Short communication: Gut microbial colonization of the mouse colon using faecal transfer was equally effective when comparing rectal inoculation and oral inoculation based on 16S rRNA sequencing.

In the present study we hypothesized that a higher degree of gut microbiota (GM) transfer and colonization could be reached by rectal inoculation compared to oral inoculation, which is commonly used in mouse studies for GM transfer. We treated C57BL/6NTac Specific Pathogen Free (SPF) mice with antibiotics and subsequently we inoculated these with GM from donor mice of the same strain by either the oral or the rectal inoculation method. 16S rRNA gene sequencing of the colon microbiota showed no difference in microbial community on account of inoculation method as determined by unweighted UniFrac distance metrics in C57BL/6NTac SPF mice. In addition, qPCR analysis on colon tissue revealed no difference in mRNA expression between the inoculation methods. Next, the SPF mice were compared to germ-free (GF)-mice to identify differences in inoculation efficacy. Whether the mice were antibiotic treated SPF or GF clearly influenced GM determined by 16S rRNA gene sequencing where the SPF mice experienced up-regulation of S24-7 (p = .0001) and a decrease in Rikenellaceae (p = .016) compared to GF mice. qPCR analysis on colon tissue revealed up-regulation in mRNA gene expression of Il6, Il10, Reg3g and transcription factor RORγt (Rorc) in GF mice compared to SPF mice on a significant level (p < .05). This gene expression profile is consistent with post colonization development of the intestinal barrier in GF mice.

Pyroptosis is involved in cryopreservation and auto-transplantation of mouse ovarian tissues and pyroptosis inhibition improves ovarian graft function.

The aim of this study was to investigate the role of pyroptosis in the cryopreservation and transplantation of mouse ovarian tissues; the effects of pyroptosis inhibitior on the ovarian graft function were also explored. ICR (institute of cancer research) mice were randomly divided into control group and experimental groups (n = 10 per group). The experimental groups included fresh graft group (autograft), cryopreserved graft group (cryopreservation + autograft), and pyroptosis inhibition group (cryopreservation + autograft + pyroptosis inhibitor). At the third day after auto-transplantation, caspase-1 and NLRP3 protein expression in grafts were assessed by Western blot; in the meantime, serum concentration of IL-1ß was examined by ELISA. After 28 days of auto-transplantation, estradiol concentrations and follicular densities of grafts were evaluated. The caspase-1 and NLRP3 protein expression in grafts from all the experimental groups were significantly higher than that from control group respectively; moreover, there was a significant increase in serum concentrations of IL-1ß in all experimental groups compared with control group. The concentration of estradiol and follicular densities of grafts in pyroptosis inhibition group were significantly higher than that in cryopreserved graft group. Pyroptosis is involved in cryopreservation and auto-transplantation of mouse ovarian tissues, and pyroptosis inhibition can improve the ovarian graft function.

Optimal tube length of orotracheal intubation for mice.

Endotracheal tube (ETT) management is an essential technique in handling mice with mechanical ventilators. Malposition into bronchi causes not only lethal complications for them but also less efficient mechanical ventilation. However, in general, it is difficult to know whether the ETT is placed with appropriate depth into the trachea of mice. We measured the distance from incisors to the bifurcation of trachea of multiple mice, and created a new estimation formula to obtain the suitable ETT length for mice with a body weight range from 17 g to 25 g: length (mm) = 0.5 × bodyweight (g) + 7. However, millimeter step adjustments are impracticable. Thus, slightly shorter than 2 cm (18-20 mm) may be the universal ETT length for mice with bodyweight > 17 g. Furthermore, their foot size may be a good alternative to predict the individual optimal ETT length for mice.

Studying Osteoarthritis Pathogenesis in Mice.

With the increasing availability and complexity of mouse models of disease, either spontaneous or induced, there is a concomitant increase in their use in the analysis of pathogenesis. Among such diseases is osteoarthritis, a debilitating disease with few treatment options. While advances in our understanding of the pathogenesis of osteoarthritis has advanced through clinical investigations and genome-wide association studies, there is still a large gap in our knowledge, hindering advances in therapy. Patient samples are available ex vivo, but these are generally in the very late stages of disease. However, with mice, we are able to induce disease at a defined time and track the progression in vivo and ex vivo, from inception to end stage, to delineate the processes involved in disease development. © 2018 by John Wiley & Sons, Inc.

Effects of nafamostat mesilate, a protease Inhibitor, on ischemia/reperfusion-induced kidney injury in mice

Ischemia is induced when blood flow to an organ is interrupted, and re-establishing blood flow is essential to prevent ongoing hypoxic injury, although it paradoxically imparts further injury. Nafamostat mesilate (NM), a synthetic serine protease inhibitor, has been used in patients undergoing hemodialysis who are at a high risk of bleeding. To determine the protective effect of NM on ischemia-reperfusion injury (IRI) in a mouse renal IRI model, NM was administered as a pre- and post-treatment or during ischemia reperfusion and compared to a control group. Mice were bilaterally nephrectomized and subjected to 40 min of renal pedicle occlusion followed by 24 h reperfusion. NM (240 μg/kg) significantly improved kidney function and lowered serum creatinine and blood urea nitrogen levels. Consistently, NM inhibited collagen formation in kidney tissues. NM treatment attenuated the effects of ischemia/reperfusion on kidney tissues and significantly inhibited activation of Toll-like receptor 4, nuclear factor kappa-light-chain-enhancer of activated B cells-phospho-65 (NF- kB-p65), phospho-inhibitor of NF-κβa, and inducible nitric oxide synthase (iNOS). NM treatment also decreased expression of Bcl-2, Caspase-3 and Bax in kidney tissues, which has been linked with induction of apoptosis in kidney tissues. Our studies suggest that NM may be a novel therapeutic agent to prevent and treat kidney IRI, in which iNOS and/or NF-κβ are upregulated. The exact regulatory mechanism and its functional significance require further elucidation

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Survival advantage depends on cecal volume rather than cecal length in a mouse model of cecal ligation and puncture.

BACKGROUND: Cecal ligation and puncture (CLP) is the most commonly used model to simulate human polymicrobial sepsis. However, the severity of CLP is difficult to be standardized across different laboratories. The aim of the present study was to evaluate the influence of ligated cecal volume and length on mortality in mouse CLP model. METHODS: Cecal length and volume were measured from 120 Kunming mice subjected to CLP or sham operation. According to cecal volume, mice were divided into three groups, volume0.0∼0.2 (0.0 cm(3)-0.2 cm(3)), volume0.2∼0.4 (0.2 cm(3)-0.4 cm(3)), and volume>0.4 (larger than 0.4 cm(3)). The contents of cytokines, including interleukin-1ß, interleukin-6, and TNF-α, were measured at 3 h after surgery. The blood bacterial load and oxidative stress indicators (including malondialdehyde and superoxide dismutase) were measured at 12 h after surgery. RESULTS: There was no significant difference on 72-h survival rate between the mice with cecum longer than 2 cm and shorter than 2 cm. Compared to the other volume groups, volume>0.4 group showed significantly increased blood bacterial load, malondialdehyde levels in lung and liver, and pro-inflammatory cytokines in serum. Surprisingly, the survival rate in volume>0.4 (0%) group showed significant difference from those of volume0.0∼0.2 group (40%) and volume0.2∼0.4 group (40%). CONCLUSIONS: The mice in volume>0.4 group have much serious inflammatory reaction and are easier to die. As the proportion of volume>0.4 mice is near 20%, it can have large influence on most of the related studies using this CLP model.

Technical Aspects and Benefits of Experimental Mouse Lung Transplantation. / Aspectos técnicos y utilidades del trasplante pulmonar experimental murino.

In recent years, the number of lung transplantations performed as the last option for many respiratory diseases has grown considerably, both in adults and children. However, the causes for the relatively short survival of lungs compared to other organ transplants still need to be studied. Techniques have improved since the 1950s when experimental lung transplantation began, and the different animal species used now include rodents. The advantage of using these small species is that the surgical model has been expanded and standardized, and different respiratory problems can be studied. In this review we examine the different technical strategies used in experimental transplantation in rats and mice, focusing on surgical techniques and anesthesia and monitoring methods, and highlighting the major contributions of mouse lung transplantation to the field.

Localization, identification, and excision of murine adipose depots.

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 reached a staggering sum of $190.2 billion in direct medical costs alone. Obesity is a major risk factor for a wide host of diseases. Historically, little was known regarding adipose and its major and essential functions in the body. Brown and white adipose are the two main types of adipose but current literature has identified a new type of fat called brite or beige adipose. Research has shown that adipose depots have specific metabolic profiles and certain depots allow for a propensity for obesity and other related disorders. The goal of this protocol is to provide researchers the capacity to identify and excise adipose depots that will allow for the analysis of different factorial effects on adipose; as well as the beneficial or detrimental role adipose plays in disease and overall health. Isolation and excision of adipose depots allows investigators to look at gross morphological changes as well as histological changes. The adipose isolated can also be used for molecular studies to evaluate transcriptional and translational change or for in vitro experimentation to discover targets of interest and mechanisms of action. This technique is superior to other published techniques due to the design allowing for isolation of multiple depots with simplicity and minimal contamination.

Effects of using the analgesic tramadol in mice undergoing embryo transfer surgery.

Embryo transfer is a surgical technique that is widely used in reproductive biotechnology. Despite the ethical obligation to relieve animals' post-operative pain, analgesia is not routinely provided after embryo transfer surgery because it has been suggested that analgesics may be detrimental to embryo survival. Studies suggest, however, that the potential for adverse effects varies depending on the type of analgesic used and the timing of its administration. The authors carried out a study to determine whether pre-operatively administered tramadol, a synthetic analogue of codeine, influenced birth rate, litter survival or the post-operative body weights of surrogate dams. Compared with controls that were not given any analgesic, surrogate dams given tramadol had similar birth rates and similar body weights at all time points. The tramadol-treated surrogate dams showed a statistically significant increase in the number of offspring that survived to weaning. The authors conclude that pre-operatively administered tramadol does not harm the success rate of embryo transfer surgery and even may improve litter survival.

Parabiosis in mice: a detailed protocol.

Parabiosis is a surgical union of two organisms allowing sharing of the blood circulation. Attaching the skin of two animals promotes formation of microvasculature at the site of inflammation. Parabiotic partners share their circulating antigens and thus are free of adverse immune reaction. First described by Paul Bert in 1864(1), the parabiosis surgery was refined by Bunster and Meyer in 1933 to improve animal survival(2). In the current protocol, two mice are surgically joined following a modification of the Bunster and Meyer technique. Animals are connected through the elbow and knee joints followed by attachment of the skin allowing firm support that prevents strain on the sutured skin. Herein, we describe in detail the parabiotic joining of a ubiquitous GFP expressing mouse to a wild type (WT) mouse. Two weeks after the procedure, the pair is separated and GFP positive cells can be detected by flow cytometric analysis in the blood circulation of the WT mouse. The blood chimerism allows one to examine the contribution of the circulating cells from one animal in the other.

In vivo serial sampling of epididymal sperm in mice.

This study was undertaken to refine the techniques of in vivo collection of sperm in the mouse. The principal objective was to offer a viable, safe and reliable method for serial collection of in vivo epididimary sperm through the direct puncture of the epididymis. Six C57Bl/6J males were subjected to the whole experiment. First we obtain a sperm sample of the right epididymis, and perform a vasectomy on the left side. This sample was used in an in vitro fertilization (IVF) experiment while the males were individually housed for 10 days to let them recover from the surgery, and then their fertility was tested with natural matings until we obtained a litter of each one. After that, the animals were subjected another time to the same process (sampling, recover and natural mating). The results of these experiments were a fertilization average value of 56.7%, and that all the males had a litter in the first month after the natural matings. This study documented the feasibility of the epididimary puncture technique to in vivo serial sampling of sperm in the mouse.

[Mice cope with parabiosis − assessment of their physiological changes of life].

The purpose of this study was to establish a parabiotic mice model and assess the physiological changes of the mice under the parabiotic state. Thirteen pairs of isogenic partners were studied. The model was created by preparing a bridge of skin and subcutaneous tissues between the two mice starting distal of the elbow joint along the humerus along the lateral costal region until the end of the waist line. Physiological, social and affective qualities of life were studied in the mice through behavioural observations for 120 days following the parabiotic surgery. During the first 2-3 days following the operation, the animals suffered from severe pain and distress. During the following days and weeks, the physiological system began to recover and the animals displayed behavioral adaptations to the parabiotic condition. All animals survived at day 120. At three days post operation, the body weight began to decrease. Following this, the animals experienced a continual body weight recovery and reached pre-surgical measures at about 30 days post op. Forty-eight h post op., faecal corticosterone-metabolites were extremely elevated, but their levels returned to two to four times of levels in control females within 72 hours post op. The faecal corticosterone-metabolite levels decreased near to control values on day 75. Out of the 13 pairs, the blood exchange rate of three parabiotic partners was tested, with the result being normal post op. After 12 weeks, the total blood exchange between both partners needed 63 or 46 or 107 min, respectively. These results demonstrated that the animals could adapt behaviourally to the parabiotic situation. Therefore, this parabiosis mouse model may provide useful insights in many research areas, such as transplantation immunity, hematological system and metabolism, etc.