Distribution of [H-3]trans-resveratrol in rat tissues following oral administration

Resveratrol has been widely investigated for its potential health properties, although little is known about its metabolism in vivo. Here we investigated the distribution of metabolic products of [H-3]trans-resveratrol, following gastric administration. At 2 h, plasma concentrations reached 1 center dot 7 % of the administered dose, whilst liver and kidney concentrations achieved 1 center dot 0 and 0 center dot 6 %, respectively. Concentrations detected at 18 h were lower, being only 0 center dot 5 % in plasma and a total of 0 center dot 35 % in tissues. Furthermore, whilst kidney and liver concentrations fell to 10 and 25 %, respectively, of concentrations at 2 h, the brain retained 43 % of that measured at 2 h. Resveratrol-glucuronide was identified as the major metabolite, reaching 7 mu m in plasma at 2 h. However, at 18 h the main form identified in liver, heart, lung and brain was native resveratrol aglycone, indicating that it is the main form retained in the tissues. No phenolic degradation products were detected in urine or tissues, indicating that, unlike flavonoids, resveratrol does not appear to serve as a substrate for colonic microflora. The present study provides additional information about the nature of resveratrol metabolites and which forms might be responsible for its in vivo biological effects.

Safety evaluation of oligofructose: 13 Week rat study and in vitro mutagenicity

Oligofructose (OF), comprised of fructose oligomers with a terminal glucose unit, is a family Of oligosaccharides derived from the hydrolysis of inulin. Consumption of OF in animals and humans increases colonic bifidobacteria levels. The present study evaluates the safety of OF in both a 13 week rat feeding Study and Using in Vitro mutagenicity tests. Fecal bifidobacteria levels were also determined by in situ hybridization to assess a biological function of OF. Rats received either a control diet OF diets containing one of four doses of OF. Total, HDL, and LDL-cholesterol levels were significantly lower at several time points during the study in groups receiving OF compared to controls with the largest effects Occurring in the high dose male animals. Weight gain in the male high dose group was significantly lower at early time points compared to controls but]lot Significantly different at the end of study. As expected, cecal weights increased in a dose-related manner and fecal bifidobacteria levels also demonstrated a dose-related increase. There were no consistent differences in gross pathology or histopathology related to dietary OF. OF did not induce a positive response in the Ames test or chromosomal aberration test with CHO cells. These results demonstrate no adverse effects of OF. (C) 2008 Elsevier Ltd. All rights reserved.

alpha-tocopherol affects androgen metabolism in male rat

The Alpha-Tocopherol Beta-Carotene Cancer Prevention Study has provided the first evidence implicating vitamin E in hormone synthesis. The effect of vitamin E on stereoidogenesis in testes and adrenal glands was assessed in growing rats using Affymetrix gene-chip technology. Dietary supplementation of rats with vitamin E (60 mg/kg feed) for a period of 429 days caused a significant repression of genes encoding for proteins centrally involved in the uptake (low-density lipoprotein receptor) and de novo synthesis (for example, 7-dehydrocholesterol reductase, 3-hydroxy-3-methylglutaryl coenzyme A synthase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase, isopentenyl-diphosphate delta-isomerase, and farnesyl pyrophosphate synthetase) of cholesterol, the precursor of all steroid hormones. The present investigation indicates that dietary vitamin E may induce changes in stereoidogenesis by affecting cholesterol homeostasis.

G protein ss gamma subunits mediate presynaptic inhibition of transmitter release from rat superior cervical ganglion neurones in culture

The activation of presynaptic G protein-coupled receptors (GPCRs) is widely reported to inhibit transmitter release; however, the lack of accessibility of many presynaptic terminals has limited direct analysis of signalling mediators. We studied GPCR-mediated inhibition of fast cholinergic transmission between superior cervical ganglion neurones (SCGNs) in culture. The adrenoceptor agonist noradrenaline (NA) caused a dose-related reduction in evoked excitatory postsynaptic potentials (EPSPs). NA-induced EPSP decrease was accompanied by effects on the presynaptic action potential (AP), reducing AP duration and amplitude of the after-hyperpolarization (AHP), without affecting the pre- and postsynaptic membrane potential. All effects of NA were blocked by yohimbine and synaptic transmission was reduced by clonidine, consistent with an action at presynaptic alpha 2-adrenoceptors. NA-induced inhibition of transmission was sensitive to pre-incubation of SCGNs with pertussis toxin (PTX), implicating the involvement of G alpha(i)/(o)beta y subunits. Expression of G alpha transducin, an agent which sequesters G protein beta gamma (G beta y) subunits, in the presynaptic neurone caused a time-dependent attenuation of NA-induced inhibition. Injection of purified G beta gamma subunits into the presynaptic neurone inhibited transmission, and also reduced the AHP amplitude. Furthermore, NA-induced inhibition was occluded by pre-injection of G beta gamma subunits. The Ca2+ channel blocker Cd2+ mimicked NA effects on transmitter release. Cd2+, NA and G beta gamma subunits also inhibited somatic Ca2+ current. In contrast to effects on AP-evoked transmitter release, NA had no clear action on AP-independent EPSPs induced by hypertonic solutions. These results demonstrate that G beta gamma subunits functionally mediate inhibition of transmitter release by alpha 2-adrenoceptors and represent important regulators of synaptic transmission at mammalian presynaptic terminals.

The Ca(v)2.3 calcium channel antagonist SNX-482 reduces dorsal horn neuronal responses in a rat model of chronic neuropathic pain

Neuropathic pain is a difficult state to treat, characterized by alterations in sensory processing that can include allodynia (touch-evoked pain). Evidence exists for nerve damage-induced plasticity in both transmission and modulatory systems, including changes in voltage-dependent calcium channel (VDCC) expression and function; however, the role of Ca(v)2.3 calcium channels has not clearly been defined. Here, the effects of SNX-482, a selective Ca(v)2.3 antagonist, on sensory transmission at the spinal cord level have been investigated in the rat. The spinal nerve ligation (SNL) model of chronic neuropathic pain [Kim & Chung, (1992) Pain, 50, 355-363] was used to induce mechanical allodynia, as tested on the ipsilateral hindpaw. In vivo electrophysiological measurements of dorsal horn neuronal responses to innocuous and noxious electrical and natural stimuli were made after SNL and compared to sham-operated animals. Spinal SNX-482 (0.5-4 mu g/50 mu L) exerted dose-related inhibitions of noxious C-fibre- and A delta-fibre-mediated neuronal responses in conditions of neuropathy, but not in sham-operated animals. Measures of spinal cord hyperexcitability and nociception were most susceptible to SNX-482. In contrast, non-noxious A beta-mediated responses were not affected by SNX-482. Moreover, responses to innocuous mechanical and also thermal stimuli were more sensitive to SNX-482 in SNL than control animals. This study is the first to demonstrate an antinociceptive role for SNX-482-sensitive channels in dorsal horn neurons during neuropathy. These data are consistent with plasticity in Ca(V)2.3 calcium channel expression and suggest a potential selective target to reduce nociceptive transmission during conditions of nerve damage.

Rat neurosphere cells protect axotomized rat retinal ganglion cells and facilitate their regeneration

We investigated the ability of a population of rat neural stem and precursor cells derived from rat embryonic spinal cord to protect injured neurons in the rat central nervous system (CNS). The neonatal rat optic pathway was used as a model of CNS injury, whereby retinal ganglion cells (RGCs) were axotomized by lesion of the lateral geniculate nucleus one day after birth. Neural stem and precursor cells derived from expanded neurospheres (NS) were transplanted into the lesion site at the time of injury. Application of Fast Blue tracer dye to the lesion site demonstrated that significant numbers of RGCs survived at 4 and 8 weeks in animals that received a transplant, with an average of 28% survival, though in some individual cases survival was greater than 50%. No RGCs survived in animals that received a lesion alone. Furthermore, labeled RGCs were also observed when Fast Blue was applied to the superior colliculus (SC) at 4 weeks, suggesting that neurosphere cells also facilitated RGC to regenerate to their normal target. Transplanted cells did not migrate or express neural markers after transplantation, and secreted several neurotrophic factors in vitro. We conclude that NS cells can protect injured CNS neurons and promote their regeneration. These effects are not attributable to cell replacement, and may be mediated via secretion of neurotrophic factors. Thus, neuroprotection by stem cell populations may be a more viable approach for treatment of CNS disorders than cell replacement therapy.

Comparison of preferences for object properties in the rat using paired- and free-choice paradigms

A common method for testing preference for objects is to determine which of a pair of objects is approached first in a paired-choice paradigm. In comparison, many studies of preference for environmental enrichment (EE) devices have used paradigms in which total time spent with each of a pair of objects is used to determine preference. While each of these paradigms gives a specific measure of the preference for one object in comparison to another, neither method allows comparisons between multiple objects simultaneously. Since it is possible that several EE objects would be placed in a cage together to improve animal welfare, it is important to determine measures for rats' preferences in conditions that mimic this potential home cage environment. While it would be predicted that each type of measure would produce similar rankings of objects, this has never been tested empirically. In this study, we compared two paradigms: EE objects were either presented in pairs (paired-choice comparison) or four objects were presented simultaneously (simultaneous presentation comparison). We used frequency of first interaction and time spent with each object to rank the objects in the paired-choice experiment, and time spent with each object to rank the objects in the simultaneous presentation experiment. We also considered the behaviours elicited by the objects to determine if these might be contributing to object preference. We demonstrated that object ranking based on time spent with objects from the paired-choice experiment predicted object ranking in the simultaneous presentation experiment. Additionally, we confirmed that behaviours elicited were an important determinant of time spent with an object. This provides convergent evidence that both paired choice and simultaneous comparisons provide valid measures of preference for EE objects in rats. (C) 2007 Elsevier B.V. All rights reserved.

Development of multi-electrode array screening for anticonvulsants in acute rat brain slices

The acute hippocampal brain slice preparation is an important in vitro screening tool for potential anticonvulsants. Application of 4-aminopyridine (4-AP) or removal of external Mg2+ ions induces epileptiform bursting in slices which is analogous to electrical brain activity seen in status epilepticus states. We have developed these epileptiform models for use with multi-electrode arrays (MEAs), allowing recording across the hippocampal slice surface from 59 points. We present validation of this novel approach and analyses using two anticonvulsants, felbamate and phenobarbital, the effects of which have already been assessed in these models using conventional extracellular recordings. In addition to assessing drug effects on commonly described parameters (duration, amplitude and frequency), we describe novel methods using the MEA to assess burst propagation speeds and the underlying frequencies that contribute to the epileptiform activity seen. Contour plots are also used as a method of illustrating burst activity. Finally, we describe hitherto unreported properties of epileptiform, bursting induced by 100 mu M 4-AP or removal of external Mg2+ ions. Specifically, we observed decreases over time in burst amplitude and increase over time in burst frequency in the absence of additional pharmacological interventions. These MEA methods enhance the depth, quality and range of data that can be derived from the hippocampal slice preparation compared to conventional extracellular recordings. it may also uncover additional modes of action that contribute to anti-epileptiform drug effects. (C) 2009 Elsevier B.V. All rights reserved.

Objects and positions in visual scenes: effects of perirhinal and postrhinal cortex lesions in the rat

The authors assessed rats' encoding of the appearance or egocentric position of objects within visual scenes containing 3 objects (Experiment 1) or I object (Experiment 2A). Experiment 2B assessed encoding of the shape and fill pattern of single objects, and encoding of configurations (object + position, shape + fill). All were assessed by testing rats' ability to discriminate changes from familiar scenes (constant-negative paradigm). Perirhinal cortex lesions impaired encoding of objects and their shape; postrhinal cortex lesions impaired encoding of egocentric position, but the effect may have been partly due to entorhinal involvement. Neither lesioned group was impaired in detecting configural change. In Experiment 1, both lesion groups were impaired in detecting small changes in relative position of the 3 objects, suggesting that more sensitive tests might reveal configural encoding deficits.

The roles of perirhinal cortex, postrhinal cortex, and the fornix in memory for objects, contexts, and events in the rat

Investigation of the anatomical substructure of the medial temporal lobe has revealed a number of highly interconnected areas, which has led some to propose that the region operates as a unitary memory system. However, here we outline the results of a number of studies from our laboratories, which investigate the contributions of the rat's perirhinal cortex and postrhinal cortex to memory, concentrating particularly on their respective roles in memory for objects. By contrasting patterns of impairment and spared abilities on a number of related tasks, we suggest that perirhinal cortex and postrhinal cortex make distinctive contributions to learning and memory: for example, that postrhinal cortex is important in learning about within-scene position and context. We also provide evidence that despite the strong connectivity between these cortical regions and the hippocampus, the hippocampus, as evidenced by lesions of the fornix, has a distinct function of its own-combining information about objects, positions, and contexts.

Induction of aquaporin 1 but not aquaporin 4 messenger RNA in rat primary brain microvessel endothelial cells in culture

Aquaporins (AQPs) are a family of proteins that mediate water transport across cells, but the extent to which they are involved in water transport across endothelial cells of the blood-brain barrier is not clear. Expression of AQP1 and AQP4 in rat brain microvessel endothelial cells was investigated in order to determine whether these isoforms were present and, in particular, to examine the hypothesis that brain endothelial expression of AQPs is dynamic and regulated by astrocytic influences. Reverse-transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemistry showed that AQP1 mRNA and protein are present at very low levels in primary rat brain microvessel endothelial cells, and are up-regulated in passaged cells. Upon passage, endothelial cell expression of mdr1a mRNA is decreased, indicating loss of blood-brain barrier phenotype. In passage 4 endothelial cells, AQP1 mRNA levels are reduced by coculture above rat astrocytes, demonstrating that astrocytic influences are important in maintaining the low levels of AQP1 characteristic of the blood-brain barrier endothelium. Reverse-transcriptase-PCR revealed very low levels of AQP1 mRNA present in the RBE4 rat brain microvessel endothelial cell line, with no expression detected in primary cultures of rat astrocytes or in the C6 rat glioma cell line. In contrast, AQP4 mRNA is strongly expressed in astrocytes, but no expression is found in primary or passaged brain microvessel endothelial cells, or in RBE4 or C6 cells. Our results support the concept that expression of AQP1, which is seen in many non-brain endothelia, is suppressed in the specialized endothelium of the blood-brain barrier.

Rat brain serotonin neurones that express neuronal nitric oxide synthase have increased sensitivity to the substituted amphetamine serotonin toxins 3,4-methylenedioxymethamphetamine and p-chloroamphetamine

Substituted amphetamines such as p-chloroamphetamine and the abused drug methylenedioxymethamphetamine cause selective destruction of serotonin axons in rats, by unknown mechanisms. Since some serotonin neurones also express neuronal nitric oxide synthase, which has been implicated in neurotoxicity, the present study was undertaken to determine whether nitric oxide synthase expressing serotonin neurones are selectively vulnerable to methylenedioxymethamphetamine or p-chloroamphetamine. Using double-labeling immunocytochemistry and double in situ hybridization for nitric oxide synthase and the serotonin transporter, it was confirmed that about two thirds of serotonergic cell bodies in the dorsal raphe nucleus expressed nitric oxide synthase, however few if any serotonin transporter immunoreactive axons in striatum expressed nitric oxide synthase at detectable levels. Methylenedioxymethamphetamine (30 mg/kg) or p-chloroamphetamine (2 x 10 mg/kg) was administered to Sprague-Dawley rats, and 7 days after drug administration there were modest decreases in the levels of serotonin transporter protein in frontal cortex, and striatum using Western blotting, even though axonal loss could be clearly seen by immunostaining. p-Chloroamphetamine or methylenedioxymethamphetamine administration did not alter the level of nitric oxide synthase in striatum or frontal cortex, determined by Western blotting. Analysis of serotonin neuronal cell bodies 7 days after p-chloroamphetamine treatment, revealed a net down-regulation of serotonin transporter mRNA levels, and a profound change in expression of nitric oxide synthase, with 33% of serotonin transporter mRNA positive cells containing nitric oxide synthase mRNA, compared with 65% in control animals. Altogether these results support the hypothesis that serotonin neurones which express nitric oxide synthase are most vulnerable to substituted amphetamine toxicity, supporting the concept that the selective vulnerability of serotonin neurones has a molecular basis.

Role of G1 phase cyclins and cyclin dependent kinases during hypertrophic growth of adult rat cardiomyocytes

Cell cycle regulatory molecules are implicated in cardiomyocyte hypertrophy. We have investigated protein expression of cyclins A, D1–3, and E and cyclin-dependent kinases (CDKs) 2, 4, 5, and 6 in left ventricular (LV) tissues during the development of LV hypertrophy in rats following aortic constriction (AC). Compared with their expression in sham-operated controls (SH), expression of cyclins D2 and D3 and of CDK4 and CDK6 increased significantly fromday 3 to day 21 after AC concomitant with increased LV mass. However, no significant difference was observed for CDK2 or CDK5. Cyclins A, D1, and E were undetectable. In vitro kinase activities of CDK4 and CDK6 increased ∼70% from day 7 to day 14 in AC myocytes compared with SH myocytes (P< 0.03). Fluorescence-activated cell sorter analysis revealed a G0/G1to G2/M phase progression in AC myocyte nuclei (22.0 ± 1.1% in G2/M) by day 7 postoperation compared with progression in SH myocyte nuclei (14.0 ± 0.8% in G2/M;P < 0.01). Thus an upregulation of certain cell cycle regulators is associated with cardiomyocyte hypertrophy.

Diacylglycerol-induced protection against injury during ischemia and reperfusion can be achieved without activation of protein kinase C in the rat heart: Comparative studies with ischemic preconditioning

The role of protein kinase C (PKC) activation in ischemic preconditioning remains controversial. Since diacylglycerol is the endogenous activator of PKC and as such might be expected cardioprotective, we have investigated whether: (i) the diacylglycerol analog 1,2-dioctanoyl-sn-glycerol (DOG) can protect against injury during ischemia and reperfusion; (ii) any effect is mediated via PKC activation; and (iii) the outcome is influenced by the time of administration. Isolated rat hearts were perfused with buffer at 37°C and paced at 400 bpm. In Study 1, hearts (n=6/group) were subjected to one of the following: (1) 36 min aerobic perfusion (controls); (2) 20 min aerobic perfusion plus ischemic preconditioning (3 min ischemia/3 min reperfusion+5 min ischemia/5 min reperfusion); (3) aerobic perfusion with buffer containing DOG (10 μM) given as a substitute for ischemic preconditioning; (4) aerobic perfusion with DOG (10 μM) during the last 2 min of aerobic perfusion. All hearts then were subjected to 35 min of global ischemia and 40 min reperfusion. A further group (5) were perfused with DOG (10 μM) for the first 2 min of reperfusion. Ischemic preconditioning improved postischemic recovery of LVDP from 24±3% in controls to 71±2% (P<0.05). Recovery of LVDP also was enhanced by DOG when given just before ischemia (54±4%), however, DOG had no effect on the recovery of LVDP when used as a substitute for ischemic preconditioning (22±5%) or when given during reperfusion (29±6%). In Study 2, the first four groups of study were repeated (n=4–5/group) without imposing the periods of ischemia and reperfusion, instead hearts were taken for the measurement of PKC activity (pmol/min/mg protein±SEM). PKC activity after 36 min in groups (1), (2), (3) and (4) was: 332±102, 299±63, 521±144, and 340±113 and the membrane:cytosolic PKC activity ratio was: 5.6±1.5, 5.3±1.8, 6.6±2.7, and 3.9±2.1 (P=NS in each instance). In conclusion, DOG is cardioprotective but under the conditions of the present study is less cardioprotective than ischemic preconditioning, furthermore the protection does not appear to necessitate PKC activation prior to ischemia.

Phorbol ester, but not ischemic preconditioning, activates protein kinase D in the rat heart

The signal transduction pathways that mediate the cardioprotective effects of ischemic preconditioning remain unclear. Here we have determined the role of a novel kinase, protein kinase D (PKD), in mediating preconditioning in the rat heart. Isolated rat hearts (n=6/group) were subjected to either: (i) 36 min aerobic perfusion (control); (ii) 20 min aerobic perfusion plus 3 min no-flow ischemia, 3 min reperfusion, 5 min no-flow ischemia, 5 min reperfusion (ischemic preconditioning); (iii) 20 min aerobic perfusion plus 200 nmol/l phorbol 12-myristate 13-acetate (PMA) given as a substitute for ischemic preconditioning. The left ventricle then was excised, homogenized and PKD immunoprecipitated from the homogenate. Activity of the purified kinase was determined following bincubation with [γ32P]-ATP±syntide-2, a substrate for PKD. Significant PKD autophosphorylation and syntide-2 phosphorylation occurred in PMA-treated hearts, but not in control or preconditioned hearts. Additional studies confirmed that recovery of LVDP was greater and initiation of ischemic contracture and time-to-peak contracture were less, in ischemic preconditioned hearts compared with controls (P<0.05). Our results suggest that the early events that mediate ischemic preconditioning in the rat heart occur via a PKD-independent mechanism.