Name: establishing a diaphyseal femur fracture model in mice
Uploaded: 2022-12-09T14:30:00
Description: Watch this Scientific Journal Video about Establishing a Diaphyseal Femur Fracture Model in Mice at JoVE.com

This work was funded by the Carlos Chagas Filho Foundation for Research Support of the State of Rio de Janeiro (FAPERJ).

All the experiments were approved by the Animal Use and Care Committee of the Center for Health Sciences of the Federal University of Rio de Janeiro (Protocol Number 101/21). Male Balb/c mice at 10-12 weeks of age (25-30 g body weight) were used in this study. The surgical procedure takes approximately 15-20 min per mouse. Before each procedure, the required instruments (listed in the Table of Materials) must be organized over a sterile surgical field covering the operating table (Fi...

<ol>
	<li>Florencio-Silva, R., Sasso, G. R., Sasso-Cerri, E., Simoes, M. J., Cerri, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+of+bone+tissue:+Structure,+function,+and+factors+that+influence+bone+cells.">Biology of bone tissue: Structure, function, and factors that influence bone cells.</a> BioMed Research International. 2015, 421746 (2015).</li><li>Bahney, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+biology+of+fracture+healing.">Cellular biology of fracture healing.</a> Journal of Orthopedic Research. 37 (1), 35-50 (2019).</li><li>Einhorn, T. A., Gerstenfeld, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fracture+healing:+Mechanisms+and+interventions.">Fracture healing: Mechanisms and interventions.</a> Nature Reviews Rheumatology. 11 (1), 45-54 (2015).</li><li>Perren, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fracture+healing:+Fracture+healing+understood+as+the+result+of+a+fascinating+cascade+of+physical+and+biological+interactions.+Part+II.">Fracture healing: Fracture healing understood as the result of a fascinating cascade of physical and biological interactions. Part II.</a> Acta Chirurgiae Orthopaedicae et Traumatologiae Cechoslovaca. 82 (1), 13-21 (2015).</li><li>Giannoudis, P. V., Krettek, C., Lowenberg, D. W., Tosounidis, T., Borrelli, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fracture+healing+adjuncts-The+world's+perspective+on+what+works.">Fracture healing adjuncts-The world's perspective on what works.</a> Journal of Orthopaedic Trauma. 32, 43-47 (2018).</li><li>Kates, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outside+the+bone:+What+is+happening+systemically+to+influence+fracture+healing.">Outside the bone: What is happening systemically to influence fracture healing.</a> Journal of Orthopaedic Trauma. 32, 33-36 (2018).</li><li>Ding, Z. C., Lin, Y. K., Gan, Y. K., Tang, T. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence,+costs+and+predictors+of+non-union,+delayed+union+and+mal-union+following+long+bone+fracture.">Incidence, costs and predictors of non-union, delayed union and mal-union following long bone fracture.</a> Internation Journal of Environmental Research and Public Health. 15 (12), 2845 (2018).</li><li>Aziziyeh, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+burden+of+osteoporosis+in+four+Latin+American+countries:+Brazil,+Mexico,+Colombia,+and+Argentina.">The burden of osteoporosis in four Latin American countries: Brazil, Mexico, Colombia, and Argentina.</a> Journal of Medical Economics. 22 (7), 638-644 (2019).</li><li>Kostenuik, P., Mirza, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fracture+healing+physiology+and+the+quest+for+therapies+for+delayed+healing+and+nonunion.">Fracture healing physiology and the quest for therapies for delayed healing and nonunion.</a> Journal of Orthopaedic Research. 35 (2), 213-223 (2017).</li><li>Gomez-Barrena, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=fracture+healing:+cell+therapy+in+delayed+unions+and+nonunions.">fracture healing: cell therapy in delayed unions and nonunions.</a> Bone. 70, 93-101 (2015).</li><li>Schlundt, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+and+research+approaches+to+treat+non-union+fracture.">Clinical and research approaches to treat non-union fracture.</a> Current Osteoporosis Reports. 16 (2), 155-168 (2018).</li><li>Gomez-Barrena, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feasibility+and+safety+of+treating+non-unions+in+tibia,+femur+and+humerus+with+autologous,+expanded,+bone+marrow-derived+mesenchymal+stromal+cells+associated+with+biphasic+calcium+phosphate+biomaterials+in+a+multicentric,+non-comparative+trial.">Feasibility and safety of treating non-unions in tibia, femur and humerus with autologous, expanded, bone marrow-derived mesenchymal stromal cells associated with biphasic calcium phosphate biomaterials in a multicentric, non-comparative trial.</a> Biomaterials. 196, 100-108 (2018).</li><li>Ryan, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemically+impaired+fracture+healing+in+small+animal+research:+A+review+of+fracture+repair+models.">Systemically impaired fracture healing in small animal research: A review of fracture repair models.</a> Journal of Orthopedic Research. 39 (7), 1359-1367 (2021).</li><li>Marmor, M. 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As the number of fractures increases worldwide9,10,25, innovative treatments for nonunion are becoming increasingly urgent. As fracture healing involves a complex and tightly orchestrated summation of events that occur over a long timescale3, the use of valid animal models is central to improving our understanding of the mechanisms that determine the success of bone repair and to selecting effective drugs...

The skeleton is a vital and functionally versatile organ. The bones of the skeleton enable body posture and movement, protect the internal organs, produce hormones that integrate physiological responses, and are the site of hematopoiesis and mineral storage1. If fractured, bones have a remarkable capacity to regenerate and fully restore their pre-injury form and function. The healing process begins with the formation of a hematoma and an inflammatory response, which induces the activation and condensation of skeletal stem/progenitor cells from the periosteum, endosteum, and bone marrow and their subsequent differentiation to form the soft cartilaginous callus. The bridging of the fractured ends then occurs through a process that resembles endochondral bone formation, in which the cartilaginous scaffold expands and then mineralizes, forming the hard osseous callus. Finally, the hard callus is gradually remodeled by osteoclasts and osteoblasts to restore the original bone structure2,3.
Although the fracture healing process is fairly robust, it involves an intricate summation of events and is significantly influenced by several individual factors, including the general health status, age, and sex of the patient, as well as injury factors, such as the mode of mechanical stabilization of the fractured bone, the occurrence of infection, and the severity of the surrounding soft tissue injury4,5,6. Therefore, failures are common, leading to the development of nonunion, which greatly impacts patient rehabilitation and quality of life7,8. Due to the increasing number of fractures as a result of high-energy trauma and aging, as well as the high costs of treatments, nonunion fractures have become a burden for health systems worldwide9,10. This increasing burden highlights the urgent need for innovative therapeutic strategies to improve bone repair11,12 based on the combination of skeletal/mesenchymal stem/stromal cells and biomimetic biomaterials13,14.
In pursuit of this goal, animal models have been widely used in studies aiming to understand the fundamental biology of fracture healing mechanisms and in proof-of-concept preclinical studies aiming to devise new therapeutic strategies to promote bone repair15,16,17. Small-animal models, such as the mouse, are excellent for fracture healing studies because of the wide availability of genetically modified strains and reagents for experimental analyses and their low maintenance costs. Additionally, mice have a rapid healing time course, which allows for the temporal analysis of all stages of the repair process15. However, the small size of the animal can pose challenges for the surgical production of fractures with fixation modes similar to those applied in humans. This protocol describes a simple and low-cost model of fracture healing in mice using an open femoral osteotomy stabilized with an intramedullary wire, which resembles the most common bone repair process, through cartilaginous callus formation, and can be used both in basic and translational investigations in which access to the fracture site is required.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td>Alcohol 70&#186;</td>
			<td>Merck</td>
			<td>109-56-8</td>
			<td>Or any general available supplier</td>
		</tr>
		<tr>
			<td>Canada balsam (mounting medium)</td>
			<td>Merck</td>
			<td>C1795</td>
			<td>Or any general available supplier</td>
		</tr>
		<tr>
			<td>Cefazoline</td>
			<td>ABL</td>
			<td>Not applicable</td>
			<td>Similar brands of the item may be used according to local availability</td>
		</tr>
		<tr>
			<td>Coverslip</td>
			<td>Merck</td>
			<td>CSL284525</td>
			<td>Or any general available supplier</td>
		</tr>
		<tr>
			<td>Dental X-Ray Generator</td>
			<td>Focus</td>
			<td>-</td>
			<td>Sold by Instrumentarium Dental Inc.&#160;</td>
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			<td>DEPC water</td>
			<td>Merck</td>
			<td>W4502</td>
			<td>Or any general available supplier</td>
		</tr>
		<tr>
			<td>Dissecting Scissor</td>
			<td>ABC Instrumentos</td>
			<td>0327</td>
			<td>Similar brands of the item may be used according to local availability</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Vetec</td>
			<td>60REAVET014340</td>
			<td>Similar brands of the item may be used according to local availability</td>
		</tr>
		<tr>
			<td>Eosin solution</td>
			<td>Laborclin</td>
			<td>EA-65</td>
			<td>Similar brands of the item may be used according to local availability</td>
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			<td>Ethanol P.A</td>
			<td>Vetec</td>
			<td>60REAVET012053</td>
			<td>Similar brands of the item may be used according to local availability</td>
		</tr>
		<tr>
			<td>Gauze&#160;pads</td>
			<td>Cremer</td>
			<td>Not applicable</td>
			<td>Or any general available supplier</td>
		</tr>
		<tr>
			<td>Harris Hematoxylin Solution</td>
			<td>Laborclin</td>
			<td>620503</td>
			<td>Similar brands of the item may be used according to local availability</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Tonkey Electrical Technology</td>
			<td>E114273</td>
			<td>Similar brands of the item may be used according to local availability</td>
		</tr>
		<tr>
			<td>Histological slides</td>
			<td>Merck</td>
			<td>CSL294875X25</td>
			<td>Or any general available supplier</td>
		</tr>
		<tr>
			<td>Histology cassettes</td>
			<td>Merck</td>
			<td>H0542-1CS</td>
			<td>Or any general available supplier</td>
		</tr>
		<tr>
			<td>Hydrochloric acid - 37%</td>
			<td>Merck</td>
			<td>258148</td>
			<td>Similar brands of the item may be used according to local availability</td>
		</tr>
		<tr>
			<td>Insulin syringe</td>
			<td>BD</td>
			<td>324918</td>
			<td>Or any general available supplier</td>
		</tr>
		<tr>
			<td>Iodopovidone&#160;sponge</td>
			<td>Rioqu&#237;mica</td>
			<td>372106</td>
			<td>Or any general available supplier</td>
		</tr>
		<tr>
			<td>Ketamine hydrochloride</td>
			<td>Ceva</td>
			<td>Not applicable</td>
			<td>Similar brands of the item may be used according to local availability</td>
		</tr>
		<tr>
			<td>Lacribel collyrium</td>
			<td>Cristalia</td>
			<td>Not applicable</td>
			<td>Similar brands of the item may be used according to local availability</td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica</td>
			<td>149AUTO00C1</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Tooth Forceps Tweezer</td>
			<td>ABC Instrumentos</td>
			<td>0164</td>
			<td>Similar brands of the item may be used according to local availability</td>
		</tr>
		<tr>
			<td>Needle 26 G</td>
			<td>BD</td>
			<td>2239</td>
			<td>Or any general available supplier</td>
		</tr>
		<tr>
			<td>Needle Holder&#160;</td>
			<td>Golgran</td>
			<td>135-18</td>
			<td>Similar brands of the item may be used according to local availability</td>
		</tr>
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			<td>Nonresorbable Nylon Suture thread n&#186; 6</td>
			<td>Atramat</td>
			<td>C1546-NT</td>
			<td>Or any general available supplier</td>
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			<td>Paraffin</td>
			<td>Exodo</td>
			<td>8002 - 74 - 2</td>
			<td>Similar brands of the item may be used according to local availability</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>30525-89-4</td>
			<td>Similar brands of the item may be used according to local availability</td>
		</tr>
		<tr>
			<td>PBS 1x&#160;</td>
			<td>Lonza</td>
			<td>&#160;BE17-516F</td>
			<td>Similar brands of the item may be used according to local availability</td>
		</tr>
		<tr>
			<td>Resorbable Nylon Suture thread n&#186; 6</td>
			<td>Atramat</td>
			<td>C1596-45B</td>
			<td>Or any general available supplier</td>
		</tr>
		<tr>
			<td>Rod Wire SS CrNi 0.016&#34;</td>
			<td>Orthometric</td>
			<td>56.50.2016</td>
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		</tr>
		<tr>
			<td>Scalpel n&#186; 11</td>
			<td>Descarpak</td>
			<td>15782</td>
			<td>Or any general available supplier</td>
		</tr>
		<tr>
			<td>Serrated Tip Tweezer</td>
			<td>Quinelato</td>
			<td>QC.404.12</td>
			<td>Similar brands of the item may be used according to local availability</td>
		</tr>
		<tr>
			<td>Shaver</td>
			<td>Phillips</td>
			<td>Not applicable</td>
			<td>Similar brands of the item may be used according to local availability</td>
		</tr>
		<tr>
			<td>Surgical tape</td>
			<td>3M</td>
			<td>2734</td>
			<td>Or any general available supplier</td>
		</tr>
		<tr>
			<td>Surgical tnt field</td>
			<td>Polarfix</td>
			<td>6153</td>
			<td>Or any general available supplier</td>
		</tr>
		<tr>
			<td>Tramadol hydrochloride</td>
			<td>Teuto&#160;</td>
			<td>Not applicable</td>
			<td>Similar brands of the item may be used according to local availability</td>
		</tr>
		<tr>
			<td>Water bath for histology</td>
			<td>Leica</td>
			<td>HI1210</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine hydrochloride</td>
			<td>Ceva</td>
			<td>Not applicable</td>
			<td>Similar brands of the item may be used according to local availability</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Dinamica</td>
			<td>60READIN001105</td>
			<td>Similar brands of the item may be used according to local availability</td>
		</tr>
	</tbody>
</table>

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.

The most simple and immediate way to evaluate the success of the surgical procedure in producing the fracture is X-ray imaging. Radiographs can be performed immediately after surgery, with the mouse still under anesthesia, and subsequently 7 days, 14 days, and 21 days after the fracture to evaluate the callus formation and progression. Acceptable fracture patterns are those in which the cortices are fully ruptured, the wires are correctly placed within the medullary canal, and the fracture lines are transverse (with an a...

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Establishing a Diaphyseal Femur Fracture Model in Mice

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 ...

The diaphyseal femur fracture model shown here is a simple and low cost murine model, suited for basic and preclinical studies of long-bone fracture healing. The open surgery technique does not require complex surgical instruments and fixation devices. It allows the visual inspection of the fracture being produced.This model can be used for local transplantation and in vivo testing of therapeutic components, including stem cells, drugs and biomaterials that cannot be delivered by percutaneous, or systemic injection. The surgery protocol requires some knowledge of basic surgery techniques and of the mouse anatomy to perform the critical steps. The visual demonstration in this work can help even for those inexperienced with animal surgery.Demonstrating the procedure will be Leonardo Muller and Bianca Frade, both master students from the Laboratory of Stem Cells and Bone Repair. After transferring the anesthetized animal to the surgical table, disinfect the area to be incised by rubbing the skin with a 10%povidone iodine sponge. Then dry the rubbed area with sterile gauze pads, wash with 70%ethanol and dry again with a sterile gauze pad.Next, place the mouse in the right lateral decubitus position and drape the mouse, making only the incision region visible. Before surgery, place eyedrops in the mouse's eyes to avoid dryness and constantly check the mouse's breathing during the surgical procedure. Make a one centimeter cutaneous lateral parapatellar incision with a scalpel blade number 11, beginning at the level of the tibial tuberosity and extending to the level of the patella, and then for an equal distance towards the distal femur.With blunt end scissors, dissect the subcutaneous fascia around the incision line to expose the fascia lata, the lateral vastus, and the femoral biceps muscles. With the scalpel blade number 11, make another incision in the fascia lata, similar to the one made in the skin, beginning at the level of the tibial tuberosity and running along the biceps femoris aponeurosis until the level of the distal femur to open the articular capsule and access the knee joint. Perform a medial luxation of the patella by placing the tip of a straight serrated precision tip tweezer under it and pushing it to the side together with the patellar and quadriceps ligaments, thus exposing the condyles of the femur.Holding the femur with a serrated tip tweezer, flex the knee at 90 degrees and manually perforate the intramedullary canal of the femur through the intercondylar fossa with a 26-gauge hypodermic needle. Maintaining the knee flexed at 90 degrees, insert a segment of 1.0 centimeters, a 0.016 inch stainless steel rod wire through the opening into the medullary canal of the femur toward the great trochanter. Adjust the prevent distal extremity of the wire with a straight serrated tip tweezer to tightly fix it in the lateral condyle.Next, separate the lateral vastus and femoral biceps muscles through blunt-end dissection with a serrated tip tweezer to access the distal diaphysis of the femur. Insert a dissecting scissor around the femur diaphysis in an angle of approximately 90 degrees, and gently perform a complete cortical osteotomy. Reposition the muscles and the patella by pushing the tip of a straight serrated precision tip tweezer over the condyle region.Close the muscle fascia with a 6-0 reabsorbable suture, and then the skin using a 6-0 nylon suture, both in simple interrupted fashion. The radiographs of acceptable fracture patterns show transverse diaphyseal fractures in which the fracture lines are at a 90 degree angle to the axis of the bone, short oblique fractures in which the fracture line is less than 30 degrees relative to the axis of the bone, reducible fragmentary fractures, where a few small fragments of bone are seen, but the anatomical alignment of the bone remains. Representative radiographs of incorrectly placed wires are shown here where the wire is not inside the medullary canal of the proximal femur fragment, thus resulting in incorrect fixation of the fractured bone, and a case where the wire did not pass through any bone fragment and the fractured bone is completely unaligned.Visible callus at the fracture site on day 14 and day 21 after surgery indicates the regenerative process of the model. Histological analysis shows the evaluation of the callus at the fracture site. The callus, initially on day seven, presents extensive areas of highline cartilage around the fracture line.On day 14, ossification fronts are observed around the cartilage area, forming trabecular bone and cavities filled with reconstituted bone marrow. Finally, on day 21, the cartilage areas are almost completely replaced by trabecular bone, indicating successful bone bridging. The success of the model depends on the correct insertion of the wire.Therefore, it's critical to keep the knee flexed at a 90 degree angle during this step. Additionally, micro-computed demography can be used to evaluate cell's evolution and the characteristics of the bone being formed in a tri-dimensional and quantitative scale.

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Intravitreous Injection for Establishing Ocular Diseases Model

Intravitreous injection is a widely used technique in visual sciences research. It can be used to establish animal models with ocular diseases or as direct application of local treatment. This video introduces how to use simple and inexpensive tools to finish the intravitreous injection procedure. Use of a 1 ml syringe, instead of a hemilton syringe, is used. Practical tips for how to make appropriate injection needles using glass pipettes with perfect tips, and how to easily connect the syringe needle with the glass pipette tightly together, are given.
To conduct a good intravitreous injection, there are three aspects to be observed: 1) injection site should not disrupt retina structure; 2) bleeding should be avoided to reduce the risk of infection; 3) lens should be untouched to avoid traumatic cataract. In brief, the most important point is to reduce the interruption of normal ocular structure. To avoid interruption of retina, the superior nasal region of rat eye was chosen. Also, the puncture point of the needle was at the par planar, which was about 1.5 mm from the limbal region of the rat eye. A small amount of vitreous is gently pushed out through the puncture hole to reduce the intraocular pressure before injection. With the 45&deg; injection angle, it is less likely to cause traumatic cataract in the rat eye, thus avoiding related complications and influence from lenticular factors. In this operation, there was no cutting of the conjunctiva and ocular muscle, no bleeding. With quick and minor injury, a successful intravitreous injection can be done in minutes. 
The injection set outlined in this particular protocol is specific for intravitreous injection. However, the methods and materials presented here can also be used for other injection procedures in drug delivery to the brain, spinal cord or other organs in small mammals.

Intravitreous injection is a widely used technique in visual sciences research for ocular diseases or as direct application of local treatment. This video demonstrated a protocol for intravitreous injection using a 1ml syringe with glass pipette. Useful tips about avoiding massive bleeding and lens damage are given.

Intravitreous injection is a widely used technique in visual sciences research. It can be used to establish animal models with ocular diseases or as ...

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 ...

Fundamental Technical Elements of Freeze-fracture/Freeze-etch in Biological Electron Microscopy

Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated to generate a carbon/platinum &#8220;cast&#8221; intended for examination by transmission electron microscopy. Specimens are subjected to ultrarapid freezing rates, often in the presence of cryoprotective agents to limit ice crystal formation, with subsequent fracturing of the specimen at liquid nitrogen cooled temperatures under high vacuum. The resultant fractured surface is replicated and stabilized by evaporation of carbon and platinum from an angle that confers surface three-dimensional detail to the cast. This technique has proved particularly enlightening for the investigation of cell membranes and their specializations and has contributed considerably to the understanding of cellular form to related cell function. In this report, we survey the instrument requirements and technical protocol for performing freeze-fracture, the associated nomenclature and characteristics of fracture planes, variations on the conventional procedure, and criteria for interpretation of freeze-fracture images. This technique has been widely used for ultrastructural investigation in many areas of cell biology and holds promise as an emerging imaging technique for molecular, nanotechnology, and materials science studies.

Basic techniques and refinements of freeze-fracture processing of biological specimens and nanomaterials for examination by transmission electron microscopy are described. This technique is a preferred method for revealing ultrastructural features and specializations of biological membranes and for obtaining ultrastructural level dimensional and spatial data in materials sciences and nanotechnology products.

Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated ...

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 ...

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 Capsule-Based Model for Immature Hard Tick Stages Infestation on Laboratory Mice

Ticks are obligatory blood feeding parasites at all stages of development (except eggs) and are recognized as vectors of various pathogens. The use of mouse models in tick research is critical for understanding their biology and tick-host-pathogen interactions. Here we demonstrate a non-laborious technique for the feeding of immature stages of hard ticks on laboratory mice. The benefit of the method is its simplicity, short duration, and the ability to monitor or collect ticks at different time points of an experiment. In addition, the technique allows attachment of two individual capsules on the same mouse, which is beneficial for a variety of experiments where two different groups of ticks are required to feed on the same animal. The non-irritating and flexible capsule is made from easily accessible materials and minimizes the discomfort of the experimental animals. Furthermore, euthanasia is not necessary, mice recover completely after the experiment and are available for re-use.

In this study, a feeding system for nymphal and larval stages of hard tick was developed using a capsule attached to laboratory mouse. The feeding capsule is made from flexible materials and remains firmly attached to the mouse for at least one week and allows comfortable monitoring of tick feeding.

Ticks are obligatory blood feeding parasites at all stages of development (except eggs) and are recognized as vectors of various pathogens. The use of ...

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 ...

Tail Vein Transection Bleeding Model in Fully Anesthetized Hemophilia A Mice

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

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

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

Transverse Fracture of the Mouse Femur with Stabilizing Pin

Fracture repair is an essential function of the skeleton that cannot be reliably modeled in vitro. A mouse injury model is an efficient approach to test whether a gene, gene product or drug influences bone repair because murine bones recapitulate the stages observed during human fracture healing. When a mouse or human breaks a bone, an inflammatory response is initiated, and the periosteum, a stem cell niche surrounding the bone itself, is activated and expands. Cells residing in the periosteum then differentiate to form a vascularized soft callus. The transition from the soft callus to a hard callus occurs as the recruited skeletal progenitor cells differentiate into mineralizing cells, and the bridging of the fractured ends results in the bone union. The mineralized callus then undergoes remodeling to restore the original shape and structure of the healed bone. Fracture healing has been studied in mice using various injury models. Still, the best way to recapitulate this entire biological process is to break through the cross-section of a long bone that encompasses both cortices. This protocol describes how a stabilized, transverse femur fracture can be safely performed to assess healing in adult mice. A surgical protocol including detailed harvesting and imaging techniques to characterize the different stages of fracture healing is also provided.

This protocol describes a method to perform fractures on adult mice and monitor the healing process.

Fracture repair is an essential function of the skeleton that cannot be reliably modeled in vitro. A mouse injury model is an efficient approach to test ...

A Simple and Inexpensive Running Wheel Model for Progressive Resistance Training in Mice

Previously developed rodent resistance-based exercise models, including synergistic ablation, electrical stimulation, weighted-ladder climbing, and most recently, weighted-sled pulling, are highly effective at providing a hypertrophic stimulus to induce skeletal muscle adaptations. While these models have proven invaluable for skeletal muscle research, they are either invasive or involuntary and labor-intensive. Fortunately, many rodent strains voluntarily run long distances when given access to a running wheel. Loaded wheel running (LWR) models in rodents are capable of inducing adaptations commonly observed with resistance training in humans, such as increased muscle mass and fiber hypertrophy, as well as stimulation of muscle protein synthesis. However, the addition of moderate wheel load either fails to deter mice from running great distances, which is more reflective of an endurance/resistance training model, or the mice discontinue running nearly entirely due to the method of load application. Therefore, a novel high-load wheel running model (HLWR) has been developed for mice where external resistance is applied and progressively increased, enabling mice to continue running with much higher loads than previously utilized. Preliminary results from this novel HLWR model suggest it provides sufficient stimulus to induce hypertrophic adaptations over the 9 week training protocol. Herein, the specific procedures to execute this simple yet inexpensive progressive resistance-based exercise training model in mice are described.

This procedure describes a translatable progressive loaded running wheel resistance training model in mice. The primary advantage of this resistance training model is that it is entirely voluntary, thus reducing stress for the animals and the burden on the researcher.

Previously developed rodent resistance-based exercise models, including synergistic ablation, electrical stimulation, weighted-ladder climbing, and most ...