Analysis of time-related metabolic fluctuations induced by ethionine in the rat

The time-course of metabolic events following response to a model hepatotoxin ethionine (800 mg/kg) was investigated over a 7 day period in rats using high-resolution (1)H NMR spectroscopic analysis of urine and multivariate statistics. Complementary information was obtained by multivariate analysis of (1)H MAS NMR spectra of intact liver and by conventional histopathology and clinical chemistry of blood plasma. (1)H MAS NMR spectra of liver showed toxin-induced lipidosis 24 h postdose consistent with the steatosis observed by histopathology, while hypertaurinuria was suggestive of liver injury. Early biochemical changes in urine included elevation of guanidinoacetate, suggesting impaired methylation reactions. Urinary increases in 5-oxoproline and glycine suggested disruption of the gamma-glutamyl cycle. Signs of ATP depletion together with impairment of the energy metabolism were given from the decreased levels in tricarboxylic acid cycle intermediates, the appearance of ketone bodies in urine, the depletion of hepatic glucose and glycogen, and also hypoglycemia. The observed increase in nicotinuric acid in urine could be an indication of an increase in NAD catabolism, a possible consequence of ATP depletion. Effects on the gut microbiota were suggested by the observed urinary reductions in the microbial metabolites 3-/4-hydroxyphenyl propionic acid, dimethylamine, and tryptamine. At later stages of toxicity, there was evidence of kidney damage, as indicated by the tubular damage observed by histopathology, supported by increased urinary excretion of lactic acid, amino acids, and glucose. These studies have given new insights into mechanisms of ethionine-induced toxicity and show the value of multisystem level data integration in the understanding of experimental models of toxicity or disease.

Evidence both L-type and non-L-type voltage-dependent calcium channels contribute to cerebral artery vasospasm following loss of NO in the rat

We recently found block of NO synthase in rat middle cerebral artery caused spasm, associated with depolarizing oscillations in membrane potential (Em) similar in form but faster in frequency (circa 1 Hz) to vasomotion. T-type voltage-gated Ca2+ channels contribute to cerebral myogenic tone and vasomotion, so we investigated the significance of T-type and other ion channels for membrane potential oscillations underlying arterial spasm. Smooth muscle cell membrane potential (Em) and tension were measured simultaneously in rat middle cerebral artery. NO synthase blockade caused temporally coupled depolarizing oscillations in cerebrovascular Em with associated vasoconstriction. Both events were accentuated by block of smooth muscle BKCa. Block of T-type channels or inhibition of Na+/K+-ATPase abolished the oscillations in Em and reduced vasoconstriction. Oscillations in Em were either attenuated or accentuated by reducing [Ca2+]o or block of KV, respectively. TRAM-34 attenuated oscillations in both Em and tone, apparently independent of effects against KCa3.1. Thus, rapid depolarizing oscillations in Em and tone observed after endothelial function has been disrupted reflect input from T-type calcium channels in addition to L-type channels, while other depolarizing currents appear to be unimportant. These data suggest that combined block of T and L-type channels may represent an effective approach to reverse cerebral vasospasm.

Thromboxane A2 inhibition of SKCa after NO synthase block in rat middle cerebral artery

NO/prostanoid independent, EDHF-mediated hyperpolarization and dilation in rat middle cerebral arteries is mediated solely by endothelial cell IK(Ca). However, when the NO-pathway is also active, both SK(Ca) and IK(Ca) contribute to EDHF responses. As the SK(Ca) component can be inhibited by stimulation of thromboxane A(2) (TxA(2)) TP receptors and NO has the potential ability to inhibit thromboxane synthesis, we investigated whether TxA(2) might explain loss of functional input from SK(Ca) during NOS inhibition in cerebral arteries. EXPERIMENTAL APPROACH: Rat middle cerebral arteries were mounted in a wire myograph. Endothelium-dependent responses to the PAR2 agonist, SLIGRL were assessed as simultaneous changes in smooth muscle membrane potential and tension. KEY RESULTS: Responses were obtained in the presence of L-NAME as appropriate. Inhibition of TP receptors with either ICI 192,605 or SQ 29,548, did not affect EDHF mediated hyperpolarization and relaxation, but in their presence neither TRAM-34 nor apamin (to block IK(Ca) and SK(Ca) respectively) individually affected the EDHF response. However, in combination they virtually abolished it. Similar effects were obtained in the presence of the thromboxane synthase inhibitor, furegrelate, which additionally revealed an iberiotoxin-sensitive residual EDHF hyperpolarization and relaxation in the combined presence of TRAM-34 and apamin. CONCLUSIONS AND IMPLICATIONS: In the rat middle cerebral artery, inhibition of NOS leads to a loss of the SK(Ca) component of EDHF responses. Either antagonism of TP receptors or block of thromboxane synthase restores an input through SK(Ca). These data indicate that NO normally enables SK(Ca) activity in rat middle cerebral arteries.

Evidence for involvement of both IKCa and SKCa channels in hyperpolarizing responses of the rat middle cerebral artery

Endothelium-derived hyperpolarizing factor responses in the rat middle cerebral artery are blocked by inhibiting IKCa channels alone, contrasting with peripheral vessels where block of both IKCa and SKCa is required. As the contribution of IKCa and SKCa to endothelium-dependent hyperpolarization differs in peripheral arteries, depending on the level of arterial constriction, we investigated the possibility that SKCa might contribute to equivalent hyperpolarization in cerebral arteries under certain conditions. METHODS: Rat middle cerebral arteries (approximately 175 microm) were mounted in a wire myograph. The effect of KCa channel blockers on endothelium-dependent responses to the protease-activated receptor 2 agonist, SLIGRL (20 micromol/L), were then assessed as simultaneous changes in tension and membrane potential. These data were correlated with the distribution of arterial KCa channels revealed with immunohistochemistry. RESULTS: SLIGRL hyperpolarized and relaxed cerebral arteries undergoing variable levels of stretch-induced tone. The relaxation was unaffected by specific inhibitors of IKCa (TRAM-34, 1 micromol/L) or SKCa (apamin, 50 nmol/L) alone or in combination. In contrast, the associated smooth-muscle hyperpolarization was inhibited, but only with these blockers in combination. Blocking nitric oxide synthase (NOS) or guanylyl cyclase evoked smooth-muscle depolarization and constriction, with both hyperpolarization and relaxation to SLIGRL being abolished by TRAM-34 alone, whereas apamin had no effect. Immunolabeling showed SKCa and IKCa within the endothelium. CONCLUSIONS: In the absence of NO, IKCa underpins endothelium-dependent hyperpolarization and relaxation in cerebral arteries. However, when NOS is active SKCa contributes to hyperpolarization, whatever the extent of background contraction. These changes may have relevance in vascular disease states where NO release is compromised and when the levels of SKCa expression may be altered.

Warfarin susceptibility in the lesser bandicoot rat (Bandicota bengalensis)

Warfarin is a first generation anticoagulant that relies on multiple feeding events to achieve lethality in susceptible rodents. For the Bandicoot rat, warfarin susceptibility baselines were established using the lethal feeding period (LFP) test methodology. Against a 0.003% warfarin formulation, LFP50 values of 2 and 4 days, and LFP99 values of 16 and 10 days were obtained for males and females respectively. However, consumption of rodenticide was significantly reduced after the 4th and 5th days of test, at a time when animals would be expected to experience symptoms of warfarin toxicity. This would seriously compromise the Probit analysis, particularly for estimates of higher percentiles. Possible modifications to the methodology are discussed to overcome this problem. Although the majority of animals were highly susceptible to warfarin, one female animal that survived a high dose of active ingredient (79.1 mg kg-1) may bode for future resistance

Calciferols and bait shyness in the laboratory rat

Rodenticides with delayed action are generally more effective than fast-acting compounds because of the phenomenon of bait shyness. Calciferols have a stop-feed effect quite soon after dosing, and physiological effects are measurable within one day of dosing. We investigated whether bait shyness might result from these fairly rapid effects in the laboratory rat. We found evidence of bait shyness following recovery from sub-lethal dosing with two forms of calciferol. Use of intubation as well as feeding showed that the response was to the bait carrier rather than to detection of calciferols per se.

Cannabinol and cannabidiol exert opposing effects on rat feeding patterns

Rationale: Increased food consumption following Δ9- tetrahydrocannabinol-induced cannabinoid type 1 receptor agonism is well documented. However, possible non-Δ9- tetrahydrocannabinol phytocannabinoid-induced feeding effects have yet to be fully investigated. Therefore, we have assessed the effects of the individual phytocannabinoids, cannabigerol, cannabidiol and cannabinol, upon feeding behaviors. Methods: Adult male rats were treated (p.o.) with cannabigerol, cannabidiol, cannabinol or cannabinol plus the CB1R antagonist, SR141716A. Prior to treatment, rats were satiated and food intake recorded following drug administration. Data were analyzed for hourly intake and meal microstructure. Results: Cannabinol induced a CB1R-mediated increase in appetitive behaviors via significant reductions in the latency to feed and increases in consummatory behaviors via increases in meal 1 size and duration. Cannabinol also significantly increased the intake during hour 1 and total chow consumed during the test. Conversely, cannabidiol significantly reduced total chow consumption over the test period. Cannabigerol administration induced no changes to feeding behavior. Conclusion: This is the first time cannabinol has been shown to increase feeding. Therefore, cannabinol could, in the future, provide an alternative to the currently used and psychotropic Δ9-tetrahydrocannabinol-based medicines since cannabinol is currently considered to be non-psychotropic. Furthermore, cannabidiol reduced food intake in line with some existing reports, supporting the need for further mechanistic and behavioral work examining possible anti-obesity effects of cannabidiol.

Natal nest locations of the Asian house rat (Rattus tanezumi) in lowland rice–coconut cropping systems: a coconut penthouse or rice bunds with water frontage?

CONTEXT. Rattus tanezumi is a serious crop pest within the island of Luzon, Philippines. In intensive flood-irrigated rice field ecosystems of Luzon, female R. tanezumi are known to primarily nest within the tillers of ripening rice fields and along the banks of irrigation canals. The nesting habits of R. tanezumi in complex rice–coconut cropping systems are unknown. AIMS. To identify the natal nest locations of R. tanezumi females in rice–coconut systems of the Sierra Madre Biodiversity Corridor (SMBC), Luzon, during the main breeding season to develop a management strategy that specifically targets their nesting habitat. METHODS. When rice was at the booting to ripening stage, cage-traps were placed in rice fields adjacent to coconut habitat. Thirty breeding adult R. tanezumi females were fitted with radio-collars and successfully tracked to their nest sites. KEY RESULTS. Most R. tanezumi nests (66.7%) were located in coconut groves, five nests (16.7%) were located in rice fields and five nests (16.7%) were located on the rice field edge. All nests were located above ground level and seven nests were located in coconut tree crowns. The median distance of nest sites to the nearest rice field was 22.5m. Most nest site locations had good cover of ground vegetation and understorey vegetation, but low canopy cover. Only one nest location had an understorey vegetation height of less than 20 cm. CONCLUSIONS. In the coastal lowland rice–coconut cropping systems of the SMBC, female R. tanezumi showed a preference for nesting in adjacent coconut groves. This is contrary to previous studies in intensive flood-irrigated rice ecosystems of Luzon, where the species nests mainly in the banks of irrigation canals. It is important to understand rodent breeding ecology in a specific ecosystem before implementing appropriate management strategies. IMPLICATIONS. In lowland rice–coconut cropping systems, coconut groves adjacent to rice fields should be targeted for the 20 management of R. tanezumi nest sites during the main breeding season as part of an integrated ecologically based approach to rodent pest management.

Relationship between dietary-induced changes in intestinal commensal microflora and duodenojejunal myoelectric activity monitored by radiotelemetry in the rat in vivo

Interdigestive intestinal motility, and especially phase III of the migrating myoelectric/motor complex (MMC), is responsible for intestinal clearance and plays an important role in prevention of bacterial overgrowth and translocation in the gut. Yet previous results from gnotobiotic rats have shown that intestinal microflora can themselves affect the characteristics of the myoelectric activity of the gut during the interdigestive state. Given that the composition of the intestinal microflora can be altered by dietary manipulations, we investigated the effect of supplementation of the diet with synbiotics on intestinal microflora structure and the duodenojejunal myoelectric activity in the rat. To reduce animal distress caused by restraint and handling, which can itself affect GI motility, we applied radiotelemetry for duodenojejunal EMG recordings in conscious, freely moving rats. Thirty 16-month-old Spraque-Dawley rats were used. The diet for 15 rats (E group) was supplemented with chicory inulin, Lactobacillus rhamnosus and Bifidobacterium lactis. The remaining 15 rats were fed control diet without supplements (C group). Three rats from each group were implanted with three bipolar electrodes positioned at 2, 14 and 28 cm distal to the pylorus. After recovery, two 6 h recordings of duodenojejunal EMG were carried out on each operated rat. Subsequently, group C rats received feed supplements and group E rats received only control diet for 1 week, and an additional two 6 h recordings were carried out on each of these rats. Non-operated C and E rats were killed and samples of GI tract were collected for microbiological analyses. Supplementation of the diet with the pro- and prebiotics mixture increased the number of bifidobacteria, whereas it decreased the number of enterobacteria in jejunum, ileum, caecum and colon. In both caecum and colon, the dietary supplementation increased the number of total anaerobes and lactobacilli. Treatment with synbiotics increased occurrence of phase III of the MMC at all three levels of the small intestine. The propagation velocity of phase III in the whole recording segment was also increased from 3.7 +/- 0.2 to 4.4 +/- 0.2 cm min(-1) by dietary treatment. Treatment with synbiotics increased the frequency of response potentials of the propagated phase III of the MMC at both levels of the jejunum, but not in the duodenum. In both parts of the jejunum, the supplementation of the diet significantly decreased the duration of phase II of the MMC, while it did not change the duration of phase I and phase III. Using the telemetry technique it was demonstrated that changes in the gastrointestinal microflora exhibited an intestinal motility response and, more importantly, that such changes can be initiated by the addition of synbiotics to the diet.

Possible role for K+ in endothelium-derived hyperpolarizing factor–linked dilatation in rat middle cerebral artery

Background and Purpose— Endothelium-derived hyperpolarizing factor (EDHF) and K+ are vasodilators in the cerebral circulation. Recently, K+ has been suggested to contribute to EDHF-mediated responses in peripheral vessels. The EDHF response to the protease-activated receptor 2 ligand SLIGRL was characterized in cerebral arteries and used to assess whether K+ contributes as an EDHF. Methods— Rat middle cerebral arteries were mounted in either a wire or pressure myograph. Concentration-response curves to SLIGRL and K+ were constructed in the presence and absence of a variety of blocking agents. In some experiments, changes in tension and smooth muscle cell membrane potential were recorded simultaneously. Results— SLIGRL (0.02 to 20 μmol/L) stimulated concentration and endothelium-dependent relaxation. In the presence of NG-nitro-L-arginine methyl ester, relaxation to SLIGRL was associated with hyperpolarization and sensitivity to a specific inhibitor of IKCa, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (1μmol/L), reflecting activation of EDHF. Combined inhibition of KIR with Ba2+ (30μmol/L) and Na+/K+-ATPase with ouabain (1 μmol/L) markedly attenuated the relaxation to EDHF. Raising extracellular [K+] to 15 mmol/L also stimulated smooth muscle relaxation and hyperpolarization, which was also attenuated by combined application of Ba2+ and ouabain. Conclusions— SLIGRL evokes EDHF-mediated relaxation in the rat middle cerebral artery, underpinned by hyperpolarization of the smooth muscle. The profile of blockade of EDHF-mediated hyperpolarization and relaxation supports a pivotal role for IKCa channels. Furthermore, similar inhibition of responses to EDHF and exogenous K+ with Ba2+ and ouabain suggests that K+ may contribute as an EDHF in the middle cerebral artery.

Affinity purification of 61- and 65-kDa rat brain corticotropin-releasing factor receptors and receptor-associated G proteins.

Corticotropin-releasing factor (CRF) has been shown to have a central role in physiological adaptation to stress. It is recognized for stimulating the release of adrenocorticotropin from the anterior pituitary gland, and has more recently been implicated as a regulator of autonomic and immunological responses to stress. Much confusion has surrounded the characterization of CRF receptors, with proteins of varying molecular weights having been identified but never purified and characterized. Recently, two CRF receptors have been cloned from brain and pituitary gland, but evidence from in-situ hybridization studies suggests that further CRF receptor types exist. We therefore developed two techniques which enable the isolation of CRF receptors from whole rat brain. The use of a solid-phase CRF analogue affinity column and elution using a competing ligand resulted in the purification of a single protein of 61 kDa. A second technique was devised which allowed the co-isolation of associated signalling proteins and the identification of CRF bound species following purification. CRF was covalently cross-linked to receptors and the complex purified using antibodies specific for the ligand. This enabled the purification of a CRF receptor of approximately 65 kDa and associated alpha and beta gamma G protein subunits. This study demonstrates the successful isolation of CRF receptors which are of different molecular weights to those previously observed from affinity cross-linking studies or predicted from cloned genes. In addition, we confirm the involvement of G proteins in CRF stimulated cell signalling by demonstrating their association with purified CRF receptor.

Cannabidivarin is anticonvulsant in mouse and rat in vitro and in seizure models

Summary Background and purpose: Phytocannabinoids in Cannabis sativa have diverse pharmacological targets extending beyond cannabinoid receptors and several exert notable anticonvulsant effects. For the first time, we investigated the anticonvulsant profile of the phytocannabinoid cannabidivarin (CBDV) in vitro and in in vivo seizure models. Experimental approach: The effect of CBDV (1-100μM) on epileptiform local field potentials (LFPs) induced in rat hippocampal brain slices by 4-AP application or Mg2+-free conditions was assessed by in vitro multi-electrode array recordings. Additionally, the anticonvulsant profile of CBDV (50-200 mg kg-1) in vivo was investigated in four rodent seizure models: maximal electroshock (mES) and audiogenic seizures in mice, and pentylenetetrazole (PTZ) and pilocarpine-induced seizures in rat. CBDV effects in combination with commonly-used antiepileptic drugs were investigated in rat seizures. Finally, the motor side effect profile of CBDV was investigated using static beam and gripstrength assays. Key results: CDBV significantly attenuated status epilepticus-like epileptiform LFPs induced by 4-AP and Mg2+-free conditions. CBDV had significant anticonvulsant effects in mES (≥100 mg kg-1), audiogenic (≥50 mg kg-1) and PTZ-induced seizures (≥100 mg kg-1). CBDV alone had no effect against pilocarpine-induced seizures, but significantly attenuated these seizures when administered with valproate or phenobarbital at 200 mg kg-1 CBDV. CBDV had no effect on motor function. Conclusions and Implications: These results indicate that CBDV is an effective anticonvulsant across a broad range of seizure models, does not significantly affect normal motor function and therefore merits further investigation in chronic epilepsy models to justify human trials.

Investigation of the impact of feeding Lactobacillus plantarum CRL 1815 encapsulated in microbially derived polymers on the rat faecal microbiota

AIMS: The aim of this study was to evaluate the impact of the administration of microencapsulated Lactobacillus plantarum CRL 1815 with two combinations of microbially derived polysaccharides, xanthan : gellan gum (1%:0·75%) and jamilan : gellan gum (1%:1%), on the rat faecal microbiota. METHODS AND RESULTS: A 10-day feeding study was performed for each polymer combination in groups of 16 rats fed either with placebo capsules, free or encapsulated Lact. plantarum or water. The composition of the faecal microbiota was analysed by fluorescence in situ hybridization and temporal temperature gradient gel electrophoresis. Degradation of placebo capsules was detected, with increased levels of polysaccharide-degrading bacteria. Xanthan : gellan gum capsules were shown to reduce the Bifidobacterium population and increase the Clostridium histolyticum group levels, but not jamilan : gellan gum capsules. Only after administration of jamilan : gellan gum-probiotic capsules was detected a significant increase in Lactobacillus-Enterococcus group levels compared to controls (capsules and probiotic) as well as two bands were identified as Lact. plantarum in two profiles of ileum samples. CONCLUSIONS: Exopolysaccharides constitute an interesting approach for colon-targeted delivery of probiotics, where jamilan : gellan gum capsules present better biocompatibility and promising results as a probiotic carrier. SIGNIFICANCE AND IMPACT OF STUDY: This study introduces and highlights the importance of biological compatibility in the encapsulating material election, as they can modulate the gut microbiota by themselves, and the use of bacterial exopolysaccharides as a powerful source of new targeted-delivery coating material.

Adhesion of Salmonella enterica var Enteritidis strains lacking fimbriae and flagella to rat ileal explants cultured at the air interface or submerged in tissue culture medium

Rat ileal air interface and submerged explant models were developed and used to compare the adhesion of Salmonella enterica var Enteritidis wild-type strains with that of their isogenic single and multiple deletion mutants. The modified strains studied were defective for fimbriae, flagella, motility or chemotaxis and binding was assessed on tissues with and without an intact mucus layer. A multiple afimbriate/aflagellate (fim(-)/fla(-)) strain, a fimbriate but aflagellate (fla(-)) strain and a fimbriate/flagellate but non-motile (mot(-)) strain bound significantly less extensively to the explants than the corresponding wild-type strains. With the submerged explant model this difference was evident in tissues with or without a mucus layer, whereas in the air interface model it was observed only in tissues,vith an intact mucus layer. A smooth swimming chemotaxis-defective (che(-)) strain and single or multiple afimbriate strains bound to explants as well as their corresponding wild-type strain. This suggests that under the present experimental conditions fimbriae were not essential for attachment of S. enterica var Enteritidis to rat ileal explants, However; the possession of active flagella did appear to be an important factor. in enabling salmonellae to penetrate the gastrointestinal mucus layer and attach specifically to epithelial cells.

Expression of type 1 fimbriae (SEF 21) of Salmonella enterica serotype Enteritidis in the early colonisation of the rat intestine

The involvement of type 1 fimbriae in colonisation of the rat gastrointestinal tract in vivo was investigated with Salmonella enterica serotype Enteritidis LA5 and a mutant of LA5 denoted EAV3 unable to elaborate type 1 fimbriae (SEF 21), Rats were given a single dose of LA5 or EAV3 or a 1:1 mixture of both, LA5 was found in higher numbers in the stomach and small intestine than EAV3 at 6 h after infection with a single strain, but not after 6 days, LA5 did not out-compete EAV3 when the strains were administered together. Indeed, after 6 and 21 days, EAV3 was found in the distal small intestine and large intestine in far higher numbers than LA5. These findings suggest that SEF 21 have an important role(s) in the early stages of infection in vivo, However, SEF 21 expression may disadvantage the pathogen in the longer term as indicated by EAV3 out-competing LA5 in the gut at 21 days.