Name: an intramedullary locking nail for standardized fixation of femur osteotomies to analyze normal and defective bone healing in mice
Uploaded: 2016-11-13T15:00:00
Description: Watch this Scientific Journal Video about An Intramedullary Locking Nail for Standardized Fixation of Femur Osteotomies to Analyze Normal and Defective Bone Healing in Mice at JoVE.com

This work was supported by RISystem AG, Davos, Switzerland.

All procedures were IACUC-approved and followed institutional guidelines (Landesamt f&#252;r Verbraucherschutz, Zentralstelle Amtstier&#228;rztlicher Dienst, Saarbr&#252;cken, Germany). Analgesia and infection prevention should be in agreement with the respective guidelines of the country and institution where the experiments are to be performed.
1. Preparation of Implants and Surgical Instruments
<ol>
	<li>Select a scalpel blade (size 15), small preparation scissors, fine forceps, dressing ...

<ol>
	<li>Histing, T., 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=Small+animal+bone+healing+models:+standards,+tips,+and+pitfalls+results+of+a+consensus+meeting.">Small animal bone healing models: standards, tips, and pitfalls results of a consensus meeting.</a> Bone. 49 (4), 591-599 (2011).</li><li>Claes, L., Augat, P., Suger, G., Wilke, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+size+and+stability+of+the+osteotomy+gap+on+the+success+of+fracture+healing.">Influence of size and stability of the osteotomy gap on the success of fracture healing.</a> J Orthop Res. 15 (4), 577-584 (1997).</li><li>Histing, T., 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=Characterization+of+the+healing+process+in+non-stabilized+and+stabilized+femur+fractures+in+mice.">Characterization of the healing process in non-stabilized and stabilized femur fractures in mice.</a> Arch Orthop Trauma Surg. 136 (2), 203-211 (2016).</li><li>Histing, T., 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=Ex+vivo+analysis+of+rotational+stiffness+of+different+osteosynthesis+techniques+in+mouse+femur+fracture.">Ex vivo analysis of rotational stiffness of different osteosynthesis techniques in mouse femur fracture.</a> J Orthop Res. 27 (9), 1152-1156 (2009).</li><li>Holstein, J. H., 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=Development+of+a+stable+closed+femoral+fracture+model+in+mice.">Development of a stable closed femoral fracture model in mice.</a> J Surg Res. 153 (1), 71-75 (2009).</li><li>Garcia, P., 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+LockingMouseNail-a+new+implant+for+standardized+stable+osteosynthesis+in+mice.">The LockingMouseNail-a new implant for standardized stable osteosynthesis in mice.</a> J Surg Res. 169 (2), 220-226 (2011).</li><li>Cheung, K. M., Kaluarachi, K., Andrew, G., Lu, W., Chan, D., Cheah, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+externally+fixed+femoral+fracture+model+for+mice.">An externally fixed femoral fracture model for mice.</a> J Orthop Res. 21 (4), 685-690 (2003).</li><li>Garcia, P., 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=A+new+technique+for+internal+fixation+of+femoral+fractures+in+mice:+impact+of+stability+on+fracture+healing.">A new technique for internal fixation of femoral fractures in mice: impact of stability on fracture healing.</a> J Biomech. 41 (8), 1689-1696 (2008).</li><li>Histing, T., 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=An+internal+locking+plate+to+study+intramembranous+bone+healing+in+a+mouse+femur+fracture+model.">An internal locking plate to study intramembranous bone healing in a mouse femur fracture model.</a> J Orthop Res. 28 (3), 397-402 (2010).</li><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 Orthop Res. 20 (5), 1091-1098 (2002).</li><li>Hiltunen, A., Vuorio, E., Aro, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+standardized+experimental+fracture+in+the+mouse+tibia.">A standardized experimental fracture in the mouse tibia.</a> J Orthop Res. 11 (2), 305-312 (1993).</li><li>Manigrasso, M. B., O'Connor, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+closed+femur+fracture+model+in+mice.">Characterization of a closed femur fracture model in mice.</a> J Orthop Trauma. 18 (10), 687-695 (2004).</li><li>Lovati, A. B., 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=Diabetic+mouse+model+of+orthopaedic+implant-related+Staphylococcus+aureus+infection.">Diabetic mouse model of orthopaedic implant-related Staphylococcus aureus infection.</a> PLoS One. 8 (6), e67628 (2013).</li><li>Slade Shantz, J. A., Yu, Y. Y., Andres, W., Miclau, T., Marcucio, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+macrophage+activity+during+fracture+repair+has+differential+effects+in+young+adult+and+elderly+mice.">Modulation of macrophage activity during fracture repair has differential effects in young adult and elderly mice.</a> J Orthop Trauma. 28, 10-14 (2014).</li><li>Claes, L. 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=Effects+of+mechanical+factors+on+the+fracture+healing+process.">Effects of mechanical factors on the fracture healing process.</a> Clin Orthop Relat Res. 355 (Suppl), 132-147 (1998).</li><li>Gr&#246;ngr&#246;ft, I., 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=Fixation+compliance+in+a+mouse+osteotomy+model+induces+two+different+processes+of+bone+healing+but+does+not+lead+to+delayed+union.">Fixation compliance in a mouse osteotomy model induces two different processes of bone healing but does not lead to delayed union.</a> J Biomech. 18 (13), 2089-2096 (2009).</li><li>Glatt, V., Matthys, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adjustable+stiffness,+external+fixator+for+the+rat+femur+osteotomy+and+segmental+bone+defect+models.">Adjustable stiffness, external fixator for the rat femur osteotomy and segmental bone defect models.</a> J Vis Exp. (9), e51558 (2014).</li><li>Manassero, 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=A+novel+murine+femoral+segmental+critical-sized+defect+model+stabilized+by+plate+osteosynthesis+for+bone+tissue+engineering+purposes.">A novel murine femoral segmental critical-sized defect model stabilized by plate osteosynthesis for bone tissue engineering purposes.</a> Tissue Eng Part C Methods. 19 (4), 271-280 (2013).</li></ol>

The most critical steps of the surgical technique are the correct positioning of the nail, the aiming device, and the pins. The nail has to be inserted completely to the marked indent at the distal end of the nail, because a protrusion of the nail into the knee joint at the level of the condyles can restrict the movement of the knee (Figure 3 A). Therefore, the size of the femur and, accordingly, the body weight of the animals, must be considered. The surgeon should also pay special attention to the fina...

The biology of bone healing may be studied in vitro using cell and spheroid cultures, but it also requires in vivo approaches using animal studies. While large-animal experiments still play an important role in preclinical testing, early stage testing of products or hypotheses has changed during the last 10 years and is nowadays often conducted in small animal models1. This switch was performed for several reasons. Production and maintenance of mice and rats are cheaper compared to pigs and sheep. In addition, small animals have shorter reproduction times and shorter normal healing periods, both of which facilitate the performance of large series of chronic experiments. Finally, the availability of gene-targeted animals and specific antibodies allows for the analysis of molecular mechanisms in bone healing. However, while the previously used osteosynthesis techniques in the larger animal models could be translated with minimal variation from similar procedures used in human or veterinary clinical patient care, the development and application of osteosynthesis techniques in the small-sized rats and mice turned out to be challenging.
It is well known that the biomechanical environment significantly influences the bone healing process2. As known from fracture healing in humans, differences in fracture stabilization result in distinct modes of healing, including intramembranous ossification after rigid fixation and endochondral ossification after less rigid fixation with micromovements. Complete axial or rotational instability may delay the healing process or may result in non-unions3. Accordingly, we feel that it is necessary to develop sophisticated implant systems and osteosynthesis techniques in mice and rats. In this way, the biomechanical conditions can be standardized appropriately, guaranteeing valid results when analyzing the healing process.
Although a considerable number of highly sophisticated murine stabilization techniques have been introduced during the last few years, the most commonly used technique is still the simple intramedullary pin. The major disadvantage of this technique, however, is the lack of rotational and axial stability4. To improve rotational and axial stability, an intramedullary screw was introduced to stabilize femur fractures in mice5. However, the screw fixation cannot be used to analyze bone-defective healing due to the need for contact and compression between the bone fragments in order to maintain rotational stability.
The intramedullary locking nail offers higher axial and rotational stability compared to the simple pin and the intramedullary screw4. A highly reproducible femur osteotomy, possible because of the guide for the Gigli saw and the ability to create defined gap sizes, allows for the analysis of both normal bone healing and bone-defective healing6. Due to the insertion of interlocking pins, the intramedullary locking nail guarantees a constant gap size during the entire healing process, even while bearing full weight. Here, we report on the design and application of the intramedullary locking nail, as well as on its advantages and disadvantages in experimental studies on normal and delayed bone healing.

<table border="0" cellpadding="0" cellspacing="0" width="476">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:187px;">MouseNail</td>
			<td style="width:179px;">RISystem AG</td>
			<td style="width:111px;">221,122</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">MouseNail aiming device</td>
			<td>RISystem AG</td>
			<td>221,201</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">MouseNail interlocking pin</td>
			<td>RISystem AG</td>
			<td>221,121</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Centering bit</td>
			<td>RISystem AG</td>
			<td>592,205</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Drill bit</td>
			<td>RISystem AG</td>
			<td>590,200</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Gigli wire saw</td>
			<td>RISystem AG</td>
			<td>590,100</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Suture (5-0 Prolene)</td>
			<td>Ethicon</td>
			<td>8614H</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Forceps</td>
			<td>Braun Aesculap AG &#38;CoKG&#160;</td>
			<td>BD520R</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Dressing forceps</td>
			<td>Braun Aesculap AG &#38;CoKG&#160;</td>
			<td>BJ009R</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Scissors</td>
			<td>Braun Aesculap AG &#38;CoKG&#160;</td>
			<td>BC100R</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Needle holder</td>
			<td>Braun Aesculap AG &#38;CoKG&#160;</td>
			<td>BM024R</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">24G needle</td>
			<td>BD Mircolance 3</td>
			<td>304100</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">27G needle</td>
			<td>Braun Melsungen AG</td>
			<td>9186182</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Scalpel blade size 15</td>
			<td>Braun Aesculap AG &#38;CoKG&#160;</td>
			<td>16600525</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Pincers</td>
			<td>Knipex</td>
			<td>7932125</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Heat radiator</td>
			<td>Sanitas</td>
			<td>605.25</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Depilatory cream</td>
			<td>Asid bonz GmbH</td>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; NDXZ10</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Eye lubricant</td>
			<td>Bayer Vital GmbH</td>
			<td>2182442</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Xylazine</td>
			<td>Bayer Vital GmbH</td>
			<td align="right">1320422</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ketamine</td>
			<td>Serumwerke Bernburg</td>
			<td align="right">7005294</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Tramadol</td>
			<td>Gr&#252;nenthal GmbH</td>
			<td align="right">2256241</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Disinfection solution (SoftaseptN)</td>
			<td>Braun Melsungen AG</td>
			<td align="right">8505018</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">CD-1 mice</td>
			<td>Charles River</td>
			<td align="right">22</td>
		</tr>
	</tbody>
</table>

an intramedullary locking nail for standardized fixation of femur osteotomies to analyze normal and defective bone healing in mice

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming more commonly used in trauma research. They offer a large number of mutant strains and antibodies for the analysis of the molecular mechanisms behind the highly differentiated process of bone healing. To control the biomechanical environment, standardized and well-characterized osteosynthesis techniques are mandatory in mice. Here, we report on the design and use of an intramedullary nail to stabilize open femur osteotomies in mice. The nail, made of medical-grade stainless steel, provides high axial and rotational stiffness. The implant further allows the creation of defined, constant osteotomy gap sizes from 0.00 mm to 2.00 mm. Intramedullary locking nail stabilization of femur osteotomies with gap sizes of 0.00 mm and 0.25 mm result in adequate bone healing through endochondral and intramembranous ossification. Stabilization of femur osteotomies with a gap size of 2.00 mm results in atrophic non-union. Thus, the intramedullary locking nail can be used in healing and non-healing models. A further advantage of the use of the nail compared to other open bone healing models is the possibility to adequately fix bone substitutes and scaffolds in order to study the process of osseous integration. A disadvantage of the use of the intramedullary nail is the more invasive surgical procedure, inherent to all open procedures compared to closed models. A further disadvantage may be the induction of some damage to the intramedullary cavity, inherent to all intramedullary stabilization techniques compared to extramedullary stabilization procedures.

The overall time for the surgical procedure was about 30 min from skin incision to wound closure. Using the surgical implants provided, surgery can be performed without a stereo-microscope. Postoperatively, the animals were monitored daily. Post-operative analgesia was terminated after 3 days because none of the animals showed evidence of pain (vocalization, restlessness, lack of mobility, failure to groom, abnormal posture, or lack of normal interest in surroundings) after this time peri...

Watch this Scientific Journal Video about An Intramedullary Locking Nail for Standardized Fixation of Femur Osteotomies to Analyze Normal and Defective Bone Healing in Mice at JoVE.com

An Intramedullary Locking Nail for Standardized Fixation of Femur Osteotomies to Analyze Normal and Defective Bone Healing in Mice

This protocol describes an osteosynthesis technique using an intramedullary locking nail for standardized fixation of femur osteotomies, which can be used to analyze normal and defective bone healing in mice.

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming ...

The overall goal of this surgical technique is to standardize the analysis of bone healing in mice. This surgical technique can help answer key questions in bone healing research about the roots of bone morphogenetic proteins and muscular grow factors and the mechanisms of bone repair. The main advantages of this technique are the reproducibility of the femur osteotomy and a high axial and rotationist ability facilitated by the use of the intramedullary locking nail.Demonstrating the procedure will be Tobias Fritz, a colleague of our department. One day before the surgery until three days after the surgery, add tramadol hydrochloride to the animal's drinking water for analgesia. On the day of the surgery, confirm the appropriate level of sedation of a 25 to 35 gram mouse, by a lack of response to toe pinch and place the animal under a heat radiator.Shave the entire right hind leg of the animal and apply depilatory cream. After five minutes, remove the cream and clean the leg with water. Then disinfect the surgical site with 96%ethanol.Under aseptic conditions, place the mouse in the supine position on the operating table with the right knee bent to allow an anterior approach of the knee condyles. Next, use a sterilized size 15 scalpel blade to make a five millimeter medial parapatellar incision in the right knee. Then use fine forceps to lift the exposed patellar ligament and use the blade to carefully mobilize the ligament.Shifting the patella laterally with the blade to expose the intercondylar notch of the femur, use a hand drill equipped with a one millimeter centering bit, and held at a 45 degree offset to the femur axis to bore the intercondylar notch. Slowly changing the direction of the bit, drill until the bit parallels the bone axis of the femur, stopping when the intramedullary cavity is reached. When the intercondylar notch is open, insert a 24 gauge needle into the cavity over the entire length of the femur and rotate the needle to manually ring the intramedullary cavity.Then replace the 24 gauge needle with a 27 gauge needle and push the 27 gauge needle forward to perforate the cortical bone of the femur proximally at the greater trochanter. Remove the 27 gauge needle and use the hand drill to implant the intramedullary nail through the intercondylar notch under continuous rotation and axial pressure until the distal end of the nail reaches the condyles. Now place the mouse in the left lateral position and use the scalpel to make a longitudinal skin incision along the diaphyseal region of the lateral femur from the hip joint to the knee joint to surgically expose the midshaft of the femur.Using small preparation scissors, split the fascia and spread the muscles toward the femur axis from the lateral side until the diaphyseal region of the femur is exposed. Undermine the bone with the dressing forceps. Then spread the dressing forceps and retract the muscles to expose the femur.Mount the aiming device to the distal end of the nail, advancing the device until it attaches to the adapter flange of the nail. Then turn the aiming device to an anterolateral angle to the femur, and use the centering one millimeter diameter drill bit to create a small cavity in the facing cortical bone at the proximal interlocking hole position. Next, with a 0.3 millimeter diameter drill bit, use the aiming device to drill a hole through both the facing and averted cortical bones.Insert the proximal interlocking pin through the aiming device. The interlocking pin driveshaft will shear off as soon as the interlocking torque is achieved. When both the proximal and distal interlocking pins have been placed, equip the aiming device with the saw guide on the lateral side, between the two interlocking pins, and saw the bone with a Gigli saw under continuous irrigation with saline.When the osteotomy is complete, cut the saw at end close to the bone and carefully remove the blade to avoid causing damage to the soft tissue. Remove the aiming device and use small pincers to clip off the remaining shaft of the intramedullary nail at the marked line. Then close the muscle layers at the lateral site of the femur, and use single sutures to close the skin.At the anterior site of the knee, reposition the patella and fix the patella tendon to the muscles with one single suture, followed by closure of the wound with single sutures. Finally, allow the animal to recover under the heat lamp with monitoring, until fully recovered. The most significant complication that can occur is the incorrect implantation of the locking nail with the protrusion of the nail level to the condyles of the knee joint.This mainly occurs due to the incorrect handling of the aiming device or the use of an animal with a too small femur, particularly in mice with body weights below 20 grams. Another complication is the dislocation of an interlocking pin, which is mainly caused by an incomplete insertion of the pin, and can be avoided by radiographic confirmation of the correct implant placement during, or immediately after, the surgery. Radiological analysis after five weeks confirms a complete healing of the 0.25 millimeter osteotomy gap, at which point, the periosteal callus is almost completely remodeled.In contrast, in femora stabilized with a 2 millimeter gap, the osteotomy does not heal, and reliably exhibits an atrophic nonunion formation for up to 10 weeks of bone healing. After stabilization with a 0.25 millimeter osteotomy gap, histological analysis reveals a typical pattern of secondary fracture healing with callus formation, including intramembranous and endochondral ossification that is remodeled into lamellar bone at five weeks. In contrast, femora stabilized with two millimeter osteotomy gaps demonstrate an atrophic nonunion after 10 weeks that is associated with a high amount of fibrous tissue within the osteotomy gap, and no evidence of bone healing or bridging.Once mastered, this technique can be completed in 20 minutes if it is performed properly. After watching this video, you should have a good understanding of how to use this technique as the standardest model for fracture healing research.

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Research

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Medicine

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Lesion Explorer: A Video-guided, Standardized Protocol for Accurate and Reliable MRI-derived Volumetrics in Alzheimer's Disease and Normal Elderly

Obtaining in vivo human brain tissue volumetrics from MRI is often complicated by various technical and biological issues. These challenges are exacerbated when significant brain atrophy and age-related white matter changes (e.g. Leukoaraiosis) are present. Lesion Explorer (LE) is an accurate and reliable neuroimaging pipeline specifically developed to address such issues commonly observed on MRI of Alzheimer's disease and normal elderly. The pipeline is a complex set of semi-automatic procedures which has been previously validated in a series of internal and external reliability tests1,2. However, LE's accuracy and reliability is highly dependent on properly trained manual operators to execute commands, identify distinct anatomical landmarks, and manually edit/verify various computer-generated segmentation outputs.
LE can be divided into 3 main components, each requiring a set of commands and manual operations: 1) Brain-Sizer, 2) SABRE, and 3) Lesion-Seg. Brain-Sizer's manual operations involve editing of the automatic skull-stripped total intracranial vault (TIV) extraction mask, designation of ventricular cerebrospinal fluid (vCSF), and removal of subtentorial structures. The SABRE component requires checking of image alignment along the anterior and posterior commissure (ACPC) plane, and identification of several anatomical landmarks required for regional parcellation. Finally, the Lesion-Seg component involves manual checking of the automatic lesion segmentation of subcortical hyperintensities (SH) for false positive errors.
While on-site training of the LE pipeline is preferable, readily available visual teaching tools with interactive training images are a viable alternative. Developed to ensure a high degree of accuracy and reliability, the following is a step-by-step, video-guided, standardized protocol for LE's manual procedures.

Lesion Explorer: A Video-guided, Standardized Protocol for Accurate and Reliable MRI-derived Volumetrics in Alzheimer&#039;s Disease and Normal Elderly

Lesion Explorer (LE) is a semi-automatic, image-processing pipeline developed to obtain regional brain tissue and subcortical hyperintensity lesion volumetrics from structural MRI of Alzheimer's disease and normal elderly. To ensure a high level of accuracy and reliability, the following is a video-guided, standardized protocol for LE's manual procedures.

Obtaining in vivo human brain tissue volumetrics from MRI is often complicated by various technical and biological issues. These challenges are ...

Adjustable Stiffness, External Fixator for the Rat Femur Osteotomy and Segmental Bone Defect Models

The mechanical environment around the healing of broken bone is very important as it determines the way the fracture will heal. Over the past decade there has been great clinical interest in improving bone healing by altering the mechanical environment through the fixation stability around the lesion. One constraint of preclinical animal research in this area is the lack of experimental control over the local mechanical environment within a large segmental defect as well as osteotomies as they heal. In this paper we report on the design and use of an external fixator to study the healing of large segmental bone defects or osteotomies. This device not only allows for controlled axial stiffness on the bone lesion as it heals, but it also enables the change of stiffness during the healing process in vivo. The conducted experiments have shown that the fixators were able to maintain a 5 mm femoral defect gap in rats in vivo during unrestricted cage activity for at least 8 weeks. Likewise, we observed no distortion or infections, including pin infections during the entire healing period. These results demonstrate that our newly developed external fixator was able to achieve reproducible and standardized stabilization, and the alteration of the mechanical environment of in vivo rat large bone defects and various size osteotomies. This confirms that the external fixation device is well suited for preclinical research investigations using a rat model in the field of bone regeneration and repair.

One constraint of preclinical research in the field of bone repair is the lack of experimental control over the local mechanical environment within a healing bone lesion. We report the design and use of an external fixator for bone repair with the ability to change fixator stiffness in vivo.

The mechanical environment around the healing of broken bone is very important as it determines the way the fracture will heal. Over the past decade there ...

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and &#956;CT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, &#956;CT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical &#956;CT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with &#956;CT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Combined In vivo Optical and  &amp;#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined optical and &#956;CT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

An Improved Method for Rapid Intubation of the Trachea in Mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

Visualization of Vascular and Parenchymal Regeneration after 70% Partial Hepatectomy in Normal Mice

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the silicone compound, via a 26 G catheter, into the portal or hepatic vein. After silicone injection, organs were explanted and prepared for ex-vivo micro-CT (&#181;CT) scanning. The silicone injection procedure is technically challenging. Achieving a successful outcome requires extensive microsurgical experience from the surgeon. One of the challenges of this procedure involves determining the adequate perfusion rate for the silicone compound. The perfusion rate for the silicone compound needs to be defined based on the hemodynamic of the vascular system of interest. Inappropriate perfusion rate can lead to an incomplete perfusion, artificial dilation and rupturing of vascular trees.
The 3D reconstruction of the vascular system was based on CT scans and was achieved using preclinical software such as HepaVision. The quality of the reconstructed vascular tree was directly related to the quality of silicone perfusion. Subsequently computed vascular parameters indicative of vascular growth, such as total vascular volume, were calculated based on the vascular reconstructions. Contrasting the vascular tree with silicone allowed for subsequent histological work-up of the specimen after &#181;CT scanning. The specimen can be subjected to serial sectioning, histological analysis and whole slide scanning, and thereafter to 3D reconstruction of the vascular trees based on histological images. This is the prerequisite for the detection of molecular events and their distribution with respect to the vascular tree. This modified silicone injection procedure can also be used to visualize and reconstruct the vascular systems of other organs. This technique has the potential to be extensively applied to studies concerning vascular anatomy and growth in various animal and disease models.

Tools used for visualizing vascular regeneration require methods for contrasting the vascular trees. This film demonstrated a delicate injection technique used to achieve optimal contrasting of the vascular trees and illustrate the potential benefits resulting from a detailed analysis of the resulting specimen using &#181;CT and histological serial sections.

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the ...

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The amount of the different tissues in the callus provides important information on the fracture healing progress. Available in vivo techniques to longitudinally monitor the callus tissue development in preclinical fracture-healing studies using small animals include digital radiography and &#181;CT imaging. However, both techniques are only able to distinguish between mineralized and non-mineralized tissue. Consequently, it is impossible to discriminate cartilage from fibrous tissue. In contrast, magnetic resonance imaging (MRI) visualizes anatomical structures based on their water content and might therefore be able to noninvasively identify soft tissue and cartilage in the fracture callus. Here, we report the use of an MRI-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice. The experiments demonstrated that the fixator and a custom-made mounting device allow repetitive MRI scans, thus enabling longitudinal analysis of fracture-callus tissue development.

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

The evaluation of tissue development in the fracture callus during endochondral bone healing is essential to monitor the healing process. Here, we report the use of a magnetic resonance imaging (MRI)-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice.

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The ...

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an increased use of mouse models in orthopedic research was noted, most probably because mouse models offer a large number of genetically-modified strains and special antibodies for the analysis of molecular mechanisms of fracture healing. To control the biomechanical conditions, well-characterized osteosynthesis techniques are mandatory, also in mice. Here, we report on the design and use of a closed bone healing model to stabilize femur fractures in mice. The intramedullary screw, made of medical-grade stainless steel, provides through fracture compression an axial and rotational stability compared to the mostly used simple intramedullary pins, which show a complete lack of axial and rotational stability. The stability achieved by the intramedullary screw allows the analysis of endochondral healing. A large amount of callus tissue, received after stabilization with the screw, offers ideal conditions to harvest tissue for biochemical and molecular analyses. A further advantage of the use of the screw is the fact that the screw can be inserted into the femur with a minimally invasive technique without inducing damage to the soft tissue. In conclusion, the screw is a unique implant that can ideally be used in closed fracture healing models offering standardized biomechanical conditions.

This protocol describes a minimally invasive osteosynthesis technique using an intramedullary screw for standardized stabilization of femur fractures, which can be used to analyze endochondral bone healing in mice.

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an ...

The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies that promote efficient bone healing are non-existent and in high demand. Effective and reproducible animal models of fractures healing are needed to understand the complex biological processes associated with bone regeneration. Many animal models of fracture healing have been generated over the years; however, murine fracture models have recently emerged as powerful tools to study bone healing. A variety of open and closed models have been developed, but the closed femoral fracture model stands out as a simple method for generating rapid and reproducible results in a physiologically relevant manner. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice and facilitate a post-fracture stabilization of the femur by inserting an intramedullary steel rod. Although devices such as a nail or a screw offer greater axial and rotational stability, the use of an intramedullary rod provides a sufficient stabilization for consistent healing outcomes without producing new defects in the bone tissue or damaging nearby soft tissue. Radiographic imaging is used to monitor the progression of callus formation, bony union, and subsequent remodeling of the bony callus. Bone healing outcomes are typically associated with the strength of the healed bone and measured with torsional testing. Still, understanding the early cellular and molecular events associated with fracture repair is critical in the study of bone tissue regeneration. The closed femoral fracture model in mice with intramedullary fixation serves as an attractive platform to study bone fracture healing and evaluate therapeutic strategies to accelerate healing.

The murine closed femoral fracture model is a powerful platform to study fracture healing and novel therapeutic strategies to accelerate bone regeneration. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice using an intramedullary steel rod to stabilize the femur.

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies ...