This work was supported by the National Nature Science Foundation of China (81570399 and 81773735), the National Key Research and Development Program of China - Traditional Chinese Medicine Modernization Research project (2017YFC1702003), and Hei Long Jiang Outstanding Youth Science Fund (JC2017020).

All procedures involving animals and their care were approved by the Institutional Animal Care and Use Committee of Harbin Medical University.
NOTE: The tools and equipment required for the method are listed in the Table of Materials.
1. Preparation of materials and animals
<ol>
	<li>Order C57BL/6 male mice with weight between 20 g-25 g from a standard laboratory animal supplier.</li>
	<li>House the mice in a cycle of 12 h light: 12 h darkness at 24&#8722;26 &#176;C with ad libitum access to water and food.</li>
</ol>
2. Parabiosis
<ol>
	<li>Anesthetize the mice by intraperitoneal injection of 20 g/L 2,2,2-tribromoethanol at a concentration of 0.1 mL/10 g. Confirm proper anesthetization as indicated by muscle relaxation, slow and steady breathing, loss of skin stimulation reflex, and disappearance of corneal reflex.</li>
	<li>Apply ophthalmic ointment with a Q-tip to prevent dry eyes.</li>
	<li>Perform the parabiosis procedure as previously described5.
	<ol>
		<li>Place the mice in the supine position. Thoroughly shave the left side of one mouse and the right side of the other mouse starting at approximately 1 cm above the elbow to 1 cm below the knee with an electric shaver. Use depilatory cream to totally clear the fur on the shaved skin.</li>
		<li>Wipe the shaved areas with iodophor. Place the mice on a heated pad covered by a sterile pad.</li>
		<li>Create longitudinal skin incisions starting from 0.5 cm above the elbow to 0.5 cm below the knee joint using a pair of sharp scissors on each animal&#39;s shaved side.</li>
		<li>Detach the skin from the subcutaneous fascia gently following the incision.</li>
		<li>Connect the olecranon and knee of the parabiont with a 3-0 suture.</li>
		<li>Suture the shaved skin with a continuous 5-0 suture.</li>
	</ol>
	</li>
	<li>Inject 0.5 mL of 0.9% NaCl subcutaneously to each mouse to prevent dehydration.</li>
	<li>Inject tramadol (10 mg/25 g/day) intramuscularly to each mouse to relieve pain.</li>
</ol>
3. Validation of circulation chimerism
<ol>
	<li>Glucose fluctuation method 
	NOTE: Verify the successful construction of circulation chimerism between parabionts using the glucose fluctuation method (no fasting) on the 10th day after parabiosis surgery.
	<ol>
		<li>Anesthetize the parabionts by intraperitoneal injection of 20 g/L 2,2,2-tribromoethanol to each mouse at a concentration of 0.1 mL/10 g.</li>
		<li>Fix the donor mice in a Venous visual-mouse tail fixator.</li>
		<li>Rub the caudal veins in the flank of one of the parabiont&#39;s tail with a cotton ball soaked in 70&#8722;75% alcohol to clean the tail and dilate the blood vessels.</li>
		<li>Hold a 1.0 mL syringe containing glucose in the right hand and keep the needle parallel to the vein (less than 15&#176;).</li>
		<li>Insert the needle at a position approximately 2&#8722;4 cm from the tail tip.</li>
		<li>Inject 100 &#181;L of glucose (1.2 g/kg) into the donor within 10 s. 
		NOTE: The term donor refers to the parabiont that receives the glucose injection through the tail vein. The recipient is the other parabiont, which does not receive glucose directly.</li>
		<li>Clean the toes with iodophor. Cut off the toes of the donor and recipient mice with a scissor and collect a drop of blood at different time points after the injection of glucose (1 min, 5 min, 10 min, 15 min, 20 min, 30 min, 40 min, 50 min, and 60 min). Remove the blood clot at each collection time point to avoid more damage. 
		NOTE: The volume of collected blood was 10-25 ul for one test, and 90-225 ul in total.</li>
		<li>Drip the blood into the center of glucometer test strips for glucose level detection.</li>
	</ol>
	</li>
	<li>Use Evan&#39;s blue Counterstain as a positive control3.
	<ol>
		<li>Inject 200 &#181;L of 0.5% Evan&#39;s blue Counterstain intraperitoneally into the donor mouse.</li>
		<li>2 h later, euthanize the parabionts by intraperitoneal injection of 20 g/L 2,2,2-tribromoethanol to each mouse at a concentration of 0.2 mL/10 g, and collect blood from both parabionts by cardiac puncture.</li>
		<li>Centrifuge the blood samples at 916 x g for 15 min.</li>
		<li>Collect serum from the supernatant.</li>
		<li>Dilute the serum with 0.9% NaCl at 1:50.</li>
		<li>Measure the absorbance of the diluted serum samples at 620 nm with a spectrophotometer.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Parabiosis refers to the surgical technique of connecting two living animals to establish a common vascular system by experimental means6,7,8. The advantage of this model is that the substances produced by a single individual can act on both animals at the same time. Thus, the parabiosis model can be used to explore the role of a substance or factor in a related disease, producing many meaningful and innovative conclusions. In v...

Parabiosis is a modeling method in which two living organisms are joined together surgically and develop as a single physiological system with a shared circulatory system1. Such models have been widely used for studying physiology owing to the advantage that the substances produced by a single individual can act on both animals at the same time via the shared circulatory system. Since the mid-1800s when parabiotic experiments were pioneered by Paul Bert2, the methods for constructing parabiotic models have become standardized. However, a straightforward and convenient method for verifying the successful establishment of blood chimerism has been missing. It has been reported that cross-circulation can be successfully assessed by intraperitoneally injecting 0.5% Evans blue dye in one of the parabionts followed by measurement of the absorbance of Evans blue in the blood of both parabionts with a microplate reader3. Another method requires a specific mouse breed that contains CD45.1+- and CD45.2+-labeled monocytes in each parabiont. Cell cytometry is then used to determine blood chimerism by measuring the frequency of the two markers in monocytes from spleen or blood4. However, these methods are often lethal or cumbersome to the animals, and a safe and simple method for quick and reliable verification of parabiotic models is highly desirable. In this study, we established a new method for this purpose, which was validated in a mouse model of parabiosis. Glucose concentration in blood samples drawn from a tail vein is measured using a glucometer, and the pattern of changes of glucose level in donor and recipient mice is considered an indication of circulation chimerism. We named this method the &#34;glucose fluctuation method&#34;. The application of this validation method is not limited to mice but could be extended to diverse pathological models except for those with serious dysregulation of glucose metabolism. The procedure is simple, timesaving, and safe.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>curved forceps</td>
			<td>JZ surgical Instruments, China</td>
			<td>J31340</td>
			<td></td>
		</tr>
		<tr>
			<td>fine scissors</td>
			<td>JZ surgical Instruments, China</td>
			<td>WA1030</td>
			<td></td>
		</tr>
		<tr>
			<td>needle forcep</td>
			<td>JZ surgical Instruments, China</td>
			<td>60017961</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5 mL syringes</td>
			<td>Agilent, USA</td>
			<td>5182-9642</td>
			<td></td>
		</tr>
		<tr>
			<td>Tribromoethano</td>
			<td>Sigma-Aldrich, USA</td>
			<td>T48402-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin</td>
			<td>Solarbio, China</td>
			<td>IP0150</td>
			<td></td>
		</tr>
		<tr>
			<td>tramadol</td>
			<td>Yijishiye, China</td>
			<td>YJT712520</td>
			<td></td>
		</tr>
		<tr>
			<td>glucometer</td>
			<td>Roche Diabetes Care, Indiana</td>
			<td>Accu-Chek Active test strips</td>
			<td></td>
		</tr>
		<tr>
			<td>Evan&#39;s blue Counterstain</td>
			<td>Solarbio, China</td>
			<td>G1810</td>
			<td></td>
		</tr>
		<tr>
			<td>depilatory cream</td>
			<td>Nair, USA</td>
			<td>LL9161</td>
			<td></td>
		</tr>
		<tr>
			<td>Warming Blanket (Heating pad)</td>
			<td>Kent Scientific Corp, USA</td>
			<td>TP-22G</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrical shaver</td>
			<td>Codos, China</td>
			<td>CP-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>3-0,5-0 
			surgical suture</td>
			<td>Shanghai Medical Suture Needle Factory, China</td>
			<td>SYZ 3-0#, SYZ 5-0#</td>
			<td></td>
		</tr>
	</tbody>
</table>

detecting establishment of shared blood supply in parabiotic mice by caudal vein glucose injection

Parabiosis is an experimental method for surgically combining two parallel animals along the longitudinal axis of the body. We present a protocol for detecting the successful establishment of blood chimerism in parabionts by a caudal vein injection of glucose. Parabiotic mice were constructed. Glucose was injected into the donor mouse through the tail vein, and the fluctuation of blood glucose level was measured in both mice using a blood glucometer at different time points. Our results showed that after glucose injection, the blood glucose level in donor mice increased sharply after 1 min and decreased slowly thereafter. Meanwhile, the blood glucose level of the recipient mice peaked 15 min after injection. Similar results were obtained with Evans blue, used as a positive control for glucose. The synchronous fluctuation of blood glucose levels indicates that blood flow between the two mice was established successfully.

In six donor mice, blood glucose levels sharply increased to 26.5 &#181;mol/L (173% increase) at an average of 1 min after the injection of 100 &#181;L of glucose (1.2 g/kg) through the tail vein and then gradually decreased to 13.3 &#181;mol/L at 60 min. In recipient mice, blood glucose slowly increased after injection and reached the first peak level at 15 min (47% increase, 12.2 &#181;mol/L). Based on the above results, the standard for circulation chimerism was set as follows: 1) a sh...

Watch this Scientific Journal Video about Detecting Establishment of Shared Blood Supply in Parabiotic Mice by Caudal Vein Glucose Injection at JoVE.com

Detecting Establishment of Shared Blood Supply in Parabiotic Mice by Caudal Vein Glucose Injection

Here we describe a new method of detecting successful establishment of shared blood circulation of two parabionts through a caudal vein injection of glucose, which causes minimal damage and is not fatal to the parabionts.

Parabiosis is an experimental method for surgically combining two parallel animals along the longitudinal axis of the body. We present a protocol for ...

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8.2K Views.  Harbin Medical University. Here we describe a new method of detecting successful establishment of shared blood circulation of two parabionts through a caudal vein injection of glucose, which causes minimal damage and is not fatal to the parabionts.

Video: Detecting Establishment of Shared Blood Supply in Parabiotic Mice by Caudal Vein Glucose Injection

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.

Blood draws are necessary in a large number of studies, for example to study the pharmacokinetics profile of a compound. Here, we demonstrate how to draw blood from rats using two techniques: blood draw from the saphenous vein or 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: ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Identification of a Murine Erythroblast Subpopulation  Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not yet been fully elucidated. A common problem encountered when studying the localization of key proteins and structures within enucleating erythroblasts by microscopy is the difficulty to observe a sufficient number of cells undergoing enucleation.&#160;We have developed a novel analysis protocol using multiparameter high-speed cell imaging in flow (Multi-Spectral Imaging Flow Cytometry), a method that combines immunofluorescent microscopy with flow cytometry, in order to identify efficiently a significant number of enucleating events, that allows to obtain measurements and perform statistical analysis.
We first describe here two in vitro erythropoiesis culture methods used in order to synchronize murine erythroblasts and increase the probability of capturing enucleation at the time of evaluation.&#160;Then, we describe in detail the staining of erythroblasts after fixation and permeabilization in order to study the localization of intracellular proteins or lipid rafts during enucleation by multi-spectral imaging flow cytometry.&#160;Along with size and DNA/Ter119 staining which are used to identify the orthochromatic erythroblasts, we utilize the parameters &#8220;aspect ratio&#8221; of a cell in the bright-field channel that aids in the recognition of elongated cells and &#8220;delta centroid XY Ter119/Draq5&#8221; that allows the identification of cellular events in which the center of Ter119 staining (nascent reticulocyte) is far apart from the center of Draq5 staining (nucleus undergoing extrusion), thus indicating a cell about to enucleate.&#160;The subset of the orthochromatic erythroblast population with high delta centroid and low aspect ratio is highly enriched in enucleating cells.

The present protocol describes a novel method of identifying a population of enucleating orthochromatic erythroblasts by multi-spectral imaging flow cytometry, providing a visualization of the erythroblast enucleation process.

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. In vivo cardiac-specific gene delivery is commonly achieved by either systemic or local delivery. Systemic injection via tail vein is easy and efficient in manipulating cardiac gene expression by using recombinant adeno-associated virus 9 (AAV9). However, this method requires a relatively high amount of vector for efficient transduction, and may result in nontarget organ gene transduction. Here, we describe a simple, efficient, and time-saving method of intramyocardial injection for in vivo cardiac-specific gene manipulation in mice. Under anesthesia (without ventilation), the pectoral major and minor muscles were bluntly dissected, and the mouse heart was quickly exposed by manual externalization through a small incision at the fourth intercostal space. Subsequently, adenovirus encoding luciferase (Luc) and vitamin D receptor (VDR), or short hairpin RNA (shRNA) targeting VDR, was injected with a Hamilton syringe into the myocardium. Subsequent in vivo imaging demonstrated that luciferase was successfully overexpressed specifically in the heart. Moreover, Western blot analysis confirmed the successful overexpression or silencing of VDR in the mouse heart. Once mastered, this technique can be used for gene manipulation, as well as injection of cells or other materials such as nanogels in the mouse heart.

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Here we present a protocol for cardiac-specific gene manipulation in mice. Under anesthesia, the mouse hearts were externalized through the fourth intercostal space. Subsequently, adenoviruses encoding specific genes were injected with a syringe into the myocardium, followed by protein expression measurement via in vivo imaging and Western blot analysis.

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. ...

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from low-sensitivity, indirect read-outs, limited throughput, and/or population-level rather than single-cell resolution. We designed a flow cytometry-based single methodology that addresses all of these limitations: Distributed Amphifluoric FRET or DAmFRET. DAmFRET detects and quantifies protein self-assemblies by sensitized emission FRET in vivo, enables deployment across model systems-from yeast to human cells-and achieves sensitive, single-cell, high-throughput read-outs irrespective of protein localization or solubility.

This article describes a FRET-based flow cytometry protocol to quantify protein self-assembly in both S. cerevisiae and HEK293T cells.

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from ...

Detecting Migration and Infiltration of Neutrophils in Mice

Neutrophils are a major member of the innate immune system and play pivotal roles in host defense against pathogens and pathologic inflammatory reactions. Neutrophils can be recruited to inflammation sites via the guidance of cytokines and chemokines. Overwhelming infiltration of neutrophils can lead to indiscriminate tissue damage, such as in rheumatoid arthritis (RA). Neutrophils isolated from peritoneal exudate respond to a defined chemoattractant, N-formyl-Met-Leu-Phe (fMLP), in vitro in Transwell or Zigmond chamber assays. The air pouch experiment can be used to evaluate the chemotaxis of neutrophils towards lipopolysaccharide (LPS) in vivo. The adjuvant-induced arthritis (AA) mouse model is frequently used in RA research, and immunohistochemical staining of joint sections with anti-myeloperoxidase (MPO) or anti-neutrophil elastase (NE) antibodies is a well-established method to measure neutrophil infiltration. These methods can be used to discover promising therapies targeting neutrophil migration.

Here, we present three methods to assess neutrophil migration and infiltration both in vivo and in vitro. These methods can be used to discover promising therapeutics targeting neutrophil migration.

Neutrophils are a major member of the innate immune system and play pivotal roles in host defense against pathogens and pathologic inflammatory reactions. ...

Measurement of Insulin- and Contraction-Stimulated Glucose Uptake in Isolated and Incubated Mature Skeletal Muscle from Mice

Skeletal muscle is an insulin-responsive tissue and typically takes up most of the glucose that enters the blood after a meal. Moreover, it has been reported that skeletal muscle may increase the extraction of glucose from the blood by up to 50-fold during exercise compared to resting conditions. The increase in muscle glucose uptake during exercise and insulin stimulation is dependent on the translocation of glucose transporter 4 (GLUT4) from intracellular compartments to the muscle cell surface membrane, as well as phosphorylation of glucose to glucose-6-phosphate by hexokinase II. Isolation and incubation of mouse muscles such as m. soleus and m. extensor digitorum longus (EDL) is an appropriate ex vivo model to study the effects of insulin and electrically-induced contraction (a model for exercise) on glucose uptake in mature skeletal muscle. Thus, the ex vivo model permits evaluation of muscle insulin sensitivity and makes it possible to match muscle force production during contraction ensuring uniform recruitment of muscle fibers during measurements of muscle glucose uptake. Moreover, the described model is suitable for pharmacological compound testing that may have an impact on muscle insulin sensitivity or may be of help when trying to delineate the regulatory complexity of skeletal muscle glucose uptake.
Here we describe and provide a detailed protocol on how to measure insulin- and contraction-stimulated glucose uptake in isolated and incubated soleus and EDL muscle preparations from mice using radiolabeled [3H]2-deoxy-D-glucose and [14C]mannitol as an extracellular marker. This allows accurate assessment of glucose uptake in mature skeletal muscle in the absence of confounding factors that may interfere in the intact animal model. In addition, we provide information on metabolic viability of incubated mouse skeletal muscle suggesting that the method&#160;applied possesses some caveats under certain conditions when studying muscle energy metabolism.

Intact regulation of muscle glucose uptake is important for maintaining whole body glucose homeostasis. This protocol presents assessment of insulin- and contraction-stimulated glucose uptake in isolated and incubated mature skeletal muscle when delineating the impact of various physiological interventions on whole body glucose metabolism.

Skeletal muscle is an insulin-responsive tissue and typically takes up most of the glucose that enters the blood after a meal. Moreover, it has been ...

Tail Vein Transection Bleeding Model in Fully Anesthetized Hemophilia A Mice

Tail bleeding models are important tools in hemophilia research, specifically for the assessment of procoagulant effects. The tail vein transection (TVT) survival model has been preferred in many settings due to sensitivity to clinically relevant doses of FVIII, whereas other established models, such as the tail clip model, require higher levels of procoagulant compounds. To avoid using survival as an endpoint, we developed a TVT model establishing blood loss and bleeding time as endpoints and full anesthesia during the entire experiment. Briefly, anesthetized mice are positioned with the tail submerged in temperate saline (37&#176;C) and dosed with the test compound in the right lateral tail vein. After 5 min, the left lateral tail vein is transected using a template guide, the tail is returned to the saline, and all bleeding episodes are monitored and recorded for 40 min while collecting the blood. If no bleeding occurs at 10 min, 20 min, or 30 min post-injury, the clot is challenged gently by wiping the cut twice with a wet gauze swab. After 40 min, blood loss is quantified by the amount of hemoglobin bled into the saline. This fast and relatively simple procedure results in consistent and reproducible bleeds. Compared to the TVT survival model, it uses a more humane procedure without compromising sensitivity to pharmacological intervention. Furthermore, it is possible to use both genders, reducing the total number of animals that need to be bred, in adherence with the principles of 3R&#39;s. A potential limitation in bleeding models is the stochastic nature of hemostasis, which can reduce the reproducibility of the model. To counter this, manual clot disruption ensures that the clot is challenged during monitoring, preventing primary (platelet) hemostasis from stopping bleeding. This addition to the catalog of bleeding injury models provides an option to characterize procoagulant effects in a standardized and humane manner.

The refined tail vein transection (TVT) bleeding model in anesthetized mice is a sensitive in vivo method for the assessment of hemophilic bleeding. This optimized TVT bleeding model uses blood loss and bleeding time as endpoints, refining other models and avoiding death as an endpoint.

Tail bleeding models are important tools in hemophilia research, specifically for the assessment of procoagulant effects. The tail vein transection (TVT) ...