共查询到20条相似文献,搜索用时 31 毫秒
1.
We present a new perfusion system and surgical technique for simultaneous perfusion of 2 tissue-isolated human cancer xenografts in nude rats by using donor blood that preserves a continuous flow. Adult, athymic nude rats (Hsd:RH-Foxn1(rnu)) were implanted with HeLa human cervical or HT29 colon adenocarcinomas and grown as tissue-isolated xenografts. When tumors reached an estimated weight of 5 to 6 g, rats were prepared for perfusion with donor blood and arteriovenous measurements. The surgical procedure required approximately 20 min to complete for each tumor, and tumors were perfused for a period of 150 min. Results showed that tumor venous blood flow, glucose uptake, lactic acid release, O(2) uptake and CO(2) production, uptake of total fatty acid and linoleic acid and conversion to the mitogen 13-HODE, cAMP levels, and activation of several marker kinases were all well within the normal physiologic, metabolic, and signaling parameters characteristic of individually perfused xenografts. This new perfusion system and technique reduced procedure time by more than 50%. These findings demonstrate that 2 human tumors can be perfused simultaneously in situ or ex vivo by using either rodent or human blood and suggest that the system may also be adapted for use in the dual perfusion of other organs. Advantages of this dual perfusion technique include decreased anesthesia time, decreased surgical manipulation, and increased efficiency, thereby potentially reducing the numbers of laboratory animals required for scientific investigations. 相似文献
2.
Verena Behringer Jeroen M. G. Stevens Gottfried Hohmann Erich M?stl Dieter Selzer Tobias Deschner 《PloS one》2014,9(9)
The management of captive animals has been improved by the establishment of positive reinforcement training as a tool to facilitate interactions between caretakers and animals. In great apes, positive reinforcement training has also been used to train individuals to participate in simple medical procedures to monitor physical health. One aim of positive reinforcement training is to establish a relaxed atmosphere for situations that, without training, might be very stressful. This is especially true for simple medical procedures that can require animals to engage in behaviours that are unusual or use unfamiliar medical devices that can be upsetting. Therefore, one cannot exclude the possibility that the training itself is a source of stress. In this study, we explored the effects of medical positive reinforcement training on salivary cortisol in two groups of captive ape species, orangutans and bonobos, which were familiar to this procedure. Furthermore, we successfully biologically validated the salivary cortisol assay, which had already been validated for bonobos, for orangutans. For the biological validation, we found that cortisol levels in orangutan saliva collected during baseline conditions were lower than in samples collected during three periods that were potentially stressful for the animals. However, we did not find significant changes in salivary cortisol during medical positive reinforcement training for either bonobos or orangutans. Therefore, for bonobos and orangutans with previous exposure to medical PRT, the procedure is not stressful. Thus, medical PRT provides a helpful tool for the captive management of the two species. 相似文献
3.
A technique is described for detecting fibrinolytic activity of single cells in culture. This method was applied to the analysis of rat ovarian granulosa cells. Cells obtained from follicules shortly before ovulation show high levels of fibrinolytic activity. This activity is plasminogen-dependent, indicating that it is due to plasminogen activator. The appearance of this activity is correlated with ovulation by temporal and functional criteria, and can be demonstrated both in immature animals primed with hormones and in mature cycling animals. Granulosa cell cultures can be stimulated to release plasminogen activator by exposure in vitro either to luteinizing hormone or to dibutyryl cyclic AMP. 相似文献
4.
Simon G. Lamarre Luke MacMillan Gregory P. Morrow Edward Randell Theerawat Pongnopparat Margaret E. Brosnan John T. Brosnan 《Amino acids》2014,46(8):1885-1891
Formate, a crucial component of one-carbon metabolism, is increasingly recognized as an important intermediate in production and transport of one-carbon units. Unlike tetrahydrofolate-linked intermediates, it is not restricted to the intracellular milieu so that circulating levels of formate can provide insight into cellular events. We report a novel isotope-dilution, GC–MS assay employing derivatization by 2,3,4,5,6-pentafluorobenzyl bromide for the determination of formate in biological samples. This assay is robust and sensitive; it may be applied to the measurement of formate in serum, plasma and urine. We demonstrate how this method may be applied by providing the first characterization of formate levels in a human population; formate levels were higher in males than in females. We also show how this procedure may be applied for the measurement of in vivo kinetics of endogenous formate production in experimental animals. 相似文献
5.
Maga EA 《Cloning and stem cells》2001,3(4):233-241
The endogenous properties of recombinase proteins allow them to associate with and bind DNA to catalyze homologous recombination. These endogenous properties of cellular recombination enzymes may be useful to the field of transgenesis. The production of transgenic animals, in particular livestock, is an inefficient process by both conventional pronuclear microinjection techniques and nuclear transfer. Furthermore, the use of pronuclear microinjection is currently limited to the random addition of genes and does not allow for the replacement of an endogenous gene with a more desired one. The functions of cellular recombination enzymes have been exploited to develop a technique that is compatible with pronuclear microinjection and may make the process of generating transgenic livestock more efficient while also enabling the targeting of homologous chromosomal genes. In our hands, transgenic animals generated by the pronuclear microinjection of various recombinase protein-coated DNA fragments led to a higher than expected birth rate as well as transgene integration frequency. Most founder animals generated were likely mosaic, indicating that integration occurred after cell division. The presence of multiple related genes makes detection of any recombination event difficult. Overall, this technique is a straightforward, rapid, and efficient procedure that can be applied to any segment of DNA and any microinjection apparatus, and is less labor intensive than nuclear transfer. 相似文献
6.
Maintenance of functional estrogen receptors in culture has been accomplished in chick oviduct cells by manipulating the estrogen exposure before tissue dissociation. Tissue from chicks pre-treated with daily 17-beta-estradiol injections for 2 weeks or with 2 weekly diethylstilbestrol implants can be established in culture using a variety of enzymes. Tissue from animals with chronic estrogen stimulation must be withdrawn from hormone in culture at least 4 days before the digestion procedure. When tissue is digested using collagenase and pancreatin buffered by bovine serum albumin (Fraction V), large quantities of virtually fibroblast-free cultures can be established. The estrogen and progesterone receptors remain intact at normal levels using this procedure. The receptors have maintained biological function as evidenced by two hormone-dependent measurements. The first was an increase in the amount of ovalbumin mRNA transcribed in response to estrogen supplementation of the cultures compared to cultures with no estrogen. The second function was an increase in ovalbumin protein secreted into the medium upon estrogen stimulation. The protein increment demonstrated that the hormone-induced levels of mRNA were functional and capable of being translated. 相似文献
7.
Barbara C. Hansen Heidi K. Ortmeyer Noni L. Bodkin 《Obesity (Silver Spring, Md.)》1995,3(Z2):199s-204s
Prevention of obesity and increase in longevity in obesity-prone rodents can be achieved by long-term moderate dietary restriction. In order to examine the likelihood that caloric restriction could have similar salutary effects in humans, rhesus monkeys, after reaching mature adult stature, were placed on a protocol to clamp or stabilize body weight by weekly caloric adjustment Further weight gain was prevented by this caloric titration procedure, and thus middle-age onset obesity, which is very common in this species, was prevented. The present study analyzed daily food intake for six weight-clamped monkeys and six ad libitum fed age-matched animals over a 3- year period, ages 18.5 to 21.5 years. After approximately 9 years of caloric restriction the daily calorie load to maintain stable adult body weight proved to be 40% less than the amount ingested by ad libitum fed animals. Calories per kg body weight did not differ significantly between the groups although the ad libitum fed animals were significantly fatter than the weight-clamped group. Prevention of obesity using this weight clamp protocol has also maintained lower insulin levels and higher glucose tolerance in the restricted animals. 相似文献
8.
Alfredo Manuel Franco Pereira Alexandre Alves Paulo Infante Evaldo A. L. Titto Flávio Baccari Jr. J. A. Afonso Almeida 《International journal of biometeorology》2010,54(1):37-43
The objective of this study was to test a device developed to improve the functionality, accuracy and precision of the original
technique for sweating rate measurements proposed by Schleger and Turner [Schleger AV, Turner HG (1965) Aust J Agric Res 16:92–106]. A device was built for this purpose and tested against the original Schleger and Turner technique.
Testing was performed by measuring sweating rates in an experiment involving six Mertolenga heifers subjected to four different
thermal levels in a climatic chamber. The device exhibited no functional problems and the results obtained with its use were
more consistent than with the Schleger and Turner technique. There was no difference in the reproducibility of the two techniques
(same accuracy), but measurements performed with the new device had lower repeatability, corresponding to lower variability
and, consequently, to higher precision. When utilizing this device, there is no need for physical contact between the operator
and the animal to maintain the filter paper discs in position. This has important advantages: the animals stay quieter, and
several animals can be evaluated simultaneously. This is a major advantage because it allows more measurements to be taken
in a given period of time, increasing the precision of the observations and diminishing the error associated with temporal
hiatus (e.g., the solar angle during field studies). The new device has higher functional versatility when taking measurements
in large-scale studies (many animals) under field conditions. The results obtained in this study suggest that the technique
using the device presented here could represent an advantageous alternative to the original technique described by Schleger
and Turner. 相似文献
9.
Videoendoscopic endotracheal intubation of the rat 总被引:2,自引:0,他引:2
Mechanical ventilation is essential to the proper maintenance of anaesthesia in research animals undergoing laparoscopic research investigations with prolonged pneumoperitoneum. Ventilatory assistance is greatly aided by endotracheal intubation, which in rats can be a challenging procedure with a substantial risk of complication. The difficulty of the procedure arises primarily from the limited exposure and access to the laryngeal opening. We describe a simple and safe technique for endotracheal intubation in the rat that permits the introduction of a large-bore tube under direct visualization using equipment commonly found in the endosurgical research setting. 相似文献
10.
This paper reports the use of a procedure for collection of semen from stallions by manual stimulation of the penis while the stallion is standing. Our use of this method with 18 stallions of various ages and types of semen collection experience indicates that this method may be an efficient alternative to traditional semen collection techniques using an artificial vagina and stimulus mare or dummy mount mare. Our observations, together with those of others who have tried the manual technique, suggest that both animals and handlers can be readily trained to use this method. Limited data suggests that semen samples obtained by manual stimulation are similar to those obtained using an artificial vagina. 相似文献
11.
Previous studies have established that 17 beta-estradiol is the principal luteotropic hormone in the rabbit. However, a direct effect of 17 beta-estradiol on rabbit luteal cell progesterone production has been difficult to show in vitro. The goal of this study was to develop a system in which the effect of estrogen on luteal cell progesterone production could be studied in vitro. To that end, a dissociated rabbit luteal cell preparation was developed using collagenase and the resultant isolated cells were studied using a perifusion system. Optimization of the cell digest procedure revealed that: inclusion of 2% bovine serum albumin in our optimal dissociation medium increased cell yield; and animals killed by cervical dislocation maintained more stable levels of progesterone during a 7-h perifusion compared to animals killed with barbituate-induced euthanasia (euthobarb). When dissociated luteal cells were perifused with medium, stable progesterone output (greater than 80% of initial levels) was observed for 5-6 h, after which medium progesterone concentrations declined. The inclusion of 17 beta-estradiol (10(-8) M) in the perifusion medium maintained progesterone output at control levels for up to 15 h. However, the maintenance of progesterone was not noted until after 5 h of perifusion, suggesting that the effect of estradiol may be time dependent. Thus, this investigation describes a rabbit luteal cell dissociation technique and perifusion system that may be used to examine the mechanism through which estradiol acts to maintain rabbit luteal progesterone production. 相似文献
12.
In small animals like mice, the monitoring of endocrine functions over time is constrained seriously by the adverse effects of blood sampling. Therefore, noninvasive techniques to monitor, for example, stress hormones in these animals are highly demanded in laboratory as well as in field research. The aim of our study was to evaluate the biological relevance of a recently developed technique to monitor stress hormone metabolites in fecal samples of laboratory mice. In total, six experiments were performed using six male and six female mice each. Two adrenocorticotropic hormone (ACTH) challenge tests, two dexamethasone (Dex) suppression tests and two control experiments [investigating effects of the injection procedure itself and the diurnal variation (DV) of glucocorticoids (GCs), respectively] were conducted. The experiments clearly demonstrated that pharmacological stimulation and suppression of adrenocortical activity was reflected accurately by means of corticosterone metabolite (CM) measurements in the feces of males and females. Furthermore, the technique proved sensitive enough to detect dosage-dependent effects of the ACTH/Dex treatment and facilitated to reveal profound effects of the injection procedure itself. Even the naturally occurring DV of GCs could be monitored reliably. Thus, our results confirm that measurement of fecal CM with the recently established 5alpha-pregnane-3beta,11beta,21-triol-20-one enzyme immunoassay is a very powerful tool to monitor adrenocortical activity in laboratory mice. Since mice represent the vast majority of all rodents used for research worldwide and the number of transgenic and knockout mice utilized as animal models is still increasing, this noninvasive technique can open new perspectives in biomedical and behavioral science. 相似文献
13.
Adrenal Medullary Transplants Increase Spinal Cord Cerebrospinal Fluid Catecholamine Levels and Reduce Pain Sensitivity 总被引:3,自引:0,他引:3
Previous work in this laboratory has shown that adrenal medullary transplants into the spinal cord subarachnoid space can reduce pain sensitivity. This analgesia most likely results from the release of neuroactive substances, particularly catecholamines and opioid peptides, from the transplanted cells into the CSF of the spinal cord, since it can be attenuated or blocked by alpha-adrenergic or opiate antagonists. The purpose of the present study was to more directly measure the release of catecholamines from adrenal medullary transplants in the spinal cord CSF using a spinal superfusion technique. CSF samples from rats with 6-month-old transplants were assayed for catecholamines using HPLC with electro-chemical detection. Results indicated that norepinephrine levels were increased threefold, and epinephrine levels nearly 100-fold, in animals with adrenal medullary transplants compared with control transplanted animals. There was no apparent increase in dopamine levels. Furthermore, the increased levels of total catecholamines were correlated with decreased pain sensitivity. Results of this study indicate that adrenal medullary transplants can survive for long periods in the rat spinal CSF and continue to release high levels of catecholamines. Together, the release of catecholamines and opioid peptides from adrenal medullary transplants may provide the ideal combination for the reduction of pain. 相似文献
14.
In this study, we report a highly efficient transgenesis technique for Xenopus tropicalis based on a method described first for Medaka. This simple procedure entails co-injection of meganuclease I-SceI and a transgene construct flanked by two I-SceI sites into fertilized eggs. Approximately 30% of injected embryos express transgenes in a promoter-dependent manner. About 1/3 of such embryos show incorporation of the transgene at the one-cell stage and the remainder are 'half-transgenics' suggesting incorporation at the two-cell stage. Transgenes from both classes of embryos are shown to be transmitted and expressed in offspring. The procedure also works efficiently in Xenopus laevis. Because the needle injection procedure does not significantly damage embryos, a high fraction develop normally and can, as well, be injected with a second reagent, for example an mRNA or antisense morpholino oligonucleotide, thus allowing one to perform several genetic manipulations on embryos at one time. This simple and efficient technique will be a powerful tool for high-throughput transgenesis assays in founder animals, and for facilitating genetic studies in the fast-breeding diploid frog, X. tropicalis. 相似文献
15.
Mutagenicity studies have been used to identify specific agents as potential carconogens or other human health hazards; however, they have been used minimally for risk assessment or in determining permissible levels of human exposure. The poor predictive value of in vitro mutagenesis tests for carcinogenic activity and a lack of mechanistic understanding of the roles of mutagens in the induction of specific cancers have made these tests unattractive for the purpose of risk assessment. However, the limited resources available for carcinogen testing and large number of chemicals which need to be evaluated necessitate the incorporation of more efficient methods into the evaluation process. In vivo genetic toxicity testing can be recommended for this purpose because in vivo assays incorporate the metabolic activation pathways that are relevant to humans. We propose the use of a multiple end-point in vivo comprehensive testing protocol (CTP) using rodents. Studies using sub-acute exposure to low levels of test agents by routes consistent with human exposure can be a useful adjunct to methods currently used to provide data for risk assessment. Evaluations can include metabolic and pharmacokinetic endpoints, in addition to genetic toxicity studies, in order to provide a comprehensive examination of the mechanism of toxicity of the agent. A parallelogram approach can be used to estimate effects in non-accessible human tissues by using data from accessible human tissues and analogous tissues in animals. A categorical risk assessment procedure can be used which would consider, in order of priority, genetic damage in man, genetic damage in animals that is highly relevant to disease outcome (mutation, chromosome damage), and data from animals that is of less certain relevance to disease. Action levels of environmental exposure would be determined based on the lowest observed effect levels or the highest observed no effect levels, using sub-acute low level exposure studies in rodents. As an example, the known genotoxic effects of benzene exposure at low levels in man and animals are discussed. The lowest observed genotoxic effects were observed at about 1–10 parts per million for man and 0.04–0.1 parts per million in subacute animal studies. If genetic toxicity is to achieve a prominent role in evaluating carcinogens and characterizing germ-cell mutagens, minimal testing requirements must be established to ascertain the risk associated with environmental mutagen exposure. The use of the in vivo approach described here should provide the information needed to meet this goal. In addition, it should allow truly epigenetic or non-genotoxic carcinogens to be distinguished from the genotoxic carcinogens that are not detected by in vitro methods. 相似文献
16.
The change-in-ratio technique is a useful practical procedure for the estimation of game animal population sizes. The major problem with this technique is failure of the assumption that both types of animals are captured or sighted with equal probabilities. Here we extend the change-in-ratio technique to the case where there are two removals with emphasis on the special situation where there are two consecutive single-type removals. The advantage of this extension is that it allows an estimation procedure which is robust to unequal capture or sighting probabilities. It is also possible to test the assumption of equal sighting probabilities. Some numerical results on mean squared error of the population size estimator for the new design and the traditional design are given. The procedure is illustrated on some juvenile grass carp data collected in a small pond where the population size is known. We believe this technique is potentially useful to wildlife and fisheries biologists and that more statistical research would be beneficial. 相似文献
17.
Zentall TR 《Behavioural processes》2007,74(2):286-292
Memory for time by animals appears to undergo a systematic shortening. This so-called choose-short effect can be seen in a conditional temporal discrimination when a delay is inserted between the sample and comparison stimuli. We have proposed that this temporal shortening may result from a procedural artifact in which the delay appears similar to the intertrial interval and thus, produces an inadvertent ambiguity or 'instructional failure'. When this ambiguity is avoided by distinguishing the intertrial interval from the delay, as well as the samples from the delay, the temporal shortening effect and other asymmetries often disappear. By avoiding artifacts that can lead to a misinterpretation of results, we may understand better how animals represent time. An alternative procedure for studying temporal discriminations is with the psychophysical bisection procedure in which following conditional discrimination training, intermediate durations are presented and the point of subjective equality is determined. Research using the bisection procedure has shown that pigeons represent temporal durations not only as their absolute value but also relative to durations from which they must be discriminated. Using this procedure, we have also found that time passes subjectively slower when animals are required to respond to the to-be-timed stimulus. 相似文献
18.
Summary A comparison between two immunoperoxidase staining procedures, a triple-bridge and an indirect, antibody-conjugated method, was made to determine the relative sensitivity of each technique in the detection of carcinoembryonic antigen (CEA) in routine tissue sections. The enzyme-antibody conjugates for the indirect procedure were prepared according to the method of Nakane and Kawaoi (1974). The indirect, antibody-conjugated method, proved to be slightly more sensitive than the triple-bridge by two criteria. First, CEA could be localized using higher dilutions of the primary antiserum by the indirect technique, and secondly, tissues were shown to stain for CEA in specimens with lower tissue CEA levels by the indirect procedure than by the triple-bridge method. The preparation of enzyme-antibody conjugates is a relatively simple procedure and, in addition, the conjugates will remain stable when kept frozen or at 4°C. Background staining due to non-specific interaction of the conjugate with the tissue can be eliminated easily by incubation with normal serum. These results indicate that the indirect, antibody-conjugated method can be used to enhance the staining of CEA in routine tissue sections.Supported in part by NIH contract NCI-NO1-CB-84257 相似文献
19.
Radiotelemetry has become a very popular biotelemetric tool for measuring physiological parameters such as heart rate, blood pressure, body temperature and muscle activity, as well as general behavioural activity in undisturbed, freely moving animals. In most studies using this technique, adult subjects are used. However, sometimes an ontogenetic approach is required to clarify whether changes in one parameter are preceeded or followed by changes in another parameter. Tracking physiological changes in young, developing individuals could explain given states of these animals as adults. Implanting telemetry devices can be done subcutaneously and intraperitoneally, the former method posing less of a challenge on the animal and its recovery from surgery. Because telemetry will be used in weanling gerbils during subsequent studies, we needed to investigate whether subcutaneous implantation of telemetric devices is preferable to intraperitoneal surgery with respect to animal welfare. This is a technical paper describing anaesthetic and surgical techniques in detail during a pre-trial involving subcutaneous (n=10, aged 21-29 days) and intraperitoneal (n=10, aged 19-34 days) implantation of dummy telemetry transmitters (1.9 cm3, 3.6 g after shortening of leads) in weanling gerbils, Meriones unguiculatus. Body weight was measured and analysed over four-day intervals. Optimizing anaesthetic dosages was a first step in this pilot trial. This occurred during the first few subcutaneous implantations. Three animals died while anaesthetized during the subcutaneous procedure but none post-surgery. All animals survived anaesthesia during the intraperitoneal implantation, but two died in the first three days post-surgery. In the former method, the tension on the dermal sutures caused by the presence of the transmitters was too great, resulting in the animals opening the sutures by chewing them. The animals died during the latter procedure probably due to strangulation of the intestine by the excess lead that was coiled in the abdomen. Furthermore, placement of the exposed negative lead of the transmitter on the underlying muscle had to be done on the m. pectoralis transversus in order for it to stay in place as the animal developed. This paper showed that the implantation of a telemetric device in weanling gerbils is feasible and is best executed through the intraperitoneal technique. 相似文献
20.
Suganuma R Pelczar P Spetz JF Hohn B Yanagimachi R Moisyadi S 《Biology of reproduction》2005,73(6):1157-1163
We have developed a novel method for mouse transgenesis. The procedure relies on a hyperactive Tn5 transposase to insert a transgene into mouse chromosomes during intracytoplasmic sperm injection. This procedure integrates foreign DNA into the mouse genome with dramatically increased effectiveness as compared to conventional methods such as pronuclear microinjection and traditional sperm injection-mediated transgenesis. Our data indicate that with this method, transgenic mice, both hybrids and inbreds, can be produced more consistently and with lower numbers of manipulated oocytes required for traditional microinjection methods. The transposase-mediated transgenesis technique is also effective with round spermatids, offering the potential for rescuing the fertility of azoospermic animals using sperm precursor cells. 相似文献