The authors thank Andrea Axtell, Satoshi Itoh, MD, Jeff Velotta, MD, Grant Hoyt, Robert C. Robbins, MD, Jin Yu, MD, Tim Doyle, PhD, and the Stanford Small Animal Imaging Core for technical assistance. The authors also thank A.M. Bickford, Inc. for support of veterinary equipment. This research was supported by research grants from the National Institutes of Health (R01 HL-75774, R01 CA098303, R21 HL085743, 1K12 HL087746), the California Tobacco Related Disease Research Program of the University of California (15IT-0257 and 1514RT-0169), and the California Institute for Regenerative Medicine (RS1-00183). N.H. is supported by a fellowship from the American Heart Association.

1. Induction of Unilateral Hindlimb Ischemia
<ol>
<li> The surgical tools needed for this operation include: fine pointed forceps, pointed forceps, spring scissors, surgical scissors, needle holder, and retractor. We make our own retractor using a paperclip because it is smaller than commercially available retractors. Sterilize these tools prior to surgery by an autoclave or a hot-bead sterilizer. A cautery tool and sterile fine pointed cotton swabs will also be needed for this surgery. It is recommended that the tools be re-sterilized at the tips as needed during the procedure.</li>
<li>When the tools are ready, place the mouse into the anesthesia induction chamber containing 1&ndash;3% isoflurane in 100% oxygen at a flow rate of 1L/min.</li>
<li>Leave the mouse in the induction chamber until it is unresponsive to external stimuli. Then remove the animal from the induction chamber. It is recommended to flush the anesthetic from the box prior to opening the lid, to decrease operator exposure to isoflurane.</li>
<li>Then place the animal in the supine position onto the pre-operating table and connect it to a continuous flow of isoflurane. Using an electric shaver, remove the hair from the hindlimb. Apply hair removal cream to thoroughly remove hair.</li>
<li>Place the mouse in the supine position over a draped heated pad on the operating table, and connect it to a continuous flow of isoflurane. Extend and secure the hindlimb with a piece of tape. Once the hindlimb is secure, wipe the skin with three alternating betadine and alcohol scrubs. For the remainder of the surgical procedure, use a dissection microscope at 10X or 20X magnification to obtain an enlarged view of the hindlimb region.</li>
<li>Using fine forceps and surgical scissors, make an incision of the skin, approximately 1 cm long, from the knee towards the medial thigh.</li>
<li>Using phosphate buffered saline (PBS)-moistened fine pointed cotton swabs, gently brush away subcutaneous fat tissue surrounding the thigh muscle.</li>
<li>Next, apply the cautery transversely to incise and dissect through the subcutaneous fat tissue to reveal the underlying femoral artery.</li>
<li>Use a retractor to open the wound and to obtain a better view of the lower extremity vasculature.</li>
<li>Using fine forceps and a fine pointed cotton swab, gently pierce through the membranous femoral sheath to expose the neurovascular bundle.</li>
<li>Then, using a clean set of fine forceps and cotton swab, dissect and separate the femoral artery from the femoral vein and nerve at the proximal location near the groin. After the dissection, pass a strand of 7-0 silk suture underneath the proximal end of the femoral artery. Occlude the proximal femoral artery using double knots. Place the tie on the vessel as proximal in the wound as possible in order to leave length for the second tie and an intervening segment that will be transected.</li>
<li>Separate the femoral artery from the femoral vein at the distal location close to the knee. Pass a strand of 7-0 suture underneath the distal end of the femoral artery proximal to the popliteal artery. Occlude the vessel using double knots.</li>
<li>Occlude the distal femoral artery with a second set of double knots just proximal to the first set of knots. This second set of sutures will be used for gripping the artery during the transaction procedure.</li>
<li>Similarly, for gripping purposes, occlude the proximal femoral artery with a second set of double knots just distal to the first set of knots.</li>
<li>Transect the segment of femoral artery between the distal and proximal knots with a fine pointed cotton swab and a pair of spring scissors. Use caution not to pierce the femoral vein wall.</li>
<li>Remove the retractor and close the incision using 5-0 Vicryl sutures. These sutures do not need to be removed at a later time, as they dissolve on their own.</li>
<li>Once the incision is closed, place the animal on top of a draped heated pad in the recovery cage and monitor continuously until awake.</li>
<li>After animal has recovered for 1 hour, proceed with the laser Doppler blood perfusion step in order to confirm the ischemia induction.</li>
</ol>
2. Laser Doppler Blood Perfusion
<ol>
<li>To begin the laser Doppler perfusion step, place the mouse into the anesthesia induction chamber containing 1&ndash;3% isoflurane in 100% oxygen at a flow rate of 1L/min.</li>
<li>Leave the mouse in the induction chamber until it is unresponsive to external stimuli. Then remove the animal from the induction chamber. It is recommended to flush the anesthetic from the box prior to opening the lid, to decrease operator exposure to isoflurane.</li>
<li>Place the animal on the pre-operating table connected to continuous flow of isoflurane. Then remove the hair from the hindlimb using an electric shaver followed by hair removal cream as necessary.</li>
<li>After removing the hair, place the animal on a 37&deg;C heated surface for 5 minutes under continuous flow of isoflurane. Monitor the core temperature to ensure euthermia, as temperature changes will markedly affect perfusion.</li>
<li>After 5 minutes, place the animal in the supine position on a non- reflective light- absorbing surface such as green-colored cloth, connected to a continuous flow of isoflurane. Extend the hindlimbs.</li>
<li>Next, turn on the laser Doppler imager and the acquisition software and initialize the software. Specify the size of the field of view and resolution. It is best to keep the field of view and pixel density consistent between animals in order to make future analysis easier.</li>
<li>Open a new file. Press start to begin acquiring the image data. Generally, the imager automatically detects the distance of the mouse, but if prompted, specify the distance of the animal to the imager. The laser will show the borders of the field of view and then start acquiring data.</li>
<li>After acquisition is complete, the image will begin to show a range of colors that are indicative of the level of blood perfusion to the legs. The colors can be set to a specific perfusion range to better compare data between animals.</li>
<li>When the data acquisition is complete, save the file.</li>
<li>Then return the animal to the recovery cage and monitor the animal continuously until awake.</li>
<li>To analyze the data, use the threshold adjustment in the analysis software to substract any background noise. A threshold of 0.2 is usually a reasonable value. Select 2 regions of interest, or ROIs, that cover each hindlimb area. A variety of landmarks can be used to standardize the ROI between limbs and animals. Then determine the mean perfusion and variability in the ROI. The perfusion difference and perfusion ratio between the ischemic limb and control limb can then be easily determined.</li>
<li>This procedure can be repeated to follow changes in hindlimb perfusion over time. At a desired time point, the animal can be euthanized for assessment of tissue function and comparison to perfusion image data.</li>
</ol>
3. Representative Results:
The anatomy of the hindlimb vasculature is shown in Figure 12. A representative diagram of the hindlimb after femoral artery explantation is shown in Figure 2. To confirm the induction of ischemia to the hindlimb, laser Doppler perfusion image analysis demonstrates a dramatic reduction in blood flow to the ischemic limb, in comparison to the control limb, as shown in Figure 3.
<img src="/files/ftp_upload/old/1035_2009_1_26_9/figure1_1035.png" alt="Figure 1" width="600" height="406" />
Figure 1. Anatomy of the hindlimb vasculature.&nbsp; Asterisks indicate the locations of ligation for the induction of hindlimb ischemia.
<img src="/files/ftp_upload/old/1035_2009_1_20_7/Niiyama_HLI_Fig2.png" alt="Image 2" width="600" height="450" />
Figure 2. Representative diagram showing the anatomy of the hindlimb after ligation of the femoral artery at the proximal and distal sites after the removal of the femoral artery.
<img src="/files/ftp_upload/old/1035_2009_1_20_8/Niiyama_HLI_Fig3.png" alt="Figure 3" width="600" height="256" />
Figure 3. Laser Doppler images showing blood flow before and after the induction of ischemia to the left hindlimb (indicated by arrow).

<ol>
	<li>Huang, N. F., Niiyama, H., De, A., Cooke, J. P., , . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+and+non-invasive+tracking+of+embryonic+stem+cell-derived+endothelial+cells+for+treatment+of+hindlimb+ischemia.">Transplantation and non-invasive tracking of embryonic stem cell-derived endothelial cells for treatment of hindlimb ischemia.</a> J Vis Exp. , (2008).</li><li>Cook, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The anatomy of the laboratory mouse. , (1976).</li><li>Niiyama, H., Kai, H., Yamamoto, T., Shimada, T., Sasaki, K., Murohara, T., Egashira, K., Imaizumi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+endogenous+monocyte+chemoattractant+protein-1+in+ischemia-induced+neovascularization.">Roles of endogenous monocyte chemoattractant protein-1 in ischemia-induced neovascularization.</a> J. Am. Coll. Cardiol. 44, 661-666 (2004).</li><li>Dokun, A. O., Keum, S., Hazarika, S., Li, Y., Lamonte, G. M., Wheeler, F., Marchuk, D. A., Annex, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+trait+locus+(LSq-1)+on+mouse+chromosome+7+is+linked+to+the+absence+of+tissue+loss+after+surgical+hindlimb+ischemia.">A quantitative trait locus (LSq-1) on mouse chromosome 7 is linked to the absence of tissue loss after surgical hindlimb ischemia.</a> Circulation. 117, 1207-1215 (2008).</li></ol>

There are some sources of variability to consider while planning and performing hindlimb ischemia models. First, the level of ischemia may vary according to the location of the ligation with respect to that of side branches. Therefore, for consistency of the model, the animals should undergo the same level of arterial ligation. Another source of variability in ischemic recovery is related to the age of the animals, with young animals (6&ndash;8 weeks old) having faster and more complete recovery rates than older animals (8&ndash;10 months old), as assessed by hemodynamic (i.e. laser Doppler perfusion) or functional (i.e. treadmill testing) measures. For studies in which one is assessing an angiogenic agent, it may be preferable to use older animals, because a greater difference between groups may be observed with a therapeutic intervention. Conversely, for studies in which one is assessing an anti-angiogenic factor, it may be preferable to use younger animals to maximize effect size3.
If performed correctly, there should be minimal complications such as bleeding, infection, or mortality. If bleeding occurs by accidental disruption of the femoral vein or other vessels, moderate pressure with a sterile cotton tipped applicator or gauze should be applied to the site of hemorrhage until the bleeding stops. Animals showing signs of infection should be treated with anti-infective agents. Animals exhibiting significant gangrene may need to beeuthanized. This complication is more common in older animals, and in some strains, such as BALB c mice4. In addition, the hindlimb ischemia model may cause mild to moderate pain. Therefore, analgesics such as buprenorphine or carprofen should be administered as needed for treatment of pain, according to recommendations of the IACUC.
In conclusion, we have demonstrated a simple and reproducible method for studying PAD in a murine model of hindlimb ischemia. The hindlimb ischemia model with laser Doppler imaging analysis is an excellent system for studying vascular pathologies and for assessing therapeutic candidates.

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<td>Surgical Tools</td>
<td>Tool</td>
<td>Fine Science Tools</td>
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<td>Constix Cotton Swabs</td>
<td>Tool</td>
<td>Contec</td>
<td>SC-4</td>
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<td>Betadine (Povidone-iodine)</td>
<td>Reagent</td>
<td>PDI</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>70% alcohol</td>
<td>Reagent</td>
<td>Kendall</td>
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<td>Phosphate Buffered Saline</td>
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<td>7-0 silk suture</td>
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<td>Baxter</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Laser Doppler and PeriScan PIM 3 System</td>
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murine model of hindlimb ischemia 

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide. Medical therapies for symptomatic relief are limited. Surgical or endovascular interventions are useful for some individuals, but long-term results are often disappointing. As a result, there is a need for developing new therapies to treat PAD. The murine hindlimb ischemia preparation is a model of PAD, and is useful for testing new therapies. When compared to other models of tissue ischemia such as coronary or cerebral artery ligation, femoral artery ligation provides for a simpler model of ischemic tissue. Other advantages of this model are the ease of access to the femoral artery and low mortality rate. 
In this video, we demonstrate the methodology for the murine model of unilateral hindimb ischemia. The specific materials and procedures for creating and evaluating the model will be described, including the assessment of limb perfusion by laser Doppler imaging. This protocol can also be utilized for the transplantation and non-invasive tracking of cells, which is demonstrated by Huang et al.1.

Watch this Scientific Journal Video about Murine Model of Hindlimb Ischemia at JoVE.com

Murine Model of Hindlimb Ischemia

The surgical procedure for induction of unilateral hindlimb ischemia is demonstrated, with confirmation of ischemia by laser Doppler perfusion imaging.

Murine Model of Hindlimb Ischemia 

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide.  Medical therapies for ...

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56.9K ビュー.  Stanford University. 片側後肢虚血の誘発のための外科手術は、レーザードップラー血流イメージングによる虚血の確認を、示されている。

ビデオ: Murine Model of Hindlimb Ischemia

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The Hypoxic Ischemic Encephalopathy Model of Perinatal Ischemia

Hypoxic-Ischemic Encephalopathy (HIE) is the consequence of systemic asphyxia occurring at birth. Twenty five percent of neonates with HIE develop severe and permanent neuropsychological sequelae, including mental retardation, cerebral palsy, and epilepsy. The outcomes of HIE are devastating and permanent, making it critical to identify and develop therapeutic strategies to reduce brain injury in newborns with HIE. To that end, the neonatal rat model for hypoxic-ischemic brain injury has been developed to model this human condition. The HIE model was first validated by Vannucci et al 1 and has since been extensively used to identify mechanisms of brain injury resulting from perinatal hypoxia-ischemia 2 and to test potential therapeutic interventions 3,4. The HIE model is a two step process and involves the ligation of the left common carotid artery followed by exposure to a hypoxic environment. Cerebral blood flow (CBF) in the hemisphere ipsilateral to the ligated carotid artery does not decrease because of the collateral blood flow via the circle of Willis; however with lower oxygen tension, the CBF in the ipsilateral hemisphere decreases significantly and results in unilateral ischemic injury. The use of 2,3,5-triphenyltetrazolium chloride (TTC) to stain and identify ischemic brain tissue was originally developed for adult models of rodent cerebral ischemia 5, and is used to evaluate the extent of cerebral infarctin at early time points up to 72 hours after the ischemic event 6. In this video, we demonstrate the hypoxic-ischemic injury model in postnatal rat brain and the evaluation of the infarct size using TTC staining.

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Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This pathology causes symptoms such as intermittent claudication (pain with walking), painful ischemic ulcerations, or even limb-threatening gangrene. It is generally believed that the vascular endothelium, a monolayer of endothelial cells that invests the luminal surface of all blood and lymphatic vessels, plays a dominant role in vascular homeostasis and vascular regeneration. As a result, stem cell-based regeneration of the endothelium may be a promising approach for treating PAD.In this video, we demonstrate the transplantation of embryonic stem cell (ESC)-derived endothelial cells for treatment of unilateral hindimb ischemia as a model of PAD, followed by non-invasive tracking of cell homing and survival by bioluminescence imaging. The specific materials and procedures for cell delivery and imaging will be described. This protocol follows another publication in describing the induction of hindlimb ischemia by Niiyama et al.1

The surgical procedure for delivery of embryonic stem cell-derived endothelial cells to the ischemic hindlimb is demonstrated, with non-invasive tracking by bioluminescence imaging.

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Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes in myocardial ischemia and to develop and optimize future treatment.
Two different methods of creating myocardial ischemia are performed in laboratory rodents. The first method is to create cryo infarction, a fast but inaccurate technique, where a cryo-pen is applied on the surface of the heart (1-3). Using this method the scientist can not guarantee that the cryo-scar leads to ischemia, also a vast myocardial injury is created that shows pathophysiological side effects that are not related to myocardial infarction. The second method is the permanent ligation of the left anterior descending artery (LAD). Here the LAD is ligated with one single stitch, forming an ischemia that can be seen almost immediately. By closing the LAD, no further blood flow is permitted in that area, while the surrounding myocardial tissue is nearly not affected. This surgical procedure imitates the pathobiological and pathophysiological aspects occurring in infarction-related myocardial ischemia.
The method introduced in this video demonstrates the surgical procedure of a mouse infarction model by ligating the LAD. This model is convenient for pathobiological and pathophysiological as well as immunobiological studies on cardiac infarction. The shown technique provides high accuracy and correlates well with histological sections.

This video demonstrates how to use a fast and reliable model to study pathobiological and pathophysiological processes of myocardial ischemia.

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes ...

An Alternant Method to the Traditional NASA Hindlimb Unloading Model in Mice

The Morey-Holton hindlimb unloading (HU) method is a widely accepted National Aeronautics and Space Administration (NASA) ground-based model for studying disuse-atrophy in rodents 4-6. Our study evaluated an alternant method to the gold-standard Morey-Holton HU tail-traction technique in mice. Fifty-four female mice (4-8 mo.) were HU for 14 days (n=34) or 28 days (n=20). Recovery from HU was assessed after 3 days of normal cage ambulation following HU (n=22). Aged matched mice (n=76) served as weight-bearing controls.
Prior to HU a tail ring was formed with a 2-0 sterile surgical steel wire that was passed through the 5th, 6th, or 7th inter-vertebral disc space and shaped into a ring from which the mice were suspended. Vertebral location for the tail-ring was selected to appropriately balance animal body weight without interfering with defecation.
We determined the success of this novel HU technique by assessing body weight before and after HU, degree of soleus atrophy, and adrenal mass following HU. Body weight of the mice prior to HU (24.3 &plusmn; 2.9g) did not significantly decline immediately after 14d of HU (22.7 &plusmn; 1.9g), 28d of HU (21.3 + 2.1g) or after 3 days recovery (24.0 &plusmn; 1.8g). Soleus muscle mass significantly declined (-39.1%, and -46.6%) following HU for 14 days and 28 days respectively (p&lt;0.001). Following 3 days of recovery soleus mass significantly increased to 74% of control values. Adrenal weights of HU mice were not different compared to control mice.
The success of our novel HU method is evidenced by the maintenance of animal body weight, comparable adrenal gland weights, and soleus atrophy following HU, corresponding to expected literature values 2, 7, 8. The primary advantages of this HU method include: 1) ease of tail examination during suspension; 2) decreased likelihood of cyanotic, inflamed, and/or necrotic tails frequently observed with tail-taping and HU; 3) no possibility of mice chewing the traction tape and coming out of the suspension apparatus; and 4) rapid recovery and normal cage activity immediately after HU.

We developed an alternant hindlimb unloading model in mice. The primary advantage of our hindlimb unloading tail-ring method over the conventional Morey-Holton tail-traction technique is a simple straightforward procedure that minimizes stress upon the animal.

The Morey-Holton hindlimb unloading (HU) method is a widely accepted National Aeronautics and Space Administration (NASA) ground-based model for studying ...

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed under anoxia for 20 hr&#160;(simulated ischemia), and subsequently moved to a normal atmosphere with food (simulated reperfusion). This experimental paradigm results in increased death and neuronal damage, and techniques are presented to assess organism viability, alterations to the morphology of touch neuron processes, as well as touch sensitivity, which represents the behavioral output of neuronal function. Finally, a method for constructing hypoxic incubators using common kitchen storage containers is described. The addition of a mass flow control unit allows for alterations to be made to the gas mixture in the custom incubators, and a circulating water bath allows for both temperature control and makes it easy to identify leaks. This method provides a low cost alternative to commercially available units.

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

A protocol is described that uses anoxia/starvation in C. elegans to model ischemia/reperfusion. Functional outcomes include increased mortality, visible abnormalities in GFP-labeled neuronal processes, and impaired behavioral responses that require neuronal function.

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed ...

The Murine Choline-Deficient, Ethionine-Supplemented (CDE) Diet Model of Chronic Liver Injury

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual inflammation, progressive destruction and regeneration of the hepatic parenchyma, liver progenitor cell proliferation, and fibrosis. The end-stage of every chronic liver disease is cirrhosis, a major risk factor for the development of hepatocellular carcinoma. To study processes regulating disease initiation, establishment, and progression, several animal models are used in laboratories. Here we describe a six-week time course of the choline-deficient and ethionine-supplemented (CDE) mouse model, which involves feeding six-week old male C57BL/6J mice with choline-deficient chow and 0.15% DL-ethionine-supplemented drinking water. Monitoring of animal health and a typical body weight loss curve are explained. The protocol demonstrates the gross examination of a CDE-treated liver and blood collection by cardiac puncture for subsequent serum analyses. Next, the liver perfusion technique and collection of different hepatic lobes for standard evaluations are shown, including liver histology assessments by hematoxylin and eosin or Sirius Red stainings, immunofluorescent detection of hepatic cell populations as well as transcriptome profiling of the liver microenvironment. This mouse model is suitable for studying inflammatory, fibrogenic, and liver progenitor cell dynamics induced through chronic liver disease and can be used to test potential therapeutic agents that may modulate these processes.

The Murine Choline-Deficient, Ethionine-Supplemented CDE Diet Model of Chronic Liver Injury

Here we describe a common method to induce chronic liver injury in mice by feeding of a choline-deficient and ethionine-supplemented (CDE) diet. We demonstrate health monitoring, liver perfusion, isolation, and preservation. A time course of six weeks can inform about liver injury, pathohistology, fibrosis, inflammatory, and liver progenitor cell responses.

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual ...

A Murine Model of Vertical Sleeve Gastrectomy

Bariatric surgery, such as vertical sleeve gastrectomy (VSG), is a surgery of the gastrointestinal tract that is performed for the purpose of weight loss. Bariatric surgery is currently the most effective long-term treatment for obesity. In addition to weight loss, bariatric surgery produces additional health benefits such as remission of type 2 diabetes, remission of hypertension, and decreased risk of developing certain types of cancer. The mechanisms beyond weight loss for these benefits remain incompletely defined. Therefore, animal models of bariatric surgery are being developed and validated to identify the mechanisms leading to these benefits, with the goal of improving understanding of gastrointestinal physiology and identifying new therapeutic targets. VSG has become the most commonly performed bariatric procedure in the clinic in the United States because it is highly effective at producing weight loss and metabolic improvement, and is simpler to perform than other bariatric procedures. Therefore, we have developed and validated a murine model of VSG. This murine VSG model recapitulates many of the effects of VSG seen in humans, including improved glucose and blood pressure regulation. The method is based on isolation of the stomach, ligation of gastric vessels, and removal of 70% of the stomach by transecting along the greater curvature of the stomach. We have successfully applied this surgical protocol to various genetically modified mouse lines to define the mechanistic contributors to the benefits of VSG. Furthermore, this murine VSG model has been combined with other surgical techniques, to achieve deeper mechanistic insight. Therefore, this is a simple and versatile model for studying gastrointestinal physiology and the health benefits of bariatric surgery.

The following describes the performance of vertical sleeve gastrectomy in mice. This is a type of weight-loss surgery that involves removal of approximately 70% of the stomach.

Bariatric surgery, such as vertical sleeve gastrectomy (VSG), is a surgery of the gastrointestinal tract that is performed for the purpose of weight loss. ...

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 Murine Model of a Burn Wound Reconstructed with an Allogeneic Skin Graft

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and require surgical debridement and skin grafting. Successful integration of the donor graft into a recipient wound bed depends on timely recruitment of immune cells, robust angiogenic response and new extracellular matrix formation. The development of novel therapeutic agents, which target some key processes involved in wound healing, are hindered by the lack of reliable preclinical models with optimized objective assessment of wound closure. Here, we describe an inexpensive and reproducible model of experimental full thickness burn wound reconstructed with an allogeneic skin graft. The wound is induced on the dorsum surface of anaesthetized inbred wild type mice from the BALB/C and SKH1-Hrhr backgrounds. The burn is produced using a brass template measuring 10 mm in diameter, which is preheated to 80 &#176;C and delivered at a constant pressure for 20 s. Burn eschar is excised 24 hours after the injury and replaced with a full thickness graft harvested from the tail of a genetically similar donor mouse. No specialized equipment is required for the procedure and surgical techniques are straightforward to follow. The method may be effortlessly implemented and reproduced in most research settings. Certain limitations are associated with the model. Due to technical difficulties, the harvest of thinner split thickness skin grafts is not possible. The surgical method we describe here allows for the reconstruction of burn wounds using full thickness skin grafts. It may be used to carry out preclinical therapeutic testing.

The aim of this study was to develop a murine model of burn wound healing. A thermal burn was induced on the dorsal skin of mice using a preheated brass template. Burned tissue was debrided and overlaid with a skin graft harvested from the tail of a genetically similar donor mouse.

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and ...

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.

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 ...