Comparison of clinical pathology parameters with two different blood sampling techniques in rats: retrobulbar plexus versus sublingual vein

Blood samples were taken from the retrobulbar venous plexus or the sublingual vein of male HamIbm:Wist rats to compare clinical pathology parameters between the two sampling techniques. By analogy with a pharmacokinetic study, blood was sampled six times during one day from unfasted animals. After 3 weeks of recovery, blood was taken from fasted animals on a single occasion. In addition, prolactin and corticosterone levels were determined to compare stress-related effects between the two sampling methods. Body weight development and food consumption were similar after single as well as after repeated blood sampling for the two blood sampling techniques. Haemotological evaluation showed a gradual decrease in erythrocyte count, haemoglobin concentration and haematocrit after repeated blood sampling. Repeated withdrawal of blood samples over 24 h corresponding to approximately 22% of the total blood volume resulted in a decrease in red blood cell parameters by up to 30%. The withdrawal of approximately 10% of the total blood volume was associated with a decrease in these parameters by up to 10% and should not be exceeded for animal welfare reasons and to allow a reliable evaluation of data in a study. Repeated blood sampling was associated with an initial decrease in the number of white blood cells, mainly due to a reduction in lymphocytes; white blood cell counts were slightly increased one day after. The decrease in lymphocytes and the increase in neutrophils after repeated sampling were generally slightly more pronounced in the blood from the retrobulbar plexus than from the sublingual vein. Comparison of serum clinical chemistry data showed significantly higher activities of creatine kinase and aspartate aminotransferase in samples from the retrobulbar plexus. These findings suggest a higher degree of tissue damage with blood sampling from the retrobulbar plexus than from the sublingual vein. Despite a large inter-individual variability, higher mean values of prolactin on each occasion and corticosterone after a single sample in fasted animals indicate a higher stress associated with blood sampling from the retrobulbar plexus.     

一项小鼠采血新技术——颌下静脉丛采血法

目的介绍小鼠颌下静脉丛采血方法。方法选用2月龄昆明小鼠,固定小鼠后,将采血注射针头刺入颌下静脉丛血管取血。结果单人操作约1 min内可完成小鼠颌下静脉丛采血,采血量达到0.3-0.5 mL。结论此方法无需麻醉、创伤小、采血量大并可重复采血,是一种简便、安全、快速、采血量多的采血方法。     

Blood collection from the tail of a rat

Techniques for blood collection from the rat include puncture of the heart, retro-orbital plexus, jugular vein, saphenous vein, tail blood vessels, carotid artery, abdominal aorta, and vena cava. Most techniques (except saphenous vein and tail blood vessel puncture) require anesthesia. The following discussion focuses on two methods of blood collection - ventral tail artery puncture and dorsal or lateral tail vein puncture.     

Drawing blood from rats through the saphenous vein and by cardiac puncture

Drawing blood from rodents is necessary for a large number of both in vitro and in vivo studies. Sites of blood draws are numerous in rodents: retro-orbital sinus, jugular vein, maxillary vein, saphenous vein, heart. Each technique has its advantages and disadvantages, and some are not approved any more in some countries (e.g., retro-orbital draws in Holland). A discussion of different techniques for drawing blood are available (1-3). Here, we present two techniques for drawing blood from rats, each with its specific applications. Blood draw from the saphenous vein, provided it is done properly, induces minimal distress in animals and does not require anesthesia. This technique allows repeated draws of small amounts of blood, such as needed for pharmacokinetic studies (4,5), determining plasma chemistry, or blood counts (6). Cardiac puncture allows the collection of large amounts of blood from a single animal (up to 10 ml of blood can be drawn from a 150 g rat). This technique is therefore very useful as a terminal procedure when drawing blood from the saphenous would not provide a large enough sample. We use cardiac puncture when we need sufficient amounts of serum from a specific strain of rats to grow T lymphocyte lines in vitro (4-9).     

An improved simple technique for the collection of blood samples from rats and mice

The technique of blood collection from the lateral caudal vein has been improved. The method requires only moderate skill and no anaesthesia is necessary. Collection of blood samples causes little trauma and can be repeated at frequent (8 h) intervals thus making the method particularly suitable for pharmacokinetic and hormonal studies. Blood samples are uncontaminated by tissue fluids. Large volumes can be obtained, in mice up to 1.5 ml and in Wistar rats over 4 ml.     

Blood sampling from the retro-orbital plexus, the saphenous vein and the tail vein in rats: comparative effects on selected behavioural and blood variables

We compared the behaviours of rats, and measured various blood parameters, after three blood sampling techniques: orbital puncture while they were under diethyl-ether anaesthesia, blood collection by tail vein puncture under O2-N2O-halothane anaesthesia and puncture of the saphenous vein without anaesthesia. Twelve rats were subjected to the three treatments according to a Latin square design. After each treatment, the behaviour of the rats was automatically monitored using the so-called LABORAS method, which discriminates between grooming, locomotion and inactivity in rats. Based on excitation scores and urine production, it was found that induction of diethyl-ether anaesthesia combined with orbital puncture caused more distress than did the other two blood sampling techniques. The three techniques had no differential effects on the behaviours of grooming, locomotion and inactivity. Collecting 0.5 ml of blood by orbital puncture was +/-7 times faster than doing so by saphenous vein puncture and +/- 15 times faster than collecting blood by tail vein puncture while the rats were under O2-N2O-halothane anaesthesia. The levels of some haematological and plasma variables differed significantly between the three blood collection techniques. These observations may help to select the most appropriate technique of blood sampling with respect to anticipated discomfort in the animals.     

Physiological and Pathological Impact of Blood Sampling by Retro-Bulbar Sinus Puncture and Facial Vein Phlebotomy in Laboratory Mice

Retro-bulbar sinus puncture and facial vein phlebotomy are two widely used methods for blood sampling in laboratory mice. However, the animal welfare implications associated with these techniques are currently debated, and the possible physiological and pathological implications of blood sampling using these methods have been sparsely investigated. Therefore, this study was conducted to assess and compare the impacts of blood sampling by retro-bulbar sinus puncture and facial vein phlebotomy. Blood was obtained from either the retro-bulbar sinus or the facial vein from male C57BL/6J mice at two time points, and the samples were analyzed for plasma corticosterone. Body weights were measured at the day of blood sampling and the day after blood sampling, and the food consumption was recorded automatically during the 24 hours post-procedure. At the end of study, cheeks and orbital regions were collected for histopathological analysis to assess the degree of tissue trauma. Mice subjected to facial vein phlebotomy had significantly elevated plasma corticosterone levels at both time points in contrast to mice subjected to retro-bulbar sinus puncture, which did not. Both groups of sampled mice lost weight following blood sampling, but the body weight loss was higher in mice subjected to facial vein phlebotomy. The food consumption was not significantly different between the two groups. At gross necropsy, subcutaneous hematomas were found in both groups and the histopathological analyses revealed extensive tissue trauma after both facial vein phlebotomy and retro-bulbar sinus puncture. This study demonstrates that both blood sampling methods have a considerable impact on the animals'' physiological condition, which should be considered whenever blood samples are obtained.     

Cannulation of the jugular vein in mice: a method for serial withdrawal of blood samples

We have developed and validated a method that allows serial drawing of blood samples in freely moving mice using a cannula that is inserted via the jugular vein into the right atrium of the heart. The cannula was tunnelled subcutaneously to the head of the animal and attached to the skin by sutures, together with a metal spring, which was covered with PVC tubing for protection of the outer part of the cannula. Samples of blood up to 250 micro l could be taken at serial time points. The blood volume in the circulation was maintained by replacement with an equal volume of blood obtained from donor animals. The applicability of this method of blood sampling for pharmacokinetic purposes was validated by comparing plasma concentrations-time curves in six cannulated animals after receiving an intravenous bolus dose of 10 mg/kg of the anti-cancer agent docetaxel versus the results in plasma samples obtained by cardiac puncture of non-cannulated mice. The presented method may lead to improved pharmacokinetic data produced from a reduced number of mice.     

Biomechanical response of femoral vein to chronic elevation of blood pressure in rabbits

Venous diseases like iliofemoral deep vein thrombosis and valvular dysfunction induce venous hypertension. To know the effects of the hypertension on venous mechanics, blood pressure in the left femoral vein in the rabbit was chronically elevated by the constriction of the left external iliac vein. Wall dimensions and biomechanical properties of the femoral vein were studied in vitro at 1, 2, or 4 wk after surgery. Blood pressure measured immediately before the animal was killed was significantly higher in the left femoral vein than in the sham-operated, contralateral vein. Wall thickness was increased by blood pressure elevation even at 1 wk, which restored circumferential wall stress to a control level. The stress was kept at normal up to 4 wk. Vascular tone and vascular contractility were increased by the elevation of blood pressure; however, wall elasticity and compliance were kept at a normal level. These results are very similar to those observed in hypertensive arteries, indicating that not only arteries but veins optimally operate against blood pressure elevation.     

Intermittent sampling of portal venous blood and bile from guinea pigs

A technique is described for intermittent collection of portal venous blood from guinea pigs through a catheter advanced from an ileal tributary of the cranio-mesenteric vein into the portal vein and for the collection of bile from a catheter in the gallbladder after ligature obstruction of the common bile duct.     

Functional arterio-venous anastomoses between the testicular artery and the pampiniform plexus in the spermatic cord of rams

Blood flow to the testis, haemoglobin oxygen saturation and testosterone concentration in arterial and venous testicular blood vessels were studied in Texel rams in the breeding and non-breeding season. Blood flow in the proximal and distal testicular artery was measured electromagnetically. The mean flow in the proximal testicular artery was 18.5 ml/min and in the distal testicular artery 7.5 ml/min, and there was no detectable seasonal influence. Haemoglobin oxygen saturation and testosterone concentration were measured in the saphenous artery and vein, the distal testicular artery and vein, and in the proximal testicular vein. The haemoglobin oxygen saturation in the proximal testicular vein was significantly higher than in the distal testicular vein in both seasons. The mean testosterone concentration was significantly lower in the proximal testicular vein than in the distal testicular vein in both seasons. Based on haemoglobin oxygen saturation and testosterone data, it was calculated that between 28 and 46% of the testicular arterial blood was bypassing the testis and was directly flowing through arterio-venous anastomoses towards the pampiniform plexus in the spermatic cord of conscious rams. In anaesthetized rams 55 and 64% of the blood was flowing directly from the testicular artery to the pampiniform plexus based on blood flow data. Transfer of testosterone and oxygen by passive diffusion from the testicular artery to the pampiniform plexus and vice versa in the spermatic cord was not detected.     

Effect of ageing on the murine venous circulation

The effect of ageing on the morphology of veins, venous valves and arteries was investigated in male wild-type mice using an adapted procedure with injection of a silicone polymer Microfil^? that preserves morphology of the vasculature. Throughout the hind limb the arterial, but not the venous, lumen area and wall thickness were significantly greater in 24-month as compared to 10-week-old C57BL/6 mice. Venous valves were most frequently located at the sapheno-femoral vein junction in the lower extremities, and appeared thicker at the base supported by structurally intact collagen fibers, and thinner towards the proximal end of the valve leaflet, with less organized collagen. Overall, valves were less supported by structurally intact collagen at 24 months as compared to 10 weeks. Endothelial expression of CD31, endothelial protein C receptor or von Willebrand factor (VWF) was not affected by age, while thrombomodulin expression was lower in aged versus young arteries. At both ages, expression of VWF was lower at venous valves versus veins. Evaluation of the blood coagulation profile revealed that aged mice had shortened prothrombin time, elevated plasma levels of factor (F)VII, FVIII and VWF and increased neutrophil and platelet counts. Thus, our data indicate that in mice with ageing, venous valves become more fragile, in association with a procoagulant and inflammatory blood phenotype. Taken together, we found that the procoagulant state in ageing, is accompanied by mild vascular changes.     

Inter-chain disulfide bond improved protein trans-splicing increases plasma coagulation activity in C57BL/6 mice following portal vein FVIII gene delivery by dual vectors

Protein trans-splicing based dual-vector factor VIII(FVIII) gene delivery is adversely affected by less efficiency of protein splicing.We sought to increase the amount of spliced FVIII protein and plasma coagulation activity in dual-vector FVIII transgene in mice by means of strengthening the interaction of inteins,protein splicing elements,thereby facilitating protein trans-splicing.Dual-vector delivery of the FVIII gene in cultured cells showed that replacement of Met226 in the heavy chain and Asp1828 in the light chain with Cys residues could facilitate inter-chain disulfide linking and improve protein trans-splicing,increasing the levels of spliced FVIII protein.In this study,C57BL/6 mice were coadministered dual vectors of intein-fused human FVIII heavy chain and light chain with Cys mutations via portal vein injection into the liver.Forty-eight hours post-injection,plasma was collected and analyzed for FVIII antigen concentration and coagulation activity.These mice showed increased circulating FVIII heavy chain polypeptide(442±151 ng mL-1 vs.305±103 ng mL-1) and coagulation activity(1.46±0.37 IU mL-1 vs.0.85±0.23 IU mL-1) compared with control mice co-administered dual vectors expressing the heavy and light chains of wild-type FVIII.Moreover,coagulation activity was similar to that of mice receiving a single vector expressing FVIII(1.79±0.59 IU mL-1).These findings indicate that improving protein trans-splicing by inter-chain disulfide bonding is a promising approach for increasing the efficacy of dual-vector based FVIII gene transfer.     

Statins Improve the Resolution of Established Murine Venous Thrombosis: Reductions in Thrombus Burden and Vein Wall Scarring

Despite anticoagulation therapy, up to one-half of patients with deep vein thrombosis (DVT) will develop the post-thrombotic syndrome (PTS). Improving the long-term outcome of DVT patients at risk for PTS will therefore require new approaches. Here we investigate the effects of statins—lipid-lowering agents with anti-thrombotic and anti-inflammatory properties—in decreasing thrombus burden and decreasing vein wall injury, mediators of PTS, in established murine stasis and non-stasis chemical-induced venous thrombosis (N = 282 mice). Treatment of mice with daily atorvastatin or rosuvastatin significantly reduced stasis venous thrombus burden by 25% without affecting lipid levels, blood coagulation parameters, or blood cell counts. Statin-driven reductions in VT burden (thrombus mass for stasis thrombi, intravital microscopy thrombus area for non-stasis thrombi) compared similarly to the therapeutic anticoagulant effects of low molecular weight heparin. Blood from statin-treated mice showed significant reductions in platelet aggregation and clot stability. Statins additionally reduced thrombus plasminogen activator inhibitor-1 (PAI-1), tissue factor, neutrophils, myeloperoxidase, neutrophil extracellular traps (NETs), and macrophages, and these effects were most notable in the earlier timepoints after DVT formation. In addition, statins reduced DVT-induced vein wall scarring by 50% durably up to day 21 in stasis VT, as shown by polarized light microscopy of picrosirius red-stained vein wall collagen. The overall results demonstrate that statins improve VT resolution via profibrinolytic, anticoagulant, antiplatelet, and anti-vein wall scarring effects. Statins may therefore offer a new pharmacotherapeutic approach to improve DVT resolution and to reduce the post-thrombotic syndrome, particularly in subjects who are ineligible for anticoagulation therapy.     

Microvasculature of the mouse eye: Scanning electron microscopy of vascular corrosion casts

Purpose

For drug safety assessment, ophthalmic fundus examination is of diagnostic importance in experimental animals. Interim blood samples are usually collected from the orbital venous sinus in the mouse. This report characterizes the angioarchitecture of the mouse eye.

Methods

In 10 mice, the microvasculature of the eyes of was investigated using scanning electron micrographs of corrosion casts.

Results

The mouse eye was characterized as having a rich vasculature with many thick retinal arteries and a well-developed orbital venous sinus. The eye receives its primary blood supply from the external ophthalmic artery, which is divided into three branches: the central retinal artery, as well as the medial and lateral long posterior ciliary arteries. The central retinal artery is divided into 8-9 radiating retinal arteries. The mouse has an orbital venous sinus around the orbit rather than a well-developed orbital venous plexus in the retrobulbar space as is in the rat. The orbital venous sinus is formed by the episcleral veins, the ophthalmic vein, the superior palpebral vein, inferior palpebral vein and numerous anastomotic veins among these veins.

Conclusions

The mouse ocular vasculature is quite similar to that of rats. It is recommended that the best location for insertion of a capillary tube for collecting blood is in the lateral canthus around the eye where the sinus is larger and is most readily accessible. Functional significance of the vascular patterns of the eye is discussed.     

Effects of preeclampsia and eclampsia on cord blood coagulation tests

Blood coagulation tests were determined in fifty-three paired umbilical cord blood and maternal venous blood samples originating from term singleton vaginal cephalic deliveries. The index group comprised seventeen deliveries complicated by preeclampsia or eclampsia, and the control group comprised thirty-six healthy women with uneventful pregnancies and deliveries. Mean values obtained from the coagulation and fibrinolytic assays did not significantly differ between study groups, except for antithrombin III levels in index group of neonates, which were significantly lower. Comparison of coagulation and fibrinolytic characteristics between mothers and their neonates produced expected level of difference due to immaturity of their haemostatic mechanisms. We found alterations in maternal blood coagulation and fibrinolysis and evidence of increased intravascular coagulation with severe preeclampsia and IUGR.     

Influence of tail versus cardiac sampling on blood glucose and lipid profiles in mice

Blood is collected during animal experimentation to measure haematological and metabolic parameters. It cannot be assumed that circulating blood has the same composition irrespective of its location, and indeed, differences in the composition of blood sampled from the arterial and venous compartments have been reported. Here we investigated whether blood collected by cardiac puncture (CP) versus that collected following removal of the distal 1 mm of the tail tip (TT) differs with respect to glucose and lipid profiles in male C57BL/6J mice at 4, 7, 20 and 28 weeks of age. Blood was first collected from the TT of unanaesthetized mice, which were then immediately anaesthetized using ketamine/xylazine, and a second blood sample was collected by CP. The CP glucose concentration was significantly higher than TT glucose by a positive bias averaging +80% (P < 0.01), irrespective of the age of the mice. Conversely, the concentrations of the CP lipids, including total cholesterol, high-density lipoprotein cholesterol and triglyceride were lower than TT lipids by a negative bias averaging -25% (P < 0.05). These observations highlight the difficulty in measuring and comparing metabolic parameters such as glucose and lipid between one blood compartment and another. They illustrate the need to standardize sampling sites, especially when repeated blood sampling is required.     

The extrinsic blood vessels of the ovary of the sheep.

The extrinsic ovarian blood vessels were studied in 134 ewes. In view of recent evidence that uterine luteolysis may involve venoarterial transfer of prostaglandin F2alpha in the ovarian pedicle, particular attention was paid to the interrelationships between veins and arteries. The ovarian artery and utero-ovarian vein are large vessels of conventional structure and lie in close apposition. Their walls are slightly thinner on their apposing sides. The ovarian branches of the ovarian artery are very tortuous, and closely intertwined with the plexiform ovarian branches of the utero-ovarian vein. An extensive plexus of small veins surrounds the ovarian artery and its ovarian branches. Within this plexus are many thin-walled, dilated regions, interspersed with narrow, thick-walled segments. Valves are inconstantly present at sites of entry of branches of the plexus into the major veins. Small numbers of arterio-venous anastomoses are present in the distal part of the ovarian pedicle. Unless blood can flow in a veno-arterial direction through arterio-venous anastomoses or capillary beds, the structural barrier between uterine venous and ovarian arterial blood is substantial.     

Effects of blood collection on wild birds: an update

Blood sampling is often critical for answering a variety of questions about wild birds. However, it is important to assess the impacts, if any, of blood collection on wild birds. Here, we examined the effects of blood sampling on adults or nestlings in three species of free-living birds. First, we examined the effects of blood collection on annual survival and reproductive success in adult buff-breasted wrens Thryothorus leucotis in Panama. In adult wrens, blood collection from the brachial vein during the breeding season had no effect on annual survival or reproductive success. Second, we examined whether blood collection influenced mass gain in developing smooth-billed anis Crotophaga ani in Puerto Rico. In developing anis, blood collection from the femoral or jugular veins had no effect on mass gain of nestlings. Third, in developing European starlings Sturnus vulgaris in British Columbia, Canada, blood collection from the brachial vein had no effect of body condition. Blood collection from the jugular vein had a transient effect on body condition during the first week post-hatch, but this effect disappeared by the second week of age. Lastly, we present an extensive up-to-date review of the literature on the effects of blood collection on free-living avian species. Taken together, these data show that blood collection has no major negative effects on developing or adult birds in the wild.     

The impact of different blood sampling methods on laboratory rats under different types of anaesthesia

Rats with implanted telemetry transponders were blood sampled by jugular puncture, periorbital puncture or tail vein puncture, or sampled by jugular puncture in carbon dioxide (CO2), isoflurane or without anaesthesia in a crossover design. Heart rate, blood pressure and body temperature were registered for three days after sampling. Initially blood pressure increased, but shortly after sampling it decreased, which led to increased heart rate. Sampling induced rapid fluctuations in body temperature, and an increase in body temperature. Generally, rats recovered from sampling within 2-3 h, except for rats sampled from the tail vein, which showed fluctuations in body temperature in excess of 30 h after sampling. Increases in heart rate and blood pressure within the first hours after sampling indicated that periorbital puncture was the method that had the largest acute impact on the rats and that it might take an extra hour to recover from it. CO2 anaesthesia seemed unable to prevent the increase in blood pressure and the fluctuations in body temperature induced by blood sampling, and up to 10 h after sampling, the rats were still affected by CO2 anaesthesia. Rats anaesthetized with isoflurane showed lower increases in blood pressure after, and fewer fluctuations in body temperature during sampling, and the post-anaesthetic effects of isoflurane, if any, seemed to disappear immediately after sampling. It is, therefore, concluded that blood sampling in rats by jugular puncture seems to be the method from which rats most rapidly recover when compared with periorbital puncture and tail vein puncture, and that for anaesthesia, isoflurane is recommended in preference to CO2.