The study is supported in part by the Philanthropy Department of Mutuelles AXA through the Head and Heart Chair and the ANR BonyBrain. We acknowledge all the members of the platform SFR Necker INSERM US24, LEAT Imagine, Paris, France, for their contribution to the realization of the procedure.

This protocol follows the animal care guidelines of Imagine Institute and has been validated by ethical committees and the French ministry (APAFIS 26995).
1. Animals
<ol>
	<li>Determine the number of animals per group. We recommend 12 animals per group. Determine the sex and age of mice. This protocol is adapted for 6-week-old C57BL/6J mice (male or female).</li>
	<li>Give animals, at least 1 week before the procedure, nutritional supplement gel for acclimation.</li>
</ol>
2. Surgical procedure
<ol>
	<li>Prepare the material required. Autoclave the instruments: scissors, needle driver, forceps, insert and handpiece of the piezoelectric bone surgery device, and elevator. Prepare sterile gloves, silk sutures (4/0), shaver, cotton gauze, 10% povidone-iodine solution, heating table, isoflurane, and the snout mask for isoflurane inhalation.</li>
	<li>Prepare buprenorphine at 0.1 mg/kg, diluted in sterile injectable physiological serum. Administer buprenorphine by subcutaneous injection with a 25G needle 30 min before surgery.</li>
	<li>Prepare the heating plate (37 &#176;C), place foam support on it, and fix the snout mask with small needles or adhesive tape.</li>
	<li>Place the mouse in the isoflurane tank for induction, with isoflurane diffusion (4%). Test anesthesia depth by performing a toe pinch. In case of sufficient induction, a lack of pedal reflex will be observed.</li>
	<li>Place the anesthetized mouse on its back on the heating plate covered by a sterile field kept at 37 &#176;C during the entire procedure. Position the mask on the snout for continuous isoflurane inhalation (maintenance at 2.5%). Fix the mouse extremities with adhesive tape.</li>
	<li>Apply eye lubricant bilaterally to prevent eye dryness induced by general anesthesia. Shave the submandibular hair, wear sterile gloves, and disinfect the skin with several swipes of 10% povidone-iodine solution and alcohol.</li>
	<li>Perform the submandibular cutaneous approach as described below.
	<ol>
		<li>Perform a skin incision from the mandibular angle to the 1/3 posterior - 2/3 anterior junction of the horizontal branch of the mandible with micro scissors.</li>
		<li>Dissect the subcutaneous tissues to expose the masseter muscle.</li>
	</ol>
	</li>
	<li>Disinsert the masseter muscle attaches with the use of a periosteal elevator from the inferior border of the mandible.</li>
	<li>Elevate the masseter muscle in a subperiosteal plane along the mandibular ramus with the elevator.</li>
	<li>Create a small osteotomy (3 mm length) at the inferior border of the mandible using the Piezoelectric bone surgery device under saline serum irrigation (10 mL/min maximum) at the inferior border of the mandible, anteriorly to the mandibular angle, in the posterior part of the concavity (Figure 1, Figure 2).</li>
	<li>Locate the posterior border of the mandible and the coronoid notch (with the micro scissors or the forceps) and complete the fracture with the use of small straight scissors inserted along the mandibular ramus to avoid additional masseter muscle aperture and injury. To allow correct orientation of the fracture, place the scissors perpendicularly to the inferior border of the mandible.</li>
	<li>Verify that the fracture is totally complete: total mobility between the two bone segments should be observed during soft mobilization of the 2 segments with forceps.</li>
	<li>Close the skin approach with silk sutures (4/0) with separated sutures. No suture is needed in the periosteum. 
	NOTE: Alternatively, the skin approach may be sutured with other non-adsorbable sutures (e.g., Polypropylene sutures).</li>
	<li>Perform cephalic lateral X-rays to confirm the direction of the fracture (Figure 3).
	<ol>
		<li>Place the mouse again in the isoflurane tank for short reinduction before performing the X-rays. Then, place the mouse in extension, on the lateral side, with the head slightly in hyperextension for X-ray analysis.</li>
		<li>In case of fracture too anteriorly placed at the level of a mandibular molar or when the fracture detached the mandibular angle (Figure 4), observe an abnormal increase in the volume of the bone callus. In these cases, exclude the animals from the study to avoid any bias in analysis.</li>
	</ol>
	</li>
	<li>Check the mice until they regain consciousness before placing them back in the cage.</li>
</ol>
3. Post-operative care
<ol>
	<li>Prepare a mouse cage without kibbles and hard enrichment to avoid any masticatory forces.</li>
	<li>Isolate the operated mouse in a single cage for the 1st week until the skin has completely healed. At 1 week post-operatively, group operated female mice together in the same cage.</li>
	<li>Administer buprenorphine (0.1 mg/kg) by subcutaneous injections at 4 h, 24 h, 48 h, and 72 h after the procedure.</li>
	<li>Feed mice with soft diet gel during all post-operative periods from day 0 to the end of the consolidation period (i.e., 28 days) to avoid pain and secondary displacement of the fracture.</li>
	<li>Daily weigh the mice from day 0 to day 4 post fracture, in the morning, before buprenorphine injection, and then 3x per week until animal sacrifice (14 days post-fracture).</li>
	<li>Evaluate post-operative animal well-being to optimize post-operative animal care. Use the score scale in Table 1 to evaluate the postoperative state of the animal and adapt the follow-up. When the score is &#62; 1, buprenorphine injection is given, and when the score is &#62; 3 and/or in case of weight loss &#62; 20% of preoperative weight despite food enrichment, euthanasia must be discussed (Table 1).</li>
</ol>

Standardized Mandibular Fracture Protocol for Bone Repair in Mice

<ol>
	<li>Thompson, Z., Miclau, T., Hu, D., Helms, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+for+intramembranous+ossification+during+fracture+healing.">A model for intramembranous ossification during fracture healing.</a> J Ortho Res. 20 (5), 1091-1098 (2002).</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> J Ortho Res. 37 (1), 35-50 (2019).</li><li>Yu, Y. Y., Lieu, S., Hu, D., Miclau, T., Colnot, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site+specific+effects+of+zoledronic+acid+during+tibial+and+mandibular+fracture+repair.">Site specific effects of zoledronic acid during tibial and mandibular fracture repair.</a> PLoS One. 7 (2), e31771 (2012).</li><li>Landry, P. S., Marino, A. A., Sadasivan, K. K., Albright, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+soft-tissue+trauma+on+the+early+periosteal+response+of+bone+to+injury.">Effect of soft-tissue trauma on the early periosteal response of bone to injury.</a> J Trauma. 48 (3), 479-483 (2000).</li><li>Colnot, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+cell+fate+decisions+within+periosteum+and+bone+marrow+during+bone+regeneration.">Skeletal cell fate decisions within periosteum and bone marrow during bone regeneration.</a> J Bone Miner Res. 24 (2), 274-282 (2009).</li><li>Julien, A., 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=Direct+contribution+of+skeletal+muscle+mesenchymal+progenitors+to+bone+repair.">Direct contribution of skeletal muscle mesenchymal progenitors to bone repair.</a> Nat Commun. 12 (1), 2860 (2021).</li><li>McGuire, C., Boudreau, C., Prabhu, N., Hong, P., Bezuhly, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Piezosurgery+versus+conventional+cutting+techniques+in+craniofacial+surgery:+A+systematic+review+and+meta-analysis.">Piezosurgery versus conventional cutting techniques in craniofacial surgery: A systematic review and meta-analysis.</a> Plast Reconstr Surg. 149 (1), 183-195 (2022).</li><li>Wong, S. A., 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=Chondrocyte-to-osteoblast+transformation+in+mandibular+fracture+repair.">Chondrocyte-to-osteoblast transformation in mandibular fracture repair.</a> J Orthop Res. 39 (8), 1622-1632 (2021).</li><li>Vercellotti, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technological+characteristics+and+clinical+indications+of+piezoelectric+bone+surgery.">Technological characteristics and clinical indications of piezoelectric bone surgery.</a> Minerva Stomatol. 53, 207-214 (2004).</li><li>Wong, S. A., 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=Chondrocyte-to-osteoblast+transformation+in+mandibular+fracture+repair.">Chondrocyte-to-osteoblast transformation in mandibular fracture repair.</a> J Orthop Res. 39 (8), 1622-1632 (2021).</li><li>Tian, Y., 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=HIF-1&#945;+regulates+osteoclast+activation+and+mediates+osteogenesis+during+mandibular+bone+repair+via+CT-1.">HIF-1&#945; regulates osteoclast activation and mediates osteogenesis during mandibular bone repair via CT-1.</a> Oral Dis. 28 (2), 428-441 (2022).</li></ol>

The authors declare no conflicts of interest.

The technique allows the realization of non-stabilized mandibular fractures in a mouse model within a minimal open approach in a short procedure time (around 10 min or less). This short-time procedure limits the risk of animal morbidity and death, especially in small animals such as mice. To obtain comparable results in the analysis of the endochondral bone repair process, particular care should be taken to perform the mandibular fractures in the same direction without abnormal split of the mandibular angle, as this unde...

Bone repair implies both membranous and endochondral ossification processes, depending on the fracture or osteotomy stabilization. Bone repair following stabilized bone osteotomies or fractures relies on membranous ossification, whereas non-stabilized fractures or poorly stabilized osteotomies consolidate according to a mode of endochondral ossification1. Membranous bone repair is characterized by bone formation by direct differentiation of mesenchymal cells into osteoblasts, whereas endochondral bone repair involves the formation of a transient cartilaginous template, which differentiates into osteoblasts to form the bone callus2.
Bone repair depends on multiple tissue and cellular interactions; it is also influenced by site-specific factors and by the force exerted on the callus3. Mandibular bone is a mobile bone supporting masticatory forces, mostly formed by cortical bone. The process of mandibular bone repair is influenced by the site and the direction of the fracture. It is well known that muscular and periosteum injuries may impact bone consolidation as well as important bone loss at fracture site4,5,6.
Analyzing endochondral mandibular bone repair implies standardized and reproducible protocols for valuable, non-biased comparisons. Regarding literature, only a few authors report protocols for non-stabilized mandibular fractures in mice models3,7, and mostly with an anterior direction at molar level8. By developing this technique, we aim to avoid muscular and periosteum injuries and limit bone loss at the fracture site to best analyze the process of endochondral bone repair without confounding factors. In addition, the purpose of developing a new technique for mandibular fracture in mice was to minimize post-operative pain and animal death frequent in such surgery in small animals. For the first time, we describe a less-invasive protocol for non-stabilized mandibular fracture at the ramus level in mice. This in vivo protocol avoids excessive muscular and periosteum damage or bone loss at the fracture site and helps reduce the level of postoperative pain and the risk of death of the animal.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Buprenorphine : Buprecare 0.3 mg/kg</td>
			<td>Animalcare</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Elevator OBWEGESER width 6mm Length 17,5cm&#160;</td>
			<td>Collin Medical</td>
			<td>HA 5905</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps : Pad Plate MORIA 11 cm N&#176;5</td>
			<td>MORIA</td>
			<td>9906</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating table Bioseblab 55 cm x 33 cm</td>
			<td>Bioseblab</td>
			<td>707</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle HOLDER : Micro Halsey Needle Holder - Metal&#160;</td>
			<td>LABODERM</td>
			<td>21,100</td>
			<td></td>
		</tr>
		<tr>
			<td>Neo Clear&#160;</td>
			<td>Merck Millipore, Darmstadt, Germany</td>
			<td>109843&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Ocry Gel Tube 10 g (eye lubricant)</td>
			<td>tvm lab&#160;&#160;</td>
			<td>3.70045E+12</td>
			<td></td>
		</tr>
		<tr>
			<td>Piezotome 2 ASS FINAL</td>
			<td>ACTEON</td>
			<td>X57402</td>
			<td></td>
		</tr>
		<tr>
			<td>Piezotome insert Piezocision PZ3</td>
			<td>ACTEON</td>
			<td>F87574</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors micro&#160;</td>
			<td>MORIA</td>
			<td>MC52</td>
			<td></td>
		</tr>
		<tr>
			<td>Silk sutures PERMA HAND SEIDE&#160; / MERSILK</td>
			<td>ETHICON</td>
			<td>REF 18501G&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight scissors</td>
			<td>MORIA</td>
			<td>4877A</td>
			<td></td>
		</tr>
		<tr>
			<td>Vetflurane (isoflurane) 250 ml</td>
			<td>VIRBAC</td>
			<td>VET066 (Centravet)</td>
			<td></td>
		</tr>
	</tbody>
</table>

less-invasive technique for non-stabilized mandibular fracture in mouse models

Non-stabilized fractures can be made at mandibular sites in mice, thus making it possible to analyze bone repair using an endochondral ossification mode. To most accurately reflect this process in vivo, it is necessary to have a standardized protocol to avoid excessive bone loss and soft tissue damage, particularly at the mandibular site, an anatomical site characterized by minimal access. To our knowledge, we describe for the first time a less-invasive protocol of non-stabilized mandibular fracture in mice. Adult mice are anesthetized with isoflurane and receive a preoperative dose of buprenorphine by subcutaneous injection. A submandibular approach is performed, with a skin incision made along the inferior border of the mandible. The masseter muscle is elevated in a subperiosteal plane along the mandibular ramus with a periosteal elevator. A vertical and complete fracture of the ramus is performed from the basilar border to the coronoid notch (between condylar and coronoid processes), using a piezoelectric bone surgery device under saline serum irrigation at the basilar edge and scissors to complete the fracture of the mandibular ramus. The skin approach is closed by silk sutures. This detailed procedure for non-stabilized mandibular fractures offers an efficient procedure allowing minimal bone loss and soft tissue damage. This technique ensures successful and consistent results to accurately reflect the process of endochondral bone repair in mouse models.

As for in vivo surgical procedures, to optimally perform this procedure of non-stabilized mandibular fracture in a mouse model, specific learning and training are required.
This technique for non-stabilized mandibular fracture allows the study of the endochondral bone repair process. Following euthanasia, mandibular samples were collected in 4% paraformaldehyde (24 h). After full decalcification with EDTA pH 8, sections were embedded in paraffin. Sagittal serial sections were performe...

Less-Invasive Technique for Non-stabilized Mandibular Fracture in Mouse Models

This protocol presents a comprehensive and efficient method for analyzing mandibular bone repair. We describe a reproducible technique for non-stabilized mandibular fracture in a mouse model, allowing analysis of the process of endochondral bone repair with minimal tissue damage and bone loss.

Non-stabilized fractures can be made at mandibular sites in mice, thus making it possible to analyze bone repair using an endochondral ossification mode. ...

less-invasive-technique-for-non-stabilized-mandibular-fracture-mouse

Research

JoVE Journal

Biology

240 Views.  Université de Paris Cité. This protocol presents a comprehensive and efficient method for analyzing mandibular bone repair. We describe a reproducible technique for non-stabilized mandibular fracture in a mouse model, allowing analysis of the process of endochondral bone repair with minimal tissue damage and bone loss. 

Video: Less-Invasive Technique for Non-stabilized Mandibular Fracture in Mouse Models

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Principles of Rodent Surgery for the New Surgeon

For both scientific and animal welfare reasons, training in basic surgical concepts and techniques should be undertaken before ever seeking to perform surgery on a rodent. Students, post-doctoral scholars, and others interested in performing surgery on rodents as part of a research protocol may not have had formal surgical training as part of their required coursework. Surgery itself is a technical skill, and one that will improve with practice. The principles of aseptic technique, however, often remain unexplained or untaught. For most new surgeons, this vital information is presented in piecemeal fashion or learned on the job, neither of which is ideal. It may also make learning how to perform a particular surgery difficult, as the new surgeon is learning both a surgical technique and the principles of asepsis at the same time. This article summarizes and makes recommendations for basic surgical skills and techniques necessary for successful rodent surgery. This article is designed to supplement hands-on training by the user's institution.

Before attempting surgery, a new surgeon should have training in basic surgical techniques and concepts. This article will present basic surgical considerations with an emphasis on rodents.

For both scientific and animal welfare reasons, training in basic surgical concepts and techniques should be undertaken before ever seeking to perform ...

Manual Restraint and Common Compound Administration Routes in Mice and Rats

Being able to safely and effectively restrain mice and rats is an important part of conducting research. Working confidently and humanely with mice and rats requires a basic competency in handling and restraint methods. This article will present the basic principles required to safely handle animals. One-handed, two-handed, and restraint with specially designed restraint objects will be illustrated. Often, another part of the research or testing use of animals is the effective administration of compounds to mice and rats. Although there are a large number of possible administration routes (limited only by the size and organs of the animal), most are not used regularly in research. This video will illustrate several of the more common routes, including intravenous, intramuscular, subcutaneous, and oral gavage. The goal of this article is to expose a viewer unfamiliar with these techniques to basic restraint and substance administration routes. This video does not replace required hands-on training at your facility, but is meant to augment and supplement that training.

Working safely and humanely with research rodents requires a core competency in handling and restraint methods. This article will present the basic principles required to safely handle and effectively administer compounds to mice and rats.

Being able to safely and effectively restrain mice and rats is an important part of conducting research.  Working confidently and humanely with mice and ...

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts1. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane2. At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy2. At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups 8. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

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 ...

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.
This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney&#39;s preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

A Morphometric and Cellular Analysis Method for the Murine Mandibular Condyle

The temporomandibular joint (TMJ) has the capacity to adapt to external stimuli, and loading changes can affect the position of condyles, as well as the structural and cellular components of the mandibular condylar cartilage (MCC). This manuscript describes methods for analyzing these changes and a method for altering the loading of the TMJ in mice (i.e., compressive static TMJ loading). The structural evaluation illustrated here is a simple morphometric approach that uses the Digimizer software and is performed in radiographs of small bones. In addition, the analysis of cellular changes leading to alterations in collagen expression, bone remodeling, cell division, and proteoglycan distribution in the MCC is described. The quantification of these changes in histological sections - by counting the positive fluorescent pixels using image software and measuring the distance mapping and stained area with Digimizer - is also demonstrated. The methods shown here are not limited to the murine TMJ, but could be used on additional bones of small experimental animals and in other regions of endochondral ossification.

This manuscript presents methods for analyzing morphometric and cellular changes within the mandibular condyle of rodents.

The temporomandibular joint (TMJ) has the capacity to adapt to external stimuli, and loading changes can affect the position of condyles, as well as the ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

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 ...

Less-Invasive Technique for Non-stabilized Mandibular Fracture in Mouse Models

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