Rat Blood Samples Research Articles

Development Of A Physiologically Based Pharmacokinetic Model For Chlorobenzene In F-344 Rats

A physiologically based pharmacokinetic (PBPK) model to describe the absorption, distribution, metabolism, and elimination of chlorobenzene in rats was developed. Partition coefficients were experimentally determined in rat tissues and blood samples using an in vitro vial equilibration technique. These solubility ratios were in agreement with previous reports. The in vivo metabolism of chlorobenzene was evaluated using groups of three F344 male rats exposed to initial chlorobenzene concentrations ranging from 82 to 6750 ppm in a closed, recirculating gas uptake system. An optimal fit of the family of uptake curves was obtained by adjusting Michaelis–Menten metabolic constants, Km (affinity) and Vmax (capacity), using the PBPK model. At the highest chamber concentration, the uptake curve could not be modeled without the addition of a first-order (Kfo) metabolic pathway. Pretreatment with pyrazole, an inhibitor of oxidative microsomal metabolism, had no impact on the slope of the uptake curve. The completed PBPK model was evaluated against real-time exhaled breath data collected from rats receiving either an intraperitoneal (ip) injection or oral gavage dose of chlorobenzene in corn oil. Exhaled breath profiles were evaluated and absorption rates were determined. Development of the chlorobenzene PBPK model in rats is the first step toward future extrapolations to apply to humans.

Three‐color labeling method for flow cytometric measurement of cytogenetic damage in rodent and human blood

Experiments described herein were designed to evaluate the performance characteristics of a flow cytometry-based system that scores the incidence of peripheral blood micronucleated reticulocytes (MN-RETs). These procedures represent the continued refinement of a previously reported anti-CD71-based method (Dertinger et al. [1996]: Mutat Res 371:283-292), with the following modifications: incorporation of a third fluorescent label to exclude platelets from the MN-RET region, and use of a CD71-associated fluorescence thresholding technique to increase data acquisition rates. Mouse, rat, and human blood samples were analyzed using both the previously described two-color procedure (anti-CD71-FITC and propidium iodide) and a newly developed three-color technique (which adds an antiplatelet-PE antibody). The rodent specimens were also evaluated by standard microscopy procedures (acridine orange staining). Mouse blood was collected via heart puncture of vehicle- and 5-fluorouracil-treated CD-1 mice; blood samples from saline-treated Sprague-Dawley rats were collected from the tail vein and via heart puncture. Rodent blood samples were analyzed by both the two- and three-color methods. Human blood specimens, obtained via arm venipuncture from cancer patients undergoing radiation therapy, were analyzed for MN-RETs using the two-color method. Subsequently, blood samples from a single chemotherapy patient were analyzed by both the two- and three-color methods. Finally, the chemotherapy patient blood samples and blood samples from 15 healthy volunteers were evaluated at very high densities in conjunction with a CD71-associated fluorescence thresholding technique. Results of these investigations showed that data from mouse blood analyzed by the two- and three-color procedures correlated well with microscopy data (r values = 0.917 and 0.937 for the two- and three-color methods, respectively); all three methods confirmed the genotoxicity of 5-FU. Data from rat tail vein samples showed improved reproducibility with the three-color technique, but no significant difference between the two techniques was seen with the heart puncture specimens. Human blood analyzed according to the two-color procedure produced unreliable results, as platelets and platelet aggregates impacted the rare MN-RET scoring region. The three-color technique effectively overcame this problem and produced reproducible measurements that fell within expected ranges. For human blood analyses, the high cell density/CD71-thresholding technique provided significant improvements over the low-density technique, as it allowed data acquisition to occur approximately six times faster with no loss of sensitivity.

Characterization of a circulating N-extended form of the thyrotropin-releasing hormone-like peptide pGlu-Glu-Pro amide in human plasma.

The TRH-like peptides pGlu-Glu-Pro amide, pGlu-Phe-Pro amide, and pGlu-Gln-Pro amide were isolated and identified some years ago, and these peptides have been proven to be present in many tissues and fluids. The presence of TRH-like immunoreactivity distinct from TRH in blood has been observed previously. In the present study, the presence of N-extended forms of TRH-like peptides in plasma has been investigated. Peripheral blood samples of human, rat, and rabbit were obtained and plasma was extracted. The peptides were separated in several steps of chromatography, including gel filtration, cation and anion exchange, and HPLC. The concentrations of the TRH-like peptides in the column fractions were measured by RIA with TRH antibody. The N-extended forms of TRH-like peptides were determined by RIA after trypsin digestion. In human plasma it was observed an N-extended form of TRH-like peptides in substantial concentration. After trypsin and heating, the N-extended forms of TRH-like peptides were rechromatographed on Sephadex G-50. This showed that the TRH-like peptides released have a similar size to TRH. The peptides were then separated by cation exchange chromatography, and the major fraction was unretained, indicating a neutral or acidic nature. Part of the unretained fraction was then chromatographed on anion exchange column in which the major fraction was retained, demonstrating the acidic nature of the peptides. Similar results have been observed in rat and rabbit. The other part of the unretained fraction from cation exchange chromatography of human plasma was purified on HPLC. The results demonstrated that the major component observed by HPLC cochromatographed with synthetic pGlu-Glu-Pro amide. This study represents the first demonstration of a circulating N-extended form of any TRH-like peptide.

The in vivo assessment of safety and gastrointestinal survival of an orally administered novel probiotic, Propionibacterium jensenii 702, in a male Wistar rat model

This study aimed to evaluate in vivo gastrointestinal survival and safety of orally administered probiotic bacterium, Propionibacterium jensenii 702, using a male Wistar rat model. A high dose of 10 10 cfu/rat/day of P. jensenii 702 was fed to each rat for 81 days. The repeated dose toxicity and translocation of P. jensenii 702 into rat tissues were evaluated, along with the rat faecal β-glucuronidase activities and dairy propionibacteria counts. Results showed that P. jensenii 702 had no adverse effect on general health status, body weight gain, visceral organs and faecal β-glucuronidase activities. No viable cells of P. jensenii 702 were recovered from blood and tissue samples (mesenteric lymph nodes, liver and spleen) of rats, and no treatment-associated illness or death was observed. Faecal dairy propionibacteria counts reached 10 8 cfu/g after 36 days treatment and remained between 10 8–10 9 cfu/g till the end of 81 days treatment. The results indicate that P. jensenii 702 was able to survive the in vivo gastrointestinal tract transit of rats, with no adverse affects on the animals. However, further human clinical trials are required before strain P. jensenii 702 could be incorporated into food for human consumption as probiotics.

Endotoxemia and hepatic injury in a rodent model of hindlimb unloading.

Hindlimb unloading (HU) is known to induce physiological alterations in various organ systems that mimic some responses observed after exposure to microgravity. In the present study, the effects of up to 4 wk of HU on the liver were assessed in male Wistar rats and two mouse strains: endotoxin-sensitive C57BL/6 mice and endotoxin-resistant C3H/HEJ mice. Plasma levels of endotoxin, a known stimulator of hepatic injury, were measured in portal and systemic blood samples. Endotoxin was elevated by approximately 50% in portal blood samples of mice and rats but was not detectable in systemic blood. This low-grade portal endotoxemia was associated with hepatic injury in rats and C57BL/6 mice as indicated by inflammation and elevated serum transaminase activities. Blood levels of the cytokine TNF-alpha were increased by approximately 50% in C57BL/6 mice; no significant elevation of this cytokine was detected in rats. Messenger RNA levels of the acute-phase proteins serum amyloid A, haptoglobin, and lipopolysaccharide binding protein were significantly enhanced after 3 wk of HU in endotoxin-sensitive rodents. In contrast, no histological changes or significant increases in serum enzyme activity were detected after HU in C3H/HEJ mice despite portal endotoxin levels of 222 +/- 83.4 pg/ml. At the 3-wk time point, expression of acute-phase proteins was not elevated in C3H/HEJ mice; however, expression after 4 wk of HU was similar to endotoxin-sensitive rodents. In conclusion, these findings indicate that HU induced mild portal endotoxemia, which contributed to the observed hepatic injury in endotoxin-sensitive rodents.

Jugular vein catheterization for repeated blood sampling in the unrestrained conscious rat

The ability to obtain repeated, low-stress blood samples from adult rats enables the design of complex experiments in which time course information or evaluation of repeated treatments is necessary. Furthermore, it reduces the number of animals necessary to acquire such information and, thus, facilitates compliance with the animal use 3Rs (reduction, refinement and replacement). To this end, a microsurgical technique to collect blood samples from the right atrium through a catheter (cannula) implanted into the right external jugular vein of adult rats is described. Rats tolerate this simple and efficient vascular access technique as evidenced by the absence of overt morbidity or abnormal behaviors. Blood is easily sampled while the rats reside in their home cages. Because the sample volume is replaced, repeated sampling is possible without compromising blood volume. Successful adoption of this procedure by other investigators will be aided by the photographic illustrations accompanying this detailed description of the procedure. Application of this technique to monitor temporal changes in plasma stress hormones during stressor paradigms as well as after behavioral and pharmacological challenges is discussed.

DEVELOPMENT OF A PHYSIOLOGICALLY BASED PHARMACOKINETIC MODEL FOR METHYL ETHYL KETONE IN F344 RATS

A physiologically based pharmacokinetic (PBPK) model to describe the absorption, distribution, metabolism, and elimination of methyl ethyl ketone (MEK) in rats was developed. Partition coefficients were experimentally determined in rat tissues and blood samples using an in vitro vial equilibration technique. These solubility ratios were in agreement with previous human-based estimates that MEK is uniformly soluble within all tissues. The in vivo metabolism of MEK was evaluated using groups of three F344 male rats exposed to 100-2000 ppm MEK in a closed, recirculating gas uptake system. An optimal fit of a family of uptake curves was obtained by adjusting Michaelis-Menten metabolic constants, K m (affinity), and V max (capacity) using the PBPK model. At the highest chamber concentration, the uptake curve could not be modeled without the addition of a first-order (K fo ) metabolic pathway. Pretreatment with pyrazole, an inhibitor of oxidative microsomal metabolism, decreased the slope of the gas uptake curve but did not abolish metabolism. Optimal model fit to the gas uptake curve from pyrazole-pretreated animals required the apparent K m to be increased roughly 50 times the value determined in naive rats. The completed PBPK model was evaluated against real-time exhaled breath data collected from rats receiving an intravenous (iv) injection of MEK via a jugular vein cannula. Model simulation of the ivtreated animals required alveolar ventilation to be reduced 30% in order to match the data. Exhaled breath profiles from animals treated with MEK by oral gavage or intraperitoneal (ip) injection were evaluated and absorption rates was determined. Development of a comprehensive PBPK model for MEK in rats is the first step toward future extrapolations to apply to humans.

Determination of d- and l-aspartate in cell culturing medium, within cells of MPT1 cell line and in rat blood by a column-switching high-performance liquid chromatographic method

HPLC fluorometric methods have been used to analyze trace amounts of d-amino acids in biological samples. In this study, we established an expedient column-switching fluorometric HPLC system that would improve the analysis of d-amino acids, in particular d-aspartate (Asp). Our system consists of the fluorogenic derivatization of amino acids with NBD-F and two chromatographic steps, one that separates individual amino acids in reverse phase mode and another that separates the chiral forms of each amino acid in normal-phase mode. The two separation steps are linked through a trapping column by an automated column-switching system. In addition, sample preparation is simplified and improved, where trichloroacetic acid is used for deproteinization, and borate buffer, pH 9.5 is employed for the fluorescent derivatization. The detection limit for d-Asp in culturing medium is 5 n M. The resulting peak heights correlated well with concentrations that ranged from 12.5 to 250 n M for both d- and l-Asp. The present method was applied to determine d- and l-Asp levels in cell culturing medium, and within cells of MPT1 cell line. The detected cellular levels of d- and l-Asp agree with those detected by our previous method. In addition, this method was used to measure d- and l-Asp levels in rat blood samples, and the results are consistent with the reported values.

Determination of perfluorobutane in rat blood by automatic headspace capillary gas chromatography and selected ion monitoring mass spectrometry.

A new contrast agent (Sonazoid; NC100100) for ultrasound imaging has been developed. It is an aqueous suspension of lipid stabilised perfluorobutane (PFB) gas microbubbles. An automatic headspace capillary gas-chromatographic mass spectrometric method using electron impact ionisation was developed for analysis of Sonazoid PFB in rat blood. The calibration standards were gaseous PFB dissolved in ethanol in the range of 0.5-5000 ng PFB. Fluorotrichloromethane (CFC 11) was used as an internal standard of the method and the MS detector was set to single ion monitoring of the base fragment ions of PFB (m/z 69 and 119) and CFC 11 (m/z 101). The calibration graph, made by plotting the peak area ratios of PFB (m/z 69) to CFC 11(m/z 101) against the amount of PFB, was fitted to a second-order polynomial equation with weighting 1/y2 and found to be reproducible. The limit of quantification of the method was set to 0.4 ng PFB. The between-day variation of the method was below 9.2% relative standard deviation (RSD) and the within-day variation of the method was below 7.6% RSD. The accuracy of the method, as compared to Coulter counter, was estimated by determination of PFB in samples where Sonazoid was added to saline and found to range from 91.5% to 105.2%. PFB, added as Sonazoid, was found to be stable for at least 7 months in rat blood samples when stored at -20 degrees C.

Study of nitric oxide (NO) distribution in the rat after a glyceryl trinitrate patch treatment using a spin trapping technique

Glyceryl Trinitrate (GTN) is commonly delivered by patch in the treatment for angina pectoris. This drug acts via the formation of Nitric Oxide (NO); however, the pharmacokinetic of NO delivery from GTN patch still remains obscure. In the present study, NO levels were evaluated after 2 hours of treatment with a 10 mg GTN patch (GTN group; n = 8) and compared with control anesthetized Wistar rats (CTL group; n = 8). Nitrosylhemoglobin (HbNO) was evaluated by Electron Spin Resonance (ESR) spectroscopy. NO levels were evaluated in the liver, kidney, lung, aorta and heart using ESR and the spin trap FeMGD. The characteristic sextuplet ESR spectrum corresponding to HbNO was not found in CTL rats. In the contrary, high levels of HbNO could be detected in arterial and venous blood samples of GTN rats. The triplet ESR spectrum corresponding to the NO-FeMGD complex was absent from control tissues (except from the kidney), and from aorta and lung in GTN-treated rats. In return, the signals were significantly increased in the liver, kidney and heart in GTN-treated rats. These data suggest that hemoglobin may play a role in the transport of NO delivered from GTN patch. On the other hand, GTN-NO pathway is likely to be largely involved in organs such as liver, kidney, and heart.(See Figure)Open in new tabDownload slideOpen in new tabDownload slide

Orbital sinus blood sampling in rats: effects upon selected behavioural variables.

The question addressed was whether the behaviour of rats is changed after orbital sinus blood sampling while they are under diethyl-ether anaesthesia. Twelve rats were subjected to sham anaesthesia, diethyl-ether anaesthesia and anaesthesia plus orbital puncture according to a Latin square. After each treatment, the behaviour of the rats was automatically monitored using the so-called LABORAS method, which discriminates between grooming, locomotion and inactivity. Treatment ended, and behaviour monitoring began, when the light period changed over to the dark period. The various behaviours were quantified as relative duration and frequency. Anaesthesia versus sham anaesthesia reduced the relative duration of grooming during the first 5 h after treatment. Anaesthesia plus orbital puncture versus anaesthesia alone did not significantly influence grooming, but orbital puncture did reduce the relative duration and the frequency of locomotion during the entire 20 h period, which was mainly due to a decrease in the dark period. After orbital puncture, the animals were also less frequently inactive. It is concluded that orbital puncture has an effect on behaviour superimposed on that of diethyl-ether anaesthesia. This observation may contribute new arguments to the debate on the acceptability of the orbital puncture technique.

Effects of chronic alcohol consumption on neutrophilic host defense in rats

Human alcohol abusers have increased vulnerability to infections. It has been hypothesized that one contributing cause of this vulnerability is the detrimental effect of alcohol on neutrophil function. In order to test this hypothesis, we have evaluated the chemotactic responsiveness of the neutrophils of rats (n=12; 7 12 at 150g starting weight/young and 5 12 at 250g starting weight/mature) fed with a 33% chronic alcohol diet (BIO-SERV liquid alcohol diet #F1697/LD'82), with weight-matched pair-fed (n=12) and weight-matched normal ad libitum diet (n=12) rats serving as controls. The differential count for the neutrophils in rats fed with a chronic alcohol diet was low for both young (6.4±0.3%) and mature (7.0±0.2%) rats. These low counts were similar to the neutrophil count of young pair-fed isoenergetic rats (6.5±0.2%), but differed significantly (P<0.001) from mature pair-fed isoenergetic (11.0±0.4%) and normal ad libitum (young: 10.7±0.2%; mature: 10.9±0.8%) controls. In addition, there was a significant presence of band neutrophils in the rats fed with the chronic alcohol diet ( n= 11 12 ; 15.0±2.1%), and this differed significantly (P<0.001) from pair-fed ( n= 5 12 ; 2.5±0.4%) and normal ad libitum ( n= 7 12 ; 2.2±0.3%) controls. Neutrophils isolated from the blood samples of all alcohol diet rats (blood alcohol levels at 359±127 mg/dl range) were viable (trypan-blue exclusion ≥97%); however, these neutrophils were chemotactically non-responsive (2.8±2.3 neutrophils/100x oil field using LPS-endotoxin activated plasma as chemoattractant) and differed significantly (P<0.001) from viable (≥97%) pair-fed (10.3±12.0 neutrophils/100x oil field) and normal ad libitum (26.3±3.0 neutrophils/100x oil field) controls. A detailed analysis revealed a unique and significant (P<0.001) dichotomy in the chemotactic responsiveness in the pair-fed rats (a low chemotactic score of 0.7±1.1 neutrophils/100x oil field for young rats and a normal chemotactic score of 23.5±3.4 neutrophils/100x oil field for mature rats). The limited diet resulting from pair-feeding apparently exerted a significant negative influence (P<0.001) on the chemotactic responsiveness of the young, but not mature, rats. This dichotomy could be explained by the nutritional impact of a pair-fed limiting diet on the young rats which were in an early stage of rapid growth and development. This study presents a mechanism (neutrophilic abnormalities) to explain the vulnerability to infections in chronic alcohol abusers, and identifies a variable in utilizing very young animals for pair-fed control in nutrition and development research.

Dusky-footed wood rats (Neotoma fuscipes) as reservoirs of granulocytic Ehrlichiae (Rickettsiales: Ehrlichieae) in northern California.

Dusky-footed wood rats (Neotoma fuscipes) and Peromyscus sp. mice (P. maniculatus and P. truei) were collected from one site in Placer County, one site in Santa Cruz County, and two sites in Sonoma County in northern California. Serum or plasma samples from 260 rodents were tested for antibodies to the agent of human granulocytic ehrlichiosis. Of these, samples from 25 wood rats (34% of those tested) and 10 (8%) Peromyscus sp. mice were found to be seropositive, but only those from one site. PCR assays targeting the groESL heat shock operon were conducted on all seropositive specimens and a subset of seronegative blood specimens. Ehrlichial DNA was identified in 17 (68%) of the 25 seropositive wood rat blood samples and in 1 of the 10 (10%) Peromyscus sp. specimens. None of 40 seronegative blood samples was PCR positive. Both seropositive and PCR-positive animals were collected during each trapping period. One male tick out of 84 Ixodes pacificus adults collected was PCR positive; samples of Dermacentor occidentalis nymphs and adults were negative. Nucleotide sequences of amplicons from three wood rat blood specimens and from the single PCR-positive tick differed by one and two bases, respectively, from a sequence previously obtained from Ehrlichia equi. At one site in Sonoma County, wood rats had a concurrent high prevalence of seropositivity and PCR positivity, while other sigmodontine rodents collected at the site were only occasionally infected. We suggest that dusky-footed wood rats serve as reservoirs of granulocytic ehrlichial agents in certain areas of northern California. The tick species involved in the transmission of granulocytic ehrlichiae among wood rats remains unknown.

Adaptation of corticosterone-but not β-endorphin-secretion to repeated blood sampling in rats

Effects of short-term repeated blood sampling on the secretion of corticosterone (CORT) and beta-endorphin (beta-END) were evaluated in male Wistar rats. Blood was drawn from the tail vein of conscious rats four times within 2 h both at the peak and trough period of the diurnal corticosterone secretion cycle. All rats were well accustomed to the procedure. The main findings were: (1) At both sampling intervals, CORT increased significantly in response to the first sampling and declined to baseline values in successive samples. (2) beta-END also increased significantly in response to the first sampling but remained elevated in successive samples. (3) Intensities of initial CORT and beta-END responses correlated positively with each other and with the baseline beta-END values. Feedback inhibition of CORT secretion with sustained elevation of beta-END titres suggests a moderate stress intensity of the repeated blood sampling procedures. In general, due to lack of short-term feedback inhibition, beta-END seems to reflect the effects of repeated administration of moderate intense stressors more closely than CORT.

A shear-induced in vitro platelet function test can assess clinically relevant anti-thrombotic effects

Morphological features of haemostatic plugs formed in vitro under high shear forces were investigated. Electron microscopy confirmed the relevance of such haemostatic plug to a platelet-rich arterial thrombus, which is formed in vivo. In rat blood samples, the effects of anticoagulants and various antiplatelet agents on platelet reactivity (rate of haemostatic plug formation) and subsequent coagulation of the flowing blood were investigated. Haemostasis did not occur in citrated blood, and heparin greatly inhibited the shear-induced platelet reaction. Aspirin (1 mM), a thromboxane A2 receptor antagonist (5 μM), a stable prostacyclin (0.55 nM), a stable prostaglandin E1 (141 nM) and a phosphodiesterase inhibitor (100 μM) were tested. All these agents exerted significant inhibitory effect on shear-induced platelet reaction, including the inhibition of the very first phase of platelet plug formation, due to aggregation of shear-activated platelets. Except for the phosphodiesterase inhibitor, which prolonged clotting time, none of the above agents affected dynamic coagulation. These results suggest that the employed in vitro shear-induced thrombosis/haemostasis test can reveal in vivo the antithrombotic effect of various agents independently of their mechanism of action.

A shear-induced in vitro platelet function test can assess clinically relevant anti-thrombotic effects

Morphological features of haemostatic plugs formed in vitro under high shear forces were investigated. Electron microscopy confirmed the relevance of such haemostatic plug to a platelet-rich arterial thrombus, which is formed in vivo . In rat blood samples, the effects of anticoagulants and various antiplatelet agents on platelet reactivity (rate of haemostatic plug formation) and subsequent coagulation of the flowing blood were investigated. Haemostasis did not occur in citrated blood, and heparin greatly inhibited the shear-induced platelet reaction. Aspirin (1 mM), a thromboxane A(2) receptor antagonist (5 microM), a stable prostacyclin (0.55 nM), a stable prostaglandin E(1) (141 nM) and a phosphodiesterase inhibitor (100 microM) were tested. All these agents exerted significant inhibitory effect on shear-induced platelet reaction, including the inhibition of the very first phase of platelet plug formation, due to aggregation of shear-activated platelets. Except for the phosphodiesterase inhibitor, which prolonged clotting time, none of the above agents affected dynamic coagulation. These results suggest that the employed in vitro shear-induced thrombosis/haemostasis test can reveal in vivo the antithrombotic effect of various agents independently of their mechanism of action.

Accuracy of repeated blood sampling in rats: a new technique applied in pharmacokinetic/pharmacodynamic studies of the interaction between warfarin and co-enzyme Q 10

In the past, combined pharmacokinetic/pharmacodynamic (PK/PD) modeling studies of oral anticoagulants in rats have been hampered by the technical problems of blood sampling. In the present study, a semi-micromethod of preparing serial plasma samples for accurate assessment of the prothrombin times (PT) and clotting factor VII activity (CFA) in rats is proposed. The method consists of orbital bleeding, blood sample weighing, gravity calculation and buffer volume adjustment. No significant differences of CFA (percentage normal) were found between citrate-diluted and undiluted plasma. This technique was employed to examine the possibility of PK/PD interaction between warfarin and Co-enzyme Q 10 (CoQ 10). Rats were given a single oral dose (1.5 mg/kg) of warfarin either alone or on day 4 of an 8-day oral dosing regimen of 10 mg/kg CoQ 10 daily. Serial plasma and serum samples, which were subjected to respectively measurements of the anticoagulant effects and concentrations of warfarin and its main metabolites, were obtained over a 96-h period following warfarin administration. All rats survived the whole experiment and maintained a stable condition except for a marked hematocrit decrease. CoQ 10 significantly augmented warfarin metabolism but showed little effect on the absorption of warfarin. CoQ 10 alone had no apparent effect on either the PT or CFA. The concomitant administration of CoQ 10 and warfarin does not significantly affect the anticoagulant effect of warfarin. In conclusion, the proposed serial orbital bleeding technique in rats to prepare an accurate citrate-diluted plasma for PT and CFA measurement is rapid and reliable.

Orbital sinus blood sampling in rats as performed by different animal technicians: the influence of technique and expertise.

In this study the influence of orbital sinus blood sampling on clinical signs was studied within the framework of various nutritional experiments. In order to assess the clinical signs in a random design, the rats were punctured in either the left or the right orbit. Thus, the effect of puncture within rats could be determined by comparing the left and right eye. Four animal technicians punctured a total of 303 rats, using different techniques. Orbital sinus blood sampling caused clinically visible alterations. The type, frequency and prognosis of the alterations differed with the person performing the puncture. Two experienced animal technicians were able to perform the technique without causing a statistically significant increase in alterations in punctured orbits. One less experienced animal technician caused severe abnormalities. The use of either a Pasteur pipette or a haematocrit capillary did not necessarily produce different results. Neither did puncturing the lateral vs the medial canthus of the orbit. By not applying chloramphenicol eye ointment in the conjunctival sac after puncture, the number of abnormalities in 'ocular discharge' and 'corneal alterations' in the punctured orbits was significantly decreased. Four punctures in the same orbit with 14-day intervals by a skilled animal technician did not cause a significant increase in abnormalities.

Quantification of the inflammatory response in exudates to three polymers implanted in vivo.

Flow cytometry was used to quantify an inflammatory reaction in vivo as a new approach to evaluating the biocompatibility of biomaterials. The exudate formed inside cylindrical tubes composed of polyvinyl chloride (PVC), silicone elastomer (SIL), or polyurethane (PU) implanted subcutaneously in the dorsal region of rats was collected over a 3-week period. The volume, number of cells, and concentration of fibrinogen were determined in the exudate for the three biomaterials. The exudate was analyzed using a flow cytometry technique after labeling of the leukocytes with a monoclonal anti-CD45 antibody. Fibrinogen rose progressively over the 3-week period for the three polymers. After the different leukocyte lines were identified in rat blood samples, their determination in the exudate revealed differences among the three biomaterials. At day 2, PVC induced a predominantly neutrophilic inflammatory reaction whereas PU and SIL gave a mixture of monocytes and neutrophils. At day 9, the aspect of the cytograms was different, but the identification of the subpopulations was still possible. At day 23, the number of cell events became too low to distinguish the subpopulations. An even more detailed approach might be possible using specific labeling for each leukocyte line to establish a comparison among the three biomaterials. Flow cytometry associated with histomorphometric assessment might provide a precise quantitative in vivo test for determining the biocompatibility of materials.

Studies on the Metabolic Fate of SS320A. (1): Absorption, Distribution, Metabolism, Excretion, Transfer into Fetus and Milk of 14C-SS320A after Single Administration in Rats.

Absorption, distribution, metabolism, excretion, transfer into the fetus and milk were investigated after a single oral administration of 14C-SS320A 25 mg/kg to rats of both sexes. 1. The blood level of radioactivity in the fasting male rats reached the Cmax of 15.03 μg/ml at 0.31 hr and then declined with t1/2z of 5.7 hr. The AUC0-∞ was 33.0 μg hr/ml. In the blood, the parameters of radioactivity in the non-fasting rats were as follows, the Cmax values was lower, the Tmax and t1/2z were longer and the AUC was 1.3 times larger than those in fasting rats. The blood radioactivity in male and female rats were similar. 2. In a study of gastro-intestinal absorption in situ, the largest amount of 14C-SS320A was rapidly absorbed from the small intestine. 3. The all of tissues in the fasting male rats showed a maximum level of radioactivity at 0.5 hr after oral administration, and it was eliminated quickly until 24 hr. The high concentration of radioactivity was observed in the kidney, liver, cerebrum and eyeball. However, the radioactivity levels in these tissues were less than 1 % of the maximum level within 120 hr after administration. Also, in majority of tissues of non-fasting rats the T/P were similar, but, the elimination of radioactivity from tissues were slower than fasting rats. 4. The protein binding ratios (in vitro) of radioactivity were low in plasma sample of rats and human at0.2-20 μg/ml. In rats, the protein binding ratios (in vivo) of radioactivity were 4.0 and 2.5 % at 0.5 and 4 hr and then increased 19.5 % at 8 hr after oral administration. 5. The distribution ratio (in vitro) of radio activity in blood cells (fasting) of 14C-SS320A was about 28 and 35% in blood samples of rats and human, respectively, at 0.2-20 μg/ml. The distribution ratio of radioactivity in blood cells was about 25% from 0.5 hr to 8 hr after oral administration of 14C-SS320A to male rats. 6. In the urine M1 and M3 were mainly found. In the plasma the SS320A, M1, M3 and M4 were mainly found at 1 hr after oral administration of 14C-SS320A in fasting and non-fasting rats. M1 was found in cerebrum, eyeball and lung as the main metabolite in fasting rats, the unchanged form, however, was found mainly in liver and kidney. 7. In male rat, 96.8, 0.7 and 0.8% of the dose was excreted in the urine, feces and expired air, respectively, within 120 hr after oral administration of 14C-SS320A. The same results were obtained after intravenous administration. The sex differences in drug excretion were not observed. 8. The excretion of radioactivity in the bile, urine and feces in male rats was 1.3, 95.4 and 0.1% of the dose, respectively, within 48 hr after oral administration of 14C-SS320A. 9. On the 12-th and 18-th days of gestation, the level of radioactivity in the fetus after oral administration of 14C-SS320A was higher than that in maternal plasma. 10. The level of radioactivity in milk, after oral adm inistration of 14C-SS320A to lactating female rats on the 11-th day after delivery, was lower than that in plasma until 2 hr. Nevertheless, it reached the same concentration at 4 hr, and it was higher than that in plasma after 8 hr.