B. Van Deuren

Flunarizine reduces cerebral infarct size after photochemically induced thrombosis in spontaneously hypertensive rats.

The cerebroprotective effect of flunarizine was studied in a minimally invasive model of photochemically induced cerebral infarction in spontaneously hypertensive rats. Intravenous administration of the photosensitizing dye rose bengal and intense focal illumination of the brain produced a deep cortical infarction that resulted from singlet oxygen-induced peroxidative injury to the endothelial membrane, subsequent platelet adhesion, and eventual thrombus formation. The infarct size was calculated from area measurements on consecutive histologic sections prepared from the brain cortex 4 hours after the onset of the insult. Oral treatment with 40 mg/kg flunarizine 3 hours before photoexcitation resulted in a significant reduction of the median infarct size from 11.75 mm3 in the untreated group to 6.40 mm3 in the treated group (n = 13, p less than 0.001). At this dose, flunarizine had no effect on systemic blood pressure. In a separate experiment the area of thrombotic obstruction was quantified 30 minutes after the onset of light exposure. Flunarizine did not significantly reduce early thrombus formation (2.28 mm3 in the untreated and 1.78 mm3 in the treated group) (n = 12, p = 0.2). The infarcted area at 4 hours was considerably larger than the initial thrombotic area. Protection with flunarizine against development of cortical infarction has been unequivocally shown. Although some effect may already be present at the early stage of lesion formation, the major protective action admittedly occurred in the later postinsult period when the lesion was expanding.(ABSTRACT TRUNCATED AT 250 WORDS)

EEG in the FEAB model: measurement of electroencephalographical burst suppression and seizure liability in safety pharmacology.

INTRODUCTIONnThe purpose of this study was to explore the integration of electroencephalographical (EEG) measurements into the fentanyl/etomidate-anaesthetised Beagle (FEAB) model in order to detect burst suppression and/or seizure development caused by compounds, prior to new molecular entity (NME) declaration. Detecting such unfavourable side effects prevents their being found in conscious animals at a later stage of safety evaluation. In addition, this has the advantage of performing safety studies on the three vital organ systems (cardiovascular system, respiratory system and central nervous system) within one and the same animal model.nnnMETHODSnDogs were anaesthetized and instrumented according to the FEAB model requirements, and in addition three needle electrodes were placed on the cranium and a one lead EEG signal was measured. The raw signal was analysed by the Narcotrend® (MonitorTechnik, Bad Bramstedt, Germany) for depth of anaesthesia registration, visually analysed for burst suppression ratio calculation after different anaesthetics (pentobarbital and etomidate), and spiking and seizure activity were quantified after intravenous administration of different proconvulsant agents: pentylenetetrazole (PTZ), bicuculline (BCC), bupropion (BUP) and pilocarpine (PIL).nnnRESULTSnHigh doses of pentobarbital (60 mg/kg over 10 min) and etomidate (6 mg/kg over 10 min) induced dose-dependent burst suppression of 98 ± 2% and 61 ± 16%, respectively. Infusions of PTZ (1.5mg/kg/min), BCC (0.0625 mg/kg/min), BUP (0.5mg/kg/min) and PIL (5mg/kg/min) induced dose-dependent spiking and seizures: the thresholds were 34 ± 2, 0.15 ± 0.03, 10.0 ± 1 and 144 ± 9 mg/kg, respectively. In PTZ-treated dogs, spiking and seizures could be abolished with diazepam (2mg/kg i.v.) or with propofol (4 mg/kg i.v.).nnnDISCUSSIONnThe present study showed that a one lead EEG can be used reliably in the FEAB model to estimate the depth of anaesthesia, and to detect burst suppression and seizure risk in safety pharmacology studies.

Brain Oxygenation after Experimental Closed Head Injury

In blunt head trauma, primary brain injury takes the form of surface contusions and fractures, diffuse axonal injury and intracranial hemorrhage. Secondary damage may result from delayed pathological events including cerebral ischemia, brain edema and increased intracranial pressure (ICP). These events reinforce one another and may lead to a fatal disturbance of the balance between oxygen delivery and oxygen demand. Improvement or maintenance of this balance is a main issue in the treatment of severe head-injured patients. In this respect reduction of ICP is a key tool as it may improve brain perfusion and attenuate the pathologic cascade. Therapeutic interventions that adequately reduce ICP do not necessarily improve and may even impair tissue oxygenation (Unterberg et al., 1997a). This addresses the need for adequate monitoring of brain oxygenation during and after treatment.

Photochemically-induced cerebral infarction in the rat: Comparison of NMR imaging and histologic changes

SummaryThe evolution of a photochemically induced cerebral thrombotic infarction was followed in rats during the first week after the insult by means of NMR imaging and histology. Heavily T2-weighted images provided an excellent lesion detection and a high specificity for the discrimination of different histological abnormalities. The T2-weighted images showed a brain lesion evolving during the first 24h from a homogeneous hyperintense area, histologically corresponding to diffuse vasogenic and cytotoxic oedema with concomitant neuronal necrosis, to an iso-intense area with a hyperintense seam, which microscopically correlated with increased vascular permeability at the periphery of the lesion. The hyperintense seam was observed up to day 7, but at that time coincided with gliomesodermal repair reaction which could be verified histochemically and ultrastructurally. It may be concluded that NMR-micro-imaging at a moderately high field, enables early detection and adequate followup of small cerebral infarctions in rats.

Intracranial Pressure in a Modified Experimental Model of Closed Head Injury

Intracranial pressure (ICP) was studied in a modified experimental model of closed head injury, in which the dynamic process of impact versus impulse loading was separately controlled. In this model, mortality of Wistar rats was considerably higher as compared to Sprague-Dawley rats subjected to similar traumatic conditions. Therefore Sprague-Dawley rats were used for all further experiments. Twenty-four rats, divided into 4 groups, underwent either sham or gradually increasing impact-acceleration trauma. Four hours after closed head injury, ICP measurements showed a significant correlation between the severity of the traumatic challenge and the resultant pressure rise (r2 = 0.731; p < 0.001). At the moment of impact there was a momentary blood pressure peak immediately followed by a transient period of hypotension. ICP measurements following directly to an impact-acceleration trauma, revealed an abrupt rise in ICP reaching pathological levels within 5 minutes. In conclusion, this modified model of closed head injury produces a predictable and reproducible pathologic ICP in Sprague-Dawley rats.

Favourable effect of flunarizine on the recovery from hemiparesis in rats with intracerebral hematomas.

In 25 rats, an intracerebral hematoma was created in the foreleg area of the motor cortex by injection of 50 microliters blood. After the lesion, 13 were treated with flunarizine and 12 with the solvent. Neurological testing was performed by measuring the running time on a rotating platform. In animals with hemiparesis, the flunarizine group (n = 7) showed a significantly (P less than 0.05) better recovery than the control group (n = 8). No significant differences occurred in animals without neurological deficits (flunarizine: n = 6, control: n = 4). So the effect of the drug is not due to a non-specific activation; it may partially cure neurological deficits caused by intracerebral hematoma.

Do in vitro assays in rat primary neurons predict drug-induced seizure liability in humans?

ABSTRACT Drug‐induced seizures contribute to the high attrition rate of pharmaceutical compounds in development. The assessment of drug‐induced seizure liability generally occurs in later phases of development using low throughput and intensive in vivo assays. In the present study, we evaluated the potential of an in vitro assay for detecting drug‐induced seizure risk compared to evaluation in rats in vivo. We investigated the effects of 8 reference drugs with a known seizurogenic risk using micro‐electrode array (MEA) recordings from freshly‐dissociated rat primary neurons cultured on 48‐well dishes for 28days, compared to their effects on the EEG in anesthetized rats. In addition, we evaluated functional responses and mRNA expression levels of different receptors in vitro to understand the potential mechanisms of drug‐induced seizure risk. Combining the functional MEA in vitro data with concomitant gene expression allowed us to identify several potential molecular targets that might explain the drug‐induced seizures occurring in both rats and humans. Our data 1) demonstrate the utility of a group of MEA parameters for detecting potential drug‐induced seizure risk in vitro; 2) suggest that an in vitro MEA assay with rat primary neurons may have advantages over an in vivo rat model; and 3) identify potential mechanisms for the discordance between rat assays and human seizure risk for certain seizurogenic drugs. HIGHLIGHTSFunctional MEA in vitro data concomitant with gene expression dataIdentification of molecular targets that might explain drug‐induced seizuresin vitro MEA assay with primary neurons may have advantages over in vivo model.Identification of mechanisms for the discordance between assays and seizure risk

Microwave-enhanced silver staining of degenerating neuronal processes

SummaryA simple and rapid method for light and electron microscopic visualization of degenerating neuronal processes and axon terminals is described. Hundred-micrometer vibratome sections of perfusion-fixed rat brain were incubated briefly in a 5% silver nitrate solution in a conventional microwave oven. After a rinse in 1% acetic acid, the sections were silver enhanced. Differentiation and counterstaining was done respectively in ethanol 100% and cresyl violet. In the light microscope, degenerating neuronal processes appeared as black dots against a clear background. Areas of calcification were also positively stained. The presence of silver deposits in degenerating presynaptic terminals and dendrites was confirmed ultrastructurally.

05 The electro-mechanical window as Torsade de Pointes risk marker in conscious and anaesthetised dogs after IKS blockade

The electro-mechanical window (EMw) is a recently proposed biomarker describing the temporal difference between electrical and mechanical events in beating hearts and is a precursor to identify Torsade de Pointes (TdP) risk in the anaesthetised dog.1 In follow-up studies, conscious dogs (n=6; telemetered) showed comparable baseline values to anaesthetised dogs (n=20): QT=250 vs 252u2005ms, QLVPend=347 vs 339u2005ms and EMw=+96 vs +87u2005ms, respectively. To compare the EMw in conscious and anaesthetised dogs after IKs blockade.2 Six conscious dogs were orally treated with JNJ303 (20u2005mg/kg) and four anaesthetised dogs received infusions of JNJ303 (cumulative 2.5u2005mg/kg). In both conditions JNJ303 induced QT prolongation, a large negative EMw and TdP appeared in 50% of the dogs in each condition (pause-dependent and adrenergic-dependent). In the conscious dogs significant differences in maximum plasma levels (PL), QT and EMw were observed between dogs that induced TdP and those without TdP: PL (4950 vs 1473u2005ng/ml), QT (427 vs 351u2005ms) and EMw (−150 vs −68u2005ms), without differences in RR (642 vs 654u2005ms) and QLVPend (273 vs 287u2005ms), respectively. Also in anaesthetised dogs long QT (766u2005ms) and large negative EMw (−445u2005ms) at similar PL (5743u2005ng/ml) were observed. In conclusion a potent IKs blocker (JNJ303) can cause TdP in conscious and anaesthetised dogs, coupled with a large negative EMw.

Phototoxic peripheral demyelination: Histopathological aspects

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