Intraperitoneal versus subcutaneous telemetry devices in young Mongolian gerbils (Meriones unguiculatus)

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.     

In vivo strain gage implantation in rats

A technique for the fabrication of encapsulated micro-miniature rosette strain gages for in vivo implantation is described. The gage units have an overall area of ten square millimeters (2.5 mm × 4.0 mm), and hence can be installed in very small experimental animals, particularly rodents. Using a rat model, strain data for up to 12 days have been obtained and in vitro studies have validified the in vivo strain recordings.     

Germfree status of mice obtained by embryo transfer in an isolator environment

The technique of embryo transfer has been evaluated for the purpose of changing the mouse stocks to a germfree (GF) status. Our results show reproducible and quality-assured conversion of animals to those which are negative for the presence of microorganisms. Rapid and easy access to GF mice is advantageous for studies of selected microflora and their cross-talks with the host, when applying, e.g. genomic, proteomic and metabolic methodology.The study involved embryo transfer in an isolator environment, thereby allowing implantation of cleansed embryos into GF recipients under well-controlled conditions. The recipient females gave birth normally and took care of the offspring as if they were their own pups, thus enhancing the survival rate. Access to full technical resources required to maintain GF isolators are, however, a prerequisite. In this study, we used stainless steel isolators designed by Gustafsson (1959), on which a stereomicroscope was mounted to facilitate embryo transfer inside the isolator.The use of embryo transfer and isolator techniques will facilitate the availability of various mouse mutant models under different gnotobiotic conditions, GF, monoxenic or polyxenic animals, to enable comparison with conventional animals for physiological and pathophysiological studies.     

A method for cerebroventricular cannulation of the rat

A convenient technique is described for the preparation and surgical implantation of permanent cannulae into the lateral ventricles of the rat brain, as well as a method for radiographic confirmation of the location of the implanted cannula. The modified implantation method described is simple, rapid and has produced low mortality in the surgically altered animals.     

EXAMINATION OF TWO DIMENSIONAL SPATIAL PATTERN OF PERIPHYTIC DIATOMS USING AN ADHESIVE SURFICIAL PEEL TECHNIQUE1

A simple surficial peel technique using adhesive tape was developed for quantitative removal of haptobenthic diatom communities from topographically simple substrata. The method combines high removal efficiency with low peel distortion, permitting the use of spatial statistics to test whether populations are distributed in the peel randomly or form aggregated or uniform patterns. Using this technique, the microdistribution of Cocconeis placentula Ehr. on a smooth acrylic rod was examined. Using conventional nearest neighbor analyses, a clonal population of C. placentula. characterized by an indentation of the value margin, was significantly aggregated, whereas the overall C. placentula population was uniform or aggregated depending on whether the method of analysis allowed for cell size. Using refined nearest neighbor analysis, the indented population was aggregated, and the overall population was random at distances greater than cell size. The results suggest that the indented clone was weakly motile following cell division and that its directional bearing was random.     

Refinement of vascular access port placement in nonhuman primates: complication rates and outcomes

Chronic vascular access is often needed in experimental animal studies, and vascular access ports (VAP) have been proposed as an alternative to conventional venipuncture. We previously reported on VAP implantation by using femoral venous cutdown (FVC) and tunneling. In an attempt to decrease the moderate complications associated with the FVC method, we developed the single-incision, peripheral-insertion (SIPI) method. In a retrospective evaluation, 92 FVC procedures were compared with 113 SIPI procedures in cynomolgus and rhesus macaques and baboons with as much as 2.5 y of follow-up. The rate of complications was significantly lower for the SIPI method than for the FVC method (19.4% versus 33.7%), particularly in regard to infectious complications (8.0% versus 27.3%, respectively). In addition, VAP patency for blood sampling and fluid infusion was significantly better for the SIPI method than for the FVC method, with 1-y patency rate of 83% and 46%, respectively, and 2-y patency rate of 74% and 36%, respectively. Additional advantages of the SIPI method include the simplified implantation of the VAP and access in the homecage without any sedation or restraint after appropriate training of animals to cooperate. We conclude that the SIPI method presents an opportunity for refinement and is superior to the FVC method for chronic vascular access.     

An alternative method to stereotactic inoculation of transplantable brain tumours in large numbers of rats

The rat 9L gliosarcoma brain tumour model has been widely used in brain cancer studies. Intracerebral implantation of the cells in the parietal lobe of the brain has been performed using the stereotactic or freehand inoculation methods. For large numbers of rats, we wished to develop a method more accurate and precise than the freehand method, but less labour intensive than the stereotactic method. A template implantation technique was developed and compared quantitatively with the stereotactic method. Rats were inoculated with either the template or stereotactic method at doses of 1000, 5000, 10000, 20000 or 40000 cells. Results of this comparison showed that the template method is precise and accurate for tumour placement within the brain cortex, and decreases labour requirements. Mean survival rates between groups were not significantly different at doses of 5000, 20000 or 40000 cells inoculated. Significance was seen at the low dose of 1000 cells (P < 0.001). This was attributable to an absence of tumour growth in five of six stereotactic rats in this group. Significance was also seen at the 10000 dose level (P < 0.05) with the stereotactic rats again surviving longer than the template rats. However, in this case all the stereotactic rats had tumour growth. Brain weights did not differ significantly between groups, except at the 1000 dose level where no growth of tumour occurred in five of the six stereotactic animals. Body weight gain within one week following surgery did not differ significantly between any of the groups at alpha = 0.05. Studies on rat cadavers showed no statistical difference in placement measurements between the stereotactic and template methods. These results indicate that the template method for intracerebrally implanting tumour cells in rats provides a precise, accurate and rapid procedure that maximizes reproducibility with a significant reduction in labour requirements, when compared with the conventional stereotactic methodology.     

The two gene pairs encoding H2A and H2B play different roles in the Saccharomyces cerevisiae life cycle.

We have isolated Saccharomyces cerevisiae mutants bearing deletions of one or the other of the two divergently transcribed gene pairs encoding H2A and H2B. The deletions produced diverse effects on the yeast life cycle. Deletion of TRT1, one of the H2A-H2B gene pair sets, affected mitotic growth, sporulation, spore germination, the heat shock response, and exit from the stationary phase; deletion of TRT2, the other H2A-H2B gene pair set, had negligible effects on these same processes. Using a genetic complementation assay, we found that the differential effects of the deletions could be attributed to two features of the gene sets: first, the expression of the TRT1 gene pair, but not the TRT2 gene pair, could compensate for the absence of its partner; second, the protein subtypes encoded by the two gene pairs appear to have different functions in the heat shock response.     

A technique for chronic intragastric drug administration in the rat

An improved method for chronic intragastric delivery of drug solutions in the rat is described. The cannula can be easily constructed while the implantation is a simple and a problem-free procedure. Animals suffered no ill effects and resumed normal weight gain profiles after the operation. Since this is a direct stomach cannulation technique, the problems associated with the nasopharyngeal method are avoided. The cannula was tested by administering methadone and observing the characteristic depression of operant responding for food reinforcement. Using the cumulative records, it was possible to detect the onset of drug effect which was found to be about 10 minutes. The cannula is relatively durable and has remained patent in some animals for as long as 10 months without maintenance.     

Noninvasive visualization of molecular events in the mammalian zygote

Following fertilization, a number of molecular events are triggered in the mammalian zygote. As biochemical studies using mammalian gametes and zygotes have inherent difficulties, the molecular nature of these processes is currently unclear. We have developed a method to visualize these events. In vitro transcribed mRNAs encoding for proteins fused with green fluorescent protein were microinjected into oocytes or embryos and fluorescence signals were observed. Using this technique we succeeded in obtaining images of the DNA methylation status in living mouse and rabbit embryos. Moreover, time-lapse images were acquired of spindle and nuclear formation during second meiosis and first mitosis. Importantly, the microinjected embryos developed to the normal offspring even after observation, suggesting that the technique is relatively noninvasive. Thus, our method may help elucidate the molecular aspects of fertilization and preimplantation development and, based on the real-time genetic and epigenetic status, could become a tool to select "good quality" embryos before implantation.     

Pedicled Greater Omentum Graft: A New Technique to Repair Recurrent Urinary Fistulae After Kidney Transplantation

Urinary fistula is the most frequent urologic complication within the first month after kidney transplantation, which often leads to graft loss and mortality. Open surgery is the most popular approach for the treatment of these fistulae; however, it is associated with high failure rates. Here, we present a new technique of pedicled greater omentum graft to repair recurrent urinary fistulae after kidney transplantation. We used this technique in the repair of recurrent urinary fistulae in 13 post-kidney transplant patients. All operations were successful at the first attempt, and there was no fistula recurrence. Further, no complications associated with the technique have been observed during the follow-up (1–7 years). In conclusion, the use of pedicled greater omentum graft for the repair of recurrent urinary fistulae after kidney transplantation is both effective and safe.     

Construction and evaluation of a coronary catheter for chronic implantation in dogs

Construction of a Silastic catheter and the procedure for chronic implantation in a coronary artery in dogs is described. In addition, studies designed to evaluate whether chronic coronary artery catheterization altered coronary vascular reactivity and myocardial function are presented. The results of these studies indicate that chronic implantation of the catheter in a coronary artery of conscious dogs does not significantly interfere with the normal reactivity of the coronary vascular bed, does not compromise regional or global left ventricular function, and does not induce collateral vessel development. This technique will be useful in studies involving the neural and metabolic regulation of the coronary circulation in animals subjected to exercise and/or exercise training.     

Establishment of a novel method for the production of chimeric mouse embryos using water-in-oil droplets

Production of chimeric animals is often a necessity for the generation of genetically modified animals and has gained popularity in recent years in regenerative medicine for the reconstruction of xenogeneic organs. Aggregation and injection methods are generally used to produce chimeric mice. In the aggregation method, the chimeras are produced by co-culturing embryos and stem cells, and keeping them physically adhered, although it may not be an assured method for producing chimeric embryos. In the injection method, the chimeras are produced by injecting stem cells into the zona pellucida using microcapillaries; however, this technique requires a high degree of skill. This study aimed to establish a novel method for producing chimeric embryos via water-in-oil droplets that differs from conventional methods. In this study, embryonic stem cells and embryos were successfully isolated in the droplets, and the emergence of chimeric embryos was confirmed by co-culture for 6 h. Using this method, the control and operability of stem cell numbers could be regulated, and reproducibility and quantification were improved during the production of chimeric embryos. In addition to the conventional methods for producing chimeric embryos, the novel method described here could be employed for the efficient production of chimeric animals.     

Usefulness of multiple chalk‐based food colorings for inducing better gene silencing by feeding RNA interference in planarians

Planarians have become widely recognized as one of the major animal models for regeneration studies in invertebrates. To induce RNA interference (RNAi) by feeding in planarians, the widely accepted protocol is one in which animals undergo two or three feedings of food containing double‐stranded RNA (dsRNA) plus visible food coloring (e.g., blood) for confirmation of feeding by individual animals. However, one possible problem is that incorporated food coloring is often retained within the gut for several days, which makes it difficult to confirm the success of each round of dsRNA feeding based on the difference of the color density within the gut before and after feeding. As a consequence, the difference of appetite levels among individuals undergoing dsRNA feeding leads to phenotypic variability among them due to insufficient knockdown. In our attempts to overcome this problem, we have developed a novel method for achieving robust confirmation of the success of dsRNA feeding in individuals fed multiple times by means of including a combination of three different colored chalks (pink, yellow and blue) as food coloring. Notably, we found that this method is superior to the conventional method for positively marking individuals that actively consumed the dsRNA‐containing food during four times of once‐daily feeding. Using these selected animals, we obtained stable and sufficiently strong RNAi‐induced phenotypes. We termed this improved multi‐colored chalk‐spiked method of feeding RNAi “Candi” and propose its benefits for gene function analysis in planarians.     

Amplification and analysis of cDNA generated from a single cell by 5'-RACE: application to isolation of antibody heavy and light chain variable gene sequences from single B cells

The technique of 5'-rapid amplification of cDNA ends (5'-RACE) is widely used to amplify unknown sequences at the 5' end of a messenger RNA (mRNA). However, conventional 5'-RACE is inappropriate for producing cDNAs from a single cell due to the small quantity of mRNA present in one cell. In this study, we report an improved 5'-RACE method that is suitable for generating cDNA from a single cell. In this method, the first-strand cDNA was directly synthesized from a single cell, and both the tailing reaction and second-strand cDNA synthesis were performed in the same tube without purifying the cDNA sample. Using this method, we were able to amplify the cDNA of the immunoglobulin (Ig) variable region gene from more than 50% of single B cells. The amplified cDNA fragment contained a full-length Ig variable region including a 5'-untranslated region, a leader sequence, and an initiation codon. This method may thus be applicable for a comprehensive analysis of the Ig variable genes of the lymphocyte repertoire in humans and animals, thereby contributing to the development of antibody-based therapeutics for infectious diseases.     

An alternative, less invasive blood sample collection technique for serologic studies utilizing triatomine bugs (Heteroptera; Insecta)

The collection of blood samples for serological studies is often stressful for the focus animal. Recently, the use of bloodsucking bugs, such as Dipetalogaster maximus or Triatoma infestans (Reduviidae; Triatominae; Heteroptera), has been suggested as a new and less invasive method for blood collection. To evaluate this technique, we collected paired blood samples from 20 domestic rabbits (Oryctolagus cuniculus) during a study of rabbit hemorrhagic disease virus (RHDV). For each rabbit, blood samples were collected by the conventional method (needle and syringe from the vena auricularis) and through feeding by D. maximus. Samples were tested for RHDV antibodies using standard test kits at three different dilutions. Antibody titers were identical for 56 paired samples and differed in only four cases. The simple matching indices were 1 for the 1:10 dilution and 0.9 for the 1:100 and 1:1000 dilutions. The major advantages of the new technique are 1) the possibility to obtain blood from animals where veins are inaccessible and 2) the fact that anesthesia of focus animals may not be necessary.     

Creation of low-copy integrated transgenic lines in Caenorhabditis elegans

In Caenorhabditis elegans, transgenic lines are typically created by injecting DNA into the hermaphrodite germline to form multicopy extrachromosomal DNA arrays. This technique is a reliable means of expressing transgenes in C. elegans, but its use has limitations. Because extrachromosomal arrays are semistable, only a fraction of the animals in a transgenic extrachromosomal array line are transformed. In addition, because extrachromosomal arrays can contain hundreds of copies of the transforming DNA, transgenes may be overexpressed, misexpressed, or silenced. We have developed an alternative method for C. elegans transformation, using microparticle bombardment, that produces single- and low-copy chromosomal insertions. Using this method, we find that it is possible to create integrated transgenic lines that reproducibly express GFP reporter constructs without the variations in expression level and pattern frequently exhibited by extrachromosomal array lines. In addition, we find that low-copy integrated lines can also be used to express transgenes in the C. elegans germline, where conventional extrachromosomal arrays typically fail to express due to germline silencing.     

The influence of motor unit composition and stature on fractionated patellar reflex times in untrained men

The purpose of the present study was to investigate the influence of muscle fibre composition and stature on fractionated patellar reflex times in ten healthy untrained men (mean age: 23.3 years, SD 3.1; mass: 65.9 kg, SD 8.5; height: 172.3 cm, SD 5.3). Biopsies were taken from the right vastus lateralis muscle. Using staining for myofibrillar adenosine triphosphatase after pre-incubation at pH 4.3 and 4.6, muscle fibres were classified into slow twitch (ST), fast twitch, oxidative-glycolytic (FTa) and fast twitch, glycolytic (FTb) fibres. Total patellar reflex time (TRT) and its fractionated components--reflex latency (LAT) and reflex motor time (MT)--were obtained from the mean of ten trials in each subject whilst performing Jendrassik's maneuvre. The TRT, LAT and MT were 77.7 ms, SD 16.5, 23.4 ms, SD 1.3 and 54.2 ms, SD 16.3, respectively. The LAT was significantly correlated to the percentage number of ST (r = 0.758, P less than 0.05) and FTa fibres (r = -0.657, P less than 0.05), fast twitch:slow twitch ratio (r = -0.799, P less than 0.01) and to the height of the subjects (r = 0.901, P less than 0.001), whereas TRT and MT were not significantly correlated with either fibre types or the height of the subjects. From these results it can be concluded that the LAT during the patellar reflex is influenced by muscle fibre composition and the length of the sensory and/or motor nerve.     

Fluorescent anticytokinins as a probe for binding. Isolation of cytokinin-binding proteins from the soluble fraction and identification of a cytokinin-binding site on ribosomes of tobacco callus cells

4-Substituted 2-methylthiopyrido[2,3-d]pyrimidines, a series of recently developed anticytokinins, have been found to fluoresce strongly in water and to be useful as probes for binding studies. The binding activity of the soluble proteins and particulate fraction of tobacco callus cells to the biologically most active member of the family, 4-n-butylamino-2-methylthiopyrido[2,3-d]pyrimidine (BAMPP), was studied fluorimetrically. We found that the binding activity is better monitored in terms of saturable binding rather than in terms of the amount of bound ligand, a conventional method used in isolation studies of hormone receptor proteins. Using this technique we isolated two kinds of high-affinity cytokinin-binding proteins from the soluble fraction and identified a high-affinity binding site on ribosomes.