Esther Bloem and Thomas Nygaard are acknowledged for support with measurements of FVIII in plasma. Bo Alsted is acknowledged for drawing and machining the template and cutting blocks.

All procedures described in this protocol have been approved by the Animal Welfare Body at Novo Nordisk A/S, and the Danish Animal Experiments Inspectorate, The Danish Ministry of Food, Agriculture, and Fisheries. The optimized 40 minutes method includes anesthesia and dosing time in the design (Figure 1). Hemophilic mice of both genders between 10-16 weeks of age are required for this procedure.
1. Preparations before the study
<ol>
	<li>Prepare the dosing solutions in the correct concentrations.</li>
	<li>Start the water bath and heat to 37 &#176;C. Fill the 15 mL centrifuge tubes for blood collection with saline (0.9% NaCl).</li>
	<li>Place the 15 mL saline tubes in the holes in the warmed base plate at least 15 min prior to the start of the experiment.</li>
	<li>Identify the mice and record their weight. Avoid handling mice more than necessary as this can cause stress and affect the study.</li>
	<li>Prepare the workstation in the fume hood before proceeding so that everything is within reach: napkins, tail holder, gauze, syringes, scalpels, stopwatches, and blood flow notation paper.</li>
	<li>Place the tail mark and cutting blocks on the heating plate - cold blocks will make the veins contract and thereby affect the bleeding.</li>
</ol>
2. Anesthesia
<ol>
	<li>Conduct the isoflurane anesthetic procedure inside a fume hood.</li>
	<li>Set the gas vaporizer to initially 5% isoflurane in 30% O2/70% N2O in the anesthesia chamber with 1 L/min flow. Allow sufficient time for the anesthesia chamber to fill (about 5 min depending on chamber volume and gas flow rate).&#160;Ensure rapid induction (less than one minute).</li>
	<li>Place the mice in the anesthesia chamber until they lose consciousness. 
	NOTE: This should occur within a minute or less if the chamber is sufficiently filled.</li>
	<li>Ensure proper anesthetization by the absence of painful response to pedal reflex (firm toe pinch).</li>
	<li>Place the mice on the heating plate, making sure that the nose is in the nose cone.</li>
	<li>Reduce the anesthesia to a maintenance level of 2% isoflurane in 30% O2/70% N2O and place a plastic cover above the mice to reduce the loss of heat. Apply a suitable eye ointment to prevent dryness while under anesthesia.</li>
	<li>Mark the tail at a diameter of 2.5 mm using the tail mark block. Do not force the tail into the slit in the block - it must fit snugly (Supplemental Figure 1)</li>
	<li>Place the tail in the saline tube for at least 5 min to ensure a warm tail vein that is optimal for intravenous (i.v.) dosing.</li>
</ol>
3. Dosing of test solution
<ol>
	<li>Place one mouse in the tail holder with the nose in an anesthesia mask.</li>
	<li>Dose the animal with the compound of interest (in this case, rFVIII) and immediately start the stopwatch (t = 0).</li>
	<li>Place the mouse back on the heating plate with the tail in the saline tube. Repeat the procedure with the other mice.</li>
</ol>
4. Performing tail vein transection
<ol>
	<li>Perform the tail vein transection exactly 5 min after dosing. Place the tail in the cutting block and turn 90&#176; to expose the vein (Supplemental Figure 2).</li>
	<li>Perform the cut on the opposite side/vein from where the test solution was&#160;dosed.</li>
	<li>Draw the #11 scalpel blade through the slit of the cutting block holding the tail to create bleeding. Reset the stopwatch and return the tail immediately to the saline.</li>
</ol>
5. Observation time and challenges
<ol>
	<li>Observe the bleeding and annotate the start and stop of the bleeding throughout 40 min; annotate it on the blood flow notation paper. 
	NOTE: This visual assessment of bleeding may vary slightly due to subjectivity.</li>
	<li>The primary bleeding must stop within 3 min after the cut is made. If this is not the case, disqualify the mouse, euthanize, and replace (failure to stop the primary bleed can indicate a too severe injury or lacking primary hemostasis, as in vWF KO mice).</li>
	<li>If there is no bleeding at 10 min, 20 min, and 30 min post-injury, challenge the tail cut as described in steps 4-5.</li>
	<li>Use a gauze swab soaked in warm saline from a separate tube kept in the water bath. Lift the tail out of the saline and softly wipe twice with the wet gauze in a distal direction over the tail cut.</li>
	<li>After each challenge, immediately re-submerge the tail into the saline tube again.</li>
	<li>At t=40 min, stop the blood collection by removing the tail from the saline tube.</li>
</ol>
6. Blood sampling
<ol>
	<li>After t = 40 min, obtain blood samples from the supraorbital vein.</li>
</ol>
7. Euthanasia
<ol>
	<li>Euthanize the mice by cervical dislocation while still under full anesthesia.</li>
</ol>
8. Treatment of samples
<ol>
	<li>Centrifuge the 15 mL blood collection tubes with saline at 4000 x g for 5 min at room temperature.</li>
	<li>Discard the supernatant from the 15 mL tubes, resuspend the pellet in 2-14 mL of erythrocytes (RBC) lysing solution, and then dilute it until it reaches a light coffee color.</li>
	<li>Note the total volume (volume of blood + volume of erythrocytes (RBC) lysing solution added using the graduation marks on the tube).</li>
	<li>Transfer 2 mL of the dilution to a hemoglobin tube and refrigerate it until the hemoglobin analysis.</li>
	<li>Determine the blood loss by measuring the hemoglobin concentration in the saline. Measure the absorbance at 550 nm on a microplate reader (Table of Materials).</li>
	<li>Convert the absorbance to nmol hemoglobin using a standard curve prepared from human hemoglobin (Table of Materials) and correct for the dilution with RBC lysing solution.</li>
</ol>
9. Statistical analyses
<ol>
	<li>Analyze the data using appropriate software. Here GraphPad Prism software was used. Over a range of studies, the following statistical methods were found to perform well. 
	NOTE: To analyze blood loss, bleeding time, exposure, platelet counts, and hematocrit; Brown-Forsythe and Welch ANOVA test was used, (as the data were continuous but without variance homogeneity of the residuals) applying Dunnett&#39;s test to adjust for multiple comparisons. A Pearson&#39;s test was used to test for correlations between bleeding time, blood loss, and doses. To determine ED50 values, a four-parameter inverse log (dose) response equation was fitted to bleeding- and blood loss data. To analyze the gender effect, a two-way ANOVA test was used, applying Bonferroni correction to adjust for multiple comparisons. The significance level was defined as P &#60; 0.05. Data are displayed as means &#177; SEM.</li>
</ol>

The authors are or were employees and/or shareholders of Novo Nordisk A/S at the time this research was carried out.

This optimized method of tail vein transection (TVT) has several advantages compared to the TVT survival method. The animals are fully anesthetized for the entire study duration, which makes mouse handling easier and increases animal wellbeing. Further, unlike the TVT survival model, overnight observation is not required, and this optimized model offers the possibility of measuring blood loss and observing the exact bleeding time over 40 min. Also, longer periods of bleeding in conscious animals can cause death by exsang...

Animal models are essential for understanding the pathogenesis of hemophilia and developing and testing treatment regimens and therapies. The Factor VIII knock-out mouse (F8-KO) is a widely used model for the study of hemophilia A1,2. These mice recapitulate key features of the disease and have been widely used for development of treatments, such as recombinant FVIII products3,4,5 and gene therapy strategies6,7.
There are various bleeding injury models for evaluating the pharmacological effects of different hemostatic compounds in vivo. One of these coagulation models is the tail vein transection survival model in mice8,9,10,11,12,13,14, measuring the ability of hemophilic mice to survive exsanguination after tail transection. This method was introduced more than four decades ago15 and is still used9,16,17. However, the model utilizes survival as an endpoint and requires observation of the animals over a period of up to 24 h, during which the animals are conscious and hence can experience pain and distress.
Bleeding models of shorter duration and under full anesthesia have been described previously, such as the tail clip model (also known as the tail tip)8,18,19,20,21,22,23,24,25,26,27,28. Nevertheless, for a complete normalization of blood loss after the bleeding challenge, these models require doses of procoagulant compounds (e.g., FVIII) far higher than those administered clinically29. A different injury model under anesthesia, the vena saphena bleeding method, is sensitive to lower doses of procoagulant compounds30 but requires a high level of experimenter intervention since the clots must be disrupted frequently (as opposed to 3 times in the presented model).
Standardization towards a common protocol to test new procoagulant compounds would greatly&#160;facilitate data comparison between laboratories31,32,33. In TVT models, there is not yet a common agreement on studied endpoints (blood loss7,26, bleeding time9,34, and survival rate35,36), and experimental length varies between studies13.
Our primary objective is to describe and characterize an optimized model with high reproducibility, the possibility to study on-demand as well as a prophylactic treatment, sensitivity to pharmacological intervention equivalent to the survival model, yet not using death or near-death as endpoints. In order to reduce pain and distress, the animals should not be conscious during bleeding and a more ethical endpoint needs to be implemented37.
Tail clip models are generally conducted in one of two variants, either amputating the tip of the tail, e.g., amputation of 1-5 mm18,19,20,21,23,24 or, in a more severe variant, transected at a tail diameter around 1-3 mm8,22,25. This causes a combined arteriovenous bleed, as the lateral and dorsal veins and ventral artery are usually severed, and in general, the larger the amputation, the lower the sensitivity to a procoagulant compound. Furthermore, since the tail tip is amputated, the arteriovenous injury is exposed without any opposing tissue; thus, at least in theory, it is dissimilar to the most common hemophilic bleeds.
As the name implies, only the vein is injured in tail vein transection models such as described in this paper, thus resulting in an exclusively&#160;venous bleed. Since the vessel is not fully severed, the injury is expected to be smaller than in the amputation models, and the tissue around the cut, which a clot may adhere to, is retained. In addition, there is lower blood pressure in the vein as opposed to the artery. These factors contribute to an increased sensitivity relative to amputation models, such that normalization of bleeding can be achieved with clinically relevant doses of replacement therapy, e.g., with rFVIII in hemophilia A, which is useful for evaluating the magnitude and durability of effects of procoagulant treatment26,38,39.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>#11 Scalpel blade</td>
			<td>Swann-Morton</td>
			<td>503</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL centrifuge tubes</td>
			<td>Greiner Bio-One, Austria</td>
			<td>188271</td>
			<td></td>
		</tr>
		<tr>
			<td>30 G needles connected to 300 &#181;L precision (insulin) syringes for dosing</td>
			<td>BD Micro-Fine + U-100 insulin syringe</td>
			<td>320830</td>
			<td></td>
		</tr>
		<tr>
			<td>Advate</td>
			<td>Takeda, Japan</td>
			<td></td>
			<td>Recombinant factor VIII replacement therapy (rFVIII)</td>
		</tr>
		<tr>
			<td>Alcohol pads 70% ethanol</td>
			<td>Hartmann, Soft-Zellin</td>
			<td>999 979</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Omnifuge 2.0 RS, Heraus Sepatech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cutting template (Stainless steel)</td>
			<td>Self produced, you are welcomed to contact the authors for the exact drawings</td>
			<td></td>
			<td>Supplementary Figure 2: Size specifications: 20 mm x 40 mm x 10 mm (L x B x H). Groove: 3 mm depth and 3 mm width; radius 1.5 mm</td>
		</tr>
		<tr>
			<td>Erythrocytes (RBC) lysing solution</td>
			<td>Lysebio, ABX Diagnostics</td>
			<td>906012</td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Haematological analyser</td>
			<td>Sysmex</td>
			<td>CT-2000iv</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating lamp on stand</td>
			<td>Phillips</td>
			<td>IR250</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pad with thermostat</td>
			<td>CMA</td>
			<td>model 150</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemoglobin standards and controls - 8.81 mmol / l batch dependent</td>
			<td>HemoCue, Denmark</td>
			<td>HemoCue calibrator, 707037</td>
			<td>Standards and controls are made from 2 different glasses of HemoCue calibrator. The value is determined against the International Reference Method for Hemoglobin (ICSH).</td>
		</tr>
		<tr>
			<td>Isofluorane anaesthesia system complete with tubes, masks and induction box</td>
			<td>Sigma Delta Dameca</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter</td>
			<td>26675-46-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnifier with lights</td>
			<td>Eschenbach</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Measuring template (Aluminum)</td>
			<td>Self produced, you are welcomed to contact the authors for the exact drawings</td>
			<td></td>
			<td>Supplementary Figure 1: Size specifications: 20 mm x 40 mm x 10 mm (L x B x H). Groove: 2.5 mm depth and 2.5 mm width; radius 1.25 mm</td>
		</tr>
		<tr>
			<td>Micropipettes + tips</td>
			<td>Finnpipette</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Photometer</td>
			<td>Molecular Devices Corporation, CA, USA</td>
			<td>SpectraMax 340 photometer</td>
			<td></td>
		</tr>
		<tr>
			<td>Prism Software</td>
			<td>GraphPad, San Diego, CA, USA</td>
			<td>Version 9.0.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline 0.9% NaCl</td>
			<td>Fresenius Kabi, Sweden</td>
			<td>883264</td>
			<td></td>
		</tr>
		<tr>
			<td>Special tail marker block for TVT tail cut</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tail holder</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum liquid suction</td>
			<td>Vacusafe comfort, IBS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Waterbath and thermostat</td>
			<td>TYP 3/8 Julabo</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

To assess the applicability of the optimized model, a study was performed in F8-KO (C57BL genetic background) mice administered with a commercially available recombinant factor VIII replacement therapy (rFVIII); four different doses were tested: 1 IU/kg, 5 IU/kg, 10 IU/kg, and 20 IU/kg. Furthermore, we tested the corresponding vehicle (negative) control in F8-KO mice and wild-type (WT) group using C57BL mice as a positive control group to assess the response range in the model.
Following the o...

Watch this Scientific Journal Video about Tail Vein Transection Bleeding Model in Fully Anesthetized Hemophilia A Mice at JoVE.com

Tail Vein Transection Bleeding Model in Fully Anesthetized Hemophilia A Mice

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) ...

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5.6K Views.  Novo Nordisk A/S. 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.

Video: Tail Vein Transection Bleeding Model in Fully Anesthetized Hemophilia A Mice

Measuring Blood Pressure in Mice using Volume Pressure Recording, a Tail-cuff Method

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. The CODA tail-cuff system uses Volume Pressure Recording (VPR) to measure the blood pressure by determining the tail blood volume. A specially designed differential pressure transducer and an occlusion tail-cuff measure the total blood volume in the tail without the need to obtain the individual pulse signal. Special attention is afforded to the length of the occlusion cuff in order to derive the most accurate blood pressure readings. VPR can easily obtain readings on dark-skinned rodents, such as C57BL6 mice and is MRI compatible. The CODA system provides you with measurements of six (6) different blood pressure parameters; systolic and diastolic blood pressure, heart rate, mean blood pressure, tail blood flow, and tail blood volume. Measurements can be made on either awake or anesthetized mice or rats. The CODA system includes a controller, laptop computer, software, cuffs, animal holders, infrared warming pads, and an infrared thermometer. There are seven different holder sizes for mice as small as 8 grams to rats as large as 900 grams.

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. This tail-cuff system uses Volume Pressure Recording (VPR) to measure the blood pressure by determining the tail blood volume.

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. The CODA ...

Right Hemihepatectomy by Suprahilar Intrahepatic Transection of the Right Hemipedicle using a Vascular Stapler

Successful hepatic resection requires profound anatomical knowledge and delicate surgical technique. Hemihepatectomies are mostly performed after preparing the extrahepatic hilar structures within the hepatoduodenal ligament, even in benign tumours or liver metastasis.1-5. Regional extrahepatic lymphadenectomy is an oncological standard in hilar cholangiocarcinoma, intrahepatic cholangio-cellular carcinoma and hepatocellular carcinoma, whereas lymph node metastases in the hepatic hilus in patients with liver metastasis are rarely occult. Major disadvantages of these procedures are the complex preparation of the hilus with the risk of injuring contralateral structures and the possibility of bleeding from portal vein side-branches or impaired perfusion of bile ducts. We developed a technique of right hemihepatectomy or resection of the left lateral segments with intrahepatic transection of the pedicle that leaves the hepatoduodenal ligament completely untouched. 6 However, if intraoperative visualization or palpation of the ligament is suspicious for tumor infiltration or lymph node metastasis, the hilus should be explored and a lymphadenectomy performed.

This video describes a right hemihepatectomy with intrahepatic transection of the right hemipedicle leaving the hepatoduodenal ligament completely untouched.

Successful hepatic resection requires profound anatomical knowledge and delicate surgical technique. Hemihepatectomies are mostly performed after ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

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, ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

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.

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 ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

A Modified Surgical Model of Hind Limb Ischemia in ApoE-/- Mice using a Miniature Incision

The purpose of this study is to introduce and evaluate a modified surgical approach to induce acute ischemia in mice that can be implemented in most animal laboratories.&#160;Contrary to the conventional approach for double ligation of the femoral artery (DLFA), a smaller incision on the right inguinal region was made to expose the proximal femoral artery (FA) to perform DLFA. Then, using a 7-0 suture, the incision was dragged to the knee region to expose the distal FA. Magnetic resonance imaging (MRI) on bilateral hind limbs was used to detect FA occlusion after the surgery. At 0, 1, 3, 5, and 7 days after the surgery, functional recovery of the hind limbs was visually assessed and graded using the Tarlov scale. Histologic evaluation was performed after euthanizing the animals 7 days after DLFA. The procedures were successfully performed on the right leg in ten ApoE-/- mice, and no mice died during subsequent observation. The incision sizes in all 10 mice were less than 5 mm (4.2 &#177; 0.63 mm). MRI results showed that FA blood flow in the ischemic side was clearly blocked. The Tarlov scale results demonstrated that hind limb function significantly decreased after the procedure and slowly recovered over the following 7 days. Histologic evaluation showed a significant inflammatory response on the ischemic side and reduced microvascular density in the ischemic hind limb. In conclusion, this study introduces a modified technique using a miniature incision to perform hind limb ischemia (HLI) using DLFA.

A Modified Surgical Model of Hind Limb Ischemia in ApoE-/- Mice using a Miniature Incision

This article demonstrates an efficient surgical approach to establish acute ischemia in mice with a small incision. This approach can be applied by most research groups without any laboratory upgrades.

The purpose of this study is to introduce and evaluate a modified surgical approach to induce acute ischemia in mice that can be implemented in most ...