Anionic metabolic profiling of urine from antibiotic-treated rats by capillary electrophoresis–mass spectrometry

A recently developed capillary electrophoresis (CE)-negative-ionisation mass spectrometry (MS) method was used to profile anionic metabolites in a microbial-host co-metabolism study. Urine samples from rats receiving antibiotics (penicillin G and streptomycin sulfate) for 0, 4, or 8 days were analysed. A quality control sample was measured repeatedly to monitor the performance of the applied CE-MS method. After peak alignment, relative standard deviations (RSDs) for migration time of five representative compounds were below 0.4 %, whereas RSDs for peak area were 7.9–13.5 %. Using univariate and principal component analysis of obtained urinary metabolic profiles, groups of rats receiving different antibiotic treatment could be distinguished based on 17 discriminatory compounds, of which 15 were downregulated and 2 were upregulated upon treatment. Eleven compounds remained down- or upregulated after discontinuation of the antibiotics administration, whereas a recovery effect was observed for others. Based on accurate mass, nine compounds were putatively identified; these included the microbial-mammalian co-metabolites hippuric acid and indoxyl sulfate. Some discriminatory compounds were also observed by other analytical techniques, but CE-MS uniquely revealed ten metabolites modulated by antibiotic exposure, including aconitic acid and an oxocholic acid. This clearly demonstrates the added value of CE-MS for nontargeted profiling of small anionic metabolites in biological samples.

Hydrophilic interaction chromatography–mass spectrometry for anionic metabolic profiling of urine from antibiotic-treated rats

Hydrophilic interaction chromatography–mass spectrometry (HILIC–MS) was used for anionic metabolic profiling of urine from antibiotic-treated rats to study microbial–host co-metabolism. Rats were treated with the antibiotics penicillin G and streptomycin sulfate for four or eight days and compared to a control group. Urine samples were collected at day zero, four and eight, and analyzed by HILIC–MS. Multivariate data analysis was applied to the urinary metabolic profiles to identify biochemical variation between the treatment groups. Principal component analysis found a clear distinction between those animals receiving antibiotics and the control animals, with twenty-nine discriminatory compounds of which twenty were down-regulated and nine up-regulated upon treatment. In the treatment group receiving antibiotics for four days, a recovery effect was observed for seven compounds after cessation of antibiotic administration. Thirteen discriminatory compounds could be putatively identified based on their accurate mass, including aconitic acid, benzenediol sulfate, ferulic acid sulfate, hippuric acid, indoxyl sulfate, penicillin G, phenol and vanillin 4-sulfate. The rat urine samples had previously been analyzed by capillary electrophoresis (CE) with MS detection and proton nuclear magnetic resonance (1H NMR) spectroscopy. Using CE–MS and 1H NMR spectroscopy seventeen and twenty-five discriminatory compounds were found, respectively. Both hippuric acid and indoxyl sulfate were detected across all three platforms. Additionally, eight compounds were observed with both HILIC–MS and CE–MS. Overall, HILIC–MS appears to be highly complementary to CE–MS and 1H NMR spectroscopy, identifying additional compounds that discriminate the urine samples from antibiotic-treated and control rats.

A role for hippocampal PSA-NCAM and NMDA-NR2B receptor function in flavonoid-induced spatial memory improvements in young rats

The increase in incidence and prevalence of neurodegenerative diseases highlights the need for a more comprehensive understanding of how food components may affect neural systems. In particular, flavonoids have been recognized as promising agents capable of influencing different aspects of synaptic plasticity resulting in improvements in memory and learning in both animals and humans. Our previous studies highlight the efficacy of flavonoids in reversing memory impairments in aged rats, yet little is known about the effects of these compounds in healthy animals, particularly with respect to the molecular mechanisms by which flavonoids might alter the underlying synaptic modifications responsible for behavioral changes. We demonstrate that a 3-week intervention with two dietary doses of flavonoids (Dose I: 8.7 mg/day and Dose II: 17.4 mg/day) facilitates spatial memory acquisition and consolidation (24 recall) (p < 0.05) in young healthy rats. We show for the first time that these behavioral improvements are linked to increased levels in the polysialylated form of the neural adhesion molecule (PSA-NCAM) in the dentate gyrus (DG) of the hippocampus, which is known to be required for the establishment of durable memories. We observed parallel increases in hippocampal NMDA receptors containing the NR2B subunit for both 8.7 mg/day (p < 0.05) and 17.4 mg/day (p < 0.001) doses, suggesting an enhancement of glutamate signaling following flavonoid intervention. This is further strengthened by the simultaneous modulation of hippocampal ERK/CREB/BDNF signaling and the activation of the Akt/mTOR/Arc pathway, which are crucial in inducing changes in the strength of hippocampal synaptic connections that underlie learning. Collectively, the present data supports a new role for PSA-NCAM and NMDA-NR2B receptor on flavonoid-induced improvements in learning and memory, contributing further to the growing body of evidence suggesting beneficial effects of flavonoids in cognition and brain health.

A toxicokinetic model for thiamethoxam in rats: implications for higher-tier risk assessment

Risk assessment for mammals is currently based on external exposure measurements, but effects of toxicants are better correlated with the systemically available dose than with the external administered dose. So for risk assessment of pesticides, toxicokinetics should be interpreted in the context of potential exposure in the field taking account of the timescale of exposure and individual patterns of feeding. Internal concentration is the net result of absorption, distribution, metabolism and excretion (ADME). We present a case study for thiamethoxam to show how data from ADME study on rats can be used to parameterize a body burden model which predicts body residue levels after exposures to LD50 dose either as a bolus or eaten at different feeding rates. Kinetic parameters were determined in male and female rats after an intravenous and oral administration of 14C labelled by fitting one-compartment models to measured pesticide concentrations in blood for each individual separately. The concentration of thiamethoxam in blood over time correlated closely with concentrations in other tissues and so was considered representative of pesticide concentration in the whole body. Body burden model simulations showed that maximum body weight-normalized doses of thiamethoxam were lower if the same external dose was ingested normally than if it was force fed in a single bolus dose. This indicates lower risk to rats through dietary exposure than would be estimated from the bolus LD50. The importance of key questions that should be answered before using the body burden approach in risk assessment, data requirements and assumptions made in this study are discussed in detail.

Intrastriatal transplantation of adult human neural crest-derived stem cells improves functional outcome in parkinsonian rats

Parkinson's disease (PD) is considered the second most frequent and one of the most severe neurodegenerative diseases, with dysfunctions of the motor system and with nonmotor symptoms such as depression and dementia. Compensation for the progressive loss of dopaminergic (DA) neurons during PD using current pharmacological treatment strategies is limited and remains challenging. Pluripotent stem cell-based regenerative medicine may offer a promising therapeutic alternative, although the medical application of human embryonic tissue and pluripotent stem cells is still a matter of ethical and practical debate. Addressing these challenges, the present study investigated the potential of adult human neural crest-derived stem cells derived from the inferior turbinate (ITSCs) transplanted into a parkinsonian rat model. Emphasizing their capability to give rise to nervous tissue, ITSCs isolated from the adult human nose efficiently differentiated into functional mature neurons in vitro. Additional successful dopaminergic differentiation of ITSCs was subsequently followed by their transplantation into a unilaterally lesioned 6-hydroxydopamine rat PD model. Transplantation of predifferentiated or undifferentiated ITSCs led to robust restoration of rotational behavior, accompanied by significant recovery of DA neurons within the substantia nigra. ITSCs were further shown to migrate extensively in loose streams primarily toward the posterior direction as far as to the midbrain region, at which point they were able to differentiate into DA neurons within the locus ceruleus. We demonstrate, for the first time, that adult human ITSCs are capable of functionally recovering a PD rat model.

The current status of anticoagulant resistance in rats and mice in the UK

Introduction: Resistance to anticoagulants in Norway rats (Rattus norvegicus) and house mice (Mus domesticus) has been studied in the UK since the early 1960s. In no other country in the world is our understanding of resistance phenomena so extensive and profound. Almost every aspect of resistance in the key rodent target species has been examined in laboratory and field trials and results obtained by independent researchers have been published. It is the principal purpose of this document to present a short synopsis of this information. More recently, however, the development of genetical techniques has provided a definitive means of detection of resistant genotypes among pest rodent populations. Preliminary information from a number of such surveys will also be presented. Resistance in Norway rats: A total of nine different anticoagulant resistance mutations (single nucleotide polymorphisms or SNPs) are found among Norway rats in the UK. In no other country worldwide are present so many different forms of Norway rat resistance. Among these nine SNPs, five are known to confer on rats that carry them a significant degree of resistance to anticoagulant rodenticides. These mutations are: L128Q, Y139S, L120Q, Y139C and Y139F. The latter three mutations confer, to varying degrees, practical resistance to bromadiolone and difenacoum, the two second-generation anticoagulants in predominant use in the UK. It is the recommendation of RRAG that bromadiolone and difenacoum should not be used against rats carrying the L120Q, Y139C and Y139F mutations because this will promote the spread of resistance and jeopardise the long-term efficacy of anticoagulants. Brodifacoum, flocoumafen and difethialone are effective against these three genotypes but cannot presently be used because of the regulatory restriction that they can only be applied against rats that are living and feeding predominantly indoors. Our understanding of the geographical distribution of Norway rat resistance in incomplete but is rapidly increasing. In particular, the mapping of the focus of L120Q Norway rat resistance in central-southern England by DNA sequencing is well advanced. We now know that rats carrying this resistance mutation are present across a large part of the counties of Hampshire, Berkshire and Wiltshire, and the resistance spreads into Avon, Oxfordshire and Surrey. It is also found, perhaps as outlier foci, in south-west Scotland and East Sussex. L120Q is currently the most severe form of anticoagulant resistance found in Norway rats and is prevalent over a considerable part of central-southern England. A second form of advanced Norway rat resistance is conferred by the Y139C mutation. This is noteworthy because it occurs in at least four different foci that are widely geographically dispersed, namely in Dumfries and Galloway, Gloucestershire, Yorkshire and Norfolk. Once again, bromadiolone and difenacoum are not recommended for use against rats carrying this genotype and a concern of RRAG is that continued applications of resisted active substances may result in Y139C becoming more or less ubiquitous across much of the UK. Another type of advanced resistance, the Y139F mutation, is present in Kent and Sussex. This means that Norway rats, carrying some degree of resistance to bromadiolone and difenacoum, are now found from the south coast of Kent, west into the city of Bristol, to Yorkshire in the north-east and to the south-west of Scotland. This difficult situation can only deteriorate further where these three genotypes exist and resisted anticoagulants are predominantly used against them. Resistance in house mice: House mouse is not so well understood but the presence in the UK of two resistant genotypes, L128S and Y139C, is confirmed. House mice are naturally tolerant to anticoagulants and such is the nature of this tolerance, and the presence of genetical resistance, that house mice resistant to the first-generation anticoagulants are considered to be widespread in the UK. Consequently, baits containing warfarin, sodium warfarin, chlorophacinone and coumatetralyl are not approved for use against mice. This regulatory position is endorsed by RRAG. Baits containing brodifacoum, flocoumafen and difethialone are effective against house mice and may be applied in practice because house mouse infestations are predominantly indoors. There are some reports of resistance among mice in some areas to the second-generation anticoagulant bromadiolone, while difenacoum remains largely efficacious. Alternatives to anticoagulants: The use of habitat manipulation, that is the removal of harbourage, denial of the availability of food and the prevention of ingress to structures, is an essential component of sustainable rodent pest management. All are of importance in the management of resistant rodents and have the advantage of not selecting for resistant genotypes. The use of these techniques may be particularly valuable in preventing the build-up of rat infestations. However, none can be used to remove any sizeable extant rat infestation and for practical reasons their use against house mice is problematic. Few alternative chemical interventions are available in the European Union because of the removal from the market of zinc phosphide, calciferol and bromethalin. Our virtual complete reliance on the use of anticoagulants for the chemical control of rodents in the UK, and more widely in the EU, calls for improved schemes for resistance management. Of course, these might involve the use of alternatives to anticoagulant rodenticides. Also important is an increasing knowledge of the distribution of resistance mutations in rats and mice and the use of only fully effective anticoagulants against them.

Human neural stem cell-derived cultures in three- dimensional substrates form spontaneously functional neuronal networks

Differentiated human neural stem cells were cultured in an inert three-dimensional (3D) scaffold and, unlike two-dimensional (2D) but otherwise comparable monolayer cultures, formed spontaneously active, functional neuronal networks that responded reproducibly and predictably to conventional pharmacological treatments to reveal functional, glutamatergic synapses. Immunocytochemical and electron microscopy analysis revealed a neuronal and glial population, where markers of neuronal maturity were observed in the former. Oligonucleotide microarray analysis revealed substantial differences in gene expression conferred by culturing in a 3D vs a 2D environment. Notable and numerous differences were seen in genes coding for neuronal function, the extracellular matrix and cytoskeleton. In addition to producing functional networks, differentiated human neural stem cells grown in inert scaffolds offer several significant advantages over conventional 2D monolayers. These advantages include cost savings and improved physiological relevance, which make them better suited for use in the pharmacological and toxicological assays required for development of stem cell-based treatments and the reduction of animal use in medical research.

Activation of G-protein-coupled receptor 30 is sufficient to enhance spatial recognition memory in ovariectomized rats

In ovariectomized rats, administration of estradiol, or selective estrogen receptor agonists that activate either the alpha or beta isoforms, have been shown to enhance spatial cognition on a variety of learning and memory tasks, including those that capitalize on the preference of rats to seek out novelty. Although the effects of the putative estrogen G-protein-coupled receptor 30 (GPR30) on hippocampus-based tasks have been reported using food-motivated tasks, the effects of activation of GPR30 receptors on tasks that depend on the preference of rats to seek out spatial novelty remain to be determined. Therefore, the aim of the current study was to determine if short-term treatment of ovariectomized rats with G-1, an agonist for GPR30, would mimic the effects on spatial recognition memory observed following short-term estradiol treatment. In Experiment 1, ovariectomized rats treated with a low dose (1mug) of estradiol 48h and 24h prior to the information trial of a Y-maze task exhibited a preference for the arm associated with the novel environment on the retention trial conducted 48h later. In Experiment 2, treatment of ovariectomized rats with G-1 (25mug) 48h and 24h prior to the information trial of a Y-maze task resulted in a greater preference for the arm associated with the novel environment on the retention trial. Collectively, the results indicated that short-term treatment of ovariectomized rats with a GPR30 agonist was sufficient to enhance spatial recognition memory, an effect that also occurred following short-term treatment with a low dose of estradiol.

Ovarian-Steroid Modulation of Locus Coeruleus Activity in Female Rats: Involvement in Luteinising Hormone Regulation

The noradrenergic nucleus locus coeruleus (LC) has been reported to regulate luteinising hormone (LH) secretion in female rats. Both oestrogen and progestin receptors have been demonstrated in LC neurones, suggesting that these cells are possibly responsive to variations in circulating levels of ovarian steroids. We therefore evaluated changes in the activity of LC neurones during the oestrous cycle and after ovarian-steroid treatment in ovariectomised (OVX) rats, as determined by immunoreactivity to Fos-related antigens (FRA), which comprises all of the known members of the Fos family. Effects of ovarian steroids on the firing rate of LC neurones were also determined in a slice preparation. The number of FRA/tyrosine hydroxylase (TH)-immunoreactive (ir) neurones in the LC increased from 14.00-16.00 h on pro-oestrus, coinciding with the onset of the LH surge and rise in plasma progesterone. FRA immunoreactivity was unaltered during dioestrus. Oestradiol-treated OVX rats (OVX+E) displayed marked reduction in FRA/TH-ir neurones in LC compared to oil-treated OVX rats. Accordingly, oestradiol superfusion significantly reduced the spontaneous firing rate of LC neurones in slices from OVX rats. Compared to OVX+E, oestradiol-treated rats injected with progesterone at 08.00 h (OVX+EP) exhibited higher number of FRA/TH-ir neurones in the LC at 10.00 h and 16.00 h, and great amplification of the LH surge. Bath application of progesterone significantly increased the spontaneous firing rate of OVX+E LC neurones. Our data suggest that ovarian steroids may physiologically modulate the activity of LC neurones in females, with possible implications for LH secretion. Moreover, oestradiol and progesterone appear to exert opposite and complementary effects (i.e. whereas oestradiol inhibits, progesterone, after oestradiol priming, stimulates LC activity).

Eag 1, Eag 2 and Kcnn3 gene brain expression of isolated reared rats

The Eag1 and Eag2, voltage-dependent potassium channels, and the small-conductance calcium-activated potassium channel (Kcnn3) are highly expressed in limbic regions of the brain, where their function is still unknown. Eag1 co-localizes with tyrosine hydroxilase enzyme in the substantia nigra and ventral tegmental area. Kcnn3 deficiency leads to enhanced serotonergic and dopaminergic neurotransmission accompanied by distinct alterations in emotional behaviors. As exposure to stress is able to change the expression and function of several ion channels, suggesting that they might be involved in the consequences of stress, we aimed at investigating Eag 1, Eag2 and Kcnn3 mRNA expression in the brains of rats submitted to isolation rearing. As the long-lasting alterations in emotional and behavioral regulation after stress have been related to changes in serotonergic neurotransmission, expressions of serotonin Htr1a and Htr2a receptors in male Wistar rats` brain were also investigated. Rats were reared in isolation or in groups of five for nine weeks after weaning. Isolated and socially reared rats were tested for exploratory activity in the open field test for 5 min and brains were processed for reverse-transcription coupled to quantitative polymerase chain reaction (qRT-PCR). Isolated reared rats showed decreased exploratory activity in the open field. Compared to socially reared rats, isolated rats showed reduced Htr2a mRNA expression in the striatum and brainstem and reduced Eag2 mRNA expression in all examined regions except cerebellum. To our knowledge, this is the first work to show that isolation rearing can change Eag2 gene expression in the brain. The involvement of this channel in stress-related behaviors is discussed.

Effects of supplementation with free glutamine and the dipeptide alanyl-glutamine on parameters of muscle damage and inflammation in rats submitted to prolonged exercise

In this study, we investigated the effect of the supplementation with the dipeptide L-alanyl-L-glutamine (DIP) and a solution containing L-glutamine and L-alanine on plasma levels markers of muscle damage and levels of pro-inflammatory cytokines and glutamine metabolism in rats submitted to prolonged exercise. Rats were submitted to sessions of swim training for 6 weeks. Twenty-one days prior to euthanasia, the animals were supplemented with DIP (n = 8) (1.5 g.kg(-1)), a solution of free L-glutamine (1 g.kg(-1)) and free L-alanine (0.61 g.kg(-1)) (G&A, n = 8) or water (control (CON), n = 8). Animals were killed at rest before (R), after prolonged exercise (PE-2 h of exercise). Plasma concentrations of glutamine, glutamate, tumour necrosis factor-alpha (TNF-alpha), prostaglandin E2 (PGE2) and activity of creatine kinase (CK), lactate dehydrogenase (LDH) and muscle concentrations Of glutamine and glutamate were measured. The concentrations of plasma TNF-alpha, PGE2 and the activity of CK were lower in the G&A-R and DIP-R groups, compared to the CON-R. Glutamine in plasma (p < 0.04) and soleus muscle (p < 0.001) was higher in the DIP-R and G&A-R groups relative to the CON-R group. G&A-PE and DIP-PE groups exhibited lower concentrations of plasma PGE2 (p < 0.05) and TNF-alpha (p < 0.05), and higher concert I rations of glutamine and glutamate in soleus (p < 0.001) and gastrocnemius muscles (p < 0.05) relative to the CON-PE group. We concluded that supplementation with free L-glutamine and the dipeptide LL-alanyl-LL-glutamine represents an effective source of glutamine, which may attenuate inflammation biomarkers after periods of training and plasma levels of CK and the inflammatory response induced by prolonged exercise. Copyright (C) 2009 John Wiley & Sons, Ltd.

Yerba Mate (Ilex paraguariensis) Aqueous Extract Decreases Intestinal SGLT1 Gene Expression but Does Not Affect Other Biochemical Parameters in Alloxan-Diabetic Wistar Rats

Yerba mate (Ilex paraguariensis) is rich in polyphenols, especially chlorogenic acids. Evidence suggests that dietary polyphenols could play a role in glucose absorption and metabolism. The aim of this study was to evaluate the antidiabetic properties of yerba mate extract in alloxan-induced diabetic Wistar rats. Animals (n = 41) were divided in four groups: nondiabetic control (NDC, n = 10), nondiabetic yerba mate (NDY, n = 10), diabetic control (DC, n = 11), and diabetic yerba mate (NDY, n = 10). The intervention consisted in the administration of yerba mate extract in a 1 g extract/kg body weight dose for 28 days; controls received saline solution only. There were no significant differences in serum glucose, insulin, and hepatic glucose-6-phosphatase activity between the groups that ingested yerba mate extract (NDY and DY) and the controls (NDC and DC). However, the intestinal SGLT1 gene expression was significantly lower in animals that received yerba mate both in upper (p = 0.007) and middle (p < 0.001) small intestine. These results indicate that bioactive compounds present in yerba mate might be capable of interfering in glucose absorption, by decreasing SGLT1 expression.

Intra-strain variations of baroreflex sensitivity in young Wistar-Kyoto rats

Purpose: To compare baroreflex sensitivity among conscious rats of the same strain. Methods: Male WKY rats (eight weeks old) were studied. Cannulas were inserted into the abdominal aortic artery through the right femoral artery to measure mean arterial pressure (MAP) and heart rate (HR). Baroreflex gain was calculated as the ratio between variation of HR in function of the MAP variation (Delta HR/Delta MAP) tested with a depressor dose of sodium nitroprusside (SNP, 50 mu g/kg, iv) and with a pressor dose of phenylephrine (PE, 8 mu g/kg, iv). We divided the rats into four groups: 1) Low bradycardic baroreflex (LB), BG between -1 and -2 bpm/mmHg tested with PE; 2) High bradycardic baroreflex (HB), BG < -2 bpm/mmHg tested with PE; 3) Low tachycardic baroreflex (LT), BG between -1 and -2 bpm/mmHg tested with SNP and; 4) High tachycardic baroreflex (HT), BG < -2 bpm/mmHg tested with SNP. Significant differences were considered for p<0.05. Results: Approximately 82% of the rats presented reduced bradycardic reflex while 22 showed attenuated tachycardic reflex. No alterations were noted regarding basal MAP and HR, tachycardic and bradycardic peak and HR range. Conclusions: There was alteration in baroreflex sensitivity among rats of the same strain. Care should be taken when interpreting studies employing WKY as a control for the SHR.