This work was supported by the AO foundation (S-08-42G) and RISystem AG.
We would like to extend a very big &#34;thank you!&#34; to Stephan Zeiter&#39;s team at the AO Research Institute Davos, Switzerland for being so accommodating in allowing us to use their OR facilities for the filming of this surgical procedure.

Animal care and experimental protocols were followed in accordance with NIH guidelines and approved by the Beth Israel Deaconess Medical Center Institutional Animal Care and Use Committee, Boston, MA. (Protocol Number: 098-2009)
1. Preparation of Surgical Materials and Instruments
<ol>
	<li>Sterilize all surgical materials and instruments used to perform surgery prior to use. Pack needed materials, with or without an instrument tray, inside a folded cloth or wrapped paper and seal with autoclave tape for steam sterilization. The temperature of the autoclave should be at 125-135 &#186;C for 20-25 min of sterilization time, and then 10-15 min of drying time.</li>
	<li>Make sure that at the time of surgery the rats are 200-250 g. This is very important because if the rats are heavier in size, then a different sized fixator should be used. For rats heavier than 250 g a larger version of the external fixator system should be used.</li>
</ol>
2. Surgical Procedure and Application of the External Fixator
<ol>
	<li>Purchase Sprague-Dawley (or any other strain) rats (male or female, 200-250 g) from any certified animal supplier. Follow the appropriate animal care and experimental protocols in accordance with national guidelines that is approved by the investigator&#8217;s Institutional Animal Care and Use Committee. Allow a minimum of 48 hr acclimatization period before the procedure.</li>
	<li>For surgery, transport the rat to a dedicated surgical procedure room.</li>
	<li>Anesthetize the rat with isoflurane first via induction chamber, and then continue with face mask connected to an anesthesia machine at a rate of 1.5-2% in 1-1.5 L of O2/min. At the start of surgery make sure the animal is under deep anesthesia. To do this, use pedal reflex technique by extend the limb and pinching the web between the toes with the fingers (not the toe itself!). The animal is not sufficiently anesthetized if the limb is withdrawn, muscle twitch occurs or if the animal makes noise.</li>
	<li>After the rat is under deep general anesthesia&#160;for surgery, inject the antibiotic (cefazolin, 20 mg/kg) and the analgesic buprenorphine (dose 0.08 mg/kg) intramuscularly in the right leg. To avoid large amount of fluid loss during the surgery administer warm sterile saline subcutaneously at 3-5% of the body weight prior to the surgery, and if it&#8217;s necessary at the end. Apply sterile eye ointment to the eyes to keep them hydrated to prevent corneal injury.</li>
	<li>Following the drug injections, shave and clean the entire right hind leg of the rat with chlorohezadine or other disinfecting solution and transfer the animal to the surgical table. (The leg operated upon must be the same as the one that was injected.)</li>
	<li>Place the animal on a heated surface in the prone position (Figure 1A). Make sure the facemask remains on the nose and mouth after the transfer to the surgical table, and maintain the anesthesia regimen mentioned in 2.3). Drape the area of the surgical procedure with a sterile fenestrated drape so that only the leg intended for surgery is exposed.</li>
	<li>Make an approximate incision of 3-4 cm (Figure 1B) through the skin running craniolateral on the surface of the right femur from the greater trochanter to the supracondylar region of the knee using a scalpel (Figure 1C). Expose the shaft of the femur by gentle dissection separating the fascia lata, and making sure that the muscle tissue is not cut. After that, separate apart the M. vastus lateralis and M. biceps femoris and lift the M. tensor fasciae latae to expose the full length of the femur (making sure that the sciatic nerve is preserved; Figure 1D).</li>
	<li>In the planned area of osteotomy, prepare the femur along the midway area of the diaphysis by releasing the surrounding muscle tissue from the femur. First, start by putting the Henahan elevator perpendicular to the exposed surface of the femur, and then using a scalpel, release the muscle in the adjacent area.
	<ol>
		<li>Continue by advancing forward and go around the femur, staying close to the bone surface, until all of the surrounding muscle tissue is released from the entire middle section of the bone (where the defect will be created), and the muscle tissue is completely cleaned from the bone. While doing this, it is very important to stay close to the bone surface to avoid cutting any major vessels.</li>
	</ol>
	</li>
	<li>For a 5 mm large bone defect, loop 2 pieces of Gigli wire saw (0.22 mm) around the bone in medio-lateral orientation (Figure 1E,F). After looping the wire saw, position one piece on the distal side of the femur, close to the knee joint, and a second piece on the proximal side close to the hip joint. Clamp the Gigli wire saw pieces on each side using S-shape curved dissecting and ligature forceps, so that it stays in the intended place. If a single cut osteotomy is planned, then only use one piece of wire saw.</li>
	<li>Use the external fixator plate as a template to determine the exact position of the implant. The position of the external fixator has to be as close as possible to the center of the femur.
	<ol>
		<li>Position the external fixator plate on the anterolateral surface of the bone. This is achieved by externally rotating the femur. In that position the soft tissue layer is at its thinnest, which prevents excessive soft tissue tension under the fixator plate after the wound is closed.</li>
		<li>Then slightly lift the external fixator plate off the bone surface to make sure that the holes of the plate are centered to the bone surface. Hold the external fixator with a small clamp for it to stay parallel to the longitudinal axis of the bone, and then use a power tool or a hand drill to predrill the first hole on the proximal side of the femur with the 0.79 mm drill bit. Before advancing forward, make sure that the tip of the drill bit is still centered on the bone surface .</li>
		<li>If the tip of the drill keeps slipping, use the 1.00 mm counter sinker (Figure 8F) to center the position of the first hole. The counter sinker should be used to position all remaining mounting pins. This will ensure a perfect alignment of the drill holes and the fixator plate relative to the bone surface.</li>
	</ol>
	</li>
</ol>
3. External Fixator Implantation Method Using Saw Guide
<ol>
	<li>Make sure that the plate of the external fixator is not mounted upside down before clipping it on the saw guide. Determine this by comparing the size of the holes on the plate. The correct side is with the larger hole&#8217;s diameter facing up. If the difference between the size of the holes within the fixator is not apparent, use the counter sinker.
	<ol>
		<li>Insert the tip of the counter sinker into one of the holes on the fixator plate, if the counter sinker easily fits into the hole then this is the upside of the fixator, however, if the tip of the counter sinker doesn&#8217;t fit then this is a bottom side of the fixator, and has to be flipped for the implantation accordingly. 
		(Important: Make sure to drill perpendicular to the longitudinal axis of the bone as this will ensure a perfect orientation of the fixator to the bone surface. The direction of the first drill hole determines the final orientation of the fixator on the bone. Remember the mounting pins are the same length, and if the fixator is not parallel to the longitudinal axis of the bone the distance between the fixator and the bone will vary too much and might prevent the ability of all four mounting pins to penetrate both cortices.)</li>
		<li>After the orientation is confirmed, clip the plate on the saw guide (Figure 2A,B) and then clip the unit on the bone so that the first predrilled hole is aligned with the first hole on the plate (Figure 2C). Use the 0.70 mm square box wrench inserted into the hand drill to drive the first mounting pin into the hole. Doing this will permit reproducible positioning for the remaining mounting pins.</li>
		<li>After the first mounting pin is in place, then drill the most distant hole from the first mounting pin on the distal side, and drive the second mounting pin into the hole. The implantation order of the two middle mounting pins is not important.</li>
	</ol>
	</li>
</ol>
4. External Fixator Implantation Method Without the Saw Guide:
The application of the external fixator can be also performed without using the saw guide. The beginning steps of the external fixator implantation are the same up until the unit with the saw guide is clipped on the bone (step 3.1). If the saw guide is not used, it is very crucial to keep the fixator plate in the correct orientation during the entire application procedure. The femur needs to be externally rotated in the anterolateral direction.
<ol>
	<li>Hold the external fixator plate with a small clamp or S-shape curved dissecting and ligature forceps so that it is parallel to the longitudinal axis of the bone (Figure 3A). The application of the first mounting pin will determine the alignment of the fixator, therefore, the rotation of bone has to be retained until the first pin is inserted (Figure 3B). After the first pin is in place, carefully use the forceps to hold the fixator plate that is acting as a drill guide.</li>
	<li>Insert the drill bit into the second hole &#8211; this is the most distal hole to the planned osteotomy gap (Figure 3C). Before drilling, check to make sure that the second hole has the same orientation as the first hole; also make sure that after the drilling is complete, both cortices are penetrated.</li>
	<li>Insert the 0.70 mm square box wrench to the hand drill and then insert the mounting pin into the tip. Carefully insert it into the plate of the external fixator without losing the alignment of the first predrilled hole.</li>
	<li>As soon as the tip is in contact with the bone, start turning the wrench under continuous axial loading applied to the proximal end of the hand drill. After about 5 full turns, make sure that the thread at the proximal end of the mounting pin catches the external fixator plate&#8217;s body. This thread locks the system. Stop turning when the end of the bone thread is close to the top surface of the bone (Figure 3D).</li>
	<li>After the pins on the most distal and proximal side are in place, predrill the remaining two middle holes. The implantation order of the two middle pins is not important (Figures 3C).</li>
	<li>After the external fixator is in place, use the 0.22 mm Gigli wire saw directed by the saw guide to make the segmental defect (Figure 4A). If the latter method is chosen, the saw guide is clipped before making a defect.
	<ol>
		<li>For this, pass a 0.22 mm Gigli wire saw through the 2 grooves underneath the femur (Figure 5A) to create a 5 mm segmental defect by reciprocal motion back and forth (Figure 5B) using sufficient irrigation (use 5 ml syringe to dispense saline at the time of the defect creation). To avoid damage to the soft tissue, cut the saw wire close to the bone on one side after completing the osteotomy. Remove the saw guide (Figure 4B).</li>
	</ol>
	</li>
	<li>After the defect or osteotomy is created, remove the saw guide and close the wound in layers, the muscle first (Figure 4C), and then the skin (Figure 4D). Before the wound is closed, treat the defect as planned in the study protocol. Close the muscular layer and the fascia lata using Ethibond vicryl suture 4-0, and the skin using Ethicon Monocryl 3-0 suture. Avoid dragging suture material over non-sterile surfaces while suturing wounds. Note: To avoid wound biting, the suture must not end distal to the lower implant. Likewise, skin glue can be used instead of a suture.</li>
	<li>On the first three postoperative days, give the rat analgesic every 12 hr and antibiotic every 24 hr. Of course, the post-operative regimen of drugs will vary depending upon the make and brand of the drugs used by each investigator (refer to drug specification instructions).</li>
	<li>Monitor animals frequently after the procedure to make sure they recover from anesthesia and only then return them to the housing facility. Provide solitary housing for the first few days after the surgery to make sure that there are no complications.</li>
	<li>Monitor water, food intake and body weight after the surgery to make sure the animal is not in pain and distress. If the animal shows a decreased activity level, difficulty moving around (possible implant failure), ataxia, unkempt greasy fur, porphyrin staining around eyes and nostrils, hunched posture, respiratory distress, reduced intake of food and water, etc. consult a veterinarian.</li>
</ol>
5. Change of External Fixator Stiffness In vivo
<ol>
	<li>If the study protocol requires the change of fixator stiffness during the healing process in vivo this is achieved by changing the connection elements secured with the special interlocking screws using 0.5 mm square wrench box attached to the hand drill. For this procedure, anesthetize the rat (refer to the 2.3 in the protocol) and give analgesia (refer to the 2.4 in the protocol) only once at the time of the procedure (Figure 6A).
	<ol>
		<li>Sedate the rat, and then insert the tip of the 0.50 mm square box wrench into the interlocking screw attached to the side of the assembled fixator, and start carefully turning it counter clockwise until the pin is half way out (Figure 6B). After this part is complete, repeat the procedure for the second pin that is on the same side of the external fixator plate (Figure 6C).</li>
		<li>When both pins on the same side are half way out, use forceps or a clamp to remove the connection element on the opposite side with a gentle motion (Figure 6D). The connection element should come off easy, if it doesn&#8217;t, then do an additional couple of turns on both interlocking screws to make sure that the tip of the interlocking screw is not imbedded in the connection element.</li>
		<li>After the connection element is removed, slide the desired stiffness connection element in place of the removed one (Figure 6E), and from the opposite side using the square box wrench start turning until the interlocking screw is half way out on the opposite side (Figure 6F). Repeat the same procedure for the second interlocking screw (Figure 6G). Important: this will require switching to the opposite side of the plate to make sure that both interlocking screws are half way out on the side where the connection element was replaced (Figure 6H,I).</li>
		<li>After this part is successfully completed, remove the second connection element (Figure 6J) and replace it with the same stiffness connection element as the one replaced on the opposite side (Figure 6K). After the second connection element is in place, start driving the interlocking screw until the interlocking screw end exits the opposite side of the plate, and the interlocking screw tip has exited the same amount on each side (Figure 6L). Repeat the same procedure for the second interlocking screw (Figure 6M,N). This procedure takes about 15 min to complete.</li>
	</ol>
	</li>
</ol>

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R., Geddis, C., Fazzalari, N., Marsh, D., Parkinson, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcomputed+tomography+imaging+in+a+rat+model+of+delayed+union/non-union+fracture.">Microcomputed tomography imaging in a rat model of delayed union/non-union fracture.</a> J Orthop Res. 26, 729-736 (2008).</li><li>Jager, M., Sager, M., Lensing-Hohn, S., Krauspe, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+critical+size+bony+defect+in+a+small+animal+for+bone+healing+studies+(II):+implant+evolution+and+surgical+technique+on+a+rat's+femur.">The critical size bony defect in a small animal for bone healing studies (II): implant evolution and surgical technique on a rat's femur.</a> Biomed Tech (Berl). 50, 137-142 (2005).</li><li>Betz, V. 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=Healing+of+segmental+bone+defects+by+direct+percutaneous+gene+delivery:+effect+of+vector+dose.">Healing of segmental bone defects by direct percutaneous gene delivery: effect of vector dose.</a> Hum Gene Ther. 18, 907-915 (2007).</li><li>Glatt, V., 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=Ability+of+recombinant+human+bone+morphogenetic+protein+2+to+enhance+bone+healing+in+the+presence+of+tobramycin:+evaluation+in+a+rat+segmental+defect+model.">Ability of recombinant human bone morphogenetic protein 2 to enhance bone healing in the presence of tobramycin: evaluation in a rat segmental defect model.</a> J Orthop Trauma. 23, 693-701 (2009).</li><li>Willie, B., Adkins, K., Zheng, X., Simon, U., Claes, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+characterization+of+external+fixator+stiffness+for+a+rat+femoral+fracture+model.">Mechanical characterization of external fixator stiffness for a rat femoral fracture model.</a> J Orthop Res. 27, 687-693 (2009).</li><li>Hess, T., Hopf, T., Fritsch, E., Mittelmeier, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+biomechanical+studies+of+conventional+and+self-tapping+cortical+bone+screws.">Comparative biomechanical studies of conventional and self-tapping cortical bone screws.</a> Z Orthop Ihre Grenzgeb. 129, 278-282 (1991).</li><li>Glatt, V., Evans, C. H., 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=Design,+characterisation+and+in+vivo+testing+of+a+new,+adjustable+stiffness,+external+fixator+for+the+rat+femur.">Design, characterisation and in vivo testing of a new, adjustable stiffness, external fixator for the rat femur.</a> Eur Cell Mater. 23, 289-298 (2012).</li><li>Glatt, V., 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=Improved+healing+of+large+segmental+defects+in+the+rat+femur+by+reverse+dynamization+in+the+presence+of+bone+morphogenetic+protein-2.">Improved healing of large segmental defects in the rat femur by reverse dynamization in the presence of bone morphogenetic protein-2.</a> J Bone Joint Surg Am. 94, 2063-2073 (2012).</li></ol>

The author Romano Matthys is an employee of RISystem AG Davos, Switzerland that produces the implants, implant specific instruments &#38; consumables used in this article. The author Vaida Glatt has no competing financial interests.

The most critical steps of a surgical procedure to create a large bone defect are: 1) choosing the appropriate body weight of the rat to match the size of the external fixator; 2) maintaining a sterile environment during the procedure; and 3) following the surgical procedure protocol.
The main goals of this study were to design, manufacture and characterize a new, variable stiffness external fixator for the rat femoral large defect model, and to use this fixator in determining the interplay be...

A number of studies have improved our understanding of the biologic mechanisms involved in bone tissue repair1-6. The effects of mechanical conditions on bone repair such as axial, shear and interfragmentary movements (IFMs) have been studied extensively7-15. In the past several years, more and more studies started to emerge describing the influence of mechanical environment on bone healing using fracture, osteotomy and large segmental bone defect in vivo models. Therefore, reliable fixation methods are needed to get reproducible and reliable study outcomes.
The mechanical environment around the healing fracture is very important as it determines the way the fracture will heal. Thus, the choice of fixation device is very important and should be carefully selected depending on the study design, and other factors such as gap size and the type of fracture. The fixation device&#8217;s mechanical properties are even more important when studying the bony healing of large bone defects to establish a fixation that provides not only a constant gap size throughout the experiment period of full weight bearing, but also an ideal mechanical environment for the healing bone. External fixators are commonly used in fracture and large bone defect experimental healing models because they have an advantage over other fixation devices. The main advantage of external fixators are that they allow for the change of the mechanical environment at the defect site in vivo without a secondary intervention, which can be achieved by changing or adjusting the stability bar of the device during the course of the experiment as the bone healing progresses. Moreover, it permits the application of specific local mechanical stimulation to enhance the repair of bone, and also provides the potential to measure the stiffness of callus tissue in vivo. Nevertheless, the devices also have a few disadvantages that include: irritation of soft tissue, infections and pin breakage.
Unfortunately, such implants were not available &#8220;off the shelf&#8221; at the time of the implant development, and investigators were forced to custom design their own fixators for an intended use. Therefore, one constraint of research in this area was the lack of experimental control over the local mechanical environment within a large segmental defect as well as osteotomies as it heals. The mechanical characteristics of an external fixator are defined by, and can be modulated by, a large number of variables which include: the distance between the pins, pin diameter, pin material, the number of pins, fixator bar length, fixator bar number, fixator bar material, fixator bar thickness and the distance from the bone surface to the fixator bar (offset). Surprisingly, only a paucity of studies could be found that have investigated the mechanical contributions of the individual components of fixators or whole frame configurations used in rodent studies16,18,28. For example, one study&#8217;s results showed that one of the main contributing factors in determining the total stiffness of the fixation construct was dominated by the flexibility of the pins in relation to their offset, diameter and material properties28. The results from the aforementioned studies clearly suggest that knowing the mechanical environment provided by the fixation device is extremely important, and yet, in many cases is not investigated in detail. The present paper reports the design, specifications, and in vivo implantation of an external fixator that addresses this issue. This fixator also allows for the modulation of the mechanical environment as healing progresses, a property that enables the study of the mechano-sensitivity of different stages of the healing process in vivo. Additionally, as well as imposing a controlled and reproducible local mechanical environment, its accessibility also allows for the modulation of this environment at different stages of bone healing.
The fixator we designed was based upon external fixation, which is widely used for fracture fixation16-21 and large defect models in experimental animals22-27. The difference between our external fixator and the other existing designs reported in the literature is that their stability bar is secured with screws to have a tight grip with Kirschner wires (K-wires). This type of design requires screws to be retightened biweekly (sometimes even weekly) to make sure that the distance of the offset is maintained as the loading is applied through weight bearing to prevent the loosening of the stability bar. If such loosening occurs, it allows for unwanted additional loading conditions such as angular, transverse and torsional shear movements to the healing bone (based on personal experience, communication with researchers). Knowing this, an external fixator was designed as such that when the stiffness of the fixator needs to be changed, it would be achieved by removing connection elements attached to the main module where the mounting pins are imbedded. The in vivo pilot experiment was performed with the new external fixator prototype to make sure that it meets all proposed demands before it is manufactured in larger quantities.
The main aim for this paper is to present a new surgical method for an external fixator used for large bone defects and osteotomies in the rat with the ability to change stiffness in vivo during the healing process. This fixation method is applied in vivo on the femora of rats.

<table border="0" cellpadding="0" cellspacing="0" width="848">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">Name of Material/ Equipment</td>
			<td style="width:283px;">Company</td>
			<td style="width:117px;">Catalog Number</td>
			<td style="width:216px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RatExFix simple 100%</td>
			<td>RISystem AG Davos, Switzerland</td>
			<td style="width:117px;">RIS.612.120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RatExFix simple 70%</td>
			<td>RISystem AG Davos, Switzerland</td>
			<td style="width:117px;">RIS.612.123</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RatExFix simple 40%</td>
			<td>RISystem AG Davos, Switzerland</td>
			<td style="width:117px;">RIS.612.121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RatExFix simple 10%</td>
			<td>RISystem AG Davos, Switzerland</td>
			<td style="width:117px;">RIS.612.122</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">RatExFix Connection element 100%</td>
			<td>RISystem AG Davos, Switzerland</td>
			<td style="width:117px;">RIS.612.130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">RatExFix Connection element 70%</td>
			<td>RISystem AG Davos, Switzerland</td>
			<td style="width:117px;">RIS.612.131</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">RatExFix Connection element 40%</td>
			<td>RISystem AG Davos, Switzerland</td>
			<td style="width:117px;">RIS.612.132</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">RatExFix Connection element 10%</td>
			<td>RISystem AG Davos, Switzerland</td>
			<td style="width:117px;">RIS.612.133</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:232px;">RatExFix Main body</td>
			<td>RISystem AG Davos, Switzerland</td>
			<td style="width:117px;">RIS.611.101</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:232px;">RatExFix InterlockingScrew</td>
			<td>RISystem AG Davos, Switzerland</td>
			<td style="width:117px;">RIS.412.110</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">RatExFix Mounting pin 0.85 mm</td>
			<td>RISystem AG Davos, Switzerland</td>
			<td style="width:117px;">RIS.412.100</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:232px;">RatExFix Saw Guide 100% 5 mm</td>
			<td>RISystem AG Davos, Switzerland</td>
			<td style="width:117px;">RIS.312.100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Accu Pen 6V+</td>
			<td>RISystem AG Davos, Switzerland</td>
			<td style="width:117px;">RIS.390.211</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:232px;">HandDrill</td>
			<td>RISystem AG Davos, Switzerland</td>
			<td style="width:117px;">RIS.390.130</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Drill Bit 0.79 mm</td>
			<td>RISystem AG Davos, Switzerland</td>
			<td style="width:117px;">RIS.593.203</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Gigly wire saw 0.22 mm</td>
			<td>RISystem AG Davos, Switzerland</td>
			<td style="width:117px;">RIS.590.100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Square box wrench 0.70 mm</td>
			<td>RISystem AG Davos, Switzerland</td>
			<td style="width:117px;">RIS.590.112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Square box wrench 0.50 mm</td>
			<td>RISystem AG Davos, Switzerland</td>
			<td style="width:117px;">RIS.590.111</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Centering bit 1.00 mm</td>
			<td>RISystem AG Davos, Switzerland</td>
			<td style="width:117px;">RIS.592.205</td>
			<td></td>
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			<td height="21" style="height:21px;width:232px;">Scalpel Blade handle</td>
			<td style="width:283px;">Fine Science tools</td>
			<td style="width:117px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Scalpel Blade (Size 15)</td>
			<td style="width:283px;">Fisher Scientific</td>
			<td style="width:117px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:232px;">Tissue Forceps</td>
			<td style="width:283px;">Fine Science tools</td>
			<td style="width:117px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:232px;">Scissors</td>
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			<td style="width:117px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Retractor</td>
			<td style="width:283px;">Fine Science tools</td>
			<td style="width:117px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Needle Holder</td>
			<td style="width:283px;">Fine Science tools</td>
			<td style="width:117px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Henahan Elevator</td>
			<td style="width:283px;">Fine Science tools</td>
			<td style="width:117px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">S-shape curved dissecting and ligature forceps&#160;</td>
			<td style="width:283px;">Fine Science tools</td>
			<td style="width:117px;"></td>
			<td>2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Dressing Forceps</td>
			<td style="width:283px;">Fine Science tools</td>
			<td style="width:117px;"></td>
			<td>2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Sterile Fenestrated drape</td>
			<td style="width:283px;">Fisher Scientific</td>
			<td style="width:117px;"></td>
			<td>for surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile gauze</td>
			<td style="width:283px;">Fisher Scientific</td>
			<td style="width:117px;"></td>
			<td>for surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5 ml syringe&#160;</td>
			<td style="width:283px;">Fisher Scientific</td>
			<td style="width:117px;"></td>
			<td>&#160;for irrigation of defect</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-27G needle&#160;</td>
			<td style="width:283px;">Fisher Scientific</td>
			<td style="width:117px;"></td>
			<td>&#160;for irrigation of defect</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1cc Insulin syringes&#160;</td>
			<td style="width:283px;">Fisher Scientific</td>
			<td style="width:117px;"></td>
			<td>for drug injections</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sterile saline&#160;</td>
			<td style="width:283px;">Fisher Scientific</td>
			<td style="width:117px;"></td>
			<td>for bone defect irrigation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sterile gloves</td>
			<td style="width:283px;">Fisher Scientific</td>
			<td style="width:117px;"></td>
			<td>to perform surgeries</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">chlorohezadine</td>
			<td style="width:283px;">Fisher Scientific</td>
			<td style="width:117px;"></td>
			<td>disinfecting solution for surgical site</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vicryl suture 4-0 with SH-1</td>
			<td style="width:283px;">Fisher Scientific</td>
			<td style="width:117px;"></td>
			<td>to suture muscle&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethibond suture 3-0&#160;</td>
			<td style="width:283px;">Fisher Scientific</td>
			<td style="width:117px;"></td>
			<td>to suture skin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isofluorine</td>
			<td style="width:283px;">Sigma-Aldrich</td>
			<td style="width:117px;"></td>
			<td>for anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Buprenorphine</td>
			<td style="width:283px;">Sigma-Aldrich</td>
			<td style="width:117px;"></td>
			<td>analgesia during and after the surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cefazolin</td>
			<td style="width:283px;">Sigma-Aldrich</td>
			<td style="width:117px;"></td>
			<td>antibiotic during and after the surgery&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sprague-Dawley Rats or any other strain</td>
			<td>Charles River Laboratories International, Inc. (Wilmington, MA USA)&#160;</td>
			<td style="width:117px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Design specifications
Stabilization of the rat femur with the external fixation system enables the creation of osteotomies from 0.5 to 5 mm. The external fixator system is a locked external fixator made of polyether ether ketone (PEEK - [the main body]) and titanium-aluminium-niobium alloy (TAN - [the mounting pins]), which offers a simple, reproducible and adjustable design, and is available in four different stiffnesses: 10, 40, 70 and 100% (100% being the standard, most rigid fixator (...

Watch this Scientific Journal Video about Adjustable Stiffness, External Fixator for the Rat Femur Osteotomy and Segmental Bone Defect Models at JoVE.com

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

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 ...

adjustable-stiffness-external-fixator-for-rat-femur-osteotomy

Research

JoVE Journal

Medicine

22.4K Views.  Queensland University of Technology. 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.

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

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal hyperplasia (IH) that eventually leads to vessel occlusion and graft failure. Mechanical forces, particularly low shear stress and high wall tension, are thought to initiate and to sustain these cellular and molecular changes, but their exact contribution remains to be unraveled. To selectively evaluate the role of pressure and shear stress on the biology of IH, an ex&#160;vivo perfusion system (EVPS) was created to perfuse segments of human saphenous veins under arterial regimen (high shear stress and high pressure). Further technical innovations allowed the simultaneous perfusion of two segments from the same vein, one reinforced with an external mesh. Veins were harvested using a no-touch technique and immediately transferred to the laboratory for assembly in the EVPS. One segment of the freshly isolated vein was not perfused (control, day 0). The two others segments were perfused for up to 7 days, one being completely sheltered with a 4 mm (diameter) external mesh. The pressure, flow velocity, and pulse rate were continuously monitored and adjusted to mimic the hemodynamic conditions prevailing in the femoral artery. Upon completion of the perfusion, veins were dismounted and used for histological and molecular analysis. Under ex&#160;vivo conditions, high pressure perfusion (arterial, mean = 100 mm Hg) is sufficient to generate IH and remodeling of human veins. These alterations are reduced in the presence of an external polyester mesh.

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

The mechanisms leading to the development of intimal hyperplasia (IH) and vein graft failure are still poorly understood. This study describes an ex&#160;vivo system to perfuse human veins under controlled flow and pressure. Furthermore the efficiency of external mesh reinforcement to limit the development of IH was evaluated.

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a model organism, the mouse has already been widely used in bone-related research. Large diaphyseal bone defect models in mice, however, are sparse and often use bone fixation which fills the bone marrow cavity and does not provide optimal mechanical stability. The objectives of the current study were to develop a critical-size, segmental, femoral defect in nude mice. A 3.5-mm mid-diaphyseal femoral ostectomy (approximately 25% of the femur length) was performed using a dedicated jig, and was stabilized with an anterior located locking plate and 4 locking screws. The bone defect was subsequently either left empty or filled with a bone substitute (syngenic bone graft or coralline scaffold). Bone healing was monitored noninvasively using radiography and in vivo micro-computed-tomography and was subsequently assessed by ex vivo micro-computed-tomography and undecalcified histology after animal sacrifice, 10 weeks postoperatively. The recovery of all mice was excellent, a full-weight-bearing was observed within one day following the surgical procedure. Furthermore, stable bone fixation and consistent fixation of the implanted materials were achieved in all animals tested throughout the study. When the bone defects were left empty, non-union was consistently obtained. In contrast, when the bone defects were filled with syngenic bone grafts, bone union was always observed. When the bone defects were filled with coralline scaffolds, newly-formed bone was observed in the interface between bone resection edges and the scaffold, as well as within a short distance within the scaffold. 
The present model describes a reproducible critical-size femoral defect stabilized by plate osteosynthesis with low morbidity in mice. The new load-bearing segmental bone defect model could be useful for studying the underlying mechanisms in bone regeneration pertinent to orthopaedic applications.

Although mouse models are invaluable tools for bone tissue engineering, models of long bone defects are sparse. This need motivated development of the present protocol which uses a locking plate with four screws and a dedicated jig to perform and stabilize a reproducible, femoral, critical-size defect with low morbidity.

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a ...

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.

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 ...

Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ischemia/reperfusion (I/R) injury with myriad negative consequences. Potential I/R injury in marginal organs destined for liver transplantation contributes to the current donor shortage secondary to a decreased organ utilization rate. A significant need exists to explore hepatic I/R injury in order to mediate its impact on graft function in transplantation. Rat liver hilar clamp models are used to investigate the impact of different molecules on hepatic I/R injury. Depending on the model, these molecules have been delivered using inhalation, epidural infusion, intraperitoneal injection, intravenous administration or injection into the peripheral superior mesenteric vein. A rat liver hilar clamp model has been developed for use in studying the impact of pharmacologic molecules in ameliorating I/R injury. The described model for rat liver hilar clamp includes direct cannulation of the portal supply to the ischemic hepatic segment via a side branch of the portal vein, allowing for direct segmental hepatic delivery. Our approach is to induce ischemia in the left lateral and median lobes for 60 min, during which time the substance under study is infused. In this case, pegylated-superoxide dismutase (PEG-SOD), a free radical scavenger, is infused directly into the ischemic segment. This series of experiments demonstrates that infusion of PEG-SOD is protective against hepatic I/R injury. Advantages of this approach include direct injection of the molecule into the ischemic segment with consequent decrease in volume of distribution and reduction in systemic side effects.

A unique rat liver hilar clamp model was developed for studying the impact of pharmacologic molecules in ameliorating ischemia-reperfusion injury. This model includes direct cannulation of the portal supply to the ischemic liver segment via a branch of the portal vein, allowing for direct hepatic delivery.

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ...

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 ...

Using a Knee Arthrometer to Evaluate Tissue-specific Contributions to Knee Flexion Contracture in the Rat

Normal knee range of motion (ROM) is critical to well-being and allows one to perform basic activities such as walking, climbing stairs and sitting. Lost ROM is called a joint contracture and results in increased morbidity. Due to the difficulty of reversing established knee contractures, early detection is important, and hence, knowing risk factors for their development is essential. The rat represents a good model with which the effect of an intervention can be studied due to the similarity of rat knee anatomy to that of humans, the rat's ability to tolerate long durations of knee immobilization in flexion, and because mechanical data can be correlated with histologic and biochemical analysis of knee tissue.
Using an automated arthrometer, we demonstrate a validated, precise, reproducible, user-independent method of measuring the extension ROM of the rat knee joint at specific torques. This arthrometer can be used to determine the effects of interventions on knee joint ROM in the rat.

The goal of the protocol is to measure the extension range of motion of the rat knee. The effects of various diseases that increase the stiffness of the knee joint and the effectiveness of treatments can be quantified.

Normal knee range of motion (ROM) is critical to well-being and allows one to perform basic activities such as walking, climbing stairs and sitting. Lost ...

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the luminal surface area in arteries thereby reducing adequate blood flow to organs and distal tissues. Clinically, revascularization procedures such as balloon angioplasty with or without stent placement aim to restore blood flow. Although these procedures reestablish blood flow by reducing plaque burden, they damage the vessel wall, which initiates the arterial healing response. The prolonged healing response causes arterial restenosis, or re-narrowing, ultimately limiting the long-term success of these revascularization procedures. Therefore, preclinical animal models are integral for analyzing the pathophysiological mechanisms driving restenosis, and provide the opportunity to test novel therapeutic strategies. Murine models are cheaper and easier to operate on than large animal models. Balloon or wire injury are the two commonly accepted injury modalities used in murine models. Balloon injury models in particular mimic the clinical angioplasty procedure and cause adequate damage to the artery for the development of restenosis. Herein we describe the surgical details for performing and histologically analyzing the modified, pressure-controlled rat carotid artery balloon injury model. Additionally, this protocol highlights how local periadventitial application of therapeutics can be used to inhibit neointimal hyperplasia. Lastly, we present light sheet fluorescence microscopy as a novel approach for imaging and visualizing the arterial injury in three-dimensions.

The rat carotid artery balloon injury mimics the clinical angioplasty procedure performed to restore blood flow in atherosclerotic vessels. This model induces the arterial injury response by distending the arterial wall, and denuding the intimal layer of endothelial cells, ultimately causing remodeling and an intimal hyperplastic response.

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the ...

Iris Fixation via External Pentagram Suturing

Extensive anterior synechia of the iris can result in the gradual disappearance of the anterior chamber. It is one of the most common outcomes of both oculopathy and complicated post anterior segment surgery. This can impact visual function and lead to bullous keratopathy, making it one of the most complex clinical problems. Conventional anterior chamber plasty will partially create the anterior chamber, but the anterior chamber disappears again in some cases. The main reasons are: (1) the iris lens diaphragm is loose with atrophied and tension-free iris so that the aqueous humor circulation will push and squeeze the iris forward; (2) the effect of "roller" formed in the process of inflammation or restoration will change the iris structure from peripheral anterior synechia (PAS) to extensive anterior synechia again; (3) the fibration will result in synechia from iris to the cornea. In such cases, corneal endothelium deficiency can't stop the aqueous humor from entering the cornea. This results in persistent corneal edema post-conventional anterior chamber plasty, resulting in progressive rubbing and lachrymation. Therefore, anterior chamber plasty is not the first choice for patients without surgical indications of Descemet's stripping automated endothelial keratoplasty (DSAEK). However, this can be performed in patients with surgical indications who plan to receive DSAEK. A unique method of iris fixation via external pentagram suturing anterior chamber plasty (PSACP) is described here. The present technique is also compared with the conventional anterior chamber plasty. PSACP and DSAEK might be an effective way to cure the bullous keratopathy with extensive anterior synechia of the iris and disappeared anterior chamber.

Iris Fixation via External Pentagram Suturing

The present protocol describes how a 10-0 polypropylene suture crosses the anterior surface of the iris, forming a pentagram to prevent both iris and pupil from moving toward the cornea. This can be combined with subsequent keratoplasty to cure bullous keratopathy with extensive anterior synechia of the iris.

Extensive anterior synechia of the iris can result in the gradual disappearance of the anterior chamber. It is one of the most common outcomes of both ...