VGB treatment did not decrease neuronal damage in various temporal lobe regions or mossy fiber sprouting. VGB treatment also did not attenuate spatial learning or memory impairments. These findings indicate that the augmentation of GABAergic neurotransmission by VGB does not prevent the development of epilepsy when treatment is started 2 days after SE.

Nitric oxide synthase immunoreactivity in the rat hippocampus after status epilepticus induced by perforant pathway stimulation.

Nitric oxide has recently been implicated in mediation of neuronal excitotoxicity and damage. This study aimed at elucidating the changes in the expression of neuronal isoform of nitric oxide synthase (nNOS) in the hippocampus after status epilepticus induced by perforant pathway stimulation. nNOS-immunoreactivity (nNOS-ir) and neuronal damage, assessed by silver staining, were evaluated separately in different hippocampal subfields 2 weeks after induction of status epilepticus. Perforant pathway stimulation resulted in an increase in the number of nNOS-immunoreactive neurons in the stratum radiatum of the CA1 and CA3 subfields of the hippocampus proper, and the hilus of the dentate gyrus. The morphology and distribution of the nNOS-ir neurons resembled that of interneurons. No correlation of the number of nNOS-ir neurons to the neuronal damage score was observed. Our results suggest that status epilepticus provokes a de novo expression of nNOS protein, and the nNOS expressing neurons may be selectively resistant to epileptic brain injury.

A new model of chronic temporal lobe epilepsy induced by electrical stimulation of the amygdala in rat.

Spontaneous seizures are the hallmark of human epilepsy but they do not occur in most of the epilepsy models that are used to investigate the mechanisms of epilepsy or to test new antiepileptic compounds. This study was designed to develop a new focal epilepsy model that mimics different aspects of human temporal lobe epilepsy (TLE), including the occurrence of spontaneous seizures. Self-sustained status epilepticus (SSSE) lasting for 6-20 h was induced by a 20-30 min stimulation of the lateral nucleus of the amygdala (100 ms train of 1 ms, 60 Hz bipolar pulses, 400 microA, every 0.5 s). Stimulated rats (n = 16) were monitored with a video-EEG recording system every other day (24 h/day) for 6 months, and every other video-EEG recording was analyzed. Spontaneous epileptic seizures (total number 3698) were detected in 13 of the 15 animals (88%) after a latency period of 6 to 85 days (median 33 days). Four animals (31%) had frequent (697-1317) seizures and 9 animals (69%) had occasional seizures (1-107) during the 6-months follow-up period. Fifty-seven percent of the seizures occurred during daytime (lights on 07:00-19:00 h). At the end of the follow-up period, epileptic animals demonstrated impaired spatial memory in the Morris water-maze. Histologic analysis indicated neuronal loss in the amygdala, hippocampus, and surrounding cortical areas, and mossy fiber sprouting in the dentate gyrus. The present data indicate that focal stimulation of the amygdala initiates a cascade of events that lead to the development of spontaneous seizures in rats. This model provides a new tool to better mimic different aspects of human TLE for investigation of the pathogenesis of TLE or the effects of new antiepileptic compounds on status epilepticus, epileptogenesis, and spontaneous seizures.

Status epilepticus-induced neuronal damage in the rat amygdaloid complex: distribution, time-course and mechanisms.

The present study was designed to elucidate the distribution, time-course and mechanism(s) of status epilepticus-induced neuronal damage in the rat amygdaloid complex. Status epilepticus was induced with kainate (9 mg/kg, i.p.), and the behavioral and electrographic seizure activity of each rat was monitored via cortical electrodes attached to a continuous video electrocorticogram system. Rats were subsequently perfused 1, 2, 4, 8, 16, 24 or 48 h after kainate injection. The first signs of amygdaloid damage were seen in rats perfused 4 h after kainate injection, though the severity and temporal appearance of damage varied substantially between the different amygdaloid nuclei and their subdivisions. Second, terminal transferase dUTP nick-end labeling (TUNEL)-positive nuclei and laddering of DNA in gel electrophoresis appeared in the amygdala 8 and 16 h after kainate, respectively. The distribution and density of TUNEL-positive nuclei in the different amygdaloid nuclei correlated with the distribution of neuronal damage in Thionin- and silver-stained sections. Third, the immunoreactivity of Bax protein, a promoter of apoptotic neuronal death, increased in the vulnerable medial division of the lateral nucleus prior to the appearance of argyrophilic neurons and TUNEL-positive nuclei. Fourth, the severity of neuronal damage progressed in some, but not all, amygdaloid regions throughout the 48-h follow-up, even though the occurrence of high-amplitude and frequency discharges, which are typically associated with behavioral seizure activity, extinguished after 7 h. These data show that status epilepticus-induced neuronal damage in the amygdala is a dynamic region-specific process, the severity of which depends on the duration of seizure activity. At least one mechanism underlying the damage involves apoptosis, which continues long after the behavioral and electrographic seizures have subsided.

Neuroprotective effect of remacemide hydrochloride in a perforant pathway stimulation model of status epilepticus in the rat.

Previous studies have demonstrated that remacemide and its desglycinyl metabolite, AR-R 2495AA, reduce neuronal damage in animal models of ischemia, subarachnoid hemorrhage, and traumatic brain injury. The aim of the present study was to investigate whether remacemide hydrochloride also alleviates seizure-induced neuronal damage in a model of status epilepticus induced by the stimulation of the perforant pathway (PP) in the rat. Chronic oral remacemide treatment (3 x 25 mg/kg/day) was started either 2 days before or 2 h after the beginning of PP stimulation (2 mA, 20 Hz, 0.1 ms pulse duration for 60 min). The effects of remacemide treatment on the severity of seizures, electroencephalogram (EEG) parameters, seizure-induced neuronal damage in the temporal lobe regions, and memory impairment were compared to unstimulated and stimulated vehicle-treated controls, and carbamazepine-pre-treated (3 x 40 mg/kg/day) rats. Both remacemide and carbamazepine pretreatments, but not remacemide posttreatment, decreased pyramidal cell damage in the CA3 and CA1 subregions of the hippocampus (P < 0.05). In addition, overall neuronal damage in the extrahippocampal temporal lobe regions (the piriform cortex, entorhinal cortex, and the amygdaloid complex) was milder in remacemide-pretreated rats compared to stimulated control rats (P < 0.01). The neuroprotective effect was most evident on the side contralateral to stimulation. Remacemide or carbamazepine pretreatment had no evident effect on the number or duration of behavioral seizures during PP stimulation. Neither drug altered the spectral parameters of the baseline EEG or prevented status epilepticus-induced EEG slowing observed 2 weeks after PP stimulation. Nor did remacemide or carbamazepine treatment alleviate spatial memory impairment determined in a Morris water-maze task 2 weeks after PP stimulation. Our data provide evidence that pretreatment with remacemide has a moderate neuroprotective effect against status epilepticus-induced neuronal damage.

Effects of vigabatrin treatment on status epilepticus-induced neuronal damage and mossy fiber sprouting in the rat hippocampus.

Selective neuronal damage and mossy fiber sprouting may underlie epileptogenesis and spontaneous seizure generation in the epileptic hippocampus. It may be beneficial to prevent their development after cerebral insults that are known to be associated with a high risk of epilepsy later in life in humans. In the present study, we investigated whether chronic treatment with an anticonvulsant, vigabatrin (gamma-vinyl GABA), would prevent the damage to hilar neurons and the development of mossy fiber sprouting. Vigabatrin treatment was started either 1 h, or 2 or 7 days after the beginning of kainic acid-induced (9 mg/kg, i.p.) status epilepticus and continued via subcutaneous osmotic minipumps for 2 months (75 mg/kg per day). Thereafter, rats were perfused for histological analyses. One series of horizontal sections was stained with thionine to estimate the total number of hilar neurons by unbiased stereology. One series was prepared for somatostatin immunohistochemistry and another for Timm histochemistry to detect mossy fiber sprouting. Our data show that vigabatrin treatment did not prevent the decrease in the total number of hilar cells, nor the decrease in hilar somatostatin-immunoreactive (SOM-ir) neurons when SOM-ir neuronal numbers were averaged from all septotemporal levels. However, when vigabatrin was administered 2 days after the onset of status epilepticus, we found a mild neuroprotective effect on SOM-ir neurons in the septal end of the hippocampus (92% SOM-ir neurons remaining; P < 0.05 compared to the vehicle group). Vigabatrin did not prevent mossy fiber sprouting regardless of when treatment was started. Rather, sprouting actually increased in the septal end of the hippocampus when vigabatrin treatment began 1 h after the onset of status epilepticus (P < 0.05 compared to the vehicle group). Our data show that chronic elevation of brain GABA levels after status epilepticus does not have any substantial effects on neuronal loss or mossy fiber sprouting in the rat hippocampus.

Hippocampal damage after injection of kainic acid into the rat entorhinal cortex.

Several experimental models of epilepsy have used kainic acid in animals to induce seizures and neuropathological changes which mimic those observed in human temporal lobe epilepsy. These models differ in the location and manner in which kainic acid is applied. In the present study, we characterized the seizure activity and neuropathological changes that occur in awake rats after kainic acid (25 ng/250 nl) is injected into the entorhinal cortex of freely moving rats. In 91% of the animals, this induced generalized motor seizures. Moreover, all of the animals survived status epilepticus. Animals were perfused two weeks after the injection for neuropathological examination. Silver-impregnation revealed that kainic acid caused pyramidal cell damage which was most severe in the CA1 subfield and to a lesser degree in the CA3c area. A loss of NADPH diaphorase-containing neurons in the hilus and the CA1 area was also consistently seen and, in most cases, a population of somatostatin-immunoreactive neurons was diminished. Our findings show that a minute amount of kainic acid delivered directly to the entorhinal cortex on unanesthetized animals reliably produces generalized seizures as well as a consistent pattern of cell damage in the hippocampus. Therefore, this model may be suitable for investigating the mechanisms underlying temporal lobe epilepsy, and may prove useful in assessing different treatment strategies for preventing seizure-induced structural damage.

Comparison of NADPH diaphorase histochemistry, somatostatin immunohistochemistry, and silver impregnation in detecting structural and functional impairment in experimental status epilepticus.

Nitric oxide has been postulated as a retrograde intercellular messenger for long-term potentiation, a form of synaptic plasticity that is associated with learning and memory processes. In the present study we investigated whether the loss or survival of nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase-containing neurons, which are known to synthesize nitric oxide, would be an useful indicator for evaluating the structural and functional state of the rat hippocampus after status epilepticus that is induced by intraperitoneal injection of kainic acid. Besides NADPH diaphorase histochemistry, two other histological parameters were studied: the grade of cell damage evaluated from silver-impregnated sections, and the number of somatostatin-containing neurons in different hippocampal subfields. We found that the number of NADPH diaphorase-containing neurons in the hilus and granule cell layer correlated well with spatial learning and memory performance as assessed by the Morris water-maze test. The extent of cell damage in the CA1 subfield analysed in silver-impregnated sections and the number of hilar somatostatin-containing neurons also significantly correlated with latencies in the water-maze test. Furthermore, linear regression analysis revealed that the number of somatostatin-containing neurons in the hilus explains about 50% of the variation in water-maze learning. These findings emphasize that although general structural preservation is of crucial importance for the function of the hippocampus also interneurons, such as somatostatin- and NADPH diaphorase-containing neurons, may play an important role during the acquisition phase and processing of information in hippocampal circuitry. Therefore, in addition to evaluating general cell damage, analysis of the cell loss that occurs in the interneuron subpopulations will be beneficial in verifying structural and functional deficits of the hippocampus after status epilepticus.

A crucial role of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid subtype of glutamate receptors in piriform and perirhinal cortex for the initiation and propagation of limbic motor seizures.

The role of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors in the initiation and propagation of limbic motor seizures in rats was examined by the intracerebral and systemic administration of 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo (f) quinoxaline (NBQX), a selective antagonist of the AMPA subtype of glutamate receptor. Limbic motor seizures were evoked focally by the application of the gamma-aminobutyric acid receptor antagonist, bicuculline, into area tempestas, an epileptogenic site in the deep anterior piriform cortex. Before eliciting seizures, NBQX was applied focally into either 1) area tempestas or 2) perirhinal or posterior piriform cortex ipsilateral to the area tempestas from which seizures were evoked. In addition, pretreatment with i.p. NBQX was evaluated for anticonvulsant actions against area tempestas-evoked clonic or systemically evoked tonic seizures. In all conditions, a dose-dependent decrease in the severity of seizures was obtained with NBQX. With focal intracerebral administration, a dose of 500 pmol of NBQX consistently protected against limbic motor seizures, with partial protection achieved with 100 pmol. After i.p. administration, 2.5 and 5.0 mg/kg significantly protected the rats from both limbic motor seizures and tonic extensor seizures. No overt disturbance of spontaneous behavior was associated with the anticonvulsant doses of NBQX. Moreover, both forebrain substrates of limbic motor seizures and hindbrain substrates of tonic extensor seizures were highly susceptible to disruption by NBQX. The results indicate that AMPA subtype of glutamate receptors are crucial mediators of seizure propagation via perirhinal and piriform cortics.

Decrease in somatostatin-immunoreactive neurons in the rat amygdaloid complex in a kindling model of temporal lobe epilepsy.

In human temporal lobe epilepsy, seizures can begin in the hippocampus, amygdala, or surrounding cortical areas. Histologically, the seizure-induced selective neuronal damage and synaptic reorganization are best documented in the hippocampus. Little information is available about the damage in the other temporal lobe structures or whether the distribution of damage depends on the location of the primary seizure focus. We used an amygdala-kindling model of temporal lobe epilepsy to study whether seizures of amygdaloid origin cause damage to the amygdala and hippocampus. All rats experienced five class 5 generalized seizures. Neuronal damage was assessed by counting the density of GABA-immunoreactive (GABA-ir) and somatostatin-immunoreactive (SOM-ir) neurons in the amygdala and hilus of the dentate gyrus six months after the last seizure. We found that the density of GABA-ir neurons did not differ from that in controls in the contralateral amygdala. The density of SOM-ir neurons was, however, decreased in the lateral (69% of neurons remaining, P < 0.01), basal (67% remaining, P < 0.05), and accessory basal (68% remaining, P < 0.05) nuclei. In the hilus, the densities of GABA-ir and SOM-ir neurons were similar to that in controls. According to our data, a few seizures of amygdaloid origin may cause more severe damage to SOM-ir neurons in the amygdala than in the hilus. Such decrease in SOM-ir neurons which form one subpopulation of GABAergic inhibitory interneurons may increase the local excitability in the amygdala and, therefore, contribute to epileptogenesis.

Status epilepticus causes selective regional damage and loss of GABAergic neurons in the rat amygdaloid complex.

In human epilepsy, the amygdala is often a primary focus for seizures. To analyse the status epilepticus-induced alterations in the amygdaloid circuitries which may later underlie epileptogenesis, we studied the amygdaloid damage in kainic acid and perforant pathway stimulation models of status epilepticus in the rat. We also studied the damage to inhibitory GABAergic neurons. In both models, the medial division of the lateral nucleus, the parvicellular division of the basal nucleus and portions of the anterior cortical and medical nuclei were damaged. In the kainate model, where the seizure activity was more severe, the accessory basal nucleus, amygdalohippocampal area, posterior cortical nucleus and periamygdaloid cortex were also damaged. Two weeks after kainate-induced seizures, 56% of the GABA-immunoreactive neurons remained in the lateral nucleus (P < 0.05) and 25% in the basal nucleus (P < 0.01). Further analysis showed that one subpopulation of damaged GABAergic neurons was immunoreactive for somatostatin (48% remaining in the lateral nucleus, P < 0.01; 33% in the basal nucleus, P < 0.01). In the perforant pathway stimulation model, the damage to somatostatin neurons was milder. According to our data, the initial insult, such as status epilepticus, selectively damages amygdaloid nuclei. The loss of inhibition may underlie the spontaneous generation of seizures and epileptogenesis. On the other hand, many amygdaloid output nuclei (magnocellular and intermediate division of the basal nucleus, the central nucleus) remained relatively undamaged, providing pathways for seizures spread and generation of seizure-related behavioural manifestations such as motor convulsions and fear response.

Seizure-induced damage to the hippocampus is prevented by modulation of the GABAergic system.

A variety of cerebral insults induce neuronal damage to the hippocampal formation. The somatostatin-immunoreactive (SOM-ir) neurones in the dentate hilus are particularly vulnerable. In the present study, we demonstrated that augmentation of hippocampal GABAergic inhibition by chronic infusion of gamma-vinyl GABA prevented the delayed seizure-induced damage to hilar SOM-ir neurones. Selective lesions of the cholinergic, serotonergic or noradrenergic pathways to the hippocampus did not attenuate the seizure-induced loss of SOM-ir neurones; rather, the damage was exacerbated by the cholinergic lesion. It is, therefore, the intrahippocampal GABAergic circuitries, rather than the selective subcortical pathways, that are critical for neuroprotection after seizures. Enhanced GABAergic inhibition in the hippocampus prevented damage to hilar SOM-ir neurones, even when started 2 days after status epilepticus. GABAergic agents may thus provide an alternative treatment for delayed neuronal damage caused by cerebral insults.

Vigabatrin and carbamazepine have different efficacies in the prevention of status epilepticus induced neuronal damage in the hippocampus and amygdala.

The present study compares the efficacy of carbamazepine (20 mg/kg/day) and vigabatrin (250 mg/kg/day) in preventing hippocampal and amygdaloid damage in the perforant pathway stimulation model of status epilepticus in the rat. One group of rats received a combination of the drugs. Drug treatments were started one week before the stimulation and continued for two weeks thereafter. Gallyas silver impregnation and somatostatin immunohistochemistry were used to detect neuronal damage. All drug treatments were equally effective in decreasing the number and severity of seizures during electrical stimulation. In the vigabatrin group, the damage to the hilar somatostatin-immunoreactive (SOM-ir) neurons and hippocampal CA3c pyramidal cells was less severe than in the vehicle (SOM-ir, P < 0.01; CA3c, P < 0.05) and carbamazepine (SOM-ir, P < 0.01; CA3c, P < 0.05) groups. In the carbamazepine and combination groups, the severity of neuronal damage in the hippocampus did not differ from that in vehicle-treated animals. The amygdaloid neurons were not protected by any of the treatments. Our results show that even though vigabatrin and carbamazepine treatments had similar anticonvulsant efficacy during the perforant pathway stimulation, only vigabatrin but not carbamazepine decreased seizure-induced neuronal damage. Vigabatrin decreased neuronal damage in the hippocampus but not in the amygdala. These results demonstrate that different brain regions and neuronal networks may be protected unequally by different anticonvulsants.

Tiagabine prevents seizures, neuronal damage and memory impairment in experimental status epilepticus.

A novel antiepileptic drug, tiagabine ((R)-N-[4,4-di-(3-methylthien-2-yl) but-3-enyl] nipecotic acid hydrochloride), was studied in rats in order to determine its efficacy in preventing seizures, seizure-induced neuronal damage and impairment of spatial memory in the perforant pathway stimulation model of status epilepticus. In pilot experiments, administration of tiagabine (50, 100 or 200 mg/kg/day) with subcutaneously implanted Alzet osmotic pumps led to a dose-dependent increase in tiagabine concentrations in the serum and brain. Two days of tiagabine treatment at a dose range of 50-200 mg/kg/day did not change the levels of gamma-aminobutyric acid (GABA), glutamate or aspartate in cisternal cerebrospinal fluid (CSF) compared to the controls. In the pentylenetetrazol test, the maximal anticonvulsive effect of tiagabine administered via osmotic pumps was achieved already with a dose of 50 mg/kg/day. In the perforant pathway model of status epilepticus, subchronic treatment with tiagabine (Alzet pumps, 50 mg/kg/day) completely prevented the appearance of generalized clonic seizures during stimulation (P < 0.001). In the same rats, tiagabine treatment reduced the loss of pyramidal cells in the CA3c and CA1 fields of the hippocampus (P < 0.05) but not the loss of somatostatin immunoreactive neurons in the hilus. Two weeks after perforant pathway stimulation, the tiagabine-treated rats performed better in the Morris water-maze test than the vehicle-treated rats did (P < 0.001). Our results show that tiagabine treatment reduces the severity of seizures in the perforant pathway stimulation model of status epilepticus. Possibly associated with the reduction in seizure number and severity, tiagabine treatment also reduced seizure-induced damage to pyramidal cells in the hippocampus as well as the impairment of the spatial memory associated with hippocampal damage.

Cerebrospinal fluid gamma-aminobutyric acid in patients with panic disorder.

Cerebrospinal fluid (CSF) gamma-aminobutyric acid (GABA) levels were measured in 11 patients with panic disorder (PD) prior to and following 7 months of treatment with alprazolam or imipramine and in six neurological control patients. Although a clear treatment response was observed in patients with PD, neither alprazolam nor imipramine significantly changed CSF GABA during the treatment period. A negative correlation was demonstrated between baseline CSF GABA and posttreatment overt psychopathology. Low pretreatment level of CSF GABA correlated significantly with poor therapeutic outcome, judged by the amount of anxiety and depression as well as by the frequency of panic attacks at the end of follow-up.

Effect of alpha 2-adrenergic drugs dexmedetomidine and atipamezole on extracellular amino acid levels in vivo.

alpha 2-Adrenoceptors are known to be involved in a variety of physiological functions and pathological conditions, including epilepsy and the extent of excitotoxin-induced cell death. In this study we evaluated whether selective alpha 2-adrenergic drugs can modulate the release of neurotransmitter amino acids. The effect of the alpha 2-adrenoceptor agonist dexmedetomidine (5 micrograms/kg, s.c.) and the alpha 2-adrenoceptor antagonist atipamezole (0.1 mg/kg and 1 mg/kg, s.c.) on the release of extracellular glutamate, aspartate and gamma-aminobutyric acid (GABA) was studied with microdialysis in the hippocampus of freely moving rats under basal and K(+)-evoked conditions. Atipamezole (1 mg/kg) decreased K(+)-evoked glutamate efflux by 30% compared to the control group (P < 0.05) but did not affect significantly the effluxes of aspartate and GABA. Dexmedetomidine and the lower dose of atipamezole (0.1 mg/kg) did not significantly alter the evoked overflow of amino acids. The results suggest that alpha 2-adrenergic drugs have only modest effects on the K(+)-stimulated overflow of extracellular neurotransmitter amino acids in rat hippocampus.