Introduction to the EQIPD quality system.

While high risk of failure is an inherent part of developing innovative therapies, it can be reduced by adherence to evidence-based rigorous research practices. Supported through the European Union's Innovative Medicines Initiative, the EQIPD consortium has developed a novel preclinical research quality system that can be applied in both public and private sectors and is free for anyone to use. The EQIPD Quality System was designed to be suited to boost innovation by ensuring the generation of robust and reliable preclinical data while being lean, effective and not becoming a burden that could negatively impact the freedom to explore scientific questions. EQIPD defines research quality as the extent to which research data are fit for their intended use. Fitness, in this context, is defined by the stakeholders, who are the scientists directly involved in the research, but also their funders, sponsors, publishers, research tool manufacturers, and collaboration partners such as peers in a multi-site research project. The essence of the EQIPD Quality System is the set of 18 core requirements that can be addressed flexibly, according to user-specific needs and following a user-defined trajectory. The EQIPD Quality System proposes guidance on expectations for quality-related measures, defines criteria for adequate processes (i.e. performance standards) and provides examples of how such measures can be developed and implemented. However, it does not prescribe any pre-determined solutions. EQIPD has also developed tools (for optional use) to support users in implementing the system and assessment services for those research units that successfully implement the quality system and seek formal accreditation. Building upon the feedback from users and continuous improvement, a sustainable EQIPD Quality System will ultimately serve the entire community of scientists conducting non-regulated preclinical research, by helping them generate reliable data that are fit for their intended use.

Where are we heading? Challenges in evidence-based severity assessment.

Evidence-based severity assessment in laboratory animals is, apart from the ethical responsibility, imperative to generate reproducible, standardized and valid data. However, the path towards a valid study design determining the degree of pain, distress and suffering experienced by the animal is lined with pitfalls and obstacles as we will elucidate in this review. Furthermore, we will ponder on the genesis of a holistic concept relying on multifactorial composite scales. These have to combine robust and reliable parameters to measure the multidimensional aspects that define the severity of animal experiments, generating a basis for the substantiation of the refinement principle.

Epileptogenesis-Associated Alterations of Heat Shock Protein 70 in a Rat Post-Status Epilepticus Model.

Temporal lobe epilepsy is triggered by an initial insult, such as status epilepticus, that initiates the process of epilepsy development. Heat shock protein 70 (Hsp70) is a ubiquitously expressed molecular chaperone, involved in the inflammatory response that is upregulated after status epilepticus. Hsp70 has been described as an endogenous intracellular ligand of Toll-like receptor 4. It is released from damaged or necrotic tissue and by activated immune cells after an inflammatory event. So far, the time course and the pattern of epileptogenesis-associated alterations in Hsp70 expression have not been described in detail. Thus, we investigated immunohistochemical expression of Hsp70 in hippocampus, parahippocampal cortex, parietal cortex, amygdala, and thalamus following status epilepticus in a rat model of temporal lobe epilepsy. The impact of status epilepticus on Hsp70 expression varied during different phases of epileptogenesis, displaying a stronger effect in the early post-insult phase, a milder and more localized effect in the latency phase and no relevant effect in the chronic phase. Cellular-level characterization revealed that Hsp70 colocalized with the neuronal marker NeuN and with Toll-like receptor 4. No colocalization with the astrocytic marker GFAP or the microglia marker Iba1 was found. The intense neuronal Hsp70 upregulation during the early post-insult phase might contribute to the onset of excessive inflammation triggering molecular and cellular reorganization and generation of a hyperexcitable epileptic network. Therefore, development of multi-targeting strategies aiming at prevention of epileptogenesis should consider Hsp70 modulation in the early days following an epileptogenic insult.

Exploratory EEG studies for the assessment of neurological liabilities in conscious dogs in early drug development.

INTRODUCTION: Convulsions in toxicology studies can be the first indication of seizure liability. Drug levels during convulsions are not usually evaluated. This, and exposure variability after oral administration, complicates estimation of safety margins. The electroencephalogram (EEG) enables symptoms to be attributed to seizures and to collect samples during epileptiform activity without clinical convulsion. We evaluated an EEG-study design for optimized detection of neurological symptoms. Additionally, we assessed whether EEG- based anticonvulsive treatment is feasible, to prevent progression to convulsions and if dogs have higher sensitivity towards neurological symptoms than non-human-primates. METHODS: Three compounds that previously were tested in non-human-primates were selected to evaluate the dog EEG-study design. Two substances were administered in escalating intravenous doses; the third was given as single oral dose. Per compound, one male and one female telemetered dog were evaluated; males also had cerebrospinal-fluid-ports. Drug levels, video-EEG and clinical symptoms were evaluated and compared to previous studies. RESULTS: While similar neurological symptoms were induced, intravenous administration reduced experimental time compared to standard toxicology studies. EEG analysis could link animal behavior to seizures but did not allow convulsion prevention. This was due to artefacts and the short latency between onset of epileptiform EEG activity and clinical convulsions. Free plasma concentrations during convulsions were comparable between dogs and non-human-primates. DISCUSSION: The findings suggest that infusion studies provide a possibility to investigate neurological adverse effects in few animals in a short time period. For candidates with a high risk for seizures, such studies can guide dose selection for longer regulatory studies and improve safety margin definition.

N-acetyl-L-leucine accelerates vestibular compensation after unilateral labyrinthectomy by action in the cerebellum and thalamus.

An acute unilateral vestibular lesion leads to a vestibular tone imbalance with nystagmus, head roll tilt and postural imbalance. These deficits gradually decrease over days to weeks due to central vestibular compensation (VC). This study investigated the effects of i.v. N-acetyl-DL-leucine, N-acetyl-L-leucine and N-acetyl-D-leucine on VC using behavioural testing and serial [18F]-Fluoro-desoxyglucose ([18F]-FDG)-µPET in a rat model of unilateral chemical labyrinthectomy (UL). Vestibular behavioural testing included measurements of nystagmus, head roll tilt and postural imbalance as well as sequential whole-brain [18F]-FDG-µPET was done before and on days 1,3,7 and 15 after UL. A significant reduction of postural imbalance scores was identified on day 7 in the N-acetyl-DL-leucine (p < 0.03) and the N-acetyl-L-leucine groups (p < 0.01), compared to the sham treatment group, but not in the N-acetyl-D-leucine group (comparison for applied dose of 24 mg i.v. per rat, equivalent to 60 mg/kg body weight, in each group). The course of postural compensation in the DL- and L-group was accelerated by about 6 days relative to controls. The effect of N-acetyl-L-leucine on postural compensation depended on the dose: in contrast to 60 mg/kg, doses of 15 mg/kg and 3.75 mg/kg had no significant effect. N-acetyl-L-leucine did not change the compensation of nystagmus or head roll tilt at any dose. Measurements of the regional cerebral glucose metabolism (rCGM) by means of µPET revealed that only N-acetyl-L-leucine but not N-acetyl-D-leucine caused a significant increase of rCGM in the vestibulocerebellum and a decrease in the posterolateral thalamus and subthalamic region on days 3 and 7. A similar pattern was found when comparing the effect of N-acetyl-L-leucine on rCGM in an UL-group and a sham UL-group without vestibular damage. In conclusion, N-acetyl-L-leucine improves compensation of postural symptoms after UL in a dose-dependent and specific manner, most likely by activating the vestibulocerebellum and deactivating the posterolateral thalamus.

Anticonvulsant effects of transcranial direct-current stimulation (tDCS) in the rat cortical ramp model of focal epilepsy.

PURPOSE: Weak direct currents induce lasting alterations of cortical excitability in animals and humans, which are controlled by polarity, duration of stimulation, and current strength applied. To evaluate its anticonvulsant potential, transcranial direct current stimulation (tDCS) was tested in a modified cortical ramp-stimulation model of focal epilepsy. METHODS: The threshold for localized seizure activity (TLS) was determined in freely moving rats by applying a single train of rising bipolar pulses through a unilateral epicranial electrode. After tDCS, TLS was determined repeatedly for 120 min at intervals of 15 min. The first group of animals received two sessions of cathodal tDCS at 100 microA, one for 30 and one for 60 min. A third session consisted of 60 min of anodal tDCS. A second group received cathodal tDCS at 200 microA for 15 and for 30 min, as well as anodal tDCS for 30 min. RESULTS: Sixty minutes of cathodal tDCS at 100 microA resulted in a TLS increase lasting for >or=2 h. When the intensity was increased to 200 microA, a similar lasting TLS elevation occurred after a stimulation of just 30-min duration. In contrast, anodal tDCS at identical stimulation durations and current strengths had no significant effect on TLS. CONCLUSIONS: The anticonvulsive effect induced by cathodal tDCS depends on stimulation duration and current strength and may be associated with the induction of alterations of cortical excitability that outlast the actual stimulation. The results lead to the reasonable assumption that cathodal tDCS could evolve as a therapeutic tool in drug-refractory partial epilepsy.