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 anesthesia procedure.</li>
	<li>Inject atropine (0.05 mg/kg) and ketamin (20 mg/kg)/ xylaxine (2 mg/kg)&#160;&#160;into the lateral cervical muscle region, behind the ear, for premedication.</li>
	<li>After sedation, tightly apply a rubber band around the base of the ear and clean the ear with topical alcohol.</li>
	<li>Place an over-the-needle plastic catheter in the ear vein and remove the rubber band. Confirm that the catheter is correctly placed. Flush the catheter with heparinized saline and fix with tape.</li>
	<li>For endotracheal intubation, position the miniature pig in sternal recumbency. With the help of an assistant, hold the jaw of the pig with a suitable sling and open its mouth.
	<ol>
		<li>Pass the tip of laryngoscope into the pharyngeal cavity to displace the epiglottis from the soft palate. Use the tip of the laryngoscope blade to make the vocal cords visible and advance the endotracheal tube into the trachea during expiration.</li>
		<li>Feel for a free passage of air for proper intubation and check the chest auscultation for respiration sounds in both left and right sides of the miniature pig.</li>
	</ol>
	</li>
	<li>Fill cuff with appropriate volume of air&#160;of the endotracheal tube with air using a 10-mL syringe and fix the tube to the snout using adhesive tape.</li>
	<li>Provide a 2.0%&#160;concentration&#160;of isoflurane, an inhalation anesthetic for prolonged anesthesia, through the endotracheal intubation tube. Test the corneal and palpebral reflexes to confirm the anesthetization and use ointment on the eyes to prevent dryness.</li>
	<li>Monitor the cardiovascular system, the respiratory system, and the body temperature during anesthesia at least every 5 min until the miniature pig recovers.</li>
	<li>30 min before the spine surgery, apply 30 mg/kg cefazolin IV slowly, the 1st generation of cephalosporin antibiotics.</li>
	<li>Administer 5 - 10 mL/kg/h of warm (37 &#176;C) saline using an IV line to maintain homeostasis and provide 50 &#181;g/kg/min of fentanyl to control pain.</li>
	<li>After the spine surgery, perform extubation when a strong swallowing reflex is apparent.</li>
	<li>Take the miniature pig into a room and monitor until it recovers from anesthesia. Provide food and water when the miniature pig is fully conscious.</li>
	<li>Apply 3 mg/kg enrofloxacin antibiotic with 4.4 mg/kg carprofen&#160;daily for pain control during the first 3 days.</li>
	<li>Monitor the pig daily until the removal of the sutures.</li>
</ol>
2. Spine Surgery
<ol>
	<li>Autoclave the transpedicular screws and posterior fixator systems for sterilization, following the manufacturer&#39;s guidelines.</li>
	<li>Shave the back of the miniature pig, approximately 10 cm from the center to the left or right, using a shaver while the pig is in a supine position. Clean the skin with povidone-iodine solution and 70% alcohol.</li>
	<li>Make a longitudinal midline incision from the second spinous process of the lumbar spine to the first median sacral crest using a scalpel. Dissect through the subcutaneous tissue and fascia to the tip of the spinous processes.</li>
	<li>Elevate the paraspinal muscles subperiosteally from the underlying laminae using a Cobb elevator. Dissect along the spinous process and lamina limited to the facet joints.</li>
	<li>Open the superficial cortex of the entry point (which is just inferior to the mammillary process from L3 to L5 on both sides) with a burr or a rongeur.</li>
	<li>Insert the guide pin at an open site, parallel to the superior endplate and at a 20&#176; angle to the spinous process. Define the ideal starting point using C-arm or portable postero-anterior/lateral X-rays.</li>
	<li>Insert a pedicle probe up to 25 mm according to the X-ray. Confirm the complete intraosseous trajectory by pedicle and body palpation using a pedicle sounding device.</li>
	<li>Insert six pedicle screws into the prepared pedicle of L3 to L5 until the screw head is well-seated. Point the side opening of the implant head in the desired direction and align the horizontal position with the rod trajectory.</li>
	<li>Insert two rods into both sides of the pedicle screw heads, respectively. Put in the sleeve and nut at the pedicle screw head using a universal handle.</li>
	<li>Loosely tighten the nut using a straight socket wrench and firmly tighten the nut using a counter torque wrench.</li>
	<li>Confirm the positions of the pedicle screws using portable postero-anterior/lateral X-rays. 
	NOTE: Wait for the miniature pig to wake up and check the gait patterns and motor function of the hind limb to determine whether a screw has been badly implanted.</li>
	<li>Irrigate the surgery site with 3 L of normal saline using a bulb irrigation syringe with suctioning.</li>
	<li>Place a silicone drain in the surgery site and take out the silicone tip. Close the paraspinal muscles and subcutaneous using 1.0 metric absorbable sutures. Close the skin with 2.0 metric non-absorbable nylon sutures.</li>
	<li>Disinfect the suture site with povidone-iodine and apply a dressing using sterilized gauze and tape.</li>
</ol>
3. Harvest Procedure
<ol>
	<li>At 12 weeks postoperatively, inject xylazine (2 mg/kg) and ketamine (10 mg/kg) into the lateral cervical muscle region, behind the ear, for premedication.</li>
	<li>After sedation, administer 15 mg/kg KCl&#160;&#160;directly into the ear vein catheter for euthanasia.</li>
	<li>Make a longitudinal midline incision at the previous surgery scar lesion. Dissect the soft tissue and paraspinal muscles.</li>
	<li>Expose the spinous process of the lumbar spines, laminas, rods, nuts, and transverse processes from L3 to L5.</li>
	<li>Remove the nuts using a counter torque wrench and rods. Cut the L2-3 disc space and the L5-S1 disc space using an oscillating saw.</li>
	<li>Dissect both sides of middle and anterior part of the L3-5 spine with a Cobb elevator and tower forceps. After harvest, if the spine cannot be tested immediately, wrap it in gauze soaked with saline and store at -20 &#176;C.</li>
</ol>

<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 authors have nothing related to this paper to disclose.

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><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&#039;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&amp;nbsp;</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>&amp;nbsp;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&amp;nbsp;</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 ...

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7.6K Views.  Chonbuk National University School of Medicine, Chonbuk National University Hospital.  Hier introduceren we een methode om transpediculaire schroeven te evalueren door gebruik te maken van een in vivo porcine lumbale ruggengraat model. 

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

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

Neuroscience

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

Laminotomy for Lumbar Dorsal Root Ganglion Access and Injection in Swine

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive targets for injection of novel therapeutics aimed at treating chronic pain. In small animal models, laminectomy has been used to facilitate DRG injection because it involves surgical removal of the vertebral bone surrounding each DRG. We demonstrate a technique for intraganglionic injection of lumbar DRG in a large animal species, namely, swine. Laminotomy is performed to allow direct access to DRG using standard neurosurgical techniques, instruments, and materials. Compared with more extensive bone removal via laminectomy, we implement laminotomy to conserve spinal anatomy while achieving sufficient DRG access. Intraoperative progress of DRG injection is monitored using a non-toxic dye. Following euthanasia on post-operative day 21, the success of injection is determined by histology for intraganglionic distribution of 4&#39;,6-diamidino-2-phenylindole (DAPI). We inject a biologically inactive solution to demonstrate the protocol. This method could be applied in future preclinical studies to target therapeutic solutions to DRG. Our methodology should facilitate testing the translatability of intraganglionic small animal paradigms in a large animal species. Additionally, this protocol may serve as a key resource for those planning preclinical studies of DRG injection in swine.

We describe a method for laminotomy in swine that provides access to lumbar dorsal root ganglia (DRG) for intraganglionic injection. Injection progress is monitored intraoperatively and histologically confirmed up to 21 days after surgery. This protocol could be used for future preclinical studies involving DRG injection.

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

In Vivo Mouse Model of Spinal Implant Infection

Spine implant infections portend poor outcomes as diagnosis is challenging and surgical eradication is at odds with mechanical spinal stability. The purpose of this method is to describe a novel mouse model of spinal implant infection (SII) that was created to provide an inexpensive, rapid, and accurate in vivo tool to test potential therapeutics and treatment strategies for spinal implant infections.
In this method, we present a model of posterior-approach spinal surgery in which a stainless-steel k-wire is transfixed into the L4 spinous process of 12-week old C57BL/6J wild-type mice and inoculated with 1 x 103 CFU of a bioluminescent strain of Staphylococcus aureus Xen36 bacteria. Mice are then longitudinally imaged for bioluminescence in vivo on post-operative days 0, 1, 3, 5, 7, 10, 14, 18, 21, 25, 28, and 35. Bioluminescence imaging (BLI) signals from a standardized field of view are quantified to measure in vivo bacterial burden.
To quantify bacteria adhering to implants and peri-implant tissue, mice are euthanized and the implant and surrounding soft tissue are harvested. Bacteria are detached from the implant by sonication, cultured overnight and then colony forming units (CFUs) are counted. The results acquired from this method include longitudinal bacterial counts as measured by in vivo S. aureus bioluminescence (mean maximum flux) and CFU counts following euthanasia.
While prior animal models of instrumented spine infection have involved invasive, ex vivo tissue analysis, the mouse model of SII presented in this paper leverages noninvasive, real time in vivo optical imaging of bioluminescent bacteria to replace static tissue study. Applications of the model are broad and may include utilizing alternative bioluminescent bacterial strains, incorporating other types of genetically engineered mice to contemporaneously study host immune response, and evaluating current or investigating new diagnostic and therapeutic modalities such as antibiotics or implant coatings.

The protocol describes a novel in vivo mouse model of spinal implant infection where a stainless-steel k-wire implant is infected with bioluminescent Staphylococcus aureus Xen36. Bacterial burden is monitored longitudinally with bioluminescent imaging and confirmed with colony forming unit counts after euthanasia.

Spine implant infections portend poor outcomes as diagnosis is challenging and surgical eradication is at odds with mechanical spinal stability. The ...

Real-Time Assessment of Spinal Cord Microperfusion in a Porcine Model of Ischemia/Reperfusion

Spinal cord injury is a devastating complication of aortic repair. Despite developments for the prevention and treatment of spinal cord injury, its incidence is still considerably high and therefore, influences patient outcome. Microcirculation plays a key role in tissue perfusion and oxygen supply and is often dissociated from macrohemodynamics. Thus, direct evaluation of spinal cord microcirculation is essential for the development of microcirculation-targeted therapies and the evaluation of existing approaches in regard to spinal cord microcirculation. However, most of the methods do not provide real-time assessment of spinal cord microcirculation. The aim of this study is to describe a standardized protocol for real-time spinal cord microcirculatory evaluation using laser-Doppler needle probes directly inserted in the spinal cord. We used a porcine model of ischemia/reperfusion to induce deterioration of the spinal cord microcirculation. In addition, a fluorescent microsphere injection technique was used. Initially, animals were anesthetized and mechanically ventilated. Thereafter, laser-Doppler needle probe insertion was performed, followed by the placement of cerebrospinal fluid drainage. A median sternotomy was performed for exposure of the descending aorta to perform aortic cross-clamping. Ischemia/reperfusion was induced by supra-celiac aortic cross-clamping for a total of 48 min, followed by reperfusion and hemodynamic stabilization. Laser-Doppler Flux was performed in parallel with macrohemodynamic evaluation. In addition, automated cerebrospinal fluid drainage was used to maintain a stable cerebrospinal pressure. After completion of the protocol, animals were sacrificed, and the spinal cord was harvested for histopathological and microsphere analysis. The protocol reveals the feasibility of spinal cord microperfusion measurements using laser-Doppler probes and shows a marked decrease during ischemia as well as recovery after reperfusion. Results showed comparable behavior to fluorescent microsphere evaluation. In conclusion, this new protocol might provide a useful large animal model for future studies using real-time spinal cord microperfusion assessment in ischemia/reperfusion conditions.

Spinal cord microcirculation plays a pivotal role in spinal cord injury. Most methods do not allow real-time assessment of spinal cord microcirculation, which is essential for the development of microcirculation-targeted therapies. Here, we propose a protocol using Laser-Doppler-Flow Needle probes in a large animal model of ischemia/reperfusion.

Spinal cord injury is a devastating complication of aortic repair. Despite developments for the prevention and treatment of spinal cord injury, its ...

Surgical Technique for Lumbar Spinal Catheter Insertion in Pigs Enabling Continuous Access to the Thecal Sac in a Terminal Setup

Pigs are increasingly used as a large animal model for pharmacologic CNS research due to the anatomical and physiological similarities between the porcine and human central nervous systems (CNS). However, accessing the cerebrospinal fluid (CSF) in larger pig breeds by conventional lumbar puncture techniques can be challenging due to an oblique orientation of the spinal spinous processes and a limited interlaminar space. Accordingly, an open surgical procedure for inserting a lumbar spinal catheter for continuous CSF sampling at the L4/L5 level in pigs is thoroughly described in this work. After positioning the pig and identifying the anatomical landmarks, a dorsal midline surgical incision is made to expose the spinous processes. By advancing the introducer needle, the spinal catheter is inserted inside the thecal sac of the spinal canal while leaving the bone structures of the spine intact. This method allows continuous infusion into or sampling from the porcine thecal sac with minimal bleeding or CSF leakage. The procedure is simple, time-efficient, and reproducible across different experimental setups, offering significant potential for various pre-clinical studies, including pharmacokinetic research, surgical training, and spinal cord injury models.

We present a technique for inserting a lumbar spinal catheter at the L4-L5 level in a 3-month-old Danish Landrace pig as part of a terminal research protocol, enabling continuous infusion or CSF sampling from the thecal sac.

Pigs are increasingly used as a large animal model for pharmacologic CNS research due to the anatomical and physiological similarities between the porcine ...

A Mouse Model of Lumbar Spine Instability

Intervertebral disc degeneration (IDD) is a common pathological change leading to low back pain. Appropriate animal models are desired for understanding the pathological processes and evaluating new drugs. Here, we introduced a surgically induced lumbar spine instability (LSI) mouse model that develops IDD starting from 1 week post operation. In detail, the mouse under anesthesia was operated by low back skin incision, L3&#8211;L5 spinous processes exposure, detachment of paraspinous muscles, resection of processes and ligaments, and skin closure. L4&#8211;L5 IVDs were chosen for the observation. The LSI model develops lumbar IDD by porosity and hypertrophy in endplates at an early stage, decrease in intervertebral disc volume, shrinkage in nucleus pulposus at an intermediate stage, and bone loss in lumbar vertebrae (L5) at a later stage. The LSI mouse model has the advantages of strong operability, no requirement of special equipment, reproducibility, inexpensive, and relatively short period of IDD development. However, LSI operation is still a trauma that causes inflammation within the first week post operation. Thus, this animal model is suitable for study of lumbar IDD.

We developed a lumbar intervertebral disc degeneration mouse model by resection of L3&#8211;L5 spinous processes along with supra- and inter-spinous ligaments and detachment of paraspinous muscles.

Intervertebral disc degeneration (IDD) is a common pathological change leading to low back pain. Appropriate animal models are desired for understanding ...

Biology

Pedicle Screw Placement Using an Augmented Reality Head-Mounted Display in a Porcine Model

This protocol helps assess the accuracy and workflow of an augmented reality (AR) hybrid navigation system using the Magic Leap head-mounted display (HMD) for minimally invasive pedicle screw placement. The cadaveric porcine specimens were placed on a surgical table and draped with sterile covers. The levels of interest were identified using fluoroscopy, and a dynamic reference frame was attached to the spinous process of a vertebra in the region of interest. Cone beam computerized tomography (CBCT) was performed, and a 3D rendering was automatically generated, which was used for the subsequent planning of the pedicle screw placements. Each surgeon was fitted with an HMD that was individually eye-calibrated and connected to the spinal navigation system.
Navigated instruments, tracked by the navigation system and displayed in 2D and 3D in the HMD, were used for 33 pedicle cannulations, each with a diameter of 4.5 mm. Postprocedural CBCT scans were assessed by an independent reviewer to measure the technical (deviation from the planned path) and clinical (Gertzbein grade) accuracy of each cannulation. The navigation time for each cannulation was measured. The technical accuracy was 1.0 mm &#177; 0.5 mm at the entry point and 0.8 mm &#177; 0.1 mm at the target. The angular deviation was 1.5&#176; &#177; 0.6&#176;, and the mean insertion time per cannulation was 141 s &#177; 71 s. The clinical accuracy was 100% according to the Gertzbein grading scale (32 grade 0; 1 grade 1). When used for minimally invasive pedicle cannulations in a porcine model, submillimeter technical accuracy and 100% clinical accuracy could be achieved with this protocol.

The augmented reality head-mounted display, Magic Leap, was used in combination with a conventional navigation system to place pedicle screws in a porcine model by adhering to a novel workflow. With a median insertion time of &lt;2.5 min, submillimeter technical accuracy and 100% clinical accuracy were achieved according to Gertzbein.

This protocol helps assess the accuracy and workflow of an augmented reality (AR) hybrid navigation system using the Magic Leap head-mounted display (HMD) ...

Laser Doppler Needle Probe Insertion: A Technique for Real-time Quantification of Blood Flow within the Spinal Cord in a Porcine Model

Source: Behem, C. R. et al., <a href="https://www.jove.com/v/62047">Real-Time Assessment of Spinal Cord Microperfusion in a Porcine Model of Ischemia/Reperfusion.</a> J. Vis. Exp. (2020).
This video demonstrates the placement of a laser Doppler needle probe in the spinal cord of a porcine model. This helps in the continuous quantification of blood supply to the spinal cord, an important assessment in spinal cord injury.

Source: Behem, C. R. et al., Real-Time Assessment of Spinal Cord Microperfusion in a Porcine Model of Ischemia/Reperfusion. J. Vis. Exp. (2020).
This ...

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

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