Rat Blood Samples Research Articles

Garlic-derived S-allylmercaptocysteine is a hepato-protective agent in non-alcoholic fatty liver disease in vivo animal model

PurposeTo investigate the hepato-protective properties and underlying mechanisms of SAMC in a non-alcoholic fatty liver disease (NAFLD) rat model.MethodsFemale rats were fed with a diet comprising highly unsaturated fat diet (30% fish oil) for 8 weeks to develop NAFLD with or without an intraperitoneal injection of 200 mg/kg SAMC three times per week. After euthanasia, blood and liver samples of rats were collected for histological and biochemical analyses.ResultsCo-treatment of SAMC attenuated NAFLD-induced liver injury, fat accumulation, collagen formation and free fatty acids (FFAs). At the molecular level, SAMC decreased the lipogenesis marker and restored the lipolysis marker. SAMC also reduced the expression levels of pro-fibrogenic factors and diminished liver oxidative stress partly through the inhibition in the activity of cytochrome P450 2E1-dependent pathway. NAFLD-induced inflammation was also partially mitigated by SAMC treatment via reduction in the pro-inflammatory mediators, chemokines and suppressor of cytokine signaling. The protective effect of SAMC is also shown partly through the restoration of altered phosphorylation status of FFAs-dependent MAP kinase pathways and diminished in the nuclear transcription factors (NF-κB and AP-1) activity during NAFLD development.ConclusionsSAMC is a novel hepato-protective agent against NAFLD caused by abnormal liver functions. Garlic or garlic derivatives could be considered as a potent food supplement in the prevention of fatty liver disease.Electronic supplementary materialThe online version of this article (doi:10.1007/s00394-012-0301-0) contains supplementary material, which is available to authorized users.

Use of pneumatic nebulization and laser ablation–inductively coupled plasma–mass spectrometry to study the distribution and bioavailability of an intraperitoneally administered Pt-containing chemotherapeutic drug

Quadrupole-based inductively coupled-mass spectrometry (ICP-MS) with pneumatic nebulization as a means of sample introduction was employed for quantification of platinum in blood and tissue samples of rats with peritoneal carcinomatosis, receiving intraperitoneal treatment with the Pt-containing chemotherapeutic drug oxaliplatin, and in the perfusate solution used for this purpose. The Pt levels were measured for various treatment conditions, i.e., with and without supporting treatment with the drug bevacizumab and at two different temperatures. Limits of detection obtained for platinum in blood and tissue samples were 0.3 and 2.0 pg g(-1) respectively. Evaluation of drug penetration into the tumor, under different conditions of treatment, was carried out via laser ablation-ICP-MS. Quantitative mapping of the Pt distribution in tissue sections of rat was attempted relying on gelatin standards. The results show an influence of the temperature at which the treatment is carried out, while supporting administration of the drug bevacizumab did not seem to affect the results.

Dynamic eicosanoid responses upon different inhibitor and combination treatments on the arachidonic acid metabolic network

The arachidonic acid (AA) metabolic network produces key inflammatory mediators which have been considered as hallmark contributors in various inflammatory related diseases. Enzymes in this network, such as 5-lipoxygenase (5-LOX), cyclooxygenase (COX), leukotriene A(4) hydrolase (LTA4H) and prostaglandin E synthase (PGES), have been used as targets for anti-inflammatory drug discovery. Multi-target drugs and drug combinations have also been developed for this network. However, how the inhibitors alter the dynamics of metabolite production and which combinatorial target intervention solutions are better needs further exploration. We did a system based intervention analysis on the AA metabolic network. Using an LC-MS/MS method, we quantitatively studied the eicosanoid metabolites responses of AA metabolic network during stimulation of Sprague Dawley rat blood samples with the calcium ionophore. Our results indicate that inhibiting the upstream rather than the downstream target of 5-LOX pathway will simultaneously alter the AA metabolism to the COX pathway (and vice versa). Therefore, single-target inhibitors cannot control all the inflammatory mediators at the same time. We also suggest that in the case of multiple-target anti-inflammatory solutions, the combination of inhibitors of the downstream enzymes may have stronger inhibition efficiency and cause less side-effects compared to the other solutions. One therapeutic strategy, LTA4H/COX inhibition solution, was found promising for the intervention of inflammatory mediator biosynthesis and at the same time stimulating the production of anti-inflammatory agents.

Study on effect of berberine on modulating lipid and CPTⅠA gene expression

To investigate the modulating effect on lipid and gene expressions of CPT I A caused by berberine (Ber) in experimental hyperlipidemia rats. Male SD rats were randomly divided into 5 groups according to the blood lipid values: normal group, hyperlipidemia group, 300 mg x kg(-1) x d(-1) Ber-treated group, 60 mg x kg(-1) x d(-1) Ber-treated group, and 7.2 mg x kg(-1) x d(-1) lovastatin-treated group. Normal group were fed with base diet and other groups were fed with high fat and cholesterol diet. 12 weeks after drugs were given the TC, TG, LDL-C, and HDL-C from rat blood samples were tested by automatic biochemistry analyzer. Gene expressions of CPT I A and PPARalpha were evaluated by RT-PCR and Western blot, respectively. It was shown that Ber significantly decreased TC and LDL-C, but increased HDL-C in dose-dependent manner, elevated expressions of CPT I A mRNA and protein without influence on PPARalpha expression. Similar effects from lovastatin on lipidemia were observed except the Ber effect on CPT I A gene expression. Ber has modulating effect on the lipid metabolism, the mechanism of which may be by promoting the CPT I A gene expression.

Fever and inflammatory cytokine response in rats injected subcutaneously with viral double-stranded RNA analog, polyinosinic:polycytidylic acid (Poly-I:C)

The objective of this study was to determine whether viral double-stranded RNA analog, polyinosinic:polycytidylic acid (Poly-I:C), is pyrogenic in rats when administered subcutaneously, thus determining whether rats can serve as an experimental model to investigate the regulation of local and systemic inflammatory responses to a Toll-like receptor(TLR)-3 agonist. Rats implanted intraperitoneally with temperature-sensitive radiotelemeters were injected subcutaneously in the skin of the tail with saline, 100 μg kg −1 Poly-I:C, or 1000 μg kg −1 Poly-I:C. In a separate group of rats blood and tail-skin samples were taken 3 and 24 h after injections to assess changes in local and systemic inflammatory cytokine release. Injection of 1000 μg kg −1 Poly-I:C induced an acute fever, which lasted for approximately seven hours. The fever was associated with elevated local tissue concentrations of interleukin(IL)-1β, IL-6, and cytokine-induced neutrophil chemoattractant (CINC)-1, and elevated plasma concentration of CINC-1. Cytokine concentrations had returned to background concentrations by 24 h after the injection. Injection of 100 μg kg −1 Poly-I:C failed to induce fever, but did induce significant increases in the local tissue, but not circulating, concentration of CINC-1 within 3 h of the injection. Tissue CINC-1 concentrations had returned to background concentrations by 24 h after the injection. In conclusion, when administered at great enough concentrations, subcutaneously injected Poly-I:C is pyretic in rats, and the pyresis is associated with elevated concentrations of local and systemic inflammatory mediators. Thus the rat can be used to study signaling pathways induced by localized, subcutaneous administration of this TLR-3 agonist that mimics viral infection.

Development of Electrochemiluminescent Biosensor for Choline Based on Carbon Nanotube Modified Platinum Electrode

Abstract An electrochemiluminescent (ECL) choline biosensor was developed by the drop coating of choline oxidase (ChOx) onto a carbon nanotubes (CNTs)/potassium ferricyanide (K 3 Fe(CN) 6 )-modified platinum electrode with ECL of luminol as readout signal. CNTs improved the loading capacity of enzyme, as well as the biocompatibility and electron transfer of the electrode surface, whereas K 3 Fe(CN) 6 facilitated activation of the enzyme and enhancement of the ECL signal, so the developed biosensor exhibited excellent analytical performance. The biosensor gave optimal results while the Pt electrode was modified with 4 μL of 0.33 g L −1 CNTs dispersoid, 2 μL of 0.1 M K 3 Fe(CN) 6 and 1.5U of ChOx. In the PBS buffer (pH 7.4) containing 8 × 10 −6 M luminol, the ECL signal linearly responded to the concentration of choline from 1 × 10 −7 M to 4 × 10 −3 M ( r = 0.994) with a detection limit of 1.21 × 10 −8 M under 30 (C. The developed biosensor was applied to measure the concentration of choline in rat blood sample and the result was satisfied, with an average recovery of 101.1%. The as-prepared biosensor showed good stability and repeatability.

Comparison of haemolytic activity of tentacle-only extract from jellyfish Cyanea capillata in diluted whole blood and erythrocyte suspension: Diluted whole blood is a valid test system for haemolysis study

In this paper, we utilized two different test systems to compare the haemolysis of tentacle-only extract (TOE) devoid of nematocysts from jellyfish Cyanea capillata, the 1% whole blood and 0.45% erythrocyte suspension approximately with the same erythrocyte concentration from the blood samples of sheep, rabbit, mouse, rat and human, respectively. Without exception, the haemolytic activity of TOE was dose-dependent in both test systems from all the five kinds of blood samples, while it was generally stronger in erythrocyte suspension than that in diluted whole blood at the relatively high concentration of TOE. When various aliquots of plasma were added into the erythrocyte suspension test system, the haemolytic activity of TOE was declined with the plasma quantity increasing, and dropped to about 20% at the presence of two aliquots of plasma. If serum albumin of 0.5 mg/ml, approximately the same albumin content in 1% whole blood, was added into the erythrocyte suspension test system instead, the haemolysis of TOE was similarly inhibited. The effects of GSH, ascorbic acid and protease inhibitor on the haemolytic activity of TOE were detected in the erythrocyte suspension and diluted whole blood simultaneously, and the test results were coincident between the two systems. These results suggested that the inconsistency of TOE haemolysis between the erythrocyte suspension and diluted whole blood is a universal occurrence in the mammals, and blood plasma plays a dose-dependent protective role against haemolysis which may be due to serum albumin. Diluted whole blood is a valid and convenient test system for haemolysis study in vitro.

Agent-based model of instant blood-mediated inflammatory reaction effects on immediate graft loss after intraportal islet cell transplantation

The purpose of this study was to investigate the mechanisms involved in the development of instant blood-mediated inflammatory reaction (IBMIR) triggered by pancreatic islet transplantation in an in vitro system.Pancreatic islets were prepared and blood was taken from male and female Wistar rats. An in vitro heparinized tubing loop model on a rocking device that was placed in a 37°C incubator was used to generate blood flow inside the loops. Rat blood was added to the device, and after a 60-minute incubation period, 800 islet equivalents were added to the system. Rat blood samples were taken for immediate hematologic analysis before circulation, before the addition of islets, and 5, 15, 30, and 60 minutes after the addition of islets. After 60 minutes, the blood in the tubing was filtered, and the filtrate was stained using hematoxylin and eosin for microscopic analysis.Following the addition of islets, the counts of platelets, white blood cells, and mononuclear cells were significantly decreased at 60 min (P < .05), and the plasma levels of thrombin-antithrombin complexes and complement C3a were significantly increased (P < .05). At 60 minutes after introduction of the islets, the thrombi and tissue in the remaining blood in the device consisted of a few islets with serious structural damage and incomplete capsules, and there were many microthrombi with a large number of infiltrating neutrophils around the islets.Our in vitro model of IBMIR was characterized by platelet consumption, activation of the coagulation and complement cascade systems, and leukocytic infiltration.

Analysis of methylene blue and its metabolites in blood by capillary electrophoresis/electrospray ionization mass spectrometry

A method for the determination of methylene blue (MB) and its metabolites (azure A, azure B and azure C) in rat blood by CE-electrospray ionization mass spectrometry (CE-ESI-MS) was developed in this paper. Different analytical parameters were investigated in detail such as pH and concentration of separation buffer, and ESI-MS instrumental parameters. Under the optimum conditions, MB and its metabolites were separated and detected in 27.3 min. LODs (defined as S/N=3) of this method were 0.22, 0.25, 0.10 and 0.30 μg/mL for MB, azure A, azure B and azure C, respectively. To get a satisfactory extraction efficiency of MB and its metabolites in rat blood, different extraction solutions were studied. By using this method, MB and its metabolites (azure A, azure B and azure C) were successfully analyzed in rat blood samples.

Effect of Tongfu granules and its constituents on barrier function of small intestine in rats with sepsis

To investigate the effects of Tongfu granules and its constituents on barrier function of small intestine in rats with sepsis. The male rats were divided into model group, Tongfu granules group, Rhubarb group and Magnoliae cortex group by random digits table, normal rats as control group. Intraperitoneal injection of lipopolysaccharide (LPS, 6 mg/kg) was used to reproduce sepsis model. After establishment of model, rats in Tongfu granules group were given Tongfu granules 28 g×kg(-1)×d(-1) by gavage, and Rhubarb group and Magnoliae cortex group rats were given Rhubarb or Magnoliae cortex 5 g×kg(-1)×d(-1) by gavage, while the model group was given normal saline in same quantity, once a day. Blood samples of rats were collected at 24, 48, 72 hours for measuring tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8) with enzyme linked immunosorbent assay (ELISA). The pathological changes in intestinal mucosa were observed, and the pathological scores was estimated at 72 hours. The levels of TNF-α and IL-8 were significantly higher in model group than those in control group at different time points. The serum levels of TNF-α and IL-8 were significantly lower in treatment groups than those in model group, and the level of TNF-α (ng/L) in Tongfu granules group was significantly lower than that in Rhubarb and Magnoliae cortex groups at different time points (24 hours: 44.64±1.48 vs. 47.18±1.83 and 46.96±2.23, 48 hours: 51.38±1.36 vs. 57.17±2.23 and 59.41±2.01, 72 hours: 55.54±2.58 vs. 64.34±1.02 and 65.96±3.45, all P<0.05), and IL-8 (ng/L) level at 72 hours was significantly lower than that in Magnoliae cortex group (65.53±4.52 vs. 69.14±2.82,P<0.05). The scores of the lesions were significantly higher in model group than that in control group (3.90±0.17 vs. 0). The scores of Rhubarb group, Magnoliae cortex group and Tongfu granules group were 3.15±0.28, 3.18±0.08, and 2.95±0.15, respectively, which were lower than those of the model group (all P<0.01), and the Tongfu granules group descended obviously than other groups. In control group, the intercellular tight junctions were normal, and the morphology of microvilli and mitochondria was also normal. In model group, the microvilli of intestinal mucosa of the small intestine were absent or disintegrated. The intercellular tight junctions were seen to be blurred in Rhubarb and Magnoliae cortex groups, and they were close to normal state in Tongfu granules group. Their integrity was better preserved compared with that of the model group. Injury of barrier function of the small intestine was found in septic rat. It was found that traditional Chinese medicine Tongfu granules, Rhubarb and Magnoliae cortex could protect the barrier function of the small intestine by decreasing the TNF-α and IL-8 levels in septic rats. Above-mentioned effects of Tongfu granules were better than Rhubarb and Magnoliae cortex.

Reticulocyte measurements in rat, dog and mouse whole blood samples using the Sysmex XT-2000iV

Immature reticulocyte measurements in peripheral blood are being used as an indicator of erythropoiesis. Some haematology analysers can now provide an indication of reticulocyte maturity by dividing the reticulocytes into three counting regions based on fluorescent intensity or absorbance. In this study, reticulocyte measurements were evaluated for three laboratory species—rat, dog and mouse—and we found the Sysmex XT-2000iV to give satisfactory analytical performance in terms of precision, carry-over between samples, linearity and sample stability. Using the XT-2000iV, results were obtained for reticulocyte absolute counts and the three regional subpopulations of reticulocytes, and these were compared with results obtained with the Siemens Advia 120™. The study highlighted differences between the analysers which may reflect the methodology of the Advia 120™ absorbance measurements and the fluorescence method used by the XT-2000iV. The different cell gating approaches and the lack of suitable calibration and control materials also contributes to these findings. It is important to establish baseline data before exploiting these measurements when investigating perturbations of erythropoiesis due to xenobiotics. We also investigated the correlations between the mean reticulocyte haemoglobin content (CHr), available on the Advia 120™ and the two parameters (Ret-Y and RBC-Y) available on XT-2000iV, as both of these measurements can be used to investigate iron deficiency anaemias.

Systemic exposure of quercetin after administration of Feng-Liao-Chang-Wei-Kang granules to rats

The objective of this study was to observe the systemic exposure of quercetin (QCT) including its free and total concentration in rat blood samples following a single p.o. dose of feng-liao-chang-wei-kang granules (FLCWKG). Six male rats were given the FLCHKG and the serial blood samples were collected. Fully or partially validated LC/MS/MS methods were developed to analyze the resulting biosamples. Various pharmacokinetic (PK) parameters were estimated from the plasma concentration versus time data using non-compartmental methods. Both methods for analysis of the free and total QCT plasma concentration are sensitive, specific, accurate and reliable. The PK parameters of free QCT after administration of FLCWKG, in comparison with total concentration, show a lower C(max) (32.4±8.2 ng/ml versus 164±49 ng/ml), a lower AUC(0→∞) (39.3±6.9 ng/ml h versus 313±16 ng/ml h), a shorter T(max) (0.17±0.00 h versus 0.83±1.01 h) and a similar t(1/2) (3.90±0.88 h versus 3.10±1.94 h), MRT(0→t) (2.27±0.10 h versus 2.86±0.41 h). Free QCT was quickly absorbed from gastrointestinal tract and circulated in blood at quite low concentration. The circulating flavonoids originating from this formula were dominantly conjugated derivatives.

Effects of chronic stress on cognitive functions and anxiety related behaviors in rats

The effects of 100 dB prenatal and chronic postnatal white noise stress (WNS) on some cognitive functions and behaviors in rats were investigated. For this purpose, 60 female Sprague-Dawley rats and their 90 male offspring were used. Pregnant rats were divided into Prenatal Stress (PS) and Prenatal Control (PC) groups. WNS was applied to PS group between the 14th and 21st days of their pregnancy, while PC rats were left undisturbed. After weaning, 40 male pups of PS dams were assigned to prenatal + chronic postnatal stress (PSCS) and prenatal stress + non-stress (PSN) groups. Pups of PC dams were divided into Control (CON) and Corticosterone (CORT) groups. During the postnatal 30th and 51st days, WNS was applied to PSCS and CORT rats everyday for 45 minutes, while PSN and CON groups were left undisturbed. The effects of stress on adult male offspring were investigated using Morris water maze and defensive withdrawal tests. Blood samples were collected after each test for serum corticosterone measurements. Blood samples of CORT rats were collected before the stress application and at the 1st, 7th, 14th, and 21st days of the stress period, immediately after cessation of the stress application. There were no significant differences among groups for learning and behavior tests. Corticosterone levels of CORT rats were significantly higher after the stress period than before stress application. These results indicate that although chronic 100 dB WNS induces a stress response by increasing corticosterone levels, it does not affect cognitive functions and anxiety related behaviors of adult male offspring.

Kinetic model‐based factor analysis of dynamic sequences for 82‐rubidium cardiac positron emission tomography

Factor analysis has been pursued as a means to decompose dynamic cardiac PET images into different tissue types based on their unique temporal signatures to improve quantification of physiological function. In this work, the authors present a novel kinetic model-based (MB) method that includes physiological models of factor relationships within the decomposition process. The physiological accuracy of MB decomposed (82)Rb cardiac PET images is evaluated using simulated and experimental data. Precision of myocardial blood flow (MBF) measurement is also evaluated. A gamma-variate model was used to describe the transport of (82)Rb in arterial blood from the right to left ventricle, and a one-compartment model to describe the exchange between blood and myocardium. Simulations of canine and rat heart imaging were performed to evaluate parameter estimation errors. Arterial blood sampling in rats and (11)CO blood pool imaging in dogs were used to evaluate factor and structure accuracy. Variable infusion duration studies in canine were used to evaluate MB structure and global MBF reproducibility. All results were compared to a previously published minimal structure overlap (MSO) method. Canine heart simulations demonstrated that MB has lower root-mean-square error (RMSE) than MSO for both factor (0.2% vs 0.5%, p < 0.001 MB vs MSO, respectively) and structure (3.0% vs 4.7%, p < 0.001) estimations, as with rat heart simulations (factors: 0.2% vs 0.9%, p < 0.001 and structures: 3.0% vs 6.7%, p < 0.001). MB blood factors compared to arterial blood samples in rats had lower RMSE than MSO (1.6% vs 2.2%, p =0.025). There was no difference in the RMSE of blood structures compared to a (11)CO blood pool image in dogs (8.5% vs 8.8%, p =0.23). Myocardial structures were more reproducible with MB than with MSO (RMSE=3.9% vs 6.2%, p < 0.001), as were blood structures (RMSE=4.9% vs 5.6%, p =0.006). Finally, MBF values tended to be more reproducible with MB compared to MSO (CV= 10% vs 18%, p =0.16). The execution time of MB was, on average, 2.4 times shorter than MSO (p < 0.001) due to fewer free parameters. Kinetic model-based factor analysis can be used to provide physiologically accurate decomposition of (82)Rb dynamic PET images, and may improve the precision of MBF quantification.

Validation of a differential in situ perfusion method with mesenteric blood sampling in rats for intestinal drug interaction profiling

The present study explored the feasibility of a differential setup for the in situ perfusion technique with mesenteric cannulation in rats to assess drug interactions at the level of intestinal absorption. In contrast to the classic, parallel in situ perfusion setup, the differential approach aims to identify intestinal drug interactions in individual animals by exposing the perfused segment to a sequence of multiple conditions. First, the setup was validated by assessing the interaction between the P-glycoprotein (P-gp) inhibitor verapamil and the transport probes atenolol (paracellular transport), propranolol (transcellular) and talinolol (P-gp mediated efflux). While transport of atenolol and propranolol remained constant for the total perfusion time (2 h), a verapamil-induced increase in talinolol transport was observed within individual rats (between 3.2- and 5.2-fold). In comparison with the parallel setup, the differential in situ perfusion approach enhances the power to detect drug interactions with compounds that exhibit strong subject-dependent permeability. This was demonstrated by identifying an interaction between amprenavir and ketoconazole (P-gp and CYP3A inhibitor) in five out of seven rats (permeability increase between 1.9- and 4.2-fold), despite high inter-individual differences in intrinsic permeability for amprenavir. In combination with an increased throughput (up to 300%) and a reduced animal use (up to 50%), the enhanced power of the differential approach improves the utility of the biorelevant in situ perfusion technique with mesenteric blood sampling to elucidate the intestinal interaction profile of drugs and drug candidates.

Recovery and metabolism of xanthohumol in germ‐free and human microbiota‐associated rats

The impact of human intestinal bacteria on the bioavailability of the prenylflavonoid xanthohumol (XN) was studied by comparing germ-free (GF) and human microbiota-associated (HMA) rats. After XN application, XN, XN conjugates, and isoxanthohumol (IX) conjugates occurred in blood samples of GF and HMA rats, whereas IX was detected only in the blood of HMA rats. Overall excretion of XN and its metabolites within 48 h was only 4.6% of the ingested dose in GF rats and 4.2% in HMA rats, feces being the major route of excretion. While both GF and HMA rats excreted XN, IX, and their conjugates with urine and feces, 8-prenylnaringenin and its corresponding conjugates were exclusively observed in the feces of HMA rats. The microbial formation of 8-prenylnaringenin was confirmed by incubation of XN and IX with human fecal slurries. The amount of conjugates excreted in urine and feces was lower in HMA rats compared to GF rats indicating their hydrolysis by human intestinal microbiota. Thus, the impact of bacteria on the XN metabolism in the gut may affect the in vivo effects of ingested XN.

Renal function evaluation in an integrative cardio‐renal pharmacology model using Culex Empis® automated drug infusion system and radio telemetry

Recently we showed utility of a novel integrative pharmacology system (CUlex TElemetry: CUTE) that allows simultaneous evaluation of CNS/CV/Renal functions with automated blood sampling in the same rat. To evaluate potential risk of drug‐induced renal and cardiac dysfunction, or their bidirectional effects, a new procedure was developed for simultaneous estimation of heart and global renal function. Briefly, inulin‐FITC (2.5 mg/mL) and p‐aminohippuric acid (PAH; 40 mg/mL) were continuously co‐administered (i.v., 0.5 mL/hour) in conscious, unrestrained Han Wistar rats using Empis® automated drug infusion system combined with CUTE. Steady state was reached after 60 min, and 11 blood samples were collected over the next 3 days. Plasma clearance of inulin and PAH were determined using Fick's principle (renal clearance = infusion rate/plasma level). There were no significant differences in estimated glomerular filtration rate (eGFR) between plasma clearance (0.95 ± 0.05) vs. reference urinary clearance method (1.07 ± 0.14 mL/min/100g BW). Mean blood pressure (102.6 ± 3.54 mmHg) and heart rate (422.9 ± 8.26 bpm) were not altered by infusion of markers for 3 days. This new approach allows simple, rapid and reproducible evaluation of renal function with respect to traditional procedures, making it a useful model for preclinical cardio‐renal safety assessment.

Pharmacokinetic Study of a Novel Recombinant Human Granulocyte Colony-stimulating Factor in Rats

To study the pharmacokinetics of a novel recombinant human granulocyte colony-stimulating factor (rhG-CSFa) in rats and to determine the proteolytic rates of rhG-CSFa in the whole blood and serum of rats in vitro. The pharmacokinetics of rhG-CSFa and conventional (wild type, WT) granulocyte colony-stimulating factor (G-CSF) were investigated in Sprague-Dawley rats which received either intravenous or subcutaneous injection of rhG-CSFa or WT G-CSF at three different doses (20, 50, or 100 microg/kg). The blood samples of rats were collected at multiple time points (from 0.08 to 12 h) and the concentrations of rhG-CSFa and WT G-CSF in serum were determined with a sandwich enzyme-linked immunosorbent assay (ELISA). For the study of proteolytic rates in vitro, the concentrations of rhG-CSFa or WT G-CSF were determined at 3-minute intervals after addition of the respective drug to rat's whole blood or serum. Pharmacokinetic analysis of serum rhG-CSFa or WT G-CSF levels indicated that, at each dose tested, for either route of drug administration, the area under concentration-time curve values and the maximum serum concentration of rhG-CSFa were higher than those of WT G-CSF, and the serum half life of rhG-CSFa was longer than that of WT G-CSF. Subsequent in vitro whole blood and serum stability study showed that the rates of drug degradation in WT G-CSF were 1.8 folds and 1.5 folds higher than those in rhG-CSFa, respectively. rhG-CSFa has better serum and whole blood stability in vitro and higher bioavailability in vivo as compared to WT G-CSF.

Safety Pharmacology Society: 9th Annual Meeting

The keynote presentation of the Safety Pharmacology (SP) Society 9th Annual Meeting addressed the urgency, for pharmaceutical organizations, to implement strategies for effectively communicating drug risks to all concerned stakeholders and, in particular, the general public. The application of chronobiology to SP investigational protocols can improve the search of drug-induced adverse effects. The Distinguished Service Award Lecture reviewed a life-long journey through trials and tribulations in the quest of the ever-distant scientific truth. The revision process of Directive 86/609/EC for improving animal welfare should be conducted with the purpose of maintaining a fair balance among animal protection, human health and research imperatives in order to prevent the migration of pharmaceutical activities outside Europe. Additional topics of interest were the behavioral, metabolic and cardiovascular problems experienced by small animals housed at the standard laboratory temperature. A technology for the automated collection of blood and urine samples in rats implanted with telemetry sensors was presented. Non-clinical, clinical, regulatory and legal aspects of abuse liability were expertly reviewed. The ‘degradability’ of pharmaceuticals into environment-friendly chemicals should be an actively searched and optimized feature of future pharmaceuticals in order to prevent drug pollution of ecosystems. Transgenic and diseased animal models should be selected whenever they can facilitate the determination of drug-induced adverse effects. SP strategies to investigate the safety of drug combination products were exemplified and analyzed in depth. The future of SP was proposed to lie not in the performance of regulatory studies of pharmacodynamic nature but in developing and early applying an array of screening assays for clearing clinical candidates against known drug-induced organ function injuries. In conclusion, the 2009 SP Society annual meeting offered a wealth of thought-provoking material to attendees for improving SP investigation strategies.

DNA damage after intracerebroventricular injection of ouabain in rats

There is an emerging body of data suggesting that bipolar disorder is associated with DNA damage. Intracerebroventricular (ICV) administration of ouabain in rats results in manic-like alterations. We evaluated DNA damage of peripheral blood, cerebrospinal fluid and hippocampus of rats after ICV ouabain injection. Ouabain-induced hyperlocomotion was examined in an open field. Additionally, we used single cell gel electrophoresis (comet assay) to measure early transient damage in cerebrospinal fluid (CSF), hippocampus and blood; and the micronucleus test to measure persistent damage in total blood samples of rats after ouabain administration. Our findings demonstrated that ouabain induced hyperlocomotion in rats, and this response remained up to 7 days following a single ICV injection. In addition, we observed that the persistent increase in the rat spontaneous locomotion is associated with increased hippocampal and peripheral index of early DNA damage in rats. No significant alterations were observed in the micronucleus frequency in total blood samples of the rats after the ouabain ICV injection. These results suggest that ouabain may induce peripheral and central early DNA damage, but this early damage may be repaired.