Effect of short- and long-term administration of baclofen on spatial learning and memory in rats.

Baclofen is the only clinically available metabotropic GABA(B) receptor agonist. In our experiment, we tested the hypothesis that long-term baclofen administration can impair learning and memory in rats. The experiment consisted of three parts. In the first part of the study the drug was administered simultaneously with the beginning of the behavioral tests. In the second and third part of the experiment baclofen was administered daily for 14 days and for one month before the tests. In each part of the experiment, adult rats were randomly divided into four treatment groups. Three groups were given an injection of baclofen at doses of 1 mg/kg, 5 mg/kg, 10 mg/kg, while the fourth group was injected with saline. The injections were given after each session. Spatial learning and memory were tested using the Morris water maze, involving three types of tests: Acquisition, Probe, and Re-acquisition. This work reveals that baclofen did not affect spatial learning at any of the tested doses and regardless of the length of administration. Memory was observed to be affected, but only at the highest dose of baclofen and only temporarily. This conclusion is in line with previously published clinical cases.

Baclofen reversed thermal place preference in rats with chronic constriction injury.

Chronic constriction injury to the sciatic nerve was used as an animal model of neuropathic pain. Instead of frequently used reflex-based tests we used an operant thermal place preference test to evaluate signs of neuropathic pain and the effect of baclofen administration in rats with neuropathy. Chronic constriction injury was induced by four loose ligations of the sciatic nerve. Thermal place preference (45 °C vs. 22 °C and 45 °C vs. 11 °C) was measured after the ligation and after the administration of baclofen in sham and experimental rats. Rats with the chronic constriction injury spent significantly less time on the colder plate compared to sham operated animals at the combination 45 °C vs. 11 °C. After administration of baclofen (10 mg/kg s.c.), the aversion to the colder plate in rats with chronic constriction injury disappeared. At the combination 45 °C vs. 22 °C, no difference in time spent on colder and/or warmer plate was found between sham and experimental animals. These findings show the importance of cold allodynia evaluation in rats with chronic constriction injury and the effectiveness of baclofen in this neuropathic pain model.

CARM1 modulators affect epigenome of stem cells and change morphology of nucleoli.

CARM1 interacts with numerous transcription factors to mediate cellular processes, especially gene expression. This is important for the maintenance of ESC pluripotency or intervention to tumorigenesis. Here, we studied epigenomic effects of two potential CARM1 modulators: an activator (EML159) and an inhibitor (ellagic acid dihydrate, EA). We examined nuclear morphology in human and mouse embryonic stem cells (hESCs, mESCs), as well as in iPS cells. The CARM1 modulators did not function similarly in all cell types. EA decreased the levels of the pluripotency markers, OCT4 and NANOG, particularly in iPSCs, whereas the levels of these proteins increased after EML159 treatment. EML159 treatment of mouse ESCs led to decreased levels of OCT4 and NANOG, which was accompanied by an increased level of Endo-A. The same trend was observed for NANOG and Endo-A in hESCs affected by EML159. Interestingly, EA mainly changed epigenetic features of nucleoli because a high level of arginine asymmetric di-methylation in the nucleoli of hESCs was reduced after EA treatment. ChIP-PCR of ribosomal genes confirmed significantly reduced levels of H3R17me2a, in both the promoter region of ribosomal genes and rDNA encoding 28S rRNA, after EA addition. Moreover, EA treatment changed the nuclear pattern of AgNORs (silver-stained nucleolus organizer regions) in all cell types studied. In EA-treated ESCs, AgNOR pattern was similar to the pattern of AgNORs after inhibition of RNA pol I by actinomycin D. Together, inhibitory effect of EA on arginine methylation and effect on related morphological parameters was especially observed in compartment of nucleoli.

Development of antibodies for determination of alkylresorcinol metabolites in human urine and elucidation of ELISA cross-reactivity.

Alkylresorcinols (ARs) are amphiphilic phenolic lipids and their two main metabolites, 3-(3,5-dihydroxyphenyl)-propanoic acid (DHPPA) and 3,5-dihydroxybenzoic acid (DHBA), can be used as biomarkers of whole grain wheat and rye intake. The aim of this study was to develop antibodies against DHBA and DHPPA for use in ELISA analysis. Good calibration curves were obtained for ELISA using alkaline phosphatase (AP) conjugates. The highest sensitivity for DHPPA was found using a reagent combination of anti-DHPPA-BSA and DHPAA-AP in a direct ELISA (IC50=1.5µmol/L), and for DHBA using a reagent combination of anti-DHBA-OV and DHBA-AP (IC50=1.3µmol/L). Calibration was conducted in the linear range (0.3-27.4µmol/L), with limit of detection (LOD) 0.1µmol/L. Intra and inter CVs was in the range of 0.7-7.2% and 5.1-11.5%, respectively, for DHPPA and 1.3-9.4% and 3.5-20%, respectively, for DHBA. Mean recovery was 104% for DHPPA and 102% for DHBA. The ELISA method developed was then used for analysis of 120 urine samples from free-living men and women that had previously been analysed by gas chromatography-mass spectrometry (GC-MS). ELISA produced several-fold higher values than GC-MS. Application of high-resolution Orbitrap mass spectrometry (HR Orbitrap MS) allowed several compounds, including novel putative AR metabolites, to be identified, synthesised and confirmed as compounds with high ELISA cross-reactivity.

Pain perception in neurodevelopmental animal models of schizophrenia.

Animal models are important for the investigation of mechanisms and therapeutic approaches in various human diseases, including schizophrenia. Recently, two neurodevelopmental rat models of this psychosis were developed based upon the use of subunit selective N-methyl-D-aspartate receptor agonists--quinolinic acid (QUIN) and N-acetyl-aspartyl-glutamate (NAAG). The aim of this study was to evaluate pain perception in these models. QUIN or NAAG was infused into lateral cerebral ventricles neonatally. In the adulthood, the pain perception was examined. The rats with neonatal brain lesions did not show any significant differences in acute mechanical nociception and in formalin test compared to controls. However, the neonatally lesioned rats exhibited significantly higher pain thresholds in thermal nociception. Increased levels of mechanical hyperalgesia, accompanying the sciatic nerve constriction (neuropathic pain), were also observed in lesioned rats. Although hyperalgesia was more pronounced in QUIN-treated animals, the number of c-Fos-immunoreactive neurons of the lumbar spinal cord was similar in experimental and control rats. We conclude that neonatal brain lesions attenuated the thermal perception in both nociceptive and neuropathic pain whereas mechanical pain was increased in the model of neuropathic pain only. Thus, nociceptive and neuropathic pain belongs--in addition to behavioral changes--among the parameters which are affected in described animal models of schizophrenia.

In-house validation of liquid chromatography tandem mass spectrometry for determination of semicarbazide in eggs and stability of analyte in matrix.

An improved LC-MS/MS method for the determination of semicarbazide in whole egg is described. Waters OASIS-MCX cation exchange purification cartridges increased the sensitivity for analysis by LC-MS/MS. The validation study was carried out according to criteria and requirements of Commission Decision 2002/657/EC for confirmatory analysis and provided the data as follows: The correlation coefficient for the matrix calibration curve, in the range of 0-5 microg kg(-1), was r=0.9968. The detection capability and decision limit, measured according to ISO11843-2, were CCalpha=0.20 microg kg(-1) and CCbeta=0.25 microg kg(-1). Repeatability (CVSr) and within-laboratory reproducibility (CVSwr) determined for the concentration levels of 0.2, 0.5 and 1.0 microg kg(-1) SEM ranged from 11.9 to 5.7% and 11.8 to 6.3%, respectively. The validated method was applied to investigate SEM stability in incurred materials (egg homogenates) during long-term storage at -20 degrees C and 4 degrees C. The study proved by a two-sampling test that SEM at levels of 17. 7, 1.2, 10.6 and 0.47 microg kg(-1) was stable for up to 12 months.

Prenatal and perinatal factors influencing nociception, addiction and behavior during ontogenetic development.

This review, which summarizes our findings concerning the long-term effects of pre-, peri- and postnatal factors affecting development, nociception and sensorimotor functions, focuses on three areas: 1) perinatal factors influencing nociception in adult rats were examined in rats with hippocampal lesions, after the administration of stress influencing and psychostimulant drugs (dexamethasone, indomethacine and methamphetamine); 2) the effect of pre- and early postnatal methamphetamine administration was shown to impair the development of sensorimotor functions tested in rat pups throughout the preweaning period; 3) the effect of extensive dorsal rhizotomy of the brachial plexus during the early postnatal period was studied with respect to neuropathic pain development and sensorimotor functions. The present study indicates that prenatal or neonatal stress, as well as various drugs, may disturb the development of the nociceptive system and cause long-term behavioral changes persisting to adulthood and that some types of neuropathic pain cannot be induced during the first two postnatal weeks at all. A mature nervous system is required for the development of the described pathological behaviors.

ELISA for semicarbazide and its application for screening in food contamination.

The development of a direct competitive enzyme-linked immunosorbent assay (ELISA) based on polyclonal antibodies specific for semicarbazide (SEM) is described. Molecular modelling of the hapten mimics and other key components of the assay system was conducted to explain antibody properties in relation to hapten design. The small aliphatic molecule SEM was coupled to 3-carboxybenzaldehyde to produce carboxyphenyl-SEM (CPSEM), for the generation of specific antibodies. Five rabbits produced antibodies against NPSEM (used in direct and indirect ELISA formats) exhibiting a 50% binding inhibition level (IC(50) values) of 0.06-2.28 microgL(-1) in assay buffer for SEM. The most sensitive indirect assay based on the antibody MVK39 showed a high dynamic range providing a linear readout in the range of 0.01-0.2 microgL(-1). Antibody MVK31 (IgG) allowed specific SEM detection at an IC(50)=0.14 microgL(-1) in direct ELISA and was evaluated using solvent extracted SEM-spiked porcine and baby food samples. Recovery levels determined from fortified samples (0.5, 1.0, 1.5, 5, 10 and 20 microgkg(-1)) of porcine and baby food ranged from 82.9 to 105.3%, respectively, with a coefficient of variation less than 15.5%. Respective detection capability and threshold of the assay for porcine muscle, set on the basis of acceptance of no false negative results, was 0.3 and 0.11 microgkg(-1).

Validation of an enzyme-linked immunosorbent assay (ELISA) for the determination of 4-nonylphenol and octylphenol in surface water samples by LC-ESI-MS.

4-Nonylphenol (NP) and octylphenol (OP) were measured by direct ELISA in both laboratory-fortified and surface water samples collected monthly from 10 rivers. In this procedure, samples were concentrated by solid phase extraction (SPE) using Lichrolut RP-18 sorbent with good recoveries obtained for both LC-MS and ELISA, giving a low level of detection (LOD) at the range of low mugL(-1) and good reproducibility. Analysis of 40 surface water samples demonstrated that the ELISA may be a useful screening tool for the determination of the alkylphenols in surface water matrices. The concentration of NP and OP in surface waters ranged from 0.11 to 6.58mugL(-1). A good correlation of results obtained by ELISA and LC-MS within the concentration range of 0.08-6.86mugL(-1) was found in the river samples [R(2)=0.924, n=39]. The influence of various factors on assay determination was also discussed.

GABA(B) receptor agonist baclofen has non-specific antinociceptive effect in the model of peripheral neuropathy in the rat.

Baclofen, which is a specific agonist of the metabotropic GABA(B) receptor, is used in clinical practice for the treatment of spasticity of skeletal muscles. It also exerts an analgesic effect, but this effect is still not clear and especially controversial in neuropathic pain. In this work, we studied the antinociceptive effects of baclofen in a model of chronic peripheral neuropathic pain - loose ligation of the sciatic nerve (chronic constriction injury, CCI). As controls we used sham-operated animals. The changes of thermal pain threshold were measured using the plantar test 15-25 days after the operation. The obtained results suggest that baclofen increases pain threshold in both groups. The antinociceptive effect of baclofen was dose-dependent and the maximum response without motor deficits was observed at a dose of 15 mg/kg s.c. In the rats with CCI, significant differences between affected (ipsilateral) and contralateral hind paw were present. This difference was dose-dependent, the highest value (6.2+/-1.37 s) was found at the dose of 20 mg/kg. Based on our results and previous findings it could be summarized that baclofen has antinociceptive action, which is attenuated in the model of chronic neuropathic pain probably due to the degeneration of GABA interneurons after chronic constriction injury.