The authors would like to thank Andrew J Dunn, an outstanding undergraduate research student in our laboratory, for his technical assistance and caring for the animals. The National Institute of Health grant HD058834 and the University of Missouri Life Sciences Fellowship funded this work.

Pre-Surgery Instruments / Materials
<ul>
<li>Wire cutters/pliers</li>
<li>Gauze 2 x 2" pads</li>
<li>Heating pad</li>
<li>Silver nitrate sticks</li>
<li>Sterile surgical gloves</li>
<li>Isoflurane inhalation anesthetic</li>
<li>Chlorhexidine Gluconate 4% (Antiseptic surgical scrub)</li>
<li>Sterile 2-0 surgical steel suture</li>
<li>25 X 5/8 A gauge needle (Autoclaved)</li>
<li>Hemostat (Autoclaved)</li>
<li>Surgical steel suture</li>
</ul>
Surgery Procedure
<ol>
<li>Create a sterile field. Follow sterile procedure.</li>
<li>Anesthetize mouse with 2 - 3% isoflurane.</li>
<li>Place mouse on the heating pad.</li>
<li>Scrub tail with Chlorhexidine solution.</li>
<li>Locate 5, 6, or 7th intervertebral space (between two bony bumps on tail, start counting from end of body hair, at the base of tail).</li>
<li>Use Hemostat and 25G needle to make a pilot hole between two vertebrae, into the intervertebral space.</li>
<li>Use the wire cutters to cut the steel suture 10 cm long.</li>
<li>Remove needle and insert steel suture through hole, 5 cm of suture on either end of tail.</li>
<li>Each side of the tail should have + 5 cm of suture.</li>
<li>Place thumb tip onto tail.</li>
<li>Use your free hand and wind the steel suture onto the tip of your thumb and twist the ends to form a big loop. The wound or circular part should be + 3 mm long.</li>
<li>Remove your thumb.</li>
<li>Clamp one strand of suture with the hemostat and bend it into a smaller loop, the end of which is attached as closely as possible to the twist on the original large loop.</li>
<li>Cut off excess wire, and bend the twisted wire back.</li>
<li>If there is any excess bleeding, you can stop it with the silver nitrate sticks.</li>
<li>Turn off the isoflurane and place mouse in a single house cage to recover for 5-7 days prior to hindlimb unloading. The tail rings get entangled if more than one mouse is housed together.</li>
</ol>
1. Hindlimb Unloading Procedure

Materials
<ul>
<li>Size 10-24 X 4 machine screws, one per animal.</li>
<li>Nylon lock nut size 10 - 24, one per animal.</li>
<li>Wing nut size 10 X 24, two per animal.</li>
<li>Washer size &frac14; X 1, two per animal.</li>
<li>Fishing swivel with snap hook size #6, 1-11/16", and one per animal.</li>
<li>Metal sewing machine bobbin. One per animal.</li>
<li>Small paper clip for S-shaped hook, one per animal</li>
<li>Pliers.</li>
<li>2 x 2" gauze.</li>
<li>2" black paper binder clamps.</li>
<li>Duct tape/ lab tape.</li>
<li>1-3/8" Plated slotted steel bar that extends slightly beyond the full length of the cage</li>
<li>50cm X 40cm X 20cm cage (rat cage).</li>
<li>Water Bottle with bended tube.</li>
<li>Stainless Steel wire, U-shaped to support water bottle.</li>
</ul>
Cage Setup
<ol>
<li>Lay the 1-38" Plated slotted steel bar over the top of the cage, and tape it temporally down with duct tape.</li>
<li>Screw 1 wing nut to the machine screw, insert a washer, and insert the machine screw trough the slotted steel plate &plusmn; 15 cm from the edge of the cage.</li>
<li>Add another washer and wing nut. Tighten both wing nuts, but do not fully tighten, as it will be necessary to do adjustments later.</li>
<li>Insert the machine screw through the center hole of the bobbin.</li>
<li>To prevent the bobbin from falling off, screw the nylon lock nut to the end of the machine screw. The bobbin should swivel freely.</li>
<li>Insert the snap hook from the fishing swivel through the lower part of the bobbin.</li>
<li>Use pliers to form an S-shaped hook from the paperclip. (Cut 2cm from the paper clip to form a hook).</li>
<li>Insert one end of the S-shaped hook through the hole of the fishing swivel and pinch it shut with the pliers.</li>
<li>Place the U-shaped wire, parallel to the machine screw, on the inside of the cage side, and bend the ends back to support a full water bottle.</li>
<li>Insert the water bottle into the U-shaped wire, with the spicket facing down, but not touching the bottom.</li>
<li>Bend the 2" binder clamp open, so that they cannot shut close. Just enough tension to hold 3 to 4 pieces of food in it without it falling out. This is preventing the force of the clamp from crushing the food, or clamping down on the mouse nose.</li>
<li>Place 3 to 4 pieces of rodent chow into the binder clamp.</li>
</ol>
Hindlimb Unloading of Mouse
<ol>
<li>Insert the open end of the S-shaped hook through the smaller ring of the tail ring.</li>
<li>Adjust the mouse height by tightening or loosening the lower wing nut on the machine screw. The lower extremities should not be weight bearing. Once the optimal height is achieved tighten the upper wing nut.</li>
<li>Reposition the water bottle, and U-shaped wire so that the mouse cannot climb onto the spicket with its hindlimbs. Secure the water bottle with Duct tape to the side of the cage.</li>
<li>Position the binder clamp with food on the bottom of the cage so that the mouse cannot climb onto it; tape it down with duct tape.</li>
<li>Repeat the same procedure if more than one animal is hindlimb unloaded on the other side of the cage. House two animals per cage; space the animals so that they can almost touch each other.</li>
<li>Use the 2 X 2 gauze to loosely wrap the tail of the mouse onto the fish swivel. Use a small piece of tape to wrap around the gauze, securing the gauze to the tail. This step is important to prevent the tail from drooping downward, leading to blood pooling in the tip of the tail and that tail becoming necrotic.</li>
<li>Add a small amount of cob bedding to the cage. Do not add too much bedding otherwise the mouse will from a mound of bedding and attempt to load hindlimbs on bedding.</li>
</ol>
2. Representative Results:

To establish the success of the tail-ring HU technique we measured animal body weight before the tail ring surgery, and prior to and following HU and recovery; soleus muscle mass; and adrenal mass in all groups. 
Body weights of the mice throughout the experiment are shown in Figure 1. Body weights of the mice prior to the tail ring surgery (24.7 &plusmn; 3.5g) were not significantly different after 5 days of recovery from the tail ring surgery (24.3 &plusmn; 2.9g) prior to HU. Nor did body weight significantly decline immediately after 14 days of HU (22.7 &plusmn; 1.9g), 28 days of HU (21.3 + 2.1g) or after 3 days recovery from HU (24.0 &plusmn; 1.8g). Although HU for 14 and 28 days resulted in a small (-6.8% and 12%) but non-significant loss in body-mass, the mice fully recovered to their pre-HU body weights after 3 days of recovery. Food consumption for ambulatory control mice and HU mice is shown in Figure 3. Average daily food intake was not different in HU mice over 28 days compared to controls.
We observed significant soleus muscle atrophy following HU for 14 and 28 days (-39.1%, and -46.6%) indicating an effective HU procedure (Figure 2). Furthermore this degree of atrophy is consistent with the expected literature values for 2-4 weeks of HU 2, 7, 8. Following 3 days of recovery from HU soleus mass significantly increased to 74% of control values. Soleus mass in the recovery group was significantly greater than the both HU groups, but still significantly less than ambulatory control animals.
Adrenal weights were measured as an indicator of the animal stress; both the left and right adrenal glands were excised and weighed. The average weights were comparable for control and HU mice mice in all groups (Figure 4).
Previously in rats we have shown that the success rate for the number of animals at the beginning of HU compared to the number of animals that completed HU without coming out of suspension apparatus (due to animals chewing through the tail-traction tape) was 10 out of 12 using the Morey-Holton technique 1. None of the mice (n=54) in this study ever came down from suspension prior to the completion of the HU period. We did however, have one mouse that that developed a severely inflamed tail. When inflammation became apparent, we removed the tail ring to allow the inflamed area to heal, and subsequently placed another tail-ring to complete the duration of HU.
In the past when we have used the Morey-Holton method of HU, we typically eliminate 1-2 mice/group because they come out of the suspension apparatus. With the trail-ring method there were no episodes of mice inadvertently coming out of the HU apparatus during the period of HU for 14 or 28 days (n=54 mice).
 <img src="/files/ftp_upload/2467/2467fig1.jpg" alt="Figure 1" /> 
Figure 1. Body Weight Changes (Mean &plusmn; SD). Abbreviations: CON: weight-bearing control mice (n=76); Pre-HU: all mice prior to HU (n=54); HU14: mice hindlimb unloaded for 14 days (n=34); HU28: mice hindlimb unloaded for 28 days (n=20); REC: hindlimb unloaded mice allowed to cage recover for 3 days (n=22). No significant differences in body weight were observed between groups.
 <img src="/files/ftp_upload/2467/2467fig2.jpg" alt="Figure 2" /> 
Figure 2. Soleus Muscle Atrophy (Mean &plusmn; SD). Abbreviations: CON: weight-bearing control mice (n=76); HU14: mice hindlimb unloaded for 14 days (n=34); HU28: mice hindlimb unloaded for 28 days (n=20); and REC: hindlimb unloaded mice allowed to cage recover for 3 days (n=22). *Significantly different (p&lt;0.001) than weight-bearing control (CON). &delta;Significantly different (p&lt;0.01) than both HU groups.
 <img src="/files/ftp_upload/2467/2467fig3.jpg" alt="Figure 3" /> 
Figure 3. Food Consumption (Mean &plusmn; SD). Average daily food intake (g/day) during weight-bearing and hindlimb unloading. Abbreviations: weight-bearing control mice (n=76); HU: mice hindlimb unloaded14 - 28 days. No significant differences between CON and HU groups were observed.
 <img src="/files/ftp_upload/2467/2467fig4.jpg" alt="Figure 4" /> 
Figure 4. Adrenal Mass (Mean &plusmn; SD). Abbreviations: CON: weight-bearing control mice (n=76); HU14: mice hindlimb unloaded for 14 days (n=34); HU28: mice hindlimb unloaded for 28 days (n=20); and REC: hindlimb unloaded mice allowed to cage recover for 3 days (n=22). No significant differences were observed between the groups.

<ol>
	<li>Brown, M., Foley, A. M., Hasser, E. M., Foley, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+alternative+method+for+hindlimb+unweighting.">An alternative method for hindlimb unweighting.</a> J Gravitational Physiol. 12, 43-50 (2005).</li><li>Fitts, R. H., Metzger, J. M., Riley, D. A., Unsworth, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+disuse:+a+comparison+of+hindlimb+suspension+and+immobilization.">Models of disuse: a comparison of hindlimb suspension and immobilization.</a> J Appl Physiol. 60, 1946-1953 (1986).</li><li>Ingalls, C. P., Warren, G. L., Armstrong, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+Ca2++transients+in+mouse+soleus+muscle+after+hindlimb+unloading+and+reloading.">Intracellular Ca2+ transients in mouse soleus muscle after hindlimb unloading and reloading.</a> Journal of Applied Physiology. 87, 386-390 (1999).</li><li>Morey, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spaceflight+and+bone+turnover:+correlation+with+a+new+rat+model+of+weightlessness.">Spaceflight and bone turnover: correlation with a new rat model of weightlessness.</a> BioScience. 29, 168-172 (1979).</li><li>Morey-Holton, E. R., Globus, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hindlimb+unloading+of+growing+rats:+a+model+for+predicting+skeletal+changes+during+space+flight.">Hindlimb unloading of growing rats: a model for predicting skeletal changes during space flight.</a> Bone. 22, 83-88 (1998).</li><li>Morey-Holton, E. R., Globus, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hindlimb+unloading+rodent+model:+technical+aspects.">Hindlimb unloading rodent model: technical aspects.</a> J Appl Physiol. 92, 1367-1377 (2002).</li><li>Thomason, D. B., Booth, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atrophy+of+the+soleus+muscle+by+hindlimb+unweighting.">Atrophy of the soleus muscle by hindlimb unweighting.</a> J Appl Physiol. 68, 1-12 (1990).</li><li>Thomason, D. B., Herrick, R. E., Surdyka, D., Baldwin, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+of+soleus+muscle+myosin+expression+during+hindlimb+suspension+and+recovery.">Time course of soleus muscle myosin expression during hindlimb suspension and recovery.</a> J Appl Physiol. 63, 130-137 (1987).</li></ol>

No conflicts of interest declared.

We evaluated an alternative method of hindlimb unloading mice, which we determined to be highly successful. The tail-ring method as described here was used for long-term hindlimb suspension, and to study recovery following HU. Under each of these conditions, there was no evidence of failure to adapt as evidenced by stable food intake, body weights, and adrenal weights of the mice. This tail-ring HU technique is easy to perform and can readily be incorporated with very little surgical practice.
There are several advantages of tail-ring method compared to the traditional Morey-Holton 5, 6 method. First, the skin and ring implantation site on that tail are always visible, so if problems arise it is evident immediately, not just at the end of the suspension period. Second, animals are not able to get out of the suspension apparatus at any time during HU, obviating the need to make a determination of whether to keep a mouse or not in the data set. Furthermore, the mice do not bother the ring apparatus and when they are released from HU for recovery, they do not tamper with their tails, allowing for a seamless recovery phase following HU. Finally, hindlimb unloaded mice with the tail-ring method maintain their body weight better than mice hindlimb unloaded by the Morey-Holton tail-traction technique. In our hands after 14 days of HU we observed a small (-6.8%) but non-significant loss in body-mass. Whereas Ingalls et al. observed a significant -12% decrease in body weight in mice after 14 days of HU by the Morey-Holton method 3. Although Ingalls did not report adrenal weights, it appears that our tail-ring method confers less stress on the mouse (adrenal weights in our HU mice are not different than controls) and better maintenance of body weight throughout the hindlimb unloading period.
In summary, a tail ring method of hindlimb unloading was developed and tested on mice HU for 14 and 28 days, and on mice returned to cage recovery for 3 days after HU. On the basis of adrenal weights, body mass and food intake, mice appear to have adapted well to the tail-ring apparatus. The tail-ring hindlimb unloading method is a useful, simple alternative to the traditional Morey-Holton hindlimb unloading technique.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Wire cutters/pliers</td>
<td>&nbsp;</td>
<td>Ace Hardware </td>
<td>84-056</td>
<td>&nbsp;</td>
<tr>
<td>Gauze</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>22-037-901</td>
<td>&nbsp;</td>
<tr>
<td>Heating pad</td>
<td>&nbsp;</td>
<td>Sunbeam</td>
<td>Z-1228-001</td>
<td>&nbsp;</td>
<tr>
<td>Silver nitrate applicators</td>
<td>&nbsp;</td>
<td>ARZOL</td>
<td>0001-2</td>
<td>&nbsp;</td>
<tr>
<td>Isoflurane,USP</td>
<td>&nbsp;</td>
<td>Novaplus</td>
<td>NDC 0409-3292-49</td>
<td>&nbsp;</td>
<tr>
<td>Chlorhexidine Gluconate 4%</td>
<td>&nbsp;</td>
<td>Purdue Products L.P</td>
<td>NDC 67618-200-01</td>
<td>&nbsp;</td>
<tr>
<td>Sterile 2-0 surgical steel suture</td>
<td>&nbsp;</td>
<td>Ethicon</td>
<td>SS-28</td>
<td>&nbsp;</td>
<tr>
<td>25 X 5/8 A gauge needle</td>
<td>&nbsp;</td>
<td>Terumo</td>
<td>NN 2516R</td>
<td>&nbsp;</td>
<tr>
<td>Hemostat/ needle holder</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>12500-12</td>
<td>&nbsp;</td>
<tr>
<td>Size 10-24 X 4 machine screws</td>
<td>&nbsp;</td>
<td>Hillman The Fastener Center</td>
<td>90272</td>
<td>&nbsp;</td>
<tr>
<td>Nylon lock nut size 10 X 24</td>
<td>&nbsp;</td>
<td>Hillman The Fastener Center</td>
<td>180141</td>
<td>&nbsp;</td>
<tr>
<td>Wing nut size 10 X 24</td>
<td>&nbsp;</td>
<td>Hillman The Fastener Center</td>
<td>180243</td>
<td>&nbsp;</td>
<tr>
<td>Fender Washer size &frac14; X 1</td>
<td>&nbsp;</td>
<td>Hillman The Fastener Center</td>
<td>290012</td>
<td>&nbsp;</td>
<tr>
<td>Fishing swivel with snap hook size #6, 1-11/16&#x201D;</td>
<td>&nbsp;</td>
<td>Bass Pro Shop</td>
<td>38-481-554-01</td>
<td>&nbsp;</td>
<tr>
<td>Metal sewing machine bobbin.</td>
<td>&nbsp;</td>
<td>Singer</td>
<td>45785</td>
<td>&nbsp;</td>
<tr>
<td>Paper clip</td>
<td>&nbsp;</td>
<td>Office Depot</td>
<td>221720</td>
<td>&nbsp;</td>
<tr>
<td>2&#x201D; black paper binder clamps.</td>
<td>&nbsp;</td>
<td>Office Depot</td>
<td>308957</td>
<td>&nbsp;</td>
<tr>
<td>Scotch Multi-Use Duct Tape</td>
<td>&nbsp;</td>
<td>Office Depot</td>
<td>790710</td>
<td>&nbsp;</td>
<tr>
<td>1-3/8&#x201D; Plated slotted steel bar</td>
<td>&nbsp;</td>
<td>Home Depot</td>
<td>548932</td>
<td>Extends slightly beyond the full length of the cage</td>
</tr>
<tr>
<td>50cm X 40cm X 20cm cage (rat cage</td>
<td>&nbsp;</td>
<td>Alternative Design Manufacturing</td>
<td>RC88D-PC</td>
<td>&nbsp;</td>
<tr>
<td>Water Bottle</td>
<td>&nbsp;</td>
<td>Alternative Design Manufacturing</td>
<td>WB16FS</td>
<td>&nbsp;</td>
<tr>
<td>Stainless Steel wire</td>
<td>&nbsp;</td>
<td>Small Parts, Inc</td>
<td>SWX-4029</td>
<td>U-shaped wire to support water bottle</td>
</tr>		</tbody>
		</table>
		</div>

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.

Watch this Scientific Journal Video about An Alternant Method to the Traditional NASA Hindlimb Unloading Model in Mice at JoVE.com

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

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-alternant-method-to-traditional-nasa-hindlimb-unloading-model

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JoVE Journal

Biology

20.3K Görüntüleme.  University of Missouri, Columbia. Biz farelerde model boşaltma alternant hindlimb geliştirdi. Geleneksel Morey Holton kuyruk çekme tekniği üzerinde hindlimb boşaltma kuyruk halka yöntemi birincil avantajı hayvan üzerine stresi en aza indirir basit bir işlemdir.

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

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.

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.

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

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 ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.

Bacteriocins are believed to play a key role in defining microbial diversity in different ecological niches. Here, we describe an efficient procedure to assess how bacteriocins affect gut microbiota composition in an animal model.

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...

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. ...

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 ...

An Antegrade Perfusion Method for Cardiomyocyte Isolation from Mice

In basic research using mouse heart, isolating viable individual cardiomyocytes is a crucial technical step to overcome. Traditionally, isolating cardiomyocytes from rabbits, guinea pigs or rats has been performed via retrograde perfusion of the heart with enzymes using a Langendorff apparatus. However, a high degree of skill is required when this method is used with a small mouse heart. An antegrade perfusion method that does not use a Langendorff apparatus was recently reported for the isolation of mouse cardiomyocytes. We herein report a complete protocol for the improved antegrade perfusion of the excised heart to isolate individual heart cells from adult mice (8 - 108 weeks old). Antegrade perfusion is performed by injecting perfusate near the apex of the left ventricle of the excised heart, the aorta of which was clamped, using an infusion pump. All procedures are carried out on a pre-warmed heater mat under a microscope, which allows for the injection and perfusion processes to be monitored. The results suggest that ventricular and atrial myocytes, and fibroblasts can be well isolated from a single adult mouse simultaneously.

We developed a simple method for isolating high quality individual mouse heart cells by the antegrade perfusion technique. This method is Langendorff-free and useful for isolating ventricular and atrial myocytes or interstitial cells, such as cardiac fibroblasts or progenitors.

In basic research using mouse heart, isolating viable individual cardiomyocytes is a crucial technical step to overcome. Traditionally, isolating ...

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of inflammation. This phenomenon occurs in postcapillary venules, where blood flow pushes leukocytes in a rolling motion on the endothelial cells. Stable rolling requires a delicate balance between adhesion bond formation and their mechanically-driven dissociation, allowing the cell to remain attached to the surface while rolling in the direction of flow. Unlike other adhesion processes occurring in relatively static environments, rolling adhesion is highly dynamic as the rolling cells travel over thousands of microns at tens of microns per second. Consequently, conventional mechanobiology methods such as traction force microscopy are unsuitable for measuring the individual adhesion events and the associated molecular forces due to the short timescale and high sensitivity required. Here, we describe our latest implementation of the adhesion footprint assay to image the P-selectin: PSGL-1 interactions in rolling adhesion at the molecular level. This method utilizes irreversible DNA-based tension gauge tethers to produce a permanent history of molecular adhesion events in the form of fluorescence tracks. These tracks can be imaged in two ways: (1) stitching together thousands of diffraction-limited images to produce a large field of view, enabling the extraction of adhesion footprint of each rolling cell over thousands of microns in length, (2) performing DNA-PAINT to reconstruct super-resolution images of the fluorescence tracks within a small field of view. In this study, the adhesion footprint assay was used to study HL-60 cells rolling at different shear stresses. In doing so, we were able to image the spatial distribution of the P-selectin: PSGL-1 interaction and gain insight into their molecular forces through fluorescence intensity. Thus, this method provides the groundwork for the quantitative investigation of the various cell-surface interactions involved in rolling adhesion at the molecular level.

This protocol presents the experimental procedures to perform the adhesion footprint assay to image the adhesion events during fast cell rolling adhesion.

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of ...

Bioluminescent Monitoring of Graft Survival in an Adoptive Transfer Model of Autoimmune Diabetes in Mice

Type 1 diabetes is characterized by the autoimmune destruction of the insulin-producing beta cells of the pancreas. A promising treatment for this disease is the transplantation of stem cell-derived beta cells. Genetic modifications, however, may be necessary to protect the transplanted cells from persistent autoimmunity. Diabetic mouse models are a useful tool for the preliminary evaluation of strategies to protect transplanted cells from autoimmune attack. Described here is a minimally invasive method for transplanting and imaging cell grafts in an adoptive transfer model of diabetes in mice. In this protocol, cells from the murine pancreatic beta cell line NIT-1 expressing the firefly luciferase transgene luc2 are transplanted subcutaneously into immunodeficient non-obese diabetic (NOD)-severe combined immunodeficient (scid) mice. These mice are simultaneously injected intravenously with splenocytes from spontaneously diabetic NOD mice to transfer autoimmunity. The grafts are imaged at regular intervals via non-invasive bioluminescent imaging to monitor the cell survival. The survival of mutant cells is compared to that of control cells transplanted into the same mouse.

This protocol describes a straightforward and minimally invasive method for transplanting and imaging NIT-1 cells in non-obese diabetic (NOD)-severe combined immunodeficient mice challenged with splenocytes purified from spontaneously diabetic NOD mice.

Type 1 diabetes is characterized by the autoimmune destruction of the insulin-producing beta cells of the pancreas. A promising treatment for this disease ...

Establishing a Diaphyseal Femur Fracture Model in Mice

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age and overall health status of the patient, failures can occur, leading to delayed union or nonunion. Due to the increasing number of fractures resulting from high-energy trauma and aging, the development of innovative therapeutic strategies to improve bone repair based on the combination of skeletal/mesenchymal stem/stromal cells and biomimetic biomaterials is urgently needed. To this end, the use of reliable animal models is fundamental to better understanding the key cellular and molecular mechanisms that determine the healing outcomes. Of all the models, the mouse is the preferred research model because it offers a wide variety of transgenic strains and reagents for experimental analysis. However, the establishment of fractures in mice may be technically challenging due to their small size. Therefore, this article aims to demonstrate the procedures for the surgical establishment of a diaphyseal femur fracture in mice, which is stabilized with an intramedullary wire and resembles the most common bone repair process, through cartilaginous callus formation.

This protocol describes a surgical procedure for the establishment of a diaphyseal fracture in the femur of mice, which is stabilized with an intramedullary wire, for fracture healing studies.

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age ...