Name: an anesthesia, surgery, and harvest method for the evaluation of transpedicular screws using an in vivo porcine lumbar spine model
Uploaded: 2017-05-31T12:30:00
Description: Watch this Scientific Journal Video about An Anesthesia, Surgery, and Harvest Method for the Evaluation of Transpedicular Screws Using an In Vivo Porcine Lumbar Spine Model at JoVE.com

This study was supported by a grant (CNUH-BRI-2012-02-005) funded by the Biomedical Research Institute of Chonbuk National University Hospital (CNUH-BRI), Republic of Korea.

The Institutional Animal Care and Use Committee of Chonbuk National University approved this study. The treatment, use, and handling of animals followed all guidelines and policies. Maintain the operating room at 24 &#176;C.
1. Anesthesia
<ol>
	<li>Acclimatize miniature pigs, aged 12 months, in the experimental unit for at least one week. Perform a clinical examination measuring the respiratory rate, heartrate, and body temperature. Do not feed each miniature pig for 12 h before the anesthes...

<ol>
	<li>Greenfield, R. T., Grant, R. E., Bryant, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pedicle+screw+fixation+in+the+management+of+unstable+thoracolumbar+spine+injuries.">Pedicle screw fixation in the management of unstable thoracolumbar spine injuries.</a> Orthop Rev. 21 (6), 701-706 (1992).</li><li>Upasani, V. 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=Pedicle+screw+surface+coatings+improve+fixation+in+nonfusion+spinal+constructs.">Pedicle screw surface coatings improve fixation in nonfusion spinal constructs.</a> Spine. 34 (4), 335-343 (2009).</li><li>Halvorson, T. L., Kelley, L. A., Thomas, K. A., Whitecloud, T. S., Cook, S. D. <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+bone+mineral+density+on+pedicle+screw+fixation.">Effects of bone mineral density on pedicle screw fixation.</a> Spine. 19 (21), 2415-2420 (1994).</li><li>Weinstein, J. N., Spratt, K. F., Spengler, D., Brick, C., Reid, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+pedicle+fixation:+reliability+and+validity+of+roentgenogram-based+assessment+and+surgical+factors+on+successful+screw+placement.">Spinal pedicle fixation: reliability and validity of roentgenogram-based assessment and surgical factors on successful screw placement.</a> Spine. 13 (9), 1012-1018 (1988).</li><li>Fini, 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=Biological+assessment+of+the+bone-screw+interface+after+insertion+of+uncoated+and+hydroxyapatite-coated+pedicular+screws+in+the+osteopenic+sheep.">Biological assessment of the bone-screw interface after insertion of uncoated and hydroxyapatite-coated pedicular screws in the osteopenic sheep.</a> J Biomed Mater Res A. 66 (1), 176-183 (2003).</li><li>Kim, D. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+Titanium-Coated+Pedicle+Screws:+In+Vivo+Porcine+Lumbar+Spine+Model.">Evaluation of Titanium-Coated Pedicle Screws: In Vivo Porcine Lumbar Spine Model.</a> World Neurosurg. 91, 163-171 (2016).</li><li>Upasani, V. 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=Pedicle+screw+surface+coatings+improve+fixation+in+nonfusion+spinal+constructs.">Pedicle screw surface coatings improve fixation in nonfusion spinal constructs.</a> Spine. 34 (4), 335-343 (2009).</li><li>Smit, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+a+quadrupted+as+an+in+vivo+model+for+the+study+of+the+spine-biomechanical+considrations.">The use of a quadrupted as an in vivo model for the study of the spine-biomechanical considrations.</a> Eur Spine J. 11 (2), 137-144 (2002).</li><li>Aldini, N. N., 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=Pedicular+fixation+in+the+osteoporotic+spine:+a+pilot+in+vivo+study+on+long-term+ovariectomized+sheep.">Pedicular fixation in the osteoporotic spine: a pilot in vivo study on long-term ovariectomized sheep.</a> J Orthop Res. 20 (6), 1217-1224 (2002).</li><li>Fini, 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=Biological+assessment+of+the+bone-screw+interface+after+insertion+of+uncoated+and+hydroxyapatite-coated+pedicular+screws+in+the+osteopenic+sheep.">Biological assessment of the bone-screw interface after insertion of uncoated and hydroxyapatite-coated pedicular screws in the osteopenic sheep.</a> J Biomed Mater Res A. 66 (1), 176-183 (2003).</li><li>Branemark, R., Ohrnell, L. O., Skalak, R., Carlsson, L., Branemark, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanical+characterization+of+osseointegration:+an+experimental+in+vivo+investigation+in+the+beagle+dog.">Biomechanical characterization of osseointegration: an experimental in vivo investigation in the beagle dog.</a> J Orthop Res. 16 (1), 61-69 (1998).</li><li>McLain, R. F., Yerby, S. A., Moseley, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+morphometry+of+L4+vertebrae:+comparison+of+large+animal+models+for+the+human+lumbar+spine.">Comparative morphometry of L4 vertebrae: comparison of large animal models for the human lumbar spine.</a> Spine. 27 (8), E200-E206 (2002).</li><li>Giavaresi, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+preclinical+evaluation+of+the+influence+of+osteoporosis+on+the+anchorage+of+different+pedicle+screw+designs.">In vivo preclinical evaluation of the influence of osteoporosis on the anchorage of different pedicle screw designs.</a> Eur Spine J. 20 (8), 1289-1296 (2011).</li><li>Hasegawa, 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=Hydroxyapatite-coating+of+pedicle+screws+improves+resistance+against+pull-out+force+in+the+osteoporotic+canine+lumbar+spine+model:+a+pilot+study.">Hydroxyapatite-coating of pedicle screws improves resistance against pull-out force in the osteoporotic canine lumbar spine model: a pilot study.</a> Spine J. 5 (3), 239-243 (2005).</li><li>Smorgick, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-+versus+multilevel+fusion+for+single-level+degenerative+spondylolisthesis+and+multilevel+lumbar+stenosis:+four-year+results+of+the+spine+patient+outcomes+research+trial.">Single- versus multilevel fusion for single-level degenerative spondylolisthesis and multilevel lumbar stenosis: four-year results of the spine patient outcomes research trial.</a> Spine. 38 (10), 797-805 (2013).</li><li>Busscher, I., Ploegmakers, J. J., Verkerke, G. J., Veldhuizen, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+anatomical+dimensions+of+the+complete+human+and+porcine+spine.">Comparative anatomical dimensions of the complete human and porcine spine.</a> Eur Spine J. 19 (7), 1104-1114 (2010).</li></ol>

The evaluation of transpedicular screws in the porcine spine requires much time and effort. First, the miniature pig is a large animal. For animal care and anesthesia, the researcher needs a specialized protocol. Second, surgery should maintain an environment similar to that of human surgery. The purpose of evaluating pedicle screws in the porcine spine is to develop an efficient screw that can be applied to humans. Third, evaluating the long-term stability of transpedicular screws requires about three months after the s...

Transpedicular screw fixation is a gold-standard treatment for degenerative lumbar spine and bursting fracture because it involves three columns of the spine and achieves stabilization1,2. However, most patients who undergo such surgery also have osteoporosis3,4. Many studies have evaluated the fixation strength and the osseointegration status of transpedicular screws, because the loosening of pedicle screws currently in use has been reported in patients with osteoporosis5,6.
The porcine spine is similar to the human spine in terms of size. It is less expensive compared to a primate model7. Furthermore, an in vivo mechanical study has demonstrated that the quadruped porcine spine is essentially loaded in the same way as that of the human spine8, which is why many researchers use porcine spines for studies on the prevention of pedicle screw loosening. However, it takes several months to study pedicle screws in the porcine spine because identifying the long-term stability of pedicle screws takes times. In order to compare different types of screws in the vertebral body, it is necessary to insert the screws in similar positions. Therefore, researchers should be well-acquainted with proper anesthesia techniques, standardized surgical protocols, and harvest procedures before performing any experiments. Here, we describe a detailed method for anesthesia, surgery, and harvest for the evaluation of pedicle screw fixation using a porcine spine model, including ex vivo imaging, histology, and strength testing.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Miniature pig</td>
			<td>OrientBio</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Atropine</td>
			<td>Jeil pharmaceutical</td>
			<td>A04900241</td>
			<td>Anesthesia</td>
		</tr>
		<tr>
			<td>Over-the needle plastic catheter</td>
			<td>BD</td>
			<td>REF382412</td>
			<td>Maintenance of IV line</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Yuhan</td>
			<td>A04502441</td>
			<td>Anesthesia</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Bayer Korea</td>
			<td>A00800071</td>
			<td>Anesthesia</td>
		</tr>
		<tr>
			<td>Laryngoscope</td>
			<td>Karl storz</td>
			<td></td>
			<td>Intubation</td>
		</tr>
		<tr>
			<td>Endotracheal tube</td>
			<td>Covidien</td>
			<td></td>
			<td>Intubation</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>JW pharmaceutical Co</td>
			<td>A02104781</td>
			<td>Anesthesia</td>
		</tr>
		<tr>
			<td>Eye ointment</td>
			<td>Hanlim pharma</td>
			<td>A37851721</td>
			<td>Protection of pig&#39;s eye</td>
		</tr>
		<tr>
			<td>Cefazolin</td>
			<td>Donga pharma</td>
			<td>A01503951</td>
			<td>Antibiotics</td>
		</tr>
		<tr>
			<td>Saline</td>
			<td>JW pharmaceutical Co</td>
			<td>A02151392</td>
			<td>Maintenance of fluid homeostasis</td>
		</tr>
		<tr>
			<td>Fentanyl</td>
			<td>Hana pharm</td>
			<td>C03200032</td>
			<td>Pain control</td>
		</tr>
		<tr>
			<td>Enrofloxacin</td>
			<td>Bayer</td>
			<td>93106-60-6&#160;</td>
			<td>Antibiotics</td>
		</tr>
		<tr>
			<td>Morphine</td>
			<td>Myungmoon pharma</td>
			<td>C03700091</td>
			<td>Pain control</td>
		</tr>
		<tr>
			<td>Meloxicam</td>
			<td>Boehringer Ingelheim</td>
			<td>A07600711</td>
			<td>Antibiotics</td>
		</tr>
		<tr>
			<td>Povidone-iodine</td>
			<td>Hyundai pharma</td>
			<td></td>
			<td>Wound dressing</td>
		</tr>
		<tr>
			<td>Scalpel blade size 15</td>
			<td>Braun</td>
			<td>&#160;I1 BB515</td>
			<td>Skin incision</td>
		</tr>
		<tr>
			<td>Cobb elevator</td>
			<td>Codman</td>
			<td>65-2546</td>
			<td>Dissection of muscle</td>
		</tr>
		<tr>
			<td>Burr</td>
			<td>Medtronic</td>
			<td></td>
			<td>Making of starting point of screw</td>
		</tr>
		<tr>
			<td>Rongeur</td>
			<td>Aesculap</td>
			<td>FO515R</td>
			<td>Making of starting point of screw</td>
		</tr>
		<tr>
			<td>Guide pin (K-wire)</td>
			<td>CE</td>
			<td>01067803</td>
			<td>Guidance of screw trajectory</td>
		</tr>
		<tr>
			<td>C-arm</td>
			<td>GE</td>
			<td>OEC 9800 plus</td>
			<td>Guidance of screw trajectory</td>
		</tr>
		<tr>
			<td>Portable X-ray</td>
			<td>Siemens</td>
			<td>Mobile XP hybrid</td>
			<td>Guidance of screw trajectory</td>
		</tr>
		<tr>
			<td>Pedicle probe</td>
			<td>OtisBiotech</td>
			<td>SPI-02-01</td>
			<td>Guidance of screw trajectory</td>
		</tr>
		<tr>
			<td>Pedicle sounding device</td>
			<td>OtisBiotech</td>
			<td>SPI-03-01</td>
			<td>Guidance of screw trajectory</td>
		</tr>
		<tr>
			<td>Pedicle screw</td>
			<td>OtisBiotech</td>
			<td>MS-40025</td>
			<td></td>
		</tr>
		<tr>
			<td>Posterior fixator systems</td>
			<td>OtisBiotech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rod&#160;</td>
			<td>OtisBiotech</td>
			<td>ROD-60140</td>
			<td>Rigid fixation between screws</td>
		</tr>
		<tr>
			<td>Universal handle</td>
			<td>OtisBiotech</td>
			<td>SPI-08-01</td>
			<td>To fix the screws to the rod</td>
		</tr>
		<tr>
			<td>Straight socket wrench</td>
			<td>OtisBiotech</td>
			<td>SPI-06-01</td>
			<td>To fix the screws to the rod</td>
		</tr>
		<tr>
			<td>counter torque wrench</td>
			<td>OtisBiotech</td>
			<td>SPI-07-01</td>
			<td>To fix the screws to the rod</td>
		</tr>
		<tr>
			<td>Bulb irrigation syringe</td>
			<td>Hyupsug medical</td>
			<td>HS-IR-140</td>
			<td>Irrigation</td>
		</tr>
		<tr>
			<td>Silicone drain</td>
			<td>Sewon medical</td>
			<td>2205-006</td>
			<td>To drain the fluid at the surgical site</td>
		</tr>
		<tr>
			<td>3.0 metric absorbable suture</td>
			<td>Ethicon</td>
			<td>BA1673H</td>
			<td>Muscle suture</td>
		</tr>
		<tr>
			<td>2.0 metric nonabsorbable nylon suture</td>
			<td>Ethicon</td>
			<td>W1626T</td>
			<td>Skin suture</td>
		</tr>
		<tr>
			<td>Gauze</td>
			<td>Kingphar Korea</td>
			<td>KP120-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Pentobarbital</td>
			<td>Hanlim pharma</td>
			<td>645301221</td>
			<td>Euthanasia</td>
		</tr>
		<tr>
			<td>Oscillating saw</td>
			<td>Zimmer</td>
			<td></td>
			<td>Harvest spine</td>
		</tr>
		<tr>
			<td>Tower forceps</td>
			<td>Aesculap</td>
			<td>BF461R</td>
			<td>Harvest spine</td>
		</tr>
	</tbody>
</table>

an anesthesia, surgery, and harvest method for the evaluation of transpedicular screws using an in vivo porcine lumbar spine model

Pedicle screw fixation is the gold standard for the treatment of spinal diseases. However, many studies have reported the issue of loosening pedicle screws after spinal surgery, which is a serious concern. To address this problem, diverse types of pedicle screws have been examined to identify those with good fixation strength and osseointegration in spine bone. The porcine spine is a good alternative for the human spine in the evaluation of pedicle screws due to the anatomical size, mechanical characteristics, and cost. Although several studies have reported that pedicle screws are efficient in the porcine model, no study has described detailed protocols for the evaluation of a pedicle screw using the porcine model. Here, we describe a detailed method for evaluating transpedicular screws using an in vivo porcine lumbar spine model. The technical details for anesthesia, spine surgery, and harvest provided here will facilitate with the evaluation of the transpedicular screw fixation model.

A detailed protocol for anesthesia, surgery, and harvest for the evaluation of transpedicular screws using an in vivo porcine lumbar spine model is described here. This protocol is suitable for a number of downstream analyses, including mechanical testing (Figure 1), quantitative micro-CT evaluation (Figure 2), and histology (Figure 3). Representative mechanical testing (Figure 1) shows the mean extraction torsio...

Watch this Scientific Journal Video about An Anesthesia, Surgery, and Harvest Method for the Evaluation of Transpedicular Screws Using an In Vivo Porcine Lumbar Spine Model at JoVE.com

An Anesthesia, Surgery, and Harvest Method for the Evaluation of Transpedicular Screws Using an In Vivo Porcine Lumbar Spine Model

Here, we introduce a method to evaluate transpedicular screws by using an in vivo porcine lumbar spine model.

An Anesthesia, Surgery, and Harvest Method for the Evaluation of Transpedicular Screws Using an In Vivo Porcine Lumbar Spine Model

Pedicle screw fixation is the gold standard for the treatment of spinal diseases. However, many studies have reported the issue of loosening pedicle ...

The overall goal of this surgical intervention is to evaluate transpedicular screws using an in vivo porcine lumbar spine model. This method can help answer key questions about such complications of the spine, such as the loosening of pedicle screw. The main advantage of this technique is that we can compare long-term stability of different type of screws.Maintain 12-month-old miniature pigs in the experimental unit for at least one week. After this acclamation period, give each pig a clinical exam, measure respiratory rate, heart rate, and body temperature. Prior to the anesthesia, starve the pigs for 12 hours.To sedate a pig, inject atropine into the lateral cervical muscle region behind the ear. Once sedated, press a hand around the base of the ear and then clean the ear with alcohol. Then, place an over the needle plastic catheter into the ear vein.Then, flush the catheter with heparinized saline and secure it with tape. Now, induce anesthesia using ketamine and xylazine via the catheter. To intubate the pig, position it in sternal recumbency.And with the help of an assistant, hold open the jaw using a suitable sling. Then, pass the tip of the laryngoscope into the pharyngeal cavity to displace the epiglottis from the soft palate. Once intubated, listen to the respiration in both lungs.Then, fill the cup of the endotracheal tube with air using a 10 milliliter syringe, and fix the tube to the snout, using adhesive tape. Next, begin providing 2%isoflurane through the tube. Then, test the pig's corneal and palpebral reflexes to confirm a surgical plane of anesthetization.Now, apply ointment on the eyes and set up vital sign monitors, which should be checked at least every five minutes. 30 minutes before the surgery, provide a bolus of cefazolin via the ear vein catheter. Then, switch the IV line to administer 37 degree Celsius saline and fentanyl for pain management.To begin, shave the pig's back 10 centimeters to either side of the midline. Then, clean the skin with alternating scrubs of povidone iodine solution and 70%ethanol. Now, make a longitudinal midline incision from the second spinous process of the lumbar spine to the first median sacral crest.Then, dissect through the subcutaneous tissue and fascia down to the tip of the spinous processes. Next, using a Cobb elevator, elevate the paraspinal muscles subperiosteally from the underlying lamina. Then, dissect along the spinous process and lamina at the facet joints.At the entry point, which is just inferior to the mamillary process from L3 to L5, use a burr or rongeur to open the superficial cortex. Then, insert the guide pen parallel to the superior end plate and at a 20 degree angle to the spinous process. Now, take posterior anterior lateral X-rays to define the insertion points for the pedicle screws.Then, insert a pedicle probe up to 25 millimeters and confirm a complete intraosseous trajectory using the sounding device and palpation. Now, insert six pedicle screws. Make sure that the screw heads are well-seated.Pay attention to the orientation of the site opening and the horizontal position which must line up with the rod. Then, insert two rods into both sides of the pedicle screw heads. Next, attach sleeves and nuts to the screw's head.First tighten the nuts loosely with a straight socket wrench and then tighten them with a counter torque wrench. Then, confirm the positions of the pedicle screws using X-rays. Once the site is ready to close, irrigate it with three liters of normal saline.Then attach a silicone drain and take out the silicone tip. Now, close the paraspinal muscles and subcutaneous tissues using absorbable sutures, and then close the skin with nonabsorbable nylon sutures. After suturing, disinfect the site with povidone iodine and apply a dressing.Once a strong swallowing reflex is apparent, extubate the pig and transfer it to a recovery room. Once fully conscious, provide the pig food and water and check its gate patterns. Monitor the hind limb movement to assess the implant and provide pain relievers for three days.12 weeks after the implantation, euthanize the pig. To harvest a spinal section, make a longitudinal midline incision at the previous surgery scar legion. Then, dissect the soft tissue and paraspinal muscles to fully expose the implant from L3 to L5.Now, remove the nuts using a counter torque wrench.And then remove the rods. Next, using an oscillating saw, cut the L2-3 disc space and the L5-S1 disc space. Then, dissect both sites in the middle and anterior part of the L3-5 spine with a Cobb elevator and tower forceps.If the spinal section cannot be immediately analyzed, store it in saline-soaked gauze at minus 20 degrees Celsius. The bonding strength between the pedicle screws and bone was evaluated for three screw types. Uncoated, hydroxyapatite-coated, and titanium-coated.The main extraction torsional peak torque, or bond strength, was greatest with the titanium-coated pedicle screws. The region of interest can be evaluated by a micro-CT program for the analysis of the bone volume fraction, bone surface density, and specific bone surface. Scanning a spinal section with Goldner trichrome shows the interface between the pedicle screw and bone.The fibrous tissue is red, and the bone is blue. When using uncoated pedicle screws, only fibrous tissue was observed at the interface. However, new bone formation was found at the interface for both types of coated screws.It was even observed that with titanium-coated pedicle screws, the space between the screw threads in the bone was completely compacted. Once mastered, this technique can be done in three hours if it is performed properly. While attempting this procedure, it's important to focus on proper positioning of the the pedicle screws so the misplacement of the pedicle can be prevented.After its development, this technique paved the way for researchers in the field of the spine surgery to explore loosening of pedicle screws in patients with osteoporosis.

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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 ...

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 ...

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 Pre-Clinical Porcine Model of Orthotopic Heart Transplantation

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart failure. Multiple small-animal models of heart transplantation have been used to study the acute and long-term effects of novel therapies. However, few are tested and demonstrated success in clinical trials. It is of critical importance to evaluate new therapies in a clinically relevant large-animal model for efficient and reliable translation of basic studies&#39;&#160;findings. Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and previously used to investigate novel cardioprotective strategies. This procedure focuses on acute ischemia-reperfusion injury and is a reliable method to investigate novel interventions which have been tested and validated in smaller experimental models, such as the murine model. We demonstrate its usefulness in assessing cardiac performance during the early post-transplantation period and other potential possibilities enabled by the model.

Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and utilized to investigate novel cardioprotective strategies.

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart ...

An In Vivo Mouse Model of Total Intravenous Anesthesia During Cancer Resection Surgery

Anesthesia is a routine component of cancer care that is used for diagnostic and therapeutic procedures. The anesthetic technique has recently been implicated in impacting long-term cancer outcomes, possibly through modulation of adrenergic-inflammatory responses that impact cancer cell behavior and immune cell function. Emerging evidence suggests that propofol-based total intravenous anesthesia (TIVA) may be beneficial for long-term cancer outcomes when compared to inhaled volatile anesthesia. However, the available clinical findings are inconsistent. Preclinical studies that identify the underlying mechanisms involved are critically needed to guide the design of clinical studies that will expedite insight. Most preclinical models of anesthesia have been extrapolated from the use of anesthesia in in vivo research and are not optimally designed to study the impact of anesthesia itself as the primary endpoint. This paper describes a method for delivering propofol-TIVA anesthesia in a mouse model of breast cancer resection that replicates key aspects of clinical delivery in cancer patients. The model can be used to study mechanisms of action of anesthesia on cancer outcomes in diverse cancer types and can be extrapolated to other non-cancer areas of preclinical anesthesia research.

An In Vivo Mouse Model of Total Intravenous Anesthesia During Cancer Resection Surgery

This paper describes a method for modeling total intravenous anesthesia (TIVA) during cancer resection surgery in mice. The goal is to replicate key features of anesthesia delivery to patients with cancer. The method allows investigation of how anesthetic technique affects cancer recurrence after resection surgery.

Anesthesia is a routine component of cancer care that is used for diagnostic and therapeutic procedures. The anesthetic technique has recently been ...

Establishment and Evaluation of a Porcine Vein Graft Disease Model

Venous graft disease (VGD) is the leading cause of coronary artery bypass graft (CABG) failure. Large animal models of CABG-VGD are needed for the investigation of disease mechanisms and the development of therapeutic strategies. 
To perform the surgery, we enter the cardiac chamber through the third intercostal space and carefully dissect the internal mammary vein and immerse it in normal saline. The right main coronary artery is then treated for ischemia. The target vessel is incised, a shunt plug is placed, and the distal end of the graft vein is anastomosed. The ascending aorta is partially blocked, and the proximal end of the graft vein is anastomosed after perforation. The graft vein is checked for patency, and the proximal right coronary artery is ligated. 
CABG surgery is performed in minipigs to harvest the left internal mammary vein for its use as a vascular graft. Serum biochemical tests are used to evaluate the physiological status of the animals after surgery. Ultrasound examination shows that the proximal, middle, and distal end of the graft vessel are unobstructed. In the surgical model, turbulent blood flow in the graft is observed upon histological examination after the CABG surgery, and venous graft stenosis associated with intimal hyperplasia is observed in the graft. The study here provides detailed surgical procedures for the establishment of a repeatable CABG-induced VGD model.

In this protocol, novel pig vein bypass grafting was performed through a small incision in the left chest wall without cardiopulmonary bypass. A postoperative pathology study was done, which showed intimal thickening.

Venous graft disease (VGD) is the leading cause of coronary artery bypass graft (CABG) failure. Large animal models of CABG-VGD are needed for the ...

3D Printing Model of a Patient's Specific Lumbar Vertebra

Selective dorsal rhizotomy (SDR) is a difficult, risky, and sophisticated operation, in which a laminectomy should not only expose an adequate surgical field of view but also protect the patient&#39;s spinal nerves from injury. Digital models play an important role in the pre-and intra-operation of SDR, because they can not only make doctors more familiar with the anatomical structure of the surgical site, but also provide precise surgical navigation coordinates for the manipulator. This study aims to create a 3D digital model of a patient-specific lumbar vertebra that can be used for planning, surgical navigation, and training of the SDR operation. The 3D printing model is also manufactured for more effective work during these processes.
Traditional orthopedic digital models rely almost entirely on computed tomography (CT) data, which is less sensitive to soft tissues. Fusion of the bone structure from CT and the neural structure from magnetic resonance imaging (MRI) is the key element for the model reconstruction in this study. The patient&#39;s specific 3D digital model is reconstructed for the real appearance of the surgical area and shows the accurate measurement of inter-structural distances and regional segmentation, which can effectively help in the preoperative planning and training of SDR. The transparent bone structure material of the 3D-printed model allows surgeons to clearly distinguish the relative relationship between the spinal nerve and the vertebral plate of the operated segment, enhancing their anatomical understanding and spatial sense of the structure. The advantages of the individualized 3D digital model and its accurate relationship between spinal nerve and bone structures make this method a good choice for preoperative planning of SDR surgery.

3D Printing Model of a Patient&#039;s Specific Lumbar Vertebra

This study aims to create a 3D-printed model of a patient-specific lumbar vertebra, which contains both the vertebra and spinal nerve models fused from high-resolution computed tomography (HRCT) and MRI-Dixon data.

Selective dorsal rhizotomy (SDR) is a difficult, risky, and sophisticated operation, in which a laminectomy should not only expose an adequate surgical ...

Quantitative Analysis of Liver Stiffness Distribution

A Three-Dimensional Digital Model for Early Diagnosis of Hepatic Fibrosis Based on Magnetic Resonance Elastography

Hepatic fibrosis is an early stage of liver cirrhosis, and there are no better non-invasive and convenient methods for the detection and evaluation of the disease. Despite the good progress made with the liver stiffness map (LSM) based on magnetic resonance elastography (MRE), there are still some limitations that need to be overcome, including manual focus determination, manual selection of regions of interest (ROIs), and discontinuous LSM data without structural information, which makes it impossible to evaluate the liver as a whole. In this study, we propose a novel three-dimensional (3D) digital model for the early diagnosis of hepatic fibrosis based on MRE.
MRE is a non-invasive imaging technique that employs magnetic resonance imaging (MRI) to measure the liver stiffness at the scanning site through human-computer interaction. Studies have indicated a significant positive correlation between the LSM obtained through MRE and the degree of hepatic fibrosis. However, for clinical purposes, a comprehensive and precise quantification of the degree of hepatic fibrosis is necessary. To address this, the concept of Liver Stiffness Distribution (LSD) was proposed in this study, which refers to the 3D stiffness volume of each liver voxel obtained by the alignment of 3D liver tissue images and MRE indicators. This provides a more effective clinical tool for the diagnosis and treatment of hepatic fibrosis.

Author Spotlight: Advancing Hepatic Fibrosis Diagnosis Using Magnetic Resonance Elastography and AI 

The objective of this study was to develop a novel three-dimensional digital model for the early diagnosis of hepatic fibrosis, which includes the stiffness of each voxel in the patient's liver and can thus, be used to calculate the distribution ratio of the patient's liver at different fibrosis stages.

Hepatic fibrosis is an early stage of liver cirrhosis, and there are no better non-invasive and convenient methods for the detection and evaluation of the ...

A 3-Channel Endoscopic Technique for Lumbar Stenosis and Contralateral Herniation

The Third Channel-Assisted Unilateral Biportal Endoscopic Technique for Lumbar Spinal Stenosis Combined with Contralateral Disc Herniation

Unilateral biportal endoscopic (UBE) spine surgery is an emerging minimally invasive surgical (MIS) technique that has gained popularity for treating lumbar spinal stenosis, particularly in Eastern Asia. The traditional UBE technique, with two portals on one side, can achieve successful unilateral laminotomy for bilateral decompression (ULBD) and, therefore, demonstrates favorable clinical outcomes. However, in the case of lumbar spinal stenosis combined with contralateral disc herniation, it is very difficult to remove the contralateral disc herniation, especially the loose disc fragment within the deep disc. Here, a third channel of the traditional UBE technique was developed to do the discectomy within the ipsilateral endoscopic vision, with which the instruments can go vertically into the contralateral disc, allowing easy discectomy. This technique can not only achieve adequate decompression of the bilateral spinal canal but also effectively remove contralateral herniated disc fragments. This technique avoids performing another UBE procedure on the opposite side, which can potentially shorten the duration of the operation, minimize blood loss and tissue damage, and ensure sufficient neural decompression. This paper will introduce the indications and surgical operation procedures, as well as present a classical case report and follow-up data, to facilitate the application of the third channel-assisted UBE (T-UBE) technique for spine surgeons.

Author Spotlight: Unveiling the Potential of TUBE Technique in Spinal Surgery

Here, we present the third channel-assisted UBE technique, which allows for the vertical removal of herniated disc fragments. This technique can effectively address the limitations of traditional UBE techniques. This article will systematically elaborate on this procedure.

Unilateral biportal endoscopic (UBE) spine surgery is an emerging minimally invasive surgical (MIS) technique that has gained popularity for treating ...

Enhancing Pulmonary Infection Treatment Using M-ROSE

Microbiological Rapid On-Site Evaluation for Pulmonary Infectious Diseases

The prompt initiation of empirical anti-infective therapy is crucial in patients presenting with unexplained pulmonary infection. Although imaging acquisition is relatively straightforward in clinical practice, its lack of specificity often necessitates additional time-consuming tests such as sputum culture, bronchoalveolar-lavage fluid culture, or genetic sequencing to identify the underlying etiology of the disease accurately. Moreover, the limited efficacy of empirical anti-infective treatment may contribute to antibiotic misuse. Recent advancements in interpreting microbial background on rapid on-site evaluation (ROSE) slides have enabled clinicians to promptly obtain samples through bronchoscopy (e.g., alveolar lavage, mucosal brushing, tissue clamp), facilitating bedside staining and interpretation that provides essential microbial background information. Consequently, this establishes a foundation for developing targeted anti-infection treatment and individualized drug therapy plans. With a better understanding of which pathogens are causing infections in real-time, physicians can avoid unnecessary broad-spectrum antibiotics contributing to antibiotic resistance. Establishing a rapid and standardized M-ROSE workflow within respiratory medicine departments or intensive care units will greatly assist physicians in formulating accurate treatment strategies for patients, which holds significant clinical implications.

Author Spotlight: Advancing Pathogen Detection and Disease Assessment in Real-Time Using M-ROSE

The protocol here demonstrates a fast and standardized microbiological rapid on-site evaluation (M-ROSE) workflow, including three steps: slide making, staining, and interpretation. This protocol will help physicians make rapid clinical decisions.