Rat Blood Samples Research Articles

A survey of the values of clinical chemistry parameters obtained for a common rat blood sample in ninety-eight Japanese laboratories.

A control survey was conducted to check the accuracy of automated analyzers used in the evaluation of clinical chemistry parameters in nonclinical toxicology studies. Pooled serum samples from male Sprague-Dawley rats were delivered refrigerated to each facility 98 laboratory facilities throughout Japan within 18 hours after sample preparation and analyzed. Commercially available normal human serum samples from a single lot were also analyzed at the same time. Survey results were divided into three categories. (1) Parameters with small coefficient of variation (CV) values for both rat and human serum samples included protein, glucose, cholesterol (CHO), urea nitrogen (UN), sodium (Na), potassium (K), chloride (Cl), calcium (Ca), and inorganic phosphate (IP). Definition of normal values in rats should be straight forward for these parameters. (2) Parameters with large CV values, but with a relatively good correlation between rat and human values include triglycerides (TG), glutamic oxaloacetic transaminase/aspartate aminotransferase (GOT/AST), glutamic pyruvic transaminase/alanine aminotransferase (GPT/ALT), and alkaline phosphatase (ALP). Measurements based on different principles gave different mean values, and this values contributed to the increase in CV values. Assessment of normal values would require a consideration of the measurement principles. (3) Parameters with large CV values only in rat serum samples included albumin (albumin/globulin ratio: A/G ratio), creatinine (CRE), and total bilirubin(BIL). Reactivity was different in rat albumin (ALB), depending on the reagents used. This difference needs to be corrected with values available by electrophoresis, or adjusted by rat ALB values, because of the lack of an appropriate measurement method. The enzyme method gave low values for rat CRE, which suggests the need for further examination of this method. The BIL values were extremely low in rat samples. It seems to be necessary to select appropriate methods to measure clinical pathology parameters correctly for rats. There was no deviation in values due solely to the mechanical operations of the analytical equipment. Non-standard initial settings of the equipment (equipment originally intended for human samples, but now applied to animal samples) was the main cause of the wide range of analytical values seen.

The effect of host blood in the in vitro transformation of bloodstream trypanosomes by tsetse midgut homogenates.

Midgut homogenates prepared from Glossina morsitans morsitans, that had previously been fed on different host blood samples, were tested for their abilities to transform bloodstream Trypanosoma brucei into procyclic (midgut) forms in vitro. Compared to rat and goat blood samples, eland blood had the least capacity to support trypanosome transformation, whereas buffalo blood showed intermediate capacity. Fractionation of rat blood showed the importance of the cellular portion since both rat and eland red blood cells (RBCs) supported the process. Virtually no transformation was observed in rat and eland plasma or serum fractions. Suspending rat blood cells in eland plasma led to a reduction in parasite transformation rates. Further experiments showed that the RBC membranes were also capable of supporting the process. These results clearly show the important role played by blood, especially the red blood cells, in the transformation of bloodstream trypanosomes. In addition, the low transformation rates observed in eland blood is due to an inhibitory factor(s) present in the plasma fraction.

Genotypic Identification of Murine Typhus Rickettsia in Rats and their Fleas in an Endemic Area of Greece by the Polymerase Chain Reaction and Restriction Fragment Length Polymorphism

Forty-nine cases of murine typhus were diagnosed in recent years in residents of several communities around the city of Chalkis, the capital of the Prefecture of Evia. (Euboea) Evia is an island connected to central mainland Greece by a bridge. To investigate the endemicity of murine typhus in this area, 226 fleas (Xenopsylla cheopis) and blood samples were collected from 53 rats (Rattus norvegicus) trapped in this area. The polymerase chain reaction followed by restriction fragment length polymorphism analysis (PCR-RFLP) was used to detect and identify Rickettsia typhi, the etiologic agent murine typhus, in the rat blood samples (buffy coat cells) as well as in their fleas. An indirect immunofluorescent antibody (IFA) assay was performed to detect antibodies against R. typhi in rat serum samples. The presence of R. typhi in both fleas and rat blood samples was demonstrated. The frequency of infection for X. cheopis was 4%, while 18% of the rats had buffy coat cells infected, and 92% of the rat sera tested by IFA were positive for anti-R. typhi antibodies. The present work is the first successful application of PCR-RFLP in a field study of naturally infected rats and their fleas in Europe.

A survey of the results of haematological parameters, using a common rat blood sample in japanese laboratories

A survey was conducted to determine the accuracy and quality control of automated haematology analysers used in non-clinical toxicity studies. Pooled blood samples from male Sprague-Dawley rats were distributed to 98 laboratory facilities throughout Japan, the samples being delivered under refrigeration to each facility within 18 h of sample preparation. At each facility, the samples were analysed within 4 h of receipt. Commercially available normal human blood samples from a single lot were also analysed at the same time. Most haematological results were within the mean ±3SD (standard deviation), but some facilities gave either high or low values consistently for both human and rat samples. No facility gave high or low values to certain parameters sporadically, which suggests no problem with the accuracy of the equipment. However, it was suspected that there would be some problem in comparing analytical values determined in a unique way by specific equipment design. The use of certain equipment resulted, in rat haematocrit values in particular, being either too high or too low. In these cases, it was deemed necessary to make some adjustments or calibration changes. There were also platelet values with a ‘plus drift’ which was apparently due to contamination with, or failure to identify small red blood cells (RBC). There was no deviation in values which could be attributed solely to the mechanical operation of any of the analytical equipment.

Effects on haematology parameters during cold storage and cold transport of rat and dog blood samples

A sample surveillance programme is scheduled to be conducted on measurement methods of haematology parameters which will include the participation of over 70 facilities. In preparation for that programme, a preliminary study was conducted, at five of the facilities, on the effects of cold storage and transport on rat and dog blood samples.

Species differences in the concentration of acetylcholine, a neurotransmitter, in whole blood and plasma

Various concentrations of acetylcholine (ACh) were detected in samples of bovine, goat, horse, porcine, rat and sheep blood and plasma using a specific, sensitive radioimmunoassay. The ACh levels in whole blood in bovine and horse samples were about 40- and ten-fold higher, respectively, than in humans, but levels comparable to those in humans were measured in porcine samples. Goat, rat and sheep samples had lower whole blood ACh concentrations than those of humans. When plasma samples were assayed, the ACh contents of bovine and porcine plasma were found to be about two- to five-fold those of human. On the other hand, levels in horse, goat, rat and sheep samples were much lower than in humans. The ratio of the ACh contents of plasma to whole blood was high in porcine and rat samples, indicating that porcine and rat blood ACh is distributed mostly in the plasma, while in the other species tested most of the ACh is present in the blood cells. These results demonstrate that variable levels of ACh are present in the blood of different species, and that the distribution of ACh in the blood constituents varies according to species.

Method to estimate the rate and extent of intestinal absorption in conscious rats using an absorption probe and portal blood sampling.

A variety of methods exist which determine the rate and extent of intestinal absorption. The method described here employs an internal absorption reference probe and portal blood sampling in unanesthetized rat. Theophylline and tritiated water were selected as absorption reference probes since they are quantitatively absorbed in conscious rat. The fraction of an intestinal dose which reaches portal blood was determined from the resulting portal-systemic blood concentration gradients of the drug relative to the absorption probe. The absorption probes provide a means to calculate the drug mass reaching portal blood without the need of measuring the portal blood flow rate. The technique was evaluated with verapamil and a well-absorbed 5-lipoxygenase inhibitor, A-79035. The fraction of an intrajejunal dose of A-79035 reaching the portal vein (FG) was 0.86 using theophylline as the absorption probe. Verapamil, which is susceptible to extensive hepatic first-pass elimination, was completely absorbed (FG = 0.98) within 1 hour, but was only 21.4% bioavailable. Absorption rate constants, estimated from initial appearance rates in portal blood, were used to monitor factors that affect drug absorption. For example, with a dose solution containing 30% PEG-400, the absorption rate constants of theophylline and A-79035 were significantly reduced. Anesthesia reduced the absorption rate constant for theophylline in rats by 40% compared to conscious animals. The technique detailed here allows reliable, direct measurement of intestinal absorption which may assist in characterizing oral dosing for novel therapeutic agents.

Evaluation of the contraves AL 820 automated haematology analyser for domestic, pet and laboratory animals

A comprehensive evaluation of the automated haematology analyser Contraves AL 820 was initiated to determine the suitability of this instrument for veterinary purposes in domestic, pet and laboratory animal species. The AL 820 (AVL Medical Instruments, Schaffhausen, Switzerland) is an impedance cell counter with automated threshold setting for rapid adaptation to cell characteristics of different animal species. Storage capability for up to 200 data sets with histograms is provided. Excellent precision, linearity and carry-over features of the AL 820 have been demonstrated in tests with rat, mouse, cat, dog, cattle and horse blood samples. Accuracy of haemoglobin and haematocrit measurements with respect to reference methods was characterised by strong linear correlation. The patented cyanide-free AL 820 method for haemoglobin determination compared very well to the haemoglobincyanide reference method. Accuracy of the red blood cell, haemoglobin, haematocrit, mean corpuscular volume, white blood cell count and platelet count parameters was generally good, when compared to the established laboratory routine method (the predecessor model AL 801) or to a high end automated haematology analyser (Abbott Cell-Dyn CD 3500). Although variation of platelet count measurements was greater than variation of all other parameters, it was considered acceptable particularly with respect to the lack of excelling alternatives. Poor accuracy of feline platelet counts was attributed to overlapping size distribution of red blood cells and platelets in this species. The overall favourable acceptance of the AL 820 was based on easy handling, simple maintenance and pronounced flexibility of this instrument at an economical purchase price. A sample volume of 30 µl and a throughput of up to 60 samples per hour are distinct advantages and render the AL 820 suitable for medium-sized laboratories. With few exceptions, the instrument provides reliable results for all major animal species encountered in routine veterinary haematology.

Solid-phase extraction and reversed-phase high-performance liquid chromatographic determination of sulphur mustard in blood

A reversed-phase high-performance liquid chromatographic method is reported for the analysis of sulphur mustard in blood with the aid of solid-phase extraction sample preparation. Sulphur mustard is extracted from blood samples (both in vitro and in vivo) of rats with a solution of 0.05 M sodium dodecyl sulphate and pre-concentrated over Sep-Pak C 18 cartridges pre-coated with Tween-20. A Polygosil C 18 column is used with acetonitrile—water (52:48, v/v) as mobile phase for separation and sulphur mustard was detected at 200 nm.

High-performance liquid chromatographic determination of 3′-hydroxy-5′-(4-isobutyl-1-piperazinyl)benzoxazinorifamycin (KRM-1648) and its deacetyl metabolite in plasma, whole blood, urine and tissue samples in rats

A reversed-phase high-performance liquid chromatographic method was developed for the determination of 3′-hydroxy-5′-(4-isobutyl-1-piperazinyl)benzoxazinorifamycin (KRM-1648, I), a new rifamycin derivative, and its 25-deacetyl metabolite (KRM-1671, II) in plasma, whole blood, tissues and urine from rats. I and II were coextracted with an internal standard from each sample matrix by solid-phase extraction (Bond Elut). Plasma and urine were directly loaded onto Bond Elut, while whole blood and tissues were homogenized and extracted with methanol or dichloromethane—chloroform prior to Bond Elut extraction. The extracts were chromatographed on Shim-pack CLC-ODS(M) using acetonitrile—0.02 M citrate buffer containing 0.1 M sodium perchlorate (2:1, v/v), and peaks were detected at 643 nm. The validation data showed that the assays for I and II in plasma, whole blood, tissues and urine were selective, accurate and reproducible.

Lack of influence of circulating adrenaline on blood pressure in normotensive and hypertensive rats.

The relationship between circulating adrenaline and blood pressure was examined by manipulating plasma adrenaline levels in both normotensive and hypertensive rats: bilateral adrenalmedullectomy was performed in spontaneously hypertensive rats and stroke-prone spontaneously hypertensive rats; adrenaline bitartrate was infused chronically (25-32 micrograms/kg/h s.c.) into Wistar Kyoto, Sprague Dawley and stroke-prone rats via osmotic minipumps. Arterial and venous catheters were subsequently implanted for direct measurement of mean arterial pressure, blood sampling and drug administration in conscious rats. Adrenaline infusion for 5-6 weeks in Wistar Kyoto rats did not affect resting blood pressure (118 +/- 3 versus 119 +/- 1 mmHg in controls) even though plasma adrenaline was elevated 12-fold. Plasma noradrenaline was marginally elevated. Blood pressure was also unaffected by adrenaline infusion in Sprague Dawley or stroke-prone hypertensive rats. One week after adrenal medullectomy, plasma adrenaline was reduced 89% in spontaneously hypertensive rats, but blood pressure was unaffected. Ten weeks after adrenal medullectomy in young stroke-prone rats, resting blood pressure was slightly higher (167 +/- 2 mmHg) than in control rats (157 +/- 2 mmHg), although adrenaline was reduced by 34% in plasma and 67% in adrenal glands. Nitroprusside was infused acutely to lower blood pressure and reflexly elevate plasma noradrenaline. Neither of these responses were affected by chronic adrenaline infusion or adrenal medullectomy. In both adrenaline-infused Wistar Kyoto and medullectomised stroke-prone rats, autonomic blockade reduced blood pressure to a similar extent as in controls, indicating that the degree of sympathetic vasoconstriction was not altered by either treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative NMR studies of diffusional water permeability of erythrocytes from some animals introduced to Australia: Rat rabbit and sheep

The diffusional water permeability (P d) of the red blood cell (RBC) membrane of rat, rabbit and sheep, representing some animal species introduced to Australia, has been monitored, by a Mn2+-doping 1H nuclear magnetic resonance (NR) technique using a high-field spectometer operating at 400 MHz. In order to make comparisons with previous measurements on the same species (performed at 25 MHz) an analysis of the influence of instrumental parameters and of the state of blood oxygenation was performed on samples of rat and rabbit blood. It was found that by using a short interpulse delay (around 100 µs) in the Carr-Purcell-Meiboom-Gill sequence, and by performing the measurements of the transverse relaxation time of the water in the cell interior (T 2i) on packed cells (haematocrit >95%), the values of the water exchange time (T e) obtained with a high-field spectrometer are comparable with those obtained by using a low-field NMR spectrometer. The values of the diffusional water permeability (P d), calculated from the T e values, were, at 24.6°C around 10 × 10−3 cm/s in case of rat and rabbit RBC and around 5 × 10−3 cm/s for sheep RBC; at 37°C the P d values were around 16 × 10−3 cm/s for rat RBC, 14 × 10−3 cm/s for rabbit RBC and 7 × 10−3 cm/s for sheep RBC. These values are very close to the P d values previously reported for the European counterparts of these species. There were no significant differences in the P d values between laboratory rabbits and wild rabbits, or between castrated males, pregnant or nonpregnant females. These results suggest that no changes in the RBC water permeability are correlated with marked alterations in the habitat of animal species introduced to Australia or by sex hormone status. If the NMR instrument-parameters are carefully chosen then comparative studies of RBC water permeability in different laboratories, at separate locations and using different instruments are meaningful.

Role of lipid peroxidation in metalloporphyrin-mediated phototoxic reactions in neonatal rats.

Our group previously demonstrated dose-dependent mortality in neonatal rats treated with tin protoporphyrin and light. We hypothesize that lipid peroxidation may be responsible for the toxic effects of photosensitizing metalloporphyrins. Neonatal rat blood samples with or without metalloporphyrins (40 mM) were exposed to cool white light (20 microW/cm2/nm) for 30 min at 37 degrees C. In the in vivo model, neonatal rat pups were given injections of 40 mumol of either tin protoporphyrin (4 mM), zinc protoporphyrin/kg body weight, or saline and placed over cool white light. The control animals were similarly treated but kept in the dark. After 3 h, the animals were killed, and their tissues were analyzed for malondialdehyde, conjugated dienes, and disappearance of polyunsaturated fatty acids as indices of lipid peroxidation. In all cases, the known photosensitizer tin protoporphyrin was associated with increased conjugated dienes in the liver and disappearance of polyunsaturated fatty acids and increased malondialdehyde in the liver and brain when animals were exposed to light. Zinc protoporphyrin was not associated with increased lipid peroxidation in the light except in the case of blood in vitro where malondialdehyde levels increased. We conclude that lipid peroxidation plays a role in metalloporphyrin-mediated phototoxicity in neonatal rat tissues.

A reliable technique for chronic carotid arterial catheterization in the rat

A reliable and simple method for cannulating the carotid artery of rats is described. The rat was anesthetized with halothane. The right carotid artery located between the omohyoideus and sternohyoideus muscles was exposed through a 2-cm ventral neck incision. The catheter was made of Silastic tubing and a monofilament line, which served as an obturator and internal support. The line was then filled with a viscous mixture of heparin, polyvinylpyrrolidinone and normal saline to prevent the formation of blood clots. The catheter was advanced through the carotid artery towards the heart by a predetermined distance (15–20 mm) depending on the size of the rat. The catheter was well tolerated by the rats and the success rate was 95%. Its patency lasted for at least 7 days postsurgery without any special maintenance care. With the described method one would be able to perform repetitive blood sampling and arterial blood pressure measurements in unanesthetized and unrestrained rats for prolonged period after Catheterization.

Influence of the blood sampling site on some haematological and clinical-chemical parameters in sprague-dawley rats

Differences in haematology and serum chemistry values related to site and method of blood sampling in Sprague-Dawley rats are described. Blood was collected from the orbital sinus, the abdominal aorta, and the carotid artery under anaesthesia, and from the tail vein after prewarming the rat. Regardless of the technique used, arterial blood had lower leucocyte counts and serum protein levels and higher glucose concentrations than did venous blood. Compared to the other techniques studied, tail-vein blood yielded slightly higher erythrocyte counts and haemoglobin concentrations, and markedly higher serum potassium concentrations. Of the methods used in this study, the tail-vein technique can be recommended for routine blood sampling in rats, because this method is repeatable, yields enough blood of acceptable quality and is aesthetically acceptable.

Determination of in vitro simulated blood cell velocity by Laser-Doppler with state space methods

In this paper a signal processing method based on an important tool of numerical analysis, the singular value decomposition (SVD), is used. This method is applied to the unprocessed output of a Laser-Doppler fluxmeter to obtain parameters that are more sensitive to blood velocity than to hematocrit. The SVD-based method utilizes the exponential shape of the frequency spectrum of the laser light scattered from the moving red blood cells and demonstrates an inverse relationship between the damping constant β associated with the exponential shape and the blood velocity. This method was applied to samples of rat blood of several known hematocrit values that were rotated at different velocities on a turntable. The method extracted one dominant singular value from the spectra, indicating that the spectra can be modeled as a single exponential dominated by scatterings with a single moving red blood cell. A 68% change in inverse velocity resulted in a 50% change in the damping constant (hematocrit 29.5% vol). On the other hand, a 37% change in hematocrit resulted in a far smaller change in the damping constant β of only 17%. Analysis of the extracted parameters shows that the damping constants are far more influenced by blood velocity than by blood hematocrit.

Antithrombotic Activity of a Novel C-Terminal Hirudin Analog in Experimental Animals

SummaryA synthetic hirudinsgs55-65 C-terminal fragment analog was evaluated for anticoagulant activity and in models of experimental thrombosis in mice and rats. Intravenous injection caused doserelated inhibition of thrombin and anticoagulation in rat blood samples, protection from thromboembolism in mice and inhibition of stasis-induced venous thrombosis in rats. Antithrombotic effectiveness corresponded with anticoagulant activity. Anephric animals exhibited longer duration of activity than normal animals suggesting the kidney as a major route of elimination.

A model of reversible obstructive jaundice in the rat

A model of reversible, extrahepatic biliary obstruction is described. Vessel loop blockade of the biliary tree results in obstructive jaundice while removal of the exteriorized vessel loop provides internal biliary drainage without subsequent laparotomy. This technique combined with a system for chronic venous infusion and arterial blood sampling in the unrestrained rat is ideal for longterm metabolic studies of obstructive jaundice. Male Fisher 344 rats (275–350 g) underwent either the combined procedure of total biliary tract blockade and vascular access or sham operation. Mean serum bilirubin was significantly elevated (12.7 ± 8.9 mg/dl) in the experimental group and following relief of biliary obstruction significantly dropped below 1 mg/dl in all animals except one. Concomitant changes in alkaline phosphatase, glutamate oxaloacetate transaminase, and glutamate pyruvate transaminase were seen. Experimental and control rats initially lost weight following laparotomy; however, mean body weight stabilized by the 5th postoperative day and was similar in both groups on the 10th postoperative day. This combined procedure is a simple, effective and reproducible method of obstructive jaundice.

Storage of blood for methemoglobin determination: Comparison of storage with a cryoprotectant at −30°C and without any additions at −80°C or −196°C

Changes in methemoglobin (Met-Hb) concentrations during storage of whole blood or mixtures of blood and a cryoprotectant at refrigerated or various freezing temperatures were examined using blood samples from nitrite-administered rats and from autopsy cadavers. When whole blood was stored at 3°C, Met-Hb reduction was observed in blood samples from nitrite-administered rats and in the blood from a victim poisoned by a weed killer containing some oxidant. When samples were stored at −30°C, Met-Hb formation by autoxidation was inevitably observed in blood samples stored as whole blood, whereas addition of a cryoprotectant to whole blood could prevent Met-Hb formation in all the blood samples. When whole blood was stored at −80°C or −196°C, Met-Hb concentrations were practically stable until at least 30 days regardless of the initial values except in the control rat blood samples stored at −80°C which showed slight formation of Met-Hb. From the results obtained, both the storage with a cryoprotectant at −30°C and that without any additions at −80°C or −196°C proved to be suitable for long-term storage of blood samples from autopsy cadavers for Met-Hb determination.

A methodological improvement for experimental control and blood sampling in rats.

We have developed an apparatus and techniques that permit repeated, undisturbed blood sampling and concurrent assessment of activity in rats. The apparatus also allows experimental control of conditioning stimuli and contingencies without the need to transfer the rat to a separate chamber. In the experimental apparatus, body weights remain stable at approximately 90% of the original body weight. Plasma corticosterone and prolactin levels in the apparatus are comparable to levels obtained in undisturbed free-ranging rats and remain at basal levels for the 7 day duration of this experiment. Additionally, circadian activity rhythms in the apparatus are similar to those seen in free-ranging rats. This technique has potential applications for investigators who need to be able to control shock delivery, monitor activity, and sample blood without disturbing rats.