Contribution of haemodynamic side effects and associated autonomic reflexes to ventricular arrhythmias triggering by torsadogenic hERG blocking drugs.

BACKGROUND AND PURPOSE: HERG blocking drugs known for their propensity to trigger Torsades de Pointes (TdP) were reported to induce a sympatho-vagal coactivation and to enhance High Frequency heart rate (HFHR) and QT oscillations (HFQT) in telemetric data. The present work aimed to characterize the underlying mechanism(s) leading to these autonomic changes. EXPERIMENTAL APPROACH: Effects of 15 torsadogenic hERG blocking drugs (astemizole, chlorpromazine, cisapride, droperidol, ibutilide, dofetilide, haloperidol, moxifloxacin, pimozide, quinidine, risperidone, sotalol, sertindole, terfenadine, and thioridazine) were assessed by telemetry in beagle dogs. Haemodynamic effects on diastolic and systolic arterial pressure were analysed from the first doses causing QTc prolongation and/or HFQT oscillations enhancement. Autonomic control changes were analysed using the high frequency autonomic modulation (HFAM) model. KEY RESULTS: Except for moxifloxacin and quinidine, all torsadogenic hERG blockers induced parasympathetic activation or sympatho-vagal coactivation combined with enhancement of HFQT oscillations. These autonomic effects result from reflex compensatory mechanisms in response to mild haemodynamic side effects. These haemodynamic mechanisms were characterized by transient HR acceleration during HF oscillations. A phenomenon of concealed QT prolongation was unmasked for several torsadogenic hERG blockers under ß-adrenoceptor blockade with atenolol. Resulting enhancement of HFQT oscillations was shown to contribute directly to triggering dofetilide-induced ventricular arrhythmias. CONCLUSION AND IMPLICATIONS: This work supports for the first time a contribution of haemodynamic side properties to ventricular arrhythmias triggered by torsadogenic hERG blocking drugs. These haemodynamic side effects may constitute a second component of their arrhythmic profile, acting as a trigger alongside their intrinsic arrhythmogenic electrophysiological properties.

Analysis of COVID-19 Spread in Tokyo through an Agent-Based Model with Data Assimilation.

In this paper, we introduce an agent-based model together with a particle filter approach to study the spread of COVID-19. Investigations are mainly performed on the metropolis of Tokyo, but other prefectures of Japan are also briefly surveyed. A novel method for evaluating the effective reproduction number is one of the main outcomes of our approach. Other unknown parameters are also evaluated. Uncertain quantities, such as, for example, the probability that an infected agent develops symptoms, are tested and discussed, and the stability of our computations is examined. Detailed explanations are provided for the model and for the assimilation process.

TREK1 channel activation as a new analgesic strategy devoid of opioid adverse effects.

BACKGROUND AND PURPOSE: Opioids are effective painkillers. However, their risk-benefit ratio is dampened by numerous adverse effects and opioid misuse has led to a public health crisis. Safer alternatives are required, but isolating the antinociceptive effect of opioids from their adverse effects is a pharmacological challenge because activation of the µ opioid receptor triggers both the antinociceptive and adverse effects of opioids. EXPERIMENTAL APPROACH: The TREK1 potassium channel is activated downstream of µ receptor and involved in the antinociceptive activity of morphine but not in its adverse effects. Bypassing the µ opioid receptor to directly activate TREK1 could therefore be a safer analgesic strategy. KEY RESULTS: We developed a selective TREK1 activator, RNE28, with antinociceptive activity in naive rodents and in models of inflammatory and neuropathic pain. This activity was lost in TREK1 knockout mice or wild-type mice treated with the TREK1 blocker spadin, showing that TREK1 is required for the antinociceptive activity of RNE28. RNE28 did not induce respiratory depression, constipation, rewarding effects, or sedation at the analgesic doses tested. CONCLUSION AND IMPLICATIONS: This proof-of-concept study shows that TREK1 activators could constitute a novel class of painkillers, inspired by the mechanism of action of opioids but devoid of their adverse effects.

Design and Validation of an Automated Process for the Expansion of Peripheral Blood-Derived CD34+ Cells for Clinical Use After Myocardial Infarction.

We previously demonstrated that intracardiac delivery of autologous peripheral blood-derived CD34+ stem cells (SCs), mobilized by granulocyte-colony stimulating factor (G-CSF) and collected by leukapheresis after myocardial infarction, structurally and functionally repaired the damaged myocardial area. When used for cardiac indication, CD34+ cells are now considered as Advanced Therapy Medicinal Products (ATMPs). We have industrialized their production by developing an automated device for ex vivo CD34+ -SC expansion, starting from a whole blood (WB) sample. Blood samples were collected from healthy donors after G-CSF mobilization. Manufacturing procedures included: (a) isolation of total nuclear cells, (b) CD34+ immunoselection, (c) expansion and cell culture recovery in the device, and (d) expanded CD34+ cell immunoselection and formulation. The assessment of CD34+ cell counts, viability, and immunophenotype and sterility tests were performed as quality tests. We established graft acceptance criteria and performed validation processes in three cell therapy centers. 59.4 × 106 ± 36.8 × 106 viable CD34+ cells were reproducibly generated as the final product from 220 ml WB containing 17.1 × 106 ± 8.1 × 106 viable CD34+ cells. CD34+ identity, genetic stability, and telomere length were consistent with those of basal CD34+ cells. Gram staining and mycoplasma and endotoxin analyses were negative in all cases. We confirmed the therapeutic efficacy of both CD34+ -cell categories in experimental acute myocardial infarct (AMI) in immunodeficient rats during preclinical studies. This reproducible, automated, and standardized expansion process produces high numbers of CD34+ cells corresponding to the approved ATMP and paves the way for a phase I/IIb study in AMI, which is currently recruiting patients. Stem Cells Translational Medicine 2019;8:822&832.

Relevance of electroencephalogram assessment in amyloid and tau pathology in rat.

In order to contribute to a better knowledge on the relationship between amyloid and tau pathology, and electroencephalography (EEG) disturbances, the aim of this study was to evaluate the effects of injection of beta amyloid Abeta(1-42) peptide, tau (a recombinant AAV (Adeno-Associated Virus) containing the human transgene tau with the P301 L mutation on rats and the combination of both, on the power of brain's rhythm (delta, theta, alpha, beta and gamma waves) during the different sleep/wake states of animals by EEG recording. Currently, no preclinical studies explore the effect of the tau pathology on EEG. The experimentations were performed 3 weeks and 3 months post injections. Beta amyloid deposits and hyperphosphorylated Tau are observed by immunohistofluorescence, only in the hippocampus. Furthermore, using a radial arm water maze, the main effect was observed on working memory which was significantly impaired in Abeta-Tau group only 3 months post injections. However, on EEG, as early as the 3rd week, an overall decrease of the EEG bands power was observed in the treated groups, particularly the theta waves during the rapid eye movement (REM) sleep. Beta amyloid was mainly involved in these perturbations. Obviously, EEG seems to be an interesting tool in the early diagnostic of amyloid and tau pathologies, with a good sensitivity and the possibility to perform a follow up during a large period.

High-frequency autonomic modulation: a new model for analysis of autonomic cardiac control.

BACKGROUND AND PURPOSE: Increase in high-frequency beat-to-beat heart rate oscillations by torsadogenic hERG blockers appears to be associated with signs of parasympathetic and sympathetic co-activation which cannot be assessed directly using classic methods of heart rate variability analysis. The present work aimed to find a translational model that would allow this particular state of the autonomic control of heart rate to be assessed. EXPERIMENTAL APPROACH: High-frequency heart rate and heart period oscillations were analysed within discrete 10 s intervals in a cohort of 200 healthy human subjects. Results were compared to data collected in non-human primates and beagle dogs during pharmacological challenges and torsadogenic hERG blockers exposure, in 127 genotyped LQT1 patients on/off ß-blocker treatment and in subgroups of smoking and non-smoking subjects. KEY RESULTS: Three states of autonomic modulation, S1 (parasympathetic predominance) to S3 (reciprocal parasympathetic withdrawal/sympathetic activation), were differentiated to build a new model of heart rate variability referred to as high-frequency autonomic modulation. The S2 state corresponded to a specific state during which both parasympathetic and sympathetic systems were coexisting or co-activated. S2 oscillations were proportionally increased by torsadogenic hERG-blocking drugs, whereas smoking caused an increase in S3 oscillations. CONCLUSIONS AND IMPLICATIONS: The combined analysis of the magnitude of high-frequency heart rate and high-frequency heart period oscillations allows a refined assessment of heart rate autonomic modulation applicable to long-term ECG recordings and offers new approaches to assessment of the risk of sudden death both in terms of underlying mechanisms and sensitivity.

Inter-individual variability and modeling of electrical activity: a possible new approach to explore cardiac safety?

Safety pharmacology aims to predict rare side effects of new drugs. We explored whether rare pro-arrhythmic effects could be linked to the variability of the effects of these drugs on ion currents and whether taking into consideration this variability in computational models could help to better detect and predict cardiac side effects. For this purpose, we evaluated how intra- and inter-individual variability influences the effect of hERG inhibition on both the action potential duration and the occurrence of arrhythmias. Using two computer simulation models of human action potentials (endocardial and Purkinje cells), we analyzed the contribution of two biological parameters on the pro-arrhythmic effects of several hERG channel blockers: (i) spermine concentration, which varies with metabolic status, and (ii) L-type calcium conductance, which varies due to single nucleotide polymorphisms or mutations. By varying these parameters, we were able to induce arrhythmias in 1 out of 16 simulations although conventional modeling methods to detect pro-arrhythmic molecules failed. On the basis of our results, taking into consideration only 2 parameters subjected to intra- and inter-individual variability, we propose that in silico computer modeling may help to better define the risks of new drug candidates at early stages of pre-clinical development.

Comparison of high-definition oscillometry -- a non-invasive technology for arterial blood pressure measurement -- with a direct invasive method using radio-telemetry in awake healthy cats.

This study compared indirect blood pressure measurements using a non-invasive method, high-definition oscillometry (HDO), with direct measurements using a radio-telemetry device in awake cats. Paired measurements partitioned to five sub-ranges were collected in six cats using both methods. The results were analysed for assessment of correlation and agreement between the two methods, taking into account all pressure ranges, and with data separated in three sub-groups, low, normal and high ranges of systolic (SBP) and diastolic (DBP) blood pressure. SBP data displayed a mean correlation coefficient of 0.92 ± 0.02 that was reduced for low SBP. The agreement level evaluated from the whole data set was high and slightly reduced for low SBP values. The mean correlation coefficient of DBP was lower than for SBP (ie, 0.81 ± 0.02). The bias for DBP between the two methods was 22.3 ± 1.6 mmHg, suggesting that HDO produced lower values than telemetry. These results suggest that HDO met the validation criteria defined by the American College of Veterinary Internal Medicine consensus panel and provided a faithful measurement of SBP in conscious cats. For DBP, results suggest that HDO tended to underestimate DBP. This finding is clearly inconsistent with the good agreement reported in dogs, but is similar to outcomes achieved in marmosets and cynomolgus monkeys, suggesting that this is not related to HDO but is species related. The data support that the HDO is the first and only validated non-invasive blood pressure device and, as such, it is the only non-invasive reference technique that should be used in future validation studies.

Power spectral analysis of heart rate variability in cynomolgus monkeys in safety pharmacology studies: comparative study with beagle dogs.

INTRODUCTION: Power spectral analysis of heart rate variability is a tool known to provide information of interest on the autonomic control of heart rate in human. However, its use and its conditions of application and interpretation for safety purposes are not well defined for cardiovascular safety pharmacology studies. Likewise, data of power spectral analysis of heart rate variability in cynomolgus monkeys, a species often appropriate for use as second non rodent species in preclinical safety programmes, are not available. This study was designed to evaluate the relevance of this biomarker in this non human primate species, and to compare results with those from beagle dogs under the conditions of safety evaluation studies. METHODS: Power spectral analysis of heart rate variability was performed on data collected in both species by telemetry following a standard design for cardiovascular safety pharmacology studies. Various pharmacological agents were tested in order to compare the profile of responses in both species after modifying the autonomic nervous balance. RESULTS: Heart rate variability in cynomolgus monkeys is mainly driven by the parasympathetic nervous system as in beagle dogs although vagal tone is less than in dogs. Power spectral analysis of heart rate variability allows detection of interaction with the autonomic nervous system in both species in all investigated situations, i.e. sympatholytic/sympathomimetic and parasympatholytic/parasympathomimetic drug induced effects. However, due to species difference in the autonomic control of heart rate, cynomolgus monkeys are likely to be more sensitive than beagle dogs for assessment of sympatholytic properties. DISCUSSION: This study confirms that power spectral analysis of heart rate variability from data derived from ECG recordings in telemetry studies is applicable in cardiovascular safety pharmacology studies and may provide relevant information about possible interaction with the autonomic nervous system when new drug entities are evaluated in either species. However, interspecies differences in autonomic control must be taken into account when interpreting possible drug effects.

Non-invasive quantification of tumor vascular architecture during docetaxel-chemotherapy.

New ultrasound parameters, potentially predictive of tumor response to chemotherapy, were sought after analyzing details of vascular architecture of mammary tumors during chemotherapy. Tumor-bearing rats were separated into untreated or docetaxel-treated group (6 mg/kg/week). Power Doppler Index and vascular contrast-enhanced ultrasound (CEUS) reference endpoints (Peak, area under the curve (AUC), blood flow) were evaluated at the beginning (W (0)), and after 2 and 6 weeks of docetaxel treatment (W (+2) and W (+6)). An improved CEUS image analysis, taking advantage of individual pixel intensity, was developed to quantify large, medium, and small vessels of tumors. Standard immunohistochemistry validated this new methodology analyzing tumor vascular architecture. In rats, there was an enrichment of vascularization with large vessels during tumor growth indicative of a vascular adjustment to tumor size. Docetaxel stopped tumor growth, and showed a sequential effect on vascular parameters. After an initial enrichment in larger vessels (by threefold) at W (+2), docetaxel led to a diminution of vascular parameters at W (+6) (-46 % for peak, -55 % for AUC -31 % compared to W (0)) and a vascular remodeling in favor of small vessels. One of the CEUS parameters measured before chemotherapy, the so-called global contrast-enhanced pixels density, was predictive of rat tumor response to treatment (r = 0.80; p < 0.01). The method was then applied in a clinical setting to detect changes of vascular architecture during chemotherapy of human breast carcinoma. The docetaxel chemotherapy of breast carcinomas induced a similar sequential effect, with vessel enlargement after two cycles of docetaxel treatment and an antiangiogenic effect after six cycles. Such vascular remodeling was not noticed when patients were treated with 5-fluorouracil-epirubicin-cyclophosphamide. Taken together, the sharpened analysis of CEUS pixel intensity presented here strengthened the monitoring of breast tumor vasculature with the potential to improve the prediction of docetaxel efficacy.

Effects of a non thermal plasma treatment alone or in combination with gemcitabine in a MIA PaCa2-luc orthotopic pancreatic carcinoma model.

Pancreatic tumors are the gastrointestinal cancer with the worst prognosis in humans and with a survival rate of 5% at 5 years. Nowadays, no chemotherapy has demonstrated efficacy in terms of survival for this cancer. Previous study focused on the development of a new therapy by non thermal plasma showed significant effects on tumor growth for colorectal carcinoma and glioblastoma. To allow targeted treatment, a fibered plasma (Plasma Gun) was developed and its evaluation was performed on an orthotopic mouse model of human pancreatic carcinoma using a MIA PaCa2-luc bioluminescent cell line. The aim of this study was to characterize this pancreatic carcinoma model and to determine the effects of Plasma Gun alone or in combination with gemcitabine. During a 36 days period, quantitative BLI could be used to follow the tumor progression and we demonstrated that plasma gun induced an inhibition of MIA PaCa2-luc cells proliferation in vitro and in vivo and that this effect could be improved by association with gemcitabine possibly thanks to its radiosensitizing properties.

Reducing endothelial NOS activation and interstitial fluid pressure with n-3 PUFA offset tumor chemoresistance.

The aim of this study was to determine how n-3 polyunsaturated fatty acid (PUFAs) counteracted tumor chemoresistance by restoring a functional vascularization. Rats with chemically induced mammary tumors were divided into two nutritional groups: a control group and a group fed with an n-3 PUFA-enriched diet. Both groups were treated with docetaxel. Functional vascular parameters (ultrasounds, interstitial fluid pressure) were determined for both nutritional groups before (W(0)) and during docetaxel treatment [every 2 h up to 1 week (W(+1)) for interstitial fluid pressure, at W(+1) for Evans blue extravasation and at W(+2) and W(+6) for ultrasounds]. In vitro n-3 PUFA-induced changes in endothelial cell migration, permeability and phosphorylation of endothelial nitric oxide synthase were evaluated using human umbilical vein endothelial cells. Whereas docetaxel stabilized tumor growth in the rat control group, it induced a 50% tumor regression in the n-3 PUFA group. Ultrasounds parameters were consistently lower in the n-3 PUFA group at all time points measured, down to ∼50% at W(+6). A single dose of docetaxel in the n-3 PUFA group markedly reduced interstitial fluid pressure from 2 h after injection up to W(+1) when Evans blue extravasation was increased by 3-fold. A decreased activation of endothelial nitric oxide synthase in tumors of the n-3 PUFA group, and in human umbilical vein endothelial cell cultured with n-3 PUFA, points toward a PUFA-induced disruption of nitric oxide signaling pathway. This normalization of tumor vasculature functions under n-3 PUFA diet indicates that such a supplementation, by improving drug delivery in mammary tumors, could be a complementary clinical strategy to decrease anticancer drug resistance.

A step towards characterisation of electrophysiological profile of torsadogenic drugs.

INTRODUCTION: In a previous study, two electrophysiological patterns for torsadogenic drugs were characterised in the model of isolated canine Purkinje fibres from their respective effects on action potential. This study was designed to elucidate the possible mechanisms underlying these two electrophysiological profiles. METHODS: Effects of representative torsadogenic agents and non torsadogenic drugs on I(Kr), I(Ks), I(K1), I(Na) and I(CaL) were studied in transfected HEK 293 cells using the path-clamp method as well as in conscious beagle dogs and cynomolgus monkeys by telemetry. RESULTS: Patch-clamp studies confirmed that torsadogenic molecules could be discriminated into at least two subgroups. The first subgroup can be defined as apparently pure I(Kr) blockers. The second subgroup can be defined as I(Kr) blockers with ancillary properties on sodium and/or calcium channels which counterbalance the I(Kr) prolongation component. This discrimination is transposable to the telemetered cynomolgus monkey model in terms of QT prolongation but not to the telemetered beagle dog model. This latter inter-species difference could be related to the sympathetic/parasympathetic balance and could involve reserve repolarisation dependent mechanisms. DISCUSSION: The confirmation that torsadogenic drugs might have at least two different electrophysiological profiles should be taken into consideration in preclinical safety pharmacology studies because it increases the value of the cynomolgus monkey model in two particular situations: firstly when an NCE causes sympathetic activation and secondly, when an NCE exhibits a pure I(Kr) blocker pattern independently of its potency to block HERG channels.

Interferences of the autonomic nervous system with drug induced QT prolongation: a point to consider in non-clinical safety studies.

INTRODUCTION: QT interval assessment by telemetry has become one of the most useful models in testing strategies adopted for detection of drug induced QT prolongation in non-clinical safety pharmacology studies. This study reports experimental data showing that the autonomic nervous system might influence drug induced QT prolongation. METHODS: Animals were instrumented with telemetric transmitters and epicardial ECG leads. Effects on QT interval of reference drugs such as thioridazine and terfenadine were analysed with different approaches, the Holzgrefe's probabilistic method, the QT shift method and an individual analysis of beat-to-beat QT/RR pair distribution visualised as points-cloud. RESULTS: Two cases of unexpected absence of QT interval prolongation are reported with thioridazine and terfenadine in conscious beagle dogs under conditions of concomitant tachycardia. The pro-arrhythmic properties of these two molecules were unmasked by co-treatment with sympatholytic agents, atenolol and clonidine respectively suggesting that sympathetic activation and/or parasympathetic withdrawal might impair a drug induced QT prolongation. DISCUSSION: The apparent absence of changes in the QT interval due to novel drug candidates should be interpreted cautiously under conditions of concomitant tachycardia or elevated heart rate levels in non-clinical safety studies.

Calculation of QT shift in non clinical safety pharmacology studies.

INTRODUCTION: Drug-induced QT interval prolongation is a major concern in new drug candidate development. This study presents a method of assessment of drug-induced QT interval prolongation without need for QT correction in conscious Beagle dogs and Cynomolgus monkeys monitored by telemetry. Accuracy and reliability are analysed by comparison with a reference QT correction method (Holzgrefe) from experiments performed with reference substances terfenadine, thioridazine and sotalol. METHODS: The QT shift method principle is assessment of any drug-induced QT interval shift directly from the individual QT/RR relationship. The individual QT/RR relationship is built from a treatment-free 24-hour recording period. QT and RR intervals are determined from a beat-to-beat analysis. A probabilistic method is used to define the individual QT/RR relationships. Checks were performed to compare results obtained with the QT shift method and the QT correction methods. The robustness of the QT shift method was tested under various conditions of drug-induced heart rate change (i.e. normal, bradycardia and tachycardia). RESULTS: The extent of agreement with the used reference QT correction method, Holzgrefe formula, was excellent (3-4 ms) in both animal species under the various drug induced effects on heart rate. The statistical sensitivity threshold for detection of QT prolongation according to a standard safety pharmacology study design was 7-8 ms. DISCUSSION: When combined with the probabilistic determination of individual QT/RR relationships, this simple method provides a direct assessment of a drug-induced effect on QT interval, without any curve fitting or application of correction formula. Despite noticeably different shapes in QT/RR relationships, the QT shift method is applicable to both Beagle dogs and Cynomolgus monkeys. It is likely that the QT shift method will be particularly helpful in problematic cases, enabling detection of drug-induced prolongation of less than 10 ms.

Electrophysiological profile of propiverine--relationship to cardiac risk.

Drugs that prolong the QT interval by blocking human ether-a-go-go (HERG) channels may enhance the risk of ventricular arrhythmia. The spasmolytic drug propiverine is widely used for the therapy of overactive bladder (OAB). Here, we have investigated the effects of propiverine on cardiac ion channels and action potentials as well as on contractile properties of cardiac tissue, in order to estimate its cardiac safety profile, because other drugs used in this indication had to be withdrawn due to safety reasons. Whole-cell patch clamp technique was used to record the following cardiac ion currents: rapidly and slowly activating delayed rectifier K+ current (I(Kr), I(Ks)), ultra rapidly activating delayed rectifier K+ current (I(Kur)), inwardly rectifying K+ current I(K1), transient outward K+ current (I(to)), and L-type Ca2+ current (I(Ca,L)). Action potentials in cardiac tissue biopsies were recorded with conventional microelectrodes. The torsade de pointes screening assay (TDPScreen) was used for drug scoring. Propiverine blocked in a concentration-dependent manner HERG channels expressed in HEK293 cells, as well as native I(Kr) current in ventricular myocytes of guinea pig (IC50 values: 10 microM and 1.8 microM respectively). At high concentrations (100 microM), propiverine suppressed I(Ks). I(K1) and the transient outward current I(to) and I(Kur) were not affected. In guinea-pig ventricular and human atrial myocytes, propiverine also blocked I(Ca,L) (IC50 values: 34.7 microM and 41.7 microM, respectively) and reduced force of contraction. Despite block of I(Kr), action potential duration was not prolonged in guinea-pig and human ventricular tissue, but decreased progressively until excitation failed altogether. Similar effects were observed in dog Purkinje fibers. Propiverine obtained a low score in the TDPScreen. In conclusion, in vitro and in vivo studies of propiverine do not provide evidence for an enhanced cardiovascular safety risk. We propose that lack of torsadogenic risk of propiverine is related to enhancement of repolarization reserve by block of I(Ca,L).

Dietary long-chain n-3 fatty acids modify blood and cardiac phospholipids and reduce protein kinase-C-delta and protein kinase-C-epsilon translocation.

The effects of an n-3 PUFA-enriched diet on cardiac cell membrane phospholipid fraction compositions and associated protein kinase-C (PKC) translocation modification have never been studied in higher mammals. This is of importance since membrane fatty acid composition has been shown to influence PKC signalling pathways. In the present study, we have tested whether the incorporation of n-3 PUFA in cardiac membrane phospholipids correlated with changes in the fatty acid composition of diacylglycerols (DAG) and led to a differential translocation of PKC isoforms. Two groups of five dogs were fed the standard diet supplemented with palm oil or fish oil for 8 weeks. Dogs fed a fish oil-enriched diet showed a preferential incorporation of EPA and, to a lesser extent, of DHA, at the expense of arachidonic acid, in the circulating TAG, plasma phospholipids, erythrocyte phospholipids and cardiomyocyte phospholipid fractions. Analysis of 1,2-DAG fatty acid composition also indicated a preferential enrichment of EPA compared with DHA. Associated with these results, a reduction in the expression of PKC-delta and PKC-epsilon isoforms in the particulate fractions was observed whereas no effect was seen for PKC-alpha and PKC-zeta. We conclude that a fish oil-enriched diet induces a modification in fatty acid composition of cardiac membrane phospholipids, associated with a differential translocation of PKC isoforms. These results can be explained by the production of structurally different DAG that may participate in some of the protective effects of n-3 PUFA against various chronic diseases.

An optimised methodology for the neurobehavioural assessment in rodents.

INTRODUCTION: The most widely used test to identify undesired effects of drugs on the central and the peripheral nervous system is the neurobehavioural observation battery adapted from that first described by Irwin in mice. As a neurobehavioural assessment is based on observations; thus, all factors involved need to be controlled and standardised to make the data collected objective, reproducible, reliable and predictive of safety liabilities. METHODS: An observation battery comprising 58 signs with assigned full details of numerical scores was defined, and a standard design with associated recording, presenting and analysing data system was established. Validation studies were conducted with chlorpromazine, amphetamine, diazepam or clonidine given orally to rats or mice, in order to assess if this methodology could clearly differentiate the profile of effects produced by these compounds. The analysis of data from 80 control rats allowed for the assessment of the normal behaviour in order to characterise the inter-individual, daytime-related variability and the habituation of animals to the procedure. RESULTS: The reference compounds induced their typical and expected transient effects on neurobehaviour, observed both in the home cage and open-arena, and on body temperature. In particular, amphetamine induced a stimulation of the nervous system activities and marked hyperthermia. Chlorpromazine, diazepam and clonidine induced depressive, anxiolytic or sedative effects associated with hypothermia. The analysis of data collected in control animals allowed for the identification of 6 signs which scored differently from the assigned normality at the first handling occasion due to the characteristic fear reactions to the unknown, and 9 signs at 8 h post-dose due to the animal's habituation to experimental conditions and handling. DISCUSSION: The neurobehavioural changes expected by reference compounds administration were detected. These results confirm that by using this methodology the normal behaviour of the rat and the mouse, the daytime-related variability and the habituation of animals can be characterised, allowing a refined, reliable and reproducible neurobehavioural assessment of test substances in rodents.