The authors thank Ms. Makiko Kato for providing encouragement to complete this study. We also thank the Division of Experimental Animals and Medical Research Engineering, Nagoya University Graduate School of Medicine, for the housing of mice.

All experiments were carried out in accordance with protocols approved by the Animal Care and Use Committee of our institution. Sterilize all instruments prior to the procedure.
1. Preparation of a Mold for Creating the Custom-made Distractor
<ol>
	<li>Make two incomplete rings (outer diameter, 20 mm; inner diameter, 10 mm), which are a part of the distractor, with one sheet of paraffin wax (145 mm x 74 mm) using an Evans wax carver.
	<ol>
		<li>Make 4 of the same pieces. Use a wax spatula heated with a gas burner. Stack the four rings for a thickness of 5 mm. Provide space for the expansion screw and needle, 8 mm x 2 mm and 5 mm x 2 mm respectively with the Evans wax carver (Figures 1A, 1B).</li>
	</ol>
	</li>
	<li>Embed wax patterns in the silicone impression material and create a mold for the resin rings (Figure 1C).</li>
	<li>After curing the silicone impression material (approximately 5 min at room temperature), remove the wax patterns.</li>
</ol>
2. Production of the Custom-made Distractor
<ol>
	<li>Apply petroleum jelly thinly and evenly to the silicone mold. Mix 3 g of polymerization dental resin and immediately pour it into the silicone mold. Set for 5 min at room temperature (RT).</li>
	<li>Remove the polymerized resin ring from the mold and polish it with a carbide bar and a dental micromotor to remove the burr (about 1 min). Two resin rings are required for each custom-made distractor.
	<ol>
		<li>Repeat the procedure to create the required number of resin rings (Figure 1D).</li>
	</ol>
	</li>
	<li>Leave a gap of about 2 mm to fix the resin rings with utility wax and secure the outside of the expansion screw completely with transparent resin. Set the resin for 5 min at RT. When polymerization is complete, remove the expansion screw knob (Figure 1E).</li>
</ol>
3. Surgical Protocol
<ol>
	<li>Use 8 week-old male mice.</li>
	<li>Induce anesthesia with an intraperitoneal injection of medetomidine hydrochloride at 0.3 mg/g, midazolam at 4 mg/kg, and butorphanol tartrate at 5 mg/kg of body weight.
	<ol>
		<li>Carefully shave and disinfect the surgical area with 10% iodine solution, and then, administer 0.5% lidocaine hydrochloride at the right lower limb.</li>
	</ol>
	</li>
	<li>Make a longitudinal skin incision (approximately 15 mm in length) at the right lower leg with a No. 15 scalpel. Bluntly separate the underlying muscles, taking care not to remove all of the periosteum. Approach from the outside to easily reach the fibula. Cut the fibula with scissors.</li>
	<li>Grasp the ankle with narrow thin sterile forceps, and use a sterile 27 G needle to make a hole in the bone about 5 mm from the heel. The needle should penetrate the skin, bone, and skin in that order. When the needle penetrates, cut the tip and root with a nipper so that the needle is about 15 mm.
	<ol>
		<li>Make another hole in the same manner, about 2&#8211;3 mm proximal. Hold around the ankle and pass two 25 G needles under the knee in the same manner. After the needles penetrate, cut the tips and roots with a nipper (Figure 1F).</li>
	</ol>
	</li>
	<li>Place the custom-made distractor such that it is parallel to the extension direction. Fix the needles and device with enough polymerization dental resin to fill the grooves of the device. Wait for the polymerization to complete (approximately 5 min) (Figure 1G).</li>
	<li>Be careful not to damage the surrounding tissues. Cut the middle of the diaphysis of the tibia using a very thin cutting disc while applying a saline solution (1&#8211;2 mL).</li>
	<li>Close the wound with a 4-0 nylon suture.</li>
	<li>Give subcutaneous injections of buprenorphine (0.1 mg/kg) for analgesia immediately after operation. Continue buprenorphine every 12 h through postoperative day 7 and as needed thereafter.</li>
</ol>
4. Distraction Protocol
NOTE: There are various reports on the latency period and distraction rate, but here, representative protocols are shown.
<ol>
	<li>After a latency period of 5 days, start distraction at a rate of 0.2 mm/12 h. For extension, use the pin attached to the rapid expansion screw. Move the pin in the direction of the yellow arrow attached to the extension screw (Figure 1E).</li>
	<li>Anesthesia is not necessary during extension. Hold the tail with the little finger and palm, and fix the extension device with the forefinger and thumb.</li>
	<li>Perform extension (0.2 mm for 1/4 turn). Continue lengthening for 8 days, which will result in a total gap of 3.2 mm.</li>
</ol>
5. Analysis
<ol>
	<li>Radiography analysis: After completion of extension, evaluate bone regeneration with computed tomography under general anesthesia by using 2.0% isoflurane. 
	NOTE: If care has been taken with regard to the position of the extension device, it is possible to evaluate it with simple radiography even with the device attached.</li>
	<li>Histological analysis: Remove the apparatus carefully so as not to cause a fracture distraction site during sampling. Fix the sample in 10% neutral buffered formalin for 24 h at room temperature. Decalcify with Morse&#39;s solution (10% sodium citrate, 20% formic acid) overnight at 4 &#176;C.</li>
</ol>

<ol>
	<li>Watson, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distraction+osteogenesis.">Distraction osteogenesis.</a> The Journal of the American Academy of Orthopaedic Surgeons. 14, 168-174 (2006).</li><li>Ilizarov, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tension-stress+effect+on+the+genesis+and+growth+of+tissues:+Part+II.+The+influence+of+the+rate+and+frequency+of+distraction.">The tension-stress effect on the genesis and growth of tissues: Part II. The influence of the rate and frequency of distraction.</a> Clinical Orthopaedics and Related Research. 239, 263-285 (1989).</li><li>Wan, 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=Activation+of+the+hypoxia-inducible+factor-1+alpha+pathway+accelerates+bone+regeneration.">Activation of the hypoxia-inducible factor-1 alpha pathway accelerates bone regeneration.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (2), 686-691 (2008).</li><li>Fujio, M., 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=Stromal+cell-derived+factor-1+enhances+distraction+osteogenesis-mediated+skeletal+tissue+regeneration+through+the+recruitment+of+endothelial+precursors.">Stromal cell-derived factor-1 enhances distraction osteogenesis-mediated skeletal tissue regeneration through the recruitment of endothelial precursors.</a> Bone. 49 (4), 693-700 (2011).</li><li>Tong, 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=Focal+adhesion+kinase+expression+during+mandibular+distraction+osteogenesis:+evidence+for+mechanotransduction.">Focal adhesion kinase expression during mandibular distraction osteogenesis: evidence for mechanotransduction.</a> Plastic and reconstructive surgery. 111 (1), 211-222 (2003).</li><li>Rhee, S. T., El-Bassiony, L., Buchman, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+signal-+related+kinase+and+bone+morphogenetic+protein+expression+during+distraction+osteogenesis+of+the+mandible:+in+vivo+evidence+of+mechanotransduction+mechanism+for+differentiation+and+osteogenesis+by+mesenchymal+precursor+cells.">Extracellular signal- related kinase and bone morphogenetic protein expression during distraction osteogenesis of the mandible: in vivo evidence of mechanotransduction mechanism for differentiation and osteogenesis by mesenchymal precursor cells.</a> Plastic and reconstructive surgery. 117 (7), 2243-2249 (2006).</li><li>Isefuku, S., Joyner, C. J., Simpson, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+murine+model+of+distraction+osteogenesis.">A murine model of distraction osteogenesis.</a> Bone. 27 (5), 661-665 (2000).</li><li>Tay, B. K., Le, A. X., Gould, S. E., 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=Histochemical+and+molecular+analyses+of+distraction+osteogenesis+in+a+mouse+model.">Histochemical and molecular analyses of distraction osteogenesis in a mouse model.</a> Journal of Orthopaedic Research. 16 (5), 636-642 (1998).</li><li>Carvalho, R. 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=The+role+of+angiogenesis+in+a+murine+tibial+model+of+distraction+osteogenesis.">The role of angiogenesis in a murine tibial model of distraction osteogenesis.</a> Bone. 34 (5), 849-861 (2004).</li><li>Osawa, 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=Activated+FGFR3+promotes+bone+formation+via+accelerating+endochondral+ossification+in+mouse+model+of+distraction+osteogenesis.">Activated FGFR3 promotes bone formation via accelerating endochondral ossification in mouse model of distraction osteogenesis.</a> Bone. 105, 42-49 (2017).</li></ol>

The authors have nothing to disclose.

When a large animal is used as an experimental model, a ready-made extension device can be used, and it is easy to obtain good fixation and assess the extension operation itself and the extension amount. However, when a mouse is used as an experimental model, it is necessary to develop some or all of the equipment. Isefuku et al. and Tay et al. made the device and created a mouse model7,8. Carvahjo et al. adopted a method involving fixa...

Distraction osteogenesis (DO) is an established treatment method for a variety of skeletal disorders, such as limb length discrepancies, bone defects, and limb deformities1. This unique treatment strategy is based on the &#34;tension-stress principle&#34; proposed by Ilizarov. The method requires several days for latency, several weeks for active distraction, and several months for consolidation until the mature bone is formed2.
The local hypoxic conditions due to blockage of blood flow3,4 and mechanical stimulation5,6 are particularly important in the healing process of DO. Hypoxia-induced angiogenesis carries oxygen, nutrients, soluble factors and cells necessary for tissue repair locally through the blood flow. Mechanical stimulation by extension operation causes biological reactions such as differentiation of mesenchymal stem cells, bone formation, calcification and remodeling. Serial DO treatment allows the formation of not only hard tissues but also soft tissues, including nerves, muscles, blood vessels, and skin tissues, without the need for stem cell transplantation. Therefore, a DO model is considered to be an excellent model for analyzing the regeneration of various tissues.
Rabbits and dogs are the most widely used animals in basic research for DO; however, there are few analysis tools available for these animals. The use of a mouse DO model facilitates a more detailed analysis. It is particularly suitable for experiments using knockout mice. However, when using a mouse as an experimental animal, an extension device should be created. Here, we present a mouse tibial DO model developed using a custom-made distractor created using a dental laboratory tool and technique, which has been used in a previous study.

<table border="0" cellpadding="0" cellspacing="0" style="width:771px;" width="772">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:183px;">Paraffin wax</td>
			<td style="width:203px;">YAMAHACHI DENTAL MFG. CO.</td>
			<td style="width:147px;">-</td>
			<td style="width:239px;">For preparation a mold for resin rings</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">Labocone putty</td>
			<td style="width:203px;">GC Corporation</td>
			<td>-</td>
			<td>For preparation a mold for resin rings</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">Utility wax</td>
			<td style="width:203px;">GC Corporation</td>
			<td>-</td>
			<td>For preparation a mold for resin rings</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">Expansion screw</td>
			<td style="width:203px;">Ortho Dentaurum</td>
			<td style="width:147px;">600-301-30</td>
			<td>Component of custom-made distractor</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:183px;">Unifast III</td>
			<td style="width:203px;">GC Corporation</td>
			<td>-</td>
			<td style="width:239px;">Immediate polymerization resin Component of custom-made distractor</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:183px;">Ortho Crystal</td>
			<td style="width:203px;">NISSIN</td>
			<td>-</td>
			<td style="width:239px;">Transparent resin Component of custom-made distractor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">25-gauge needle</td>
			<td style="width:203px;">TERUMO</td>
			<td style="width:147px;">NN-2516R</td>
			<td>For custom-made distractor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">27-gauge needle</td>
			<td style="width:203px;">TERUMO</td>
			<td style="width:147px;">NN-2719S</td>
			<td>For custom-made distractor</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:183px;">ICR mouse</td>
			<td style="width:203px;">Chubu Kagaku Shizai Corporation</td>
			<td>-</td>
			<td>Experimental animal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">Somnopentyl</td>
			<td style="width:203px;">Kyoritsu Seiyaku</td>
			<td>-</td>
			<td>Pentobarbital sodium salt</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:183px;">Isoflurane</td>
			<td style="width:203px;">FUJIFILM Wako Pure Chemical Corporation</td>
			<td style="width:147px;">099-06571</td>
			<td>Isoflurane inhalation solution</td>
		</tr>
	</tbody>
</table>

a mouse distraction osteogenesis model

Distraction osteogenesis (DO) is a surgical procedure that involves skeletal tissue regeneration without cell transplantation. A DO model consists of the following three phases: the latency phase after osteotomy and placement of the external distractor; the distraction phase, wherein the separated bone ends are gradually and continuously distracted; and the consolidation phase. This custom-made distractor used for DO is comprised of two incomplete acrylic resin rings and an expansion screw. The process was initiated by making a mold with silicone impression material and then creating the custom-made distractor. Dental resin was poured into the formwork made of silicone impression material, and it was allowed to polymerize to create the incomplete resin rings required for the custom-made distractor. These rings were fixed with an expansion screw using transparent resin. The custom-made distractor created via this approach was attached to the tibia of mice. The tibia was fixed to the device using one pair of 25 G needles proximally, one pair of 27 G needles distally, and acrylic resin. After a latency period of 5 days, distraction was initiated at a rate of 0.2 mm/12 h. The lengthening was continued for 8 days, resulting in a total gap of 3.2 mm. The mice were sacrificed 4 weeks after distraction. Bone formation in the distraction gap was confirmed using both radiography and histology.

Figures 1A and 1B present incomplete rings (outer diameter, 20 mm; inner diameter, 10 mm; thickness, 5 mm) with paraffin wax. Two wax patterns were embedded in silicone impression material, and a mold for the resin rings (Figure 1C) was formed. Polymerized resin was immediately poured into this mold, and resin rings were obtained (Figure 1D). A custom-made distra...

Watch this Scientific Journal Video about A Mouse Distraction Osteogenesis Model at JoVE.com

A Mouse Distraction Osteogenesis Model

We present a mouse tibial distraction osteogenesis model developed using a custom-made distractor. The use of a mouse as an analysis target is advantageous for advancing research.

Distraction osteogenesis (DO) is a surgical procedure that involves skeletal tissue regeneration without cell transplantation. A DO model consists of the ...

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Developmental Biology

6.2K Views.  Nagoya University Graduate School of Medicine. We present a mouse tibial distraction osteogenesis model developed using a custom-made distractor. The use of a mouse as an analysis target is advantageous for advancing research.

Video: A Mouse Distraction Osteogenesis Model

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the process by which they must be harvested can be associated with significant donor site morbidity. In contrast, adipose-derived stromal cells (ASCs) are more readily abundant and more easily harvested, making them an appealing alternative to BM-MSCs. Like BM-MSCs, ASCs can differentiate into osteogenic lineage cells and can be used in tissue engineering applications, such as seeding onto scaffolds for use in craniofacial skeletal defects. ASCs are obtained from the stromal vascular fraction (SVF) of digested adipose tissue, which is a heterogeneous mixture of ASCs, vascular endothelial and mural cells, smooth muscle cells, pericytes, fibroblasts, and circulating cells. Flow cytometric analysis has shown that the surface marker profile for ASCs is similar to that for BM-MSCs. Despite several published reports establishing markers for the ASC phenotype, there is still a lack of consensus over profiles identifying osteoprogenitor cells in this heterogeneous population. This protocol describes how to isolate and use a subpopulation of ASCs with enhanced osteogenic capacity to repair critical-sized calvarial defects.

The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the ...

Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted applications, facilitate the detection and measure of vascular biomarkers at the molecular level. Within the mouse embryo, this noninvasive technique may be used to uncover basic mechanisms underlying vascular development in the early mouse circulatory system and in genetic models of cardiovascular disease. The mouse embryo also presents as an excellent model for studying the adhesion of microbubbles to angiogenic targets (including vascular endothelial growth factor receptor 2 (VEGFR2) or &#945;v&#946;3) and for assessing the quantitative nature of molecular ultrasound. We therefore developed a method to introduce ultrasound contrast agents into the vasculature of living, isolated embryos. This allows freedom in terms of injection control and positioning, reproducibility of the imaging plane without obstruction and motion, and simplified image analysis and quantification. Late gestational stage (embryonic day (E)16.6 and E17.5) murine embryos were isolated from the uterus, gently exteriorized from the yolk sac and microbubble contrast agents were injected into veins accessible on the chorionic surface of the placental disc. Nonlinear contrast ultrasound imaging was then employed to collect a number of basic perfusion parameters (peak enhancement, wash-in rate and time to peak) and quantify targeted microbubble binding in an endoglin mouse model. We show the successful circulation of microbubbles within living embryos and the utility of this approach in characterizing embryonic vasculature and microbubble behavior.

Here, we present a protocol to inject ultrasound microbubble contrast agents into living, isolated late-gestation stage murine embryos. This method enables the study of perfusion parameters and of vascular molecular markers within the embryo using contrast-enhanced high-frequency ultrasound imaging.

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted ...

Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or longitudinal bone growth. As such, it is imperative that we further our understanding of the underpinning molecular mechanisms involved in such disorders so as to provide advances towards human and animal patient benefit. The mouse metatarsal organ explant culture is a highly physiological ex vivo model for studying endochondral ossification and bone growth as the growth rate of the bones in culture mimic that observed in vivo. Uniquely, the metatarsal organ culture allows the examination of chondrocytes in different phases of chondrogenesis and maintains cell-cell and cell-matrix interactions, therefore providing conditions closer to the in vivo situation than cells in monolayer or 3D culture. This protocol describes in detail the intricate dissection of embryonic metatarsals from the hind limb of E15 murine embryos and the subsequent analyses that can be performed in order to examine endochondral ossification and longitudinal bone growth.

We present a protocol to dissect and culture embryonic day 15 (E15) murine metatarsal bones. This highly physiological ex vivo model of endochondral ossification provides conditions closer to the in vivo situation than cells in monolayer or 3D culture and is a vital tool for investigating bone growth and development.

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or ...

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

A Seminiferous Tubule Squash Technique for the Cytological Analysis of Spermatogenesis Using the Mouse Model

Meiotic progression in males is a process that requires the concerted action of a number of highly regulated cellular events. Errors occurring during meiosis can lead to infertility, pregnancy loss or genetic defects. Commencing at the onset of puberty and continuing throughout adulthood, continuous semi-synchronous waves of spermatocytes undergo spermatogenesis and ultimately form haploid sperm. The first wave of mouse spermatocytes undergoing meiotic initiation appear at day 10 post-partum (10 dpp) and are released into the lumen of seminiferous tubules as mature sperm at 35 dpp. Therefore, it is advantageous to utilize mice within this developmental time-window in order to obtain highly enriched populations of interest. Analysis of rare cell stages is more difficult in older mice due to the contribution of successive spermatogenic waves, which increase the diversity of the cellular populations within the tubules. The method described here is an easily implemented technique for the cytological evaluation of the cells found within the seminiferous tubules of mice, including spermatogonia, spermatocytes, and spermatids. The tubule squash technique maintains the integrity of isolated male germ cells and allows examination of cellular structures that are not easily visualized with other techniques. To demonstrate the possible applications of this tubule squash technique, spindle assembly was monitored in spermatocytes progressing through the prophase to metaphase I transition (G2/MI transition). In addition, centrosome duplication, meiotic sex chromosome inactivation (MSCI), and chromosome bouquet formation were assessed as examples of the cytological structures that can be observed using this tubule squash method. This technique can be used to pinpoint specific defects during spermatogenesis that are caused by mutation or exogenous perturbation, and thus, contributes to our molecular understanding of spermatogenesis.

The goal of this tubule squash technique is to rapidly assess cytological features of developing mouse spermatocytes while preserving cellular integrity. This method allows for the study of all stages of spermatogenesis, and can be easily implemented alongside other biochemical and molecular biological approaches for the study of mouse meiosis.

Meiotic progression in males is a process that requires the concerted action of a number of highly regulated cellular events. Errors occurring during ...

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset cardiovascular disease due to marked elevation of LDL cholesterol (LDL-C) in blood. Statins are the first line of lipid-lowering drugs for treating FH and other types of hypercholesterolemia, but new approaches are emerging, in particular PCSK9 antibodies, which are now being tested in clinical trials. To explore novel therapeutic approaches for FH, either new drugs or new formulations, we need appropriate in vivo models. However, differences in the lipid metabolic profiles compared to humans are a key problem of the available animal models of FH. To address this issue, we have generated a human liver chimeric mouse model using FH induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps). We used Ldlr-/-/Rag2-/-/Il2rg-/- (LRG) mice to avoid immune rejection of transplanted human cells and to assess the effect of LDLR-deficient iHeps in an LDLR null background. Transplanted FH iHeps could repopulate 5-10% of the LRG mouse liver based on human albumin staining. Moreover, the engrafted iHeps responded to lipid-lowering drugs and recapitulated clinical observations of increased efficacy of PCSK9 antibodies compared to statins. Our human liver chimeric model could thus be useful for preclinical testing of new therapies to FH. Using the same protocol, similar human liver chimeric mice for other FH genetic variants, or mutations corresponding to other inherited liver diseases, may also be generated.

Here, we present a protocol to generate a human liver chimeric mouse model of familial hypercholesterolemia using human induced pluripotent stem cell-derived hepatocytes. This is a valuable model for testing new therapies for hypercholesterolemia.

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset ...

A Hyperandrogenic Mouse Model to Study Polycystic Ovary Syndrome

Hyperandrogenemia plays a critical role in reproductive and metabolic function in females and is the hallmark of polycystic ovary syndrome. Developing a lean PCOS-like mouse model that mimics women with PCOS is clinically meaningful. In this protocol, we describe such a model. By inserting a 4 mm length of DHT (dihydrotestosterone) crystal powder pellet (total length of pellet is 8 mm), and replacing it monthly, we are able to produce a PCOS-like mouse model with serum DHT levels 2 fold higher than mice not implanted with DHT (no-DHT). We observed reproductive and metabolic dysfunction without changing body weight and body composition. While exhibiting a high degree of infertility, a small subset of these PCOS-like female mice can get pregnant and their offspring show delayed puberty and increased testosterone as adults. This PCOS-like lean mouse model is a useful tool to study the pathophysiology of PCOS and the offspring from these PCOS-like dams.

We describe the development of a lean PCOS-like mouse model with&#160;dihydrotestosterone pellet to study the pathophysiology of PCOS and the offspring from these PCOS-like dams.

Hyperandrogenemia plays a critical role in reproductive and metabolic function in females and is the hallmark of polycystic ovary syndrome. Developing a ...

Adult Mouse Digit Amputation and Regeneration: A Simple Model to Investigate Mammalian Blastema Formation and Intramembranous Ossification

Here, we present a protocol of adult mouse distal terminal phalanx (P3) amputation, a procedurally simple and reproducible mammalian model of epimorphic regeneration, which involves blastema formation and intramembranous ossification analyzed by fluorescence immunohistochemistry and sequential in-vivo microcomputed tomography (&#956;CT). Mammalian regeneration is restricted to amputations transecting the distal region of the terminal phalanx (P3); digits amputated at more proximal levels fail to regenerate and undergo fibrotic healing and scar formation. The regeneration response is mediated by the formation of a proliferative blastema, followed by bone regeneration via intramembranous ossification to restore the amputated skeletal length. P3 amputation is a preclinical model to investigate epimorphic regeneration in mammals, and is a powerful tool for the design of therapeutic strategies to replace fibrotic healing with a successful regenerative response. Our protocol uses fluorescence immunohistochemistry to 1) identify early-and-late blastema cell populations, 2) study revascularization in the context of regeneration, and 3) investigate intramembranous ossification without the need for complex bone stabilization devices. We also demonstrate the use of sequential in vivo &#956;CT to create high resolution images to examine morphological changes after amputation, as well as quantify volume and length changes in the same digit over the course of regeneration. We believe this protocol offers tremendous utility to investigate both epimorphic and tissue regenerative responses in mammals.

Here, we present a protocol of adult mouse terminal phalanx amputation to investigate mammalian blastema formation and intramembranous ossification, analyzed by fluorescent immunohistochemistry and sequential in-vivo microcomputed tomography.

Here, we present a protocol of adult mouse distal terminal phalanx (P3) amputation, a procedurally simple and reproducible mammalian model of epimorphic ...

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to ...

Vascular Casting of Adult and Early Postnatal Mouse Lungs for Micro-CT Imaging

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and behave by observing the surface of a tissue. This method provides a means to visualize the complex 3-dimensional vascular architecture of the lung.
To accomplish this, a catheter is inserted into the pulmonary artery and the vasculature is simultaneously flushed of blood and chemically dilated to limit resistance. Lungs are then inflated through the trachea at a standard pressure and the polymer compound is infused into the vascular bed at a standard flow rate. Once the entire arterial network is filled and allowed to cure, the lung vasculature may be visualized directly or imaged on a micro-CT (&#181;CT) scanner.
When performed successfully, one can appreciate the pulmonary arterial network in mice ranging from early postnatal ages to adults. Additionally, while demonstrated in the pulmonary arterial bed, this method can be applied to any vascular bed with optimized catheter placement and endpoints.

The aim of this technique is ex vivo visualization of pulmonary arterial networks of early postnatal and adult mice through lung inflation and injection of a radio-opaque polymer-based compound via the pulmonary artery. Potential applications for casted tissues are also discussed.

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and ...