Postoperative Severity Assessment in Sheep.

INTRODUCTION: Sheep are frequently used in translational surgical orthopedic studies. Naturally, a good pain management is mandatory for animal welfare, although it is also important with regard to data quality. However, methods for adequate severity assessment, especially considering pain, are rather rare regarding large animal models. Therefore, in the present study, accompanying a surgical pilot study, telemetry and the Sheep Grimace Scale (SGS) were used in addition to clinical scoring for severity assessment after surgical interventions in sheep. METHODS: Telemetric devices were implanted in a first surgery subcutaneously into four German black-headed mutton ewes (4-5 years, 77-115 kg). After 3-4 weeks of recovery, sheep underwent tendon ablation of the left M. infraspinatus. Clinical scoring and video recordings for SGS analysis were performed after both surgeries, and the heart rate (HR) and general activity were monitored by telemetry. RESULTS: Immediately after surgery, clinical score and HR were slightly increased, and activity was decreased in individual sheep after both surgeries. The SGS mildly elevated directly after transmitter implantation but increased to higher levels after tendon ablation immediately after surgery and on the following day. CONCLUSION: In summary, SGS- and telemetry-derived data were suitable to detect postoperative pain in sheep with the potential to improve individual pain recognition and postoperative management, which consequently contributes to refinement.

99mTc-HMPAO SPECT imaging reveals brain hypoperfusion during status epilepticus.

Status epilepticus (SE) is a clinical emergency with high mortality. SE can trigger neuronal death or injury and alteration of neuronal networks resulting in long-term cognitive decline or epilepsy. Among the multiple factors contributing to this damage, imbalance between oxygen and glucose requirements and brain perfusion during SE has been proposed. Herein, we aimed to quantify by neuroimaging the spatiotemporal course of brain perfusion during and after lithium-pilocarpine-induced SE in rats. To this purpose, animals underwent 99mTc-HMPAO SPECT imaging at different time points during and after SE using a small animal SPECT/CT system. 99mTc-HMPAO regional uptake was normalized to the injected dose. In addition, voxel-based statistical parametric mapping was performed. SPECT imaging showed an increase of cortical perfusion before clinical seizure activity onset followed by regional hypo-perfusion starting with the first convulsive seizure and during SE. Twenty-four hours after SE, brain 99mTc-HMPAO uptake was widely decreased. Finally, chronic epileptic animals showed regionally decreased perfusion affecting hippocampus and cortical sub-regions. Despite elevated energy and oxygen requirements, brain hypo-perfusion is present during SE. Our results suggest that insufficient compensation of required blood flow might contribute to neuronal damage and neuroinflammation, and ultimately to chronic epilepsy generated by SE.

Proof-of-concept that network pharmacology is effective to modify development of acquired temporal lobe epilepsy.

Epilepsy is a complex network phenomenon that, as yet, cannot be prevented or cured. We recently proposed network-based approaches to prevent epileptogenesis. For proof of concept we combined two drugs (levetiracetam and topiramate) for which in silico analysis of drug-protein interaction networks indicated a synergistic effect on a large functional network of epilepsy-relevant proteins. Using the intrahippocampal kainate mouse model of temporal lobe epilepsy, the drug combination was administered during the latent period before onset of spontaneous recurrent seizures (SRS). When SRS were periodically recorded by video-EEG monitoring after termination of treatment, a significant decrease in incidence and frequency of SRS was determined, indicating antiepileptogenic efficacy. Such efficacy was not observed following single drug treatment. Furthermore, a combination of levetiracetam and phenobarbital, for which in silico analysis of drug-protein interaction networks did not indicate any significant drug-drug interaction, was not effective to modify development of epilepsy. Surprisingly, the promising antiepileptogenic effect of the levetiracetam/topiramate combination was obtained in the absence of any significant neuroprotective or anti-inflammatory effects as indicated by multimodal brain imaging and histopathology. High throughput RNA-sequencing (RNA-seq) of the ipsilateral hippocampus of mice treated with the levetiracetam/topiramate combination showed that several genes that have been linked previously to epileptogenesis, were significantly differentially expressed, providing interesting entry points for future mechanistic studies. Overall, we have discovered a novel combination treatment with promise for prevention of epilepsy.

Attenuation of epileptogenesis by 2-deoxy-d-glucose is accompanied by increased cerebral glucose supply, microglial activation and reduced astrocytosis.

RATIONALE: Neuronal excitability and brain energy homeostasis are strongly interconnected and evidence suggests that both become altered during epileptogenesis. Pharmacologic modulation of cerebral glucose metabolism might therefore exert anti-epileptogenic effects. Here we provide mechanistic insights into effects of the glycolytic inhibitor 2-deoxy-d-glucose (2-DG) on experimental epileptogenesis by longitudinal 2-deoxy-2[18F]fluoro-d-glucose positron emission tomography ([18F]FDG PET) and histology. METHODS: To imitate epileptogenesis, 6 Hz-corneal kindling was performed in male NMRI mice by twice daily electrical stimulation for 21 days. Kindling groups were treated i.p. 1 min after each stimulation with either 250 mg/kg 2-DG (CoKi_2-DG) or saline (CoKi_vehicle). A separate group of unstimulated mice was treated with 2-DG (2-DG_only). Dynamic 60-min [18F]FDG PET/CT scans were acquired at baseline and interictally on days 10 and 17 of kindling. [18F]FDG uptake (%injected dose/cc) was quantified in predefined regions of interest (ROI) using a MRI-based brain atlas, and kinetic modelling was performed to evaluate glucose net influx rate Ki and glucose metabolic rate MRGlu. Furthermore, statistical parametric mapping (SPM) analysis was applied on kinetic brain maps. For histological evaluation, brain sections were stained for glucose transporter 1 (GLUT1), astrocytes, microglia, as well as dying neurons. RESULTS: Post-stimulation 2-DG treatment attenuated early kindling progression, indicated by a reduction of fully-kindled mice, and a lower overall percentage of type five seizures. While 2-DG treatment alone led to globally increased Ki and MRGlu values at day 17, kindling progression per se did not influence glucose turnover. Kindling accompanied by 2-DG treatment, however, resulted in regionally elevated [18F]FDG uptake as well as increased Ki at days 10 and 17 compared both to baseline and to the 2-DG_only group. In hippocampus and thalamus, higher MRGlu values were found in the CoKi_2-DG vs. the CoKi_vehicle group at day 17. t maps resulting from SPM analysis generally confirmed the results of the ROI analysis, and additionally revealed increased MRGlu restricted to the ventral hippocampus when comparing the CoKi_2-DG and the 2-DG_only group both at days 10 and, more distinct, day 17. Immunohistochemical staining showed an attenuated kindling-induced regional activation of astrocytes in the CoKi_2-DG group. Interestingly, 2-DG treatment alone (and also in combination with kindling, but not kindling alone) led to increased microglial activation scores, whereas neither staining of GLUT1 nor of dying neurons revealed any differences to untreated controls. CONCLUSIONS: Post-stimulation treatment with 2-DG exerts disease-modifying effects in the mouse 6 Hz corneal kindling model. The observed local increase in glucose supply and turnover, the alleviation of astroglial activation and the activation of microglia by 2-DG might contribute separately or in combination to its positive interference with epileptogenesis.

Ex vivo characterization of neuroinflammatory and neuroreceptor changes during epileptogenesis using candidate positron emission tomography biomarkers.

OBJECTIVE: Identification of patients at risk of developing epilepsy before the first spontaneous seizure may promote the development of preventive treatment providing opportunity to stop or slow down the disease. METHODS: As development of novel radiotracers and on-site setup of existing radiotracers is highly time-consuming and expensive, we used dual-centre in vitro autoradiography as an approach to characterize the potential of innovative radiotracers in the context of epilepsy development. Using brain slices from the same group of rats, we aimed to characterise the evolution of neuroinflammation and expression of inhibitory and excitatory neuroreceptors during epileptogenesis using translational positron emission tomography (PET) tracers; 18 F-flumazenil (18 F-FMZ; GABAA receptor), 18 F-FPEB (metabotropic glutamate receptor 5; mGluR5), 18 F-flutriciclamide (translocator protein; TSPO, microglia activation) and 18 F-deprenyl (monoamine oxidase B, astroglia activation). Autoradiography images from selected time points after pilocarpine-induced status epilepticus (SE; baseline, 24 and 48 hours, 5, 10 and 15 days and 6 and 12-14 weeks after SE) were normalized to a calibration curve, co-registered to an MRI-based 2D region-of-interest atlas, and activity concentration (Bq/mm2 ) was calculated. RESULTS: In epileptogenesis-associated brain regions, 18 F-FMZ and 18 F-FPEB showed an early decrease after SE. 18 F-FMZ decrease was maintained in the latent phase and further reduced in the chronic epileptic animals, while 18 F-FPEB signal recovered from day 10, reaching baseline levels in chronic epilepsy. 18 F-flutriciclamide showed an increase of activated microglia at 24 hours after SE, peaking at 5-15 days and decreasing during the chronic phase. On the other hand, 18 F-deprenyl autoradiography showed late astrogliosis, peaking in the chronic phase. SIGNIFICANCE: Autoradiography revealed different evolution of the selected targets during epileptogenesis. Our results suggest an advantage of combined imaging of inter-related targets like glutamate and GABAA receptors, or microglia and astrocyte activation, in order to identify important interactions, especially when using PET imaging for the evaluation of novel treatments.

[18 F]GE180 positron emission tomographic imaging indicates a potential double-hit insult in the intrahippocampal kainate mouse model of temporal lobe epilepsy.

OBJECTIVE: Accumulating evidence suggests that brain inflammation, elicited by epileptogenic insults, is involved in epilepsy development. Noninvasive nuclear imaging of brain inflammation in animal models of epileptogenesis represents a diagnostic in vivo approach with potential for direct translation into the clinic. Here, we investigated up-regulation of the translocator protein (TSPO) indicative of microglial activation by serial [18 F]GE180 positron emission tomographic (PET) imaging in a mouse model of temporal lobe epilepsy. METHODS: As epileptogenic insult, a status epilepticus (SE) was induced in mice by intrahippocampal injection of kainate. Post-SE mice injected with kainate and sham-injected mice were subjected to [18 F]GE180 PET scans before SE and at 2 days, 5-7 days, 2 weeks, 3 weeks, 7 weeks, and 14 weeks postinsult. For data evaluation, brain regions ipsilateral and contralateral to the injection site were outlined by coregistration with a standard mouse brain atlas, and percentage of injected dose per cubic centimeter was calculated. In addition, a statistical parametric mapping analysis, comparing post-SE mice to baseline, sham mice to baseline, and post-SE to sham mice was performed. RESULTS: Following SE, elevations in [18 F]GE180 uptake were most prominent in the ipsilateral hippocampus, occurring between 2 days and at least 7 weeks after SE, with a peak at 5-7 days after SE. In the contralateral hippocampus and other epilepsy-associated brain regions, increased tracer uptake was observed with a similar time profile but to a lesser extent. Moderate enhancement of tracer uptake was also evident in mice after sham surgery. SIGNIFICANCE: TSPO in vivo imaging reliably detects brain inflammation during epileptogenesis. These inflammatory processes most prominently affect the hippocampus ipsilateral to the injection site. Inflammation induced by the traumatic insult associated with surgery synergistically contributes to total brain inflammation and may also contribute to epileptogenesis. The revealed time course of neuroinflammation will help to identify appropriate time points for anti-inflammatory, potentially antiepileptogenic treatment.

Isoflurane prevents acquired epilepsy in rat models of temporal lobe epilepsy.

OBJECTIVE: Acquired epilepsy is a devastating long-term risk of various brain insults, including trauma, stroke, infections, and status epilepticus (SE). There is no preventive treatment for patients at risk. Attributable to the complex alterations involved in epileptogenesis, it is likely that multitargeted approaches are required for epilepsy prevention. We report novel preclinical findings with isoflurane, which exerts various nonanesthetic effects that may be relevant for antiepileptogenesis. METHODS: The effects of isoflurane were investigated in two rat models of SE-induced epilepsy: intrahippocampal kainate and systemic administration of paraoxon. Isoflurane was either administered during (kainate) or after (paraoxon) induction of SE. Magnetic resonance imaging was used to assess blood-brain barrier (BBB) dysfunction. Positron emission tomography was used to visualize neuroinflammation. Long-term electrocorticographic recordings were used to monitor spontaneous recurrent seizures. Neuronal damage was assessed histologically. RESULTS: In the absence of isoflurane, spontaneous recurrent seizures were common in the majority of rats in both models. When isoflurane was administered during kainate injection, duration and severity of SE were not affected, but only few rats developed spontaneous recurrent seizures. A similar antiepileptogenic effect was found when paraoxon-treated rats were exposed to isoflurane after SE. Moreover, in the latter model, isoflurane prevented BBB dysfunction and neurodegeneration, whereas isoflurane reduced neuroinflammation in the kainate model. INTERPRETATION: Given that isoflurane is a widely used volatile anesthetic, and is used for inhalational long-term sedation in critically ill patients at risk to develop epilepsy, our findings hold a promising potential to be successfully translated into the clinic. Ann Neurol 2016;80:896-908.

The pilocarpine model of temporal lobe epilepsy: Marked intrastrain differences in female Sprague-Dawley rats and the effect of estrous cycle.

Rat strains such as Sprague-Dawley (SD) or Wistar are widely used in epilepsy research, including popular models of temporal lobe epilepsy in which spontaneous recurrent seizures (SRS), hippocampal damage, and behavioral alterations develop after status epilepticus (SE). Such rats are randomly outbred, and outbred strains are known to be genetically heterogeneous populations with a high intrastrain variation. Intrastrain differences may be an important reason for discrepancies between studies from different laboratories, but the extent to which such differences affect the development of seizures, neurodegeneration, and psychopathology in post-SE models of epilepsy has received relatively little attention. In the present study, we induced SE by systemic administration of pilocarpine (following pretreatment with lithium) in SD rats from different breeders (Harlan, Charles River [CRL], Taconic) as well as different breeding locations of the same breeder (Harlan-Winkelmann [HW] in Germany vs. Harlan Laboratories [HL] in the Netherlands). Some experiments were also performed in Wistar rats. Pilocarpine was administered by a ramp-up dosing protocol that allows determining interindividual differences in susceptibility to the convulsant. Marked intrastrain differences in induction of SE and its long-term consequences were found. Sprague-Dawley rats from HW were significantly more sensitive to SE induction than all other SD substrains. The majority of SD rats from different vendors developed SRS after SE except SD rats from HL. The CRL-SD rats markedly differed in basal behavior and SE-induced behavioral alterations from other SD substrains. Susceptibility to pilocarpine was hardly affected by the estrous cycle. The marked intrastrain differences provide an interesting tool to study the impact of genetic and environmental factors on seizure susceptibility, epileptogenesis, and relationship between behavior and epilepsy and vice versa.

Tariquidar and elacridar are dose-dependently transported by P-glycoprotein and Bcrp at the blood-brain barrier: a small-animal positron emission tomography and in vitro study.

Elacridar (ELC) and tariquidar (TQD) are generally thought to be nontransported inhibitors of P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP), but recent data indicate that they may also be substrates of these multidrug transporters (MDTs). The present study was designed to investigate potential transport of ELC and TQD by MDTs at the blood-brain barrier at tracer doses as used in positron emission tomography (PET) studies. We performed PET scans with carbon-11-labeled ELC and TQD before and after MDT inhibition in wild-type and transporter-knockout mice as well as in in vitro transport assays in MDT-overexpressing cells. Brain entrance of [(11)C]ELC and [(11)C]TQD administered in nanomolar tracer doses was found to be limited by Pgp- and Bcrp1-mediated efflux at the mouse blood-brain barrier. At higher, MDT-inhibitory doses, i.e., 15 mg/kg for TQD and 5 mg/kg for ELC, brain activity uptake of [(11)C]ELC at 25 minutes after tracer injection was 5.8 ± 0.3, 2.1 ± 0.2, and 7.5 ± 1.0-fold higher in wild-type, Mdr1a/b((-/-),()) and Bcrp1((-/-)) mice, respectively, but remained unchanged in Mdr1a/b((-/-))Bcrp1((-/-)) mice. Activity uptake of [(11)C]TQD was 2.8 ± 0.2 and 6.8 ± 0.4-fold higher in wild-type and Bcrp1((-/-)) mice, but remained unchanged in Mdr1a/b((-/-)) and Mdr1a/b((-/-))Bcrp1((-/-)) mice. Consistent with the in vivo findings, in vitro uptake assays in Pgp- and Bcrp1-overexpressing cell lines confirmed low intracellular accumulation of ELC and TQD at nanomolar concentrations and increased uptake at micromolar concentrations. As this study shows that microdoses can behave pharmacokinetically differently from MDT-inhibitory doses if a compound interacts with MDTs, conclusions from microdose studies should be drawn carefully.

Is switching from brand name to generic formulations of phenobarbital associated with loss of antiepileptic efficacy?: a pharmacokinetic study with two oral formulations (Luminal(®) vet, Phenoleptil(®)) in dogs.

BACKGROUND: In human medicine, adverse outcomes associated with switching between bioequivalent brand name and generic antiepileptic drug products is a subject of concern among clinicians. In veterinary medicine, epilepsy in dogs is usually treated with phenobarbital, either with the standard brand name formulation Luminal(®) or the veterinary products Luminal(®) vet and the generic formulation Phenoleptil(®). Luminal(®) and Luminal(®) vet are identical 100 mg tablet formulations, while Phenoleptil(®) is available in the form of 12.5 and 50 mg tablets. Following approval of Phenoleptil(®) for treatment of canine epilepsy, it was repeatedly reported by clinicians and dog owners that switching from Luminal(®) (human tablets) to Phenoleptil(®) in epileptic dogs, which were controlled by treatment with Luminal(®), induced recurrence of seizures. In the present study, we compared bioavailability of phenobarbital after single dose administration of Luminal(®) vet vs. Phenoleptil(®) with a crossover design in 8 healthy Beagle dogs. Both drugs were administered at a dose of 100 mg/dog, resulting in 8 mg/kg phenobarbital on average. RESULTS: Peak plasma concentrations (Cmax) following Luminal(®) vet vs. Phenoleptil(®) were about the same in most dogs (10.9 ± 0.92 vs. 10.5 ± 0.77 µg/ml), and only one dog showed noticeable lower concentrations after Phenoleptil(®) vs. Luminal(®) vet. Elimination half-life was about 50 h (50.3 ± 3.1 vs. 52.9 ± 2.8 h) without differences between the formulations. The relative bioavailability of the two products (Phenoleptil(®) vs. Luminal(®) vet.) was 0.98 ± 0.031, indicating that both formulations resulted in about the same bioavailability. CONCLUSIONS: Overall, the two formulations did not differ significantly with respect to pharmacokinetic parameters when mean group parameters were compared. Thus, the reasons for the anecdotal reports, if true, that switching from the brand to the generic formulation of phenobarbital may lead to recurrence of seizures are obviously not related to a generally lower bioavailability of the generic formulation, although single dogs may exhibit lower plasma levels after the generic formulation that could be clinically meaningful.

Anesthesia and analgesia for experimental craniotomy in mice and rats: a systematic scoping review comparing the years 2009 and 2019.

Experimental craniotomies are a common surgical procedure in neuroscience. Because inadequate analgesia appears to be a problem in animal-based research, we conducted this review and collected information on management of craniotomy-associated pain in laboratory mice and rats. A comprehensive search and screening resulted in the identification of 2235 studies, published in 2009 and 2019, describing craniotomy in mice and/or rats. While key features were extracted from all studies, detailed information was extracted from a random subset of 100 studies/year. Reporting of perioperative analgesia increased from 2009 to 2019. However, the majority of studies from both years did not report pharmacologic pain management. Moreover, reporting of multimodal treatments remained at a low level, and monotherapeutic approaches were more common. Among drug groups, reporting of pre- and postoperative administration of non-steroidal anti-inflammatory drugs, opioids, and local anesthetics in 2019 exceeded that of 2009. In summary, these results suggest that inadequate analgesia and oligoanalgesia are persistent issues associated with experimental intracranial surgery. This underscores the need for intensified training of those working with laboratory rodents subjected to craniotomies. Systematic review registration: https://osf.io/7d4qe.

A novel positron emission tomography imaging protocol identifies seizure-induced regional overactivity of P-glycoprotein at the blood-brain barrier.

Approximately one-third of epilepsy patients are pharmacoresistant. Overexpression of P-glycoprotein and other multidrug transporters at the blood-brain barrier is thought to play an important role in drug-refractory epilepsy. Thus, quantification of regionally different P-glycoprotein activity in the brain in vivo is essential to identify P-glycoprotein overactivity as the relevant mechanism for drug resistance in an individual patient. Using the radiolabeled P-glycoprotein substrate (R)-[(11)C]verapamil and different doses of coadministered tariquidar, which is an inhibitor of P-glycoprotein, we evaluated whether small-animal positron emission tomography can quantify regional changes in transporter function in the rat brain at baseline and 48 h after a pilocarpine-induced status epilepticus. P-glycoprotein expression was additionally quantified by immunohistochemistry. To reveal putative seizure-induced changes in blood-brain barrier integrity, we performed gadolinium-enhanced magnetic resonance scans on a 7.0 tesla small-animal scanner. Before P-glycoprotein modulation, brain uptake of (R)-[(11)C]verapamil was low in all regions investigated in control and post-status epilepticus rats. After administration of 3 mg/kg tariquidar, which inhibits P-glycoprotein only partially, we observed increased regional differentiation in brain activity uptake in post-status epilepticus versus control rats, which diminished after maximal P-glycoprotein inhibition. Regional increases in the efflux rate constant k(2), but not in distribution volume V(T) or influx rate constant K(1), correlated significantly with increases in P-glycoprotein expression measured by immunohistochemistry. This imaging protocol proves to be suitable to detect seizure-induced regional changes in P-glycoprotein activity and is readily applicable to humans, with the aim to detect relevant mechanisms of pharmacoresistance in epilepsy in vivo.

Vigabatrin for focal drug delivery in epilepsy: bilateral microinfusion into the subthalamic nucleus is more effective than intranigral or systemic administration in a rat seizure model.

Vigabatrin is a rationally developed antiepileptic drug, which acts by increasing GABA levels in the brain by irreversibly inhibiting GABA degradation. However, its clinical use in epilepsy is restricted by severe side effects, including vision loss, which is thought to be a consequence of drug exposure of the retina and nonepileptic brain regions. Targeted delivery into brain regions involved in seizure generation and propagation would overcome this problem. Previous studies in rat models of seizures or epilepsy have shown that anticonvulsant effects can be achieved by bilateral microinjection of vigabatrin into the substantia nigra pars reticulata (SNr), a basal ganglia output structure that plays an important role in the modulation of seizures. In the present study, we compared the anticonvulsant efficacy of vigabatrin after systemic and intranigral administration in a rat model, in which seizure susceptibility can be determined by timed intravenous infusion of pentylenetetrazol (PTZ) before and after drug injection in individual animals. Furthermore, because the subthalamic nucleus (STN) plays a crucial role as a regulator of basal ganglia outflow by providing excitatory glutamatergic input into the two output nuclei of the basal ganglia, SNr and entopeduncular nucleus, we evaluated the effects of bilateral focal delivery of vigabatrin into the STN on PTZ seizure threshold. A significant increase in seizure threshold was observed following systemic (i.p.) administration of high (600 or 1200 mg/kg) doses of vigabatrin. Bilateral microinjection of vigabatrin (10 µg) into either the anterior or posterior SNr also increased seizure threshold, but less markedly than systemic treatment. In contrast, focal delivery into the STN increased seizure threshold more markedly than either intranigral or systemic administration of vigabatrin. Furthermore, focal inhibition of STN was not associated with the severe adverse effects associated with systemic treatment. The data demonstrate that vigabatrin is an interesting substance for focal drug delivery in epilepsy and may be advantageous compared to more commonly evaluated compounds such as muscimol.

A comparative small-animal PET evaluation of [11C]tariquidar, [11C]elacridar and (R)-[11C]verapamil for detection of P-glycoprotein-expressing murine breast cancer.

PURPOSE: One important mechanism for chemoresistance of tumours is overexpression of the adenosine triphosphate-binding cassette transporter P-glycoprotein (Pgp). Pgp reduces intracellular concentrations of chemotherapeutic drugs. The aim of this study was to compare the suitability of the radiolabelled Pgp inhibitors [(11)C]tariquidar and [(11)C]elacridar with the Pgp substrate radiotracer (R)-[(11)C]verapamil for discriminating tumours expressing low and high levels of Pgp using small-animal PET imaging in a murine breast cancer model. METHODS: Murine mammary carcinoma cells (EMT6) were continuously exposed to doxorubicin to generate a Pgp-overexpressing, doxorubicin-resistant cell line (EMT6AR1.0 cells). Both cell lines were subcutaneously injected into female athymic nude mice. One week after implantation, animals underwent PET scans with [(11)C]tariquidar (n = 7), [(11)C]elacridar (n = 6) and (R)-[(11)C]verapamil (n = 7), before and after administration of unlabelled tariquidar (15 mg/kg). Pgp expression in tumour grafts was evaluated by Western blotting. RESULTS: [(11)C]Tariquidar showed significantly higher retention in Pgp-overexpressing EMT6AR1.0 compared with EMT6 tumours: the mean ± SD areas under the time-activity curves in scan 1 from time 0 to 60 min (AUC(0-60)) were 38.8 ± 2.2 min and 25.0 ± 5.3 min (p = 0.016, Wilcoxon matched pairs test). [(11)C]Elacridar and (R)-[(11)C]verapamil were not able to discriminate Pgp expression in tumour models. Following administration of unlabelled tariquidar, both EMT6Ar1.0 and EMT6 tumours showed increases in uptake of [(11)C]tariquidar, [(11)C]elacridar and (R)-[(11)C]verapamil. CONCLUSION: Among the tested radiotracers, [(11)C]tariquidar performed best in discriminating tumours expressing high and low levels of Pgp. Therefore [(11)C]tariquidar merits further investigation as a PET tracer to assess Pgp expression levels in solid tumours.

The novel dual-mechanism Kv7 potassium channel/TSPO receptor activator GRT-X is more effective than the Kv7 channel opener retigabine in the 6-Hz refractory seizure mouse model.

Epilepsy, one of the most common and most disabling neurological disorders, is characterized by spontaneous recurrent seizures, often associated with structural brain alterations and cognitive and psychiatric comorbidities. In about 30% of patients, the seizures are resistant to current treatments; so more effective treatments are urgently needed. Among the ∼30 clinically approved antiseizure drugs, retigabine (ezogabine) is the only drug that acts as a positive allosteric modulator (or opener) of voltage-gated Kv7 potassium channels, which is particularly interesting for some genetic forms of epilepsy. Here we describe a novel dual-mode-of-action compound, GRT-X (N-[(3-fluorophenyl)-methyl]-1-(2-methoxyethyl)-4-methyl-2-oxo-(7-trifluoromethyl)-1H-quinoline-3-carboxylic acid amide) that activates both Kv7 potassium channels and the mitochondrial translocator protein 18 kDa (TSPO), leading to increased synthesis of brain neurosteroids. TSPO activators are known to exert anti-inflammatory, neuroprotective, anxiolytic, and antidepressive effects, which, together with an antiseizure effect (mediated by Kv7 channels), would be highly relevant for the treatment of epilepsy. This prompted us to compare the antiseizure efficacy of retigabine and GRT-X in six mouse and rat models of epileptic seizures, including the 6-Hz model of difficult-to-treat focal seizures. Furthermore, the tolerability of the two compounds was compared in mice and rats. Potency comparisons were based on both doses and peak plasma concentrations. Overall, GRT-X was more effective than retigabine in three of the six seizure models used here, the most important difference being the high efficacy in the 6-Hz (32 mA) seizure model in mice. Based on drug plasma levels, GRT-X was at least 30 times more potent than retigabine in the latter model. These data indicate that GRT-X is a highly interesting novel anti-seizure drug with a unique (first-in-class) dual-mode mechanism of action.

Spatio-Temporal Alterations in Synaptic Density During Epileptogenesis in the Rat Brain.

Synaptic vesicle glycoprotein 2A (SV2A) is a transmembrane protein that binds levetiracetam and is involved in neurotransmission via an unknown mechanism. SV2A-immunoreactivity is reduced in animal models of epilepsy, and in postmortem hippocampi from patients with temporal lobe epilepsy. It is not known if other regions outside the hippocampus are affected in epilepsy, and whether SV2A expression is permanently reduced or regulated over time. In this study, we induced a generalized status epilepticus (SE) by systemic administration of lithium-pilocarpine to adult female rats. The brains from all animals experiencing SE were collected at different time points after the treatment. The radiotracer, [11C]-UCB-J, binds to SV2A with high affinity, and has been used for in vivo imaging as an a-proxy marker for synaptic density. Here we determined the level of tritiated UCB-J binding by semiquantitative autoradiography in the cerebral cortex, hippocampus, thalamus, and hypothalamus, and in cortical subregions. A prominent and highly significant reduction in SV2A binding capacity was observed over the first days after SE in the cerebral cortex and the hippocampus, but not in the thalamus and hypothalamus. The magnitude in reduction was larger and occurred earlier in the hippocampus and the piriform cortex, than in other cortical subregions. Interestingly, in all areas examined, the binding capacity returned to control levels 12 weeks after the SE comparable to the chronic epileptic phase. These data indicate that lithium-pilocarpine-induced epileptogenesis involves both loss and gain of synapses in the in a time-dependent manner.

Grimace scale, burrowing, and nest building for the assessment of post-surgical pain in mice and rats-A systematic review.

Several studies suggested an informative value of behavioral and grimace scale parameters for the detection of pain. However, the robustness and reliability of the parameters as well as the current extent of implementation are still largely unknown. In this study, we aimed to systematically analyze the current evidence-base of grimace scale, burrowing, and nest building for the assessment of post-surgical pain in mice and rats. The following platforms were searched for relevant articles: PubMed, Embase via Ovid, and Web of Science. Only full peer-reviewed studies that describe the grimace scale, burrowing, and/or nest building as pain parameters in the post-surgical phase in mice and/or rats were included. Information about the study design, animal characteristics, intervention characteristics, and outcome measures was extracted from identified publications. In total, 74 papers were included in this review. The majority of studies have been conducted in young adult C57BL/6J mice and Sprague Dawley and Wistar rats. While there is an apparent lack of information about young animals, some studies that analyzed the grimace scale in aged rats were identified. The majority of studies focused on laparotomy-associated pain. Only limited information is available about other types of surgical interventions. While an impact of surgery and an influence of analgesia were rather consistently reported in studies focusing on grimace scales, the number of studies that assessed respective effects was rather low for nest building and burrowing. Moreover, controversial findings were evident for the impact of analgesics on post-surgical nest building activity. Regarding analgesia, a monotherapeutic approach was identified in the vast majority of studies with non-steroidal anti-inflammatory (NSAID) drugs and opioids being most commonly used. In conclusion, most evidence exists for grimace scales, which were more frequently used to assess post-surgical pain in rodents than the other behavioral parameters. However, our findings also point to relevant knowledge gaps concerning the post-surgical application in different strains, age levels, and following different surgical procedures. Future efforts are also necessary to directly compare the sensitivity and robustness of different readout parameters applied for the assessment of nest building and burrowing activities.

Radiosynthesis and in vivo evaluation of 1-[18F]fluoroelacridar as a positron emission tomography tracer for P-glycoprotein and breast cancer resistance protein.

Aim of this study was to label the potent dual P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) inhibitor elacridar (1) with (18)F to provide a positron emission tomography (PET) radiotracer to visualize Pgp and BCRP. A series of new 1- and 2-halogen- and nitro-substituted derivatives of 1 (4a-e) was synthesized as precursor molecules and reference compounds for radiolabelling and shown to display comparable in vitro potency to 1 in increasing rhodamine 123 accumulation in a cell line overexpressing human Pgp (MDCKII-MDR1). 1-[(18)F]fluoroelacridar ([(18)F]4b) was synthesized in a decay-corrected radiochemical yield of 1.7±0.9% by a 1-step no-carrier added nucleophilic aromatic (18)F-substitution of 1-nitro precursor 4c. Small-animal PET imaging of [(18)F]4b was performed in naïve rats, before and after administration of unlabelled 1 (5 mg/kg, n=3), as well as in wild-type and Mdr1a/b((-/-))Bcrp1((-/-)) mice (n=3). In PET experiments in rats, administration of unlabelled 1 increased brain activity uptake by a factor of 9.5 (p=0.0002, 2-tailed Student's t-test), whereas blood activity levels remained unchanged. In Mdr1a/b((-/-))Bcrp1((-/-)) mice, the mean brain-to-blood ratio of activity at 60 min after tracer injection was 7.6 times higher as compared to wild-type animals (p=0.0002). HPLC analysis of rat brain tissue extracts collected at 40 min after injection of [(18)F]4b revealed that 93±7% of total radioactivity in brain was in the form of unchanged [(18)F]4b. In conclusion, the in vivo behavior of [(18)F]4b was found to be similar to previously described [(11)C]1 suggesting transport of [(18)F]4b by Pgp and/or BCRP at the rodent BBB. However, low radiochemical yields and a significant degree of in vivo defluorination will limit the utility of [(18)F]4b as a PET tracer.

Choice of anesthesia and data analysis method strongly increases sensitivity of 18F-FDG PET imaging during experimental epileptogenesis.

PURPOSE: Alterations in brain glucose metabolism detected by 2-deoxy-2-[18F]-fluoro-D-glucose (18F-FDG) positron emission tomography (PET) may serve as an early predictive biomarker and treatment target for epileptogenesis. Here, we aimed to investigate changes in cerebral glucose metabolism before induction of epileptogenesis, during epileptogenesis as well as during chronic epilepsy. As anesthesia is usually unavoidable for preclinical PET imaging and influences the distribution of the radiotracer, four different protocols were compared. PROCEDURES: We investigated 18F-FDG uptake phase in conscious rats followed by a static scan as well as dynamic scans under continuous isoflurane, medetomidine-midazolam-fentanyl (MMF), or propofol anesthesia. Furthermore, we applied different analysis approaches: atlas-based regional analysis, statistical parametric mapping, and kinetic analysis. RESULTS: At baseline and compared to uptake in conscious rats, isoflurane and propofol anesthesia resulted in decreased cortical 18F-FDG uptake while MMF anesthesia led to a globally decreased tracer uptake. During epileptogenesis, MMF anesthesia was clearly best distinctive for visualization of prominently increased glucometabolism in epilepsy-related brain areas. Kinetic modeling further increased sensitivity, particularly for continuous isoflurane anesthesia. During chronic epilepsy, hypometabolism affecting more or less the whole brain was detectable with all protocols. CONCLUSION: This study reveals evaluation of anesthesia protocols for preclinical 18F-FDG PET imaging as a critical step in the study design. Together with an appropriate data analysis workflow, the chosen anesthesia protocol may uncover otherwise concealed disease-associated regional glucometabolic changes.

Novel aspects of age-protection by spermidine supplementation are associated with preserved telomere length.

Ageing provokes a plethora of molecular, cellular and physiological deteriorations, including heart failure, neurodegeneration, metabolic maladaptation, telomere attrition and hair loss. Interestingly, on the molecular level, the capacity to induce autophagy, a cellular recycling and cleaning process, declines with age across a large spectrum of model organisms and is thought to be responsible for a subset of age-induced changes. Here, we show that a 6-month administration of the natural autophagy inducer spermidine in the drinking water to aged mice is sufficient to significantly attenuate distinct age-associated phenotypes. These include modulation of brain glucose metabolism, suppression of distinct cardiac inflammation parameters, decreased number of pathological sights in kidney and liver and decrease of age-induced hair loss. Interestingly, spermidine-mediated age protection was associated with decreased telomere attrition, arguing in favour of a novel cellular mechanism behind the anti-ageing effects of spermidine administration.