Name: ovine lumbar intervertebral disc degeneration model utilizing a lateral retroperitoneal drill bit injury
Uploaded: 2017-05-25T15:00:00
Description: Watch this Scientific Journal Video about Ovine Lumbar Intervertebral Disc Degeneration Model Utilizing a Lateral Retroperitoneal Drill Bit Injury at JoVE.com

Dr. Chris Daly is the recipient of the Foundation for Surgery Richard Jepson Research Scholarship. The authors would like to thank Dr. Anne Gibbon, Dr. Dong Zhang and the staff of Monash Animal Services, Monash University for their assistance with animal surgery and care.

The protocol detailed in this manuscript follows the animal care guidelines of Monash University Animal Ethics. Animal ethics approval for this protocol has been granted by Monash University Animal Ethics. Ethics approval number: MMCA/2014/55
1. Sheep Preparation
NOTE: Ewes aged two to four years were used.
<ol>
	<li>Fast sheep for 18 h prior to anesthesia. Provide animals with access to water until 6-12 h prior to operation19.</li>
	<li>Se...

<ol>
	<li>Hoy, D., March, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+global+burden+of+low+back+pain:+estimates+from+the+Global+Burden+of+Disease+2010+study.">The global burden of low back pain: estimates from the Global Burden of Disease 2010 study.</a> Ann Rheum Dis. 73 (6), 968-974 (2014).</li><li>Luoma, K., Riihim&#228;ki, H., Luukkonen, R., Raininko, R., Viikari-Juntura, E., Lammine, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+back+pain+in+relation+to+lumbar+disc+degeneration.">Low back pain in relation to lumbar disc degeneration.</a> Spine. 25 (4), 487-492 (2000).</li><li>Daly, C., Ghosh, P., Jenkin, G., Oehme, D., Goldschlager, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Review+of+Animal+Models+of+Intervertebral+Disc+Degeneration:+Pathophysiology,+Regeneration,+and+Translation+to+the+Clinic.">A Review of Animal Models of Intervertebral Disc Degeneration: Pathophysiology, Regeneration, and Translation to the Clinic.</a> BioMed Res Int. 2016 (3), 5952165 (2016).</li><li>Sahlman, J., Inkinen, R., 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=Premature+vertebral+endplate+ossification+and+mild+disc+degeneration+in+mice+after+inactivation+of+one+allele+belonging+to+the+Col2a1+gene+for+Type+II+collagen.">Premature vertebral endplate ossification and mild disc degeneration in mice after inactivation of one allele belonging to the Col2a1 gene for Type II collagen.</a> Spine. 26 (23), 2558-2565 (2001).</li><li>Melrose, J., Taylor, T., Ghosh, P., Holbert, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intervertebral+disc+reconstitution+after+chemonucleolysis+with+chymopapain+is+dependent+on+dosage:+An+experimental+study+in+beagle+dogs.">Intervertebral disc reconstitution after chemonucleolysis with chymopapain is dependent on dosage: An experimental study in beagle dogs.</a> Spine. 21 (1), (1996).</li><li>Oehme, D., Goldschlager, T., Shimon, S., Wu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiological,+Morphological,+Histological+and+Biochemical+Changes+of+Lumbar+Discs+in+an+Animal+Model+of+Disc+Degeneration+Suitable+for+Evaluating+the+potential+regenerative+capacity+of+novel+biological+agents.">Radiological, Morphological, Histological and Biochemical Changes of Lumbar Discs in an Animal Model of Disc Degeneration Suitable for Evaluating the potential regenerative capacity of novel biological agents.</a> J Tiss Sci Eng. , (2015).</li><li>Platenberg, R. C., Hubbard, G. B., Ehler, W. J., Hixson, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+disc+degeneration+in+the+baboon+model:+magnetic+resonance+imaging+and+histopathologic+correlation.">Spontaneous disc degeneration in the baboon model: magnetic resonance imaging and histopathologic correlation.</a> J Med Primatol. 30 (5), 268-272 (2001).</li><li>Iatridis, J. C., Mente, P. L., Stokes, I. A. F., Aronsson, D. D., Alini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compression-Induced+Changes+in+Intervertebral+Disc+Properties+in+a+Rat+Tail+Model.">Compression-Induced Changes in Intervertebral Disc Properties in a Rat Tail Model.</a> Spine. 24 (10), 996 (1999).</li><li>Silberberg, R., Aufdermaur, M., Adler, J. 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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimally+invasive+surgery:+lateral+approach+interbody+fusion:+results+and+review.">Minimally invasive surgery: lateral approach interbody fusion: results and review.</a> Spine. 35 (Suppl 26), S302-S311 (2010).</li><li>Zhang, Y., Drapeau, S., An, H. S., Markova, D., Lenart, B. A., Anderson, D. 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J., Matyas, J. R., Duncan, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytomorphology+of+notochordal+and+chondrocytic+cells+from+the+nucleus+pulposus:+a+species+comparison.">Cytomorphology of notochordal and chondrocytic cells from the nucleus pulposus: a species comparison.</a> J Anat. 205 (5), 357-362 (2004).</li><li>Hoogendoorn, R. J., Helder, M. N., Smit, T. H., Wuisman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notochordal+cells+in+mature+caprine+intervertebral+discs.">Notochordal cells in mature caprine intervertebral discs.</a> Eur Cells Mater. 10 (Suppl 3), (2005).</li><li>Pohlmeyer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Zur vergleichenden Anatomie von Damtier, Schaf und Ziege: Osteologie und postnatale Osteogenese. , (1985).</li><li>Pfirrmann, C. 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This minimally invasive lateral access approach is efficacious and safe with no post-operative herniae, abdominal wound dehiscence or infection observed in this series. Use of the drill bit intervertebral disc injury model with a depth stop provides a reproducible method of inducing a consistent intervertebral disc injury of known dimension (i.e. a 3.5 mm diameter x 12 mm depth injury in this study). In our experience, this method produces a less severe degree of disc degeneration than that observed in conventio...

Lower back pain is the leading cause of disability worldwide1. Lumbar intervertebral disc degeneration associated discogenic pain is considered a significant contributor to lower back pain2. There is an increasing demand for reliable animal models of intervertebral disc disease for broadening the understanding of the degenerative process and for the investigation of potential therapies.
Numerous animal models of intervertebral disc degeneration exist3. Animals models used in the investigation of degenerative disc disease range in size from mice4, to larger mammals such as dogs5, sheep6, and non-human primates7. Methods used to induce intervertebral disc degeneration can be broadly classified into the categories of mechanical (e.g. intervertebral disc compression8 or surgical injury6), chemical (e.g. chemical nucleolysis5) or, less commonly, spontaneous degeneration (e.g. the sand rat9).
Given the complexity of human intervertebral disc degeneration, a perfect animal model does not exist. However, important considerations in choosing an appropriate animal model to mimic this condition closely have been identified3. Such considerations include the absence of notochordal cells (primitive cells with possible progenitor cell function10 absent from the adult nucleus pulposus in humans, sheep, goats and chondrodystrophic dogs but present in most mammals), similarities in animal and intervertebral disc size relative to humans, comparable biomechanical forces to the clinical condition, mechanistic and ethical considerations3.
Non-human primates meet many of the above criteria. Baboon and macaque models of spontaneous intervertebral disc degeneration have been described11,12,13. Both species spend large amounts of time in erect or semi-erect postures &#8212; a distinct advantage relative to other animal models. However, ethical and practical consideration (e.g. expense, housing, delayed onset of spontaneous degeneration) restrict their use in many institutions.
The ovine spine is an established model of intervertebral disc degeneration, with advantages including cellular, biomechanical and anatomical similarities to the human spine10,14,15. Despite the quadrupedal stature of sheep the ovine lumbar intervertebral disc is exposed to similar stresses to the human disc14. The ovine model is also more widely accepted, from an ethical perspective, than non-human primate models. Varied methods have been described to initiate the degenerative process, many of which require direct access to the intervertebral disc. Due to the termination of the spinal cord in the sacral region and ossification of the posterior longitudinal ligament in the ovine lumbar spine, posterior approaches to the intervertebral disc are technically challenging and less commonly used in the sheep16. The traditional access routes to the sheep lumbar spine, i.e. via anterior or anterolateral approaches, require large abdominal incisions, are fraught with risks of hernia, and damage to internal viscera and neurovascular structures16. The use of a relatively small lateral incision away from dependent abdominal areas may decrease such risks17.
We present an ovine model of degenerative lumbar intervertebral disc disease using drill bit injury performed through a minimally invasive lateral approach, and inspired by the work of Zhang et. al18. The goal of this protocol is to enable a reliable lumbar disc injury model that is readily reproducible, produces a consistent injury, and is safe and well tolerated. This approach is well-suited to investigators seeking to induce a milder degree of lumbar intervertebral disc degeneration than that observed with traditional surgical annulotomy (unpublished data) for the investigation of either intervertebral disc degeneration or regenerative therapies. These findings will be described in a forthcoming publication.

<table border="0" cellpadding="0" cellspacing="0" width="947">
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		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Medetomidine Hydrochloride (10 mL Injection)</td>
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			<td height="42" style="height:42px;width:296px;">Chlorhexidine 5%w/v</td>
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			<td height="42" style="height:42px;width:296px;">Atipamezole (Antisedan 0.06 mg/kg - 0.08 mg/kg)</td>
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			<td style="width:119px;">PFIANT10</td>
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			<td height="21" style="height:21px;width:296px;">Drill Bit Stop 4 mm</td>
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			<td height="21" style="height:21px;width:296px;">3-0 absorbable synthetic braided sutures</td>
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ovine lumbar intervertebral disc degeneration model utilizing a lateral retroperitoneal drill bit injury

Intervertebral disc degeneration is a significant contributor to the development of back pain and the leading cause of disability worldwide. Numerous animal models of intervertebral disc degeneration have been developed. The ideal animal model should closely mimic the human intervertebral disc with regard to morphology, biomechanical properties and the absence of notochordal cells. The sheep lumbar intervertebral disc model fulfils these criteria. We present an ovine model of intervertebral disc degeneration utilizing a drill bit injury through a lateral retroperitoneal approach. The lateral approach significantly reduces the incision and potential morbidity associated with the traditional anterior approach to the ovine spine. Utilization of a drill-bit method of injury affords the ability to produce a consistent and reproducible injury, of precise dimensions, that initiates a consistent degree of intervertebral disc degeneration. The focal nature of the annular and nucleus pulposus defect more closely mimics the clinical condition of focal intervertebral disc herniation. Sheep recover rapidly following this procedure and are typically mobile and eating within the hour. Intervertebral disc degeneration ensues and sheep undergo necropsy and subsequent analysis at periods from eight weeks. We believe that the drill bit injury model of intervertebral disc degeneration offers advantages over more conventional annular injury models.

Pre-operatively, sheep underwent baseline 3T magnetic resonance imaging (MRI) for assessment of underlying intervertebral disc morphology and degeneration. Sheep underwent additional intra-operative lateral radiography for confirmation of intervertebral disc level and calculation of disc height index. A pre-operative sagittal plane slice from 3T MRI and an intra-operative radiograph are demonstrated in Figure 1.
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Watch this Scientific Journal Video about Ovine Lumbar Intervertebral Disc Degeneration Model Utilizing a Lateral Retroperitoneal Drill Bit Injury at JoVE.com

Ovine Lumbar Intervertebral Disc Degeneration Model Utilizing a Lateral Retroperitoneal Drill Bit Injury

Intervertebral disc degeneration is a significant contributor to back pain and a leading cause of disability worldwide. Numerous animal models of intervertebral disc degeneration exist. We demonstrate an ovine model of intervertebral disc degeneration, utilizing a drill bit, which achieves a consistent disc injury and reproducible level of disc degeneration.

Intervertebral disc degeneration is a significant contributor to the development of back pain and the leading cause of disability worldwide. Numerous ...

The overall goal of this Lateral Retroperitoneal approach, Drill Bit Intervertebral Disc Injury method is to create a safe, consistent, and reproducible Ovine model of degenerative intervertebral discs. This Ovine Lumbar Intervertebral Disc Degeneration model can help answer key questions in the field of translational spinal research by providing another method for investigating the path or physiology of disc degeneration and the potential investigation of regenerative therapies. The main advantage of this approach is that it allows for consistent surgical injury through a safe, surgical approach in an appropriate animal model.After anesthetizing and intubating a sheep and preparing the operating suite according to the text protocol, place the animal on the operating table in the right lateral position. Using electronic clippers, shave the region defined superiorly by the lower ribs, inferiorly by the Iliac bone, medially by the Contralateral Lumbar Transverse Processes and approximately 10 centimeters lateral to the Ipsilateral Lumbar Transverse Processes. Palpate the Iliac crest, lumbar transverse processes, and cross a vertebral angle for the landmarks for this surgical incision site.Then, use a sterile pen to mark these landmarks. Next, with chlorhexidine and alcohol iodine antiseptic wash, disinfect the lateral abdomen. Then, place a sterile fenestrated square drape over the surgical site and a large sterile square drape on the overhead table followed by the sterile instruments.Under surgical loupe magnification and a headlight, inject local anesthetic, and then use a 22 scalpel blade attached to a four scalpel handle to make a longitudinal incision parallel to and one centimeter anterior to one to two lumbar transverse processes above and below the intervertebral disc levels of interest. Using monopolar diathermy, divide the underlying subcutaneous tissue and the lateral aspect of the abdominal wall musculature. Directing the dissection towards the tips of the lumbar transverse processes, above and below the intervertebral discs of interest.Next, divide the thoracolumbar fascia longitudinally at it's attachment to the transverse processes. Visualize and preserve the underlying quadratus lumborum, soleus muscles and the traversing neurovascular bundles. Using diathermy, maintain hemostasis through the procedure.Sweep the fingers between the plane of the peritoneum and posterior abdominal wall musculature at the exposed intervertebral disc levels to perform digital blunt dissection. Now, using diva retractor, retract the quadratus lumborum and soleus muscles posterior laterally to further expose the intervertebral discs. Then, palpate for the concave intervertebral bodies and the convex intervertebral discs.Position the retractors immediately over the discs and take care to ensure lumbar vessels are not damaged. Then, using surgical loupe magnification with the headlight, identify the lumbar vessels which are located approximately one centimeter caudal to the inferior end plate. After performing an intraoperative lateral x-ray to confirm the disc level, depending on the disc levels desired, expose the intervertebral discs by separating the surrounding structures.For levels L three, four, and above, use digital blunt dissection to sweep aside the muscular attachments over the discs. For levels L four, five, and below, use bipolar diathermian scissors to sharply divide the thicker tendonus muscular attachments over the disc. Define the drill bit injury point by observing the left lateral and interior extremities at the intervertebral disc.The disc level should be confirmed with lateral x-ray prior to drill bit injury. Fit a brad point drill bit into the power drill. Ensure the diameter of the drill bit is slightly less than the intervertebral disc height.For example, 3.5 millimeters for lumbar intervertebral discs in 60 to 70 kilogram sheep. Apply a drill bit stop to provide an unprotected drill bit length of approximately half the diameter of the lumbar intervertebral disc. For example, 12 millimeters for lumbar intervertebral discs in 60 to 70 kilogram sheep.Next, apply the drill bit to the entry point and direct it in the trajectory slightly cranial to the center of the intervertebral disc, which will minimize the risk of end plate injury. With the drill on low power, slowly advance the drill bit into the intervertebral disc for one second. If excessive resistance is encountered, adjust the trajectory in a slight cranial or caudal fashion.Once hemostasis is achieved, use Ringer's solution to irrigate the wound. Then, perform layered closure, preferably using 2-0 absorbable synthetic braided sutures to the lateral abdominal wall tissues and continuous 3-0 undyed absorbable synthetic subcuticular suture to the skin. Shown here is a pre-operative sagittal plane slice from a 3T MRI of ovine lumbar one half to the lumbosacral intervertebral disc with no evidence of significant pre-operative intervertebral disc degeneration.This intraoperative lateral radiograph confirms the intervertebral disc level and enables calculation of the disc height index. The gross morphological appearance presented here demonstrates the drill bit injury tract penetrating the annulus fibrosus and extending into the nucleus pulposus. The injury is also evident in this 9.4T MRI.While attempting this approach, it's important to direct the drill bit cranially and advance the drill bit slowly to minimize the risk of end plate injury. After watching this video, you should have a good understanding of how to produce drill bit injury in the ovine model through a lateral retroperitoneal approach. Don't forget that working with scalpels and cautery can be hazardous.Personal protective equipment and appropriate safety precautions must be followed at all times. It is important to maintain surgical sterility.

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