Name: A Mouse Model of Lumbar Spine Instability (Video) | JoVE Journal
Uploaded: 2021-04-23T13:30:00.000+00:00
Duration: 5 min 28 s
Description: Learn a reproducible surgical mouse model for lumbar intervertebral disc degeneration using C57BL/6 mice. This method enables 3D histomorphometric analysis of disc volume, height changes, and vertebral bone loss for orthopedic research.

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5 minutes

This work was supported by the National Natural Science Foundation of China (81973607) and Essential Drug Research and Development (2019ZX09201004-003-032) from Ministry of Science and Technology of China.

The investigations described conform to the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health and were approved by Shanghai University of Traditional Chinese Medicine Animal Care and Use Committee. All surgical manipulations were performed under deep anesthesia and the animals did not experience pain at any stage during the procedure.
1. Pre-operation preparation
<ol>
	<li>Instrument sterilization: Steam-sterilize surgical instruments in an autoclave (121 &#176;C for 15 min) prior to the surgery. Pack instruments in a metal container and maintain them until they are used in the surgery.</li>
	<li>Surgery platform setup: Assign a bench area of at least 60 cm x 60 cm for the operation. Clean the surface of area with 75% alcohol and cover with a disposable medical towel. Place a sterile surgical instruments pack, reagents, surgical items onto a disposable medical towel within the upper 1/3 of area. Leave the remaining 2/3 of area clean for surgical operation.&#160;Add a hotpad underneath surgical pad for thermal support.</li>
	<li>Animal preparation
	<ol>
		<li>Place the animal (C57BL/6J mice, male, 8-week old) into the induction chamber. Turn on the vaporizer at an induction level of 4% for isoflurane and 4 L/min for oxygen. After the animal is fully anesthetized, maintain the anesthetic with the nose cone and the anesthetic delivery at a level of 1.5% for isoflurane and 0.4 L/min for oxygen during surgery. Monitor the animal for respiration.</li>
		<li>Apply chlortetracycline hydrochloride eye ointment to prevent corneal dryness during the surgery.</li>
		<li>Shave the surgical area on the dorsal surface from the lower thoracic region to the top of the sacral region using a small animal trimmer. Remove the shaved fur with tissue wipes.</li>
		<li>Apply depilatory cream onto the shaved area and leave it there no longer than 3 min. Remove the cream with gauze and flush with 2 mL of 0.9% sterile saline.</li>
		<li>Place a custom-made surgical cylindrical pad (Figure 2A) under the abdomen of the mouse to raise up the lumbar spine and facilitate the surgical operation.</li>
	</ol>
	</li>
</ol>
2. Exposure of the lumbar third to lumbar fifth (L3&#8211;L5) spinous processes
<ol>
	<li>Use the index finger to touch the subcutaneous spinous processes of the lumbar vertebrae, which are more outward, and compare with thoracic vertebrae and sacral vertebrae to identify the lumbar region.</li>
	<li>Rinse the skin using 75% alcohol. Perform a 3&#8211;4 cm midline skin incision over the lumber region from the mid-thoracic region to the hip using a scalpel blade to expose the fascia.</li>
	<li>Identify the lumbar spine by the morphology of the posterior fascia inserted onto the tips of the spinous processes. In detail, the third lumbar (L3) to the first sacral (S1) fasciae are distinct from other fasciae by their &#8220;V&#8221; shapes. The last &#8220;V&#8221; tip connects to the first sacral (S1) fascia and the first &#8220;V&#8221; tip corresponds to the L3 spinous process (Figure 2B).</li>
	<li>Make the posterior paraspinous muscle incisions along the spinous processes from L3 to L5 on both sides laterally with a scalpel blade (Figure 2C). Control the incision depth towards the facets to reduce hemorrhage.</li>
	<li>Separate the muscle layers using two ophthalmic forceps to expose L3 to L5 spinous processes and supraspinous ligaments.</li>
</ol>
3. Resection of L3&#8211;L5 spinous processes along with the ligaments
<ol>
	<li>Separate individual spinous processes by cutting off interspinous ligaments using Venus shears (Figure 2D).</li>
	<li>Resect the L3&#8211;L5 spinous processes along with the interspinous ligaments with Venus shears (Figure 2E).</li>
	<li>Suture the skin incision with sterile silk braided (suture size 5.0) without reattachment of the paravertebral muscles.</li>
	<li>Apply Chlortetracycline Hydrochloride Eye Ointment to the surgical site.</li>
	<li>Administer Buprenorphine-SR (25 uL per gram of mouse weight) immediately after LSI surgery for analgesia.</li>
	<li>Place the animals in a warm chamber and monitor during recovery from the anesthesia. Monitor food and water intake before returning the animals to the home cage.</li>
	<li>Monitor the animal once daily for the first 3 days after operation. The animal should be able to have a normal appetite and should heal with no sign of pus, hemorrhage, or swelling. They may have minor impairment in locomotion.</li>
	<li>Carry out sham operations only by the detachment of the posterior paravertebral muscles from the L3&#8211;L5 vertebrae.</li>
</ol>

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<li>Vo, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Accelerated+aging+of+intervertebral+discs+in+a+mouse+model+of+progeria%5BTitle%5D)+AND+%22Journal+of+Orthopaedic+Research%22%5BJournal%5D)">Accelerated aging of intervertebral discs in a mouse model of progeria.</a> Journal of Orthopaedic Research. 28 (12), 1600-1607 (2010).</li>
<li>Oichi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+mouse+intervertebral+disc+degeneration+model+by+surgically+induced+instability%5BTitle%5D)+AND+%22Spine%22%5BJournal%5D)">A mouse intervertebral disc degeneration model by surgically induced instability.</a> Spine. 43 (10), 557-564 (2018).</li>
<li>Ohnishi, T., Sudo, H., Tsujimoto, T., Iwasaki, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Age-related+spontaneous+lumbar+intervertebral+disc+degeneration+in+a+mouse+model%5BTitle%5D)+AND+%22Journal+of+Orthopaedic+Research%22%5BJournal%5D)">Age-related spontaneous lumbar intervertebral disc degeneration in a mouse model.</a> Journal of Orthopaedic Research. 36 (1), 224-232 (2018).</li>
<li>Stern, W. E., Coulson, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+collagenase+upon+the+intervertebral+disc+in+monkeys%5BTitle%5D)+AND+%22Journal+of+Neurosurgery%22%5BJournal%5D)">Effects of collagenase upon the intervertebral disc in monkeys.</a> Journal of Neurosurgery. 44 (1), 32-44 (1976).</li>
<li>Silva, M. J., Holguin, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((LRP5-deficiency+in+OsxCreERT2+mice+models+intervertebral+disc+degeneration+by+aging+and+compression%5BTitle%5D)+AND+%22bioRxiv%22%5BJournal%5D)">LRP5-deficiency in OsxCreERT2 mice models intervertebral disc degeneration by aging and compression.</a> bioRxiv. , (2019).</li>
<li>Nemoto, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Histological+changes+in+intervertebral+discs+after+smoking+and+cessation%3A+experimental+study+using+a+rat+passive+smoking+model%5BTitle%5D)+AND+%22Journal+of+Orthopaedic+Science%3A+Official+Journal+of+the+Japanese+Orthopaedic+Association%22%5BJournal%5D)">Histological changes in intervertebral discs after smoking and cessation: experimental study using a rat passive smoking model.</a> Journal of Orthopaedic Science: Official Journal of the Japanese Orthopaedic Association. 11 (2), 191-197 (2006).</li>
<li>Mulholland, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+myth+of+lumbar+instability%3A+the+importance+of+abnormal+loading+as+a+cause+of+low+back+pain%5BTitle%5D)+AND+%22European+Spine+Journal%22%5BJournal%5D)">The myth of lumbar instability: the importance of abnormal loading as a cause of low back pain.</a> European Spine Journal. 17 (5), 619-625 (2008).</li>
<li>Bian, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanosignaling+activation+of+TGF%CE%B2+maintains+intervertebral+disc+homeostasis%5BTitle%5D)+AND+%22Bone+Research%22%5BJournal%5D)">Mechanosignaling activation of TGFβ maintains intervertebral disc homeostasis.</a> Bone Research. 5, 17008(2017).</li>
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<li>Ni, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sensory+innervation+in+porous+endplates+by+Netrin-1+from+osteoclasts+mediates+PGE2-induced+spinal+hypersensitivity+in+mice%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Sensory innervation in porous endplates by Netrin-1 from osteoclasts mediates PGE2-induced spinal hypersensitivity in mice.</a> Nature Communications. 10 (1), 5643(2019).</li>
<li>Liu, S., Cheng, Y., Tan, Y., Dong, J., Bian, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ligustrazine+prevents+intervertebral+disc+degeneration+via+suppression+of+aberrant+tgf%CE%B2+activation+in+nucleus+pulposus+cells%5BTitle%5D)+AND+%22BioMed+Research+International%22%5BJournal%5D)">Ligustrazine prevents intervertebral disc degeneration via suppression of aberrant tgfβ activation in nucleus pulposus cells.</a> BioMed Research International. 2019, 5601734(2019).</li>
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</ol>

The authors have nothing to disclose.

We developed the lumbar spine instability mouse model based on the cervical spondylosis mouse model in which the posterior paravertebral muscles from the vertebrae were detached and the spinous processes along with the supraspinous and interspinous ligaments were resected25. We performed a similar operation onto the lumbar spine, which has more prominent spinous processes. The LSI mouse model developed similar IDD in the lumbar spine.
The advantages of the LSI model inc...

Intervertebral disc degeneration (IDD) is commonly seen in aging and even young people caused by many factors1. Surgery for patients who suffer from IDD, causing low back pain and impaired movement, is usually performed at a later stage or in severe cases and has potential risks such as nonunion or infection2. Ideal non-operative treatment requires comprehensive understanding of the IDD mechanism. The IDD animal model serves as a crucial tool for studies of IDD mechanism and evaluation of IDD treatment.
Larger animals have been chosen for IDD models such as primates, sheep, goats, dogs, and rabbits due to their similarity with human anatomical structure to a great extent and the strong operability in terms of size of intervertebral discs (IVDs)3,4,5,6,7,8. However, these animal models are time-consuming and cost-intensive9. Mouse IVD is a poor representation of the human IVD based on geometrical measurements of the aspect ratio, nucleus pulposus to disc area ratio, and normalized height10. Despite the difference in size, mouse lumbar IVD segment exhibits mechanical properties similar to human IVD such as compression and torsion stiffness11. In addition, mouse IDD model has the advantage of low cost, relatively short IDD development, and more options for genetically modified animals and antibodies utilized in further mechanistic studies12,13,14,15.
Experimental-induced IDD models vary from the inducers and applications. For example, collagenase-induced extracellular matrix (ECM) degeneration is appropriate for ECM regeneration research16. Genetically modified phenotype are suitable for studying the gene function in the IDD process and in genetic therapies17. Annulus fibrosus incision and smoke models mimic trauma and non-inflammation induced IDD12,18.
Spinal instability (SI) leads to an unstable spine that is not in an optimal state of equilibrium. It can be caused by abnormal movement of a lumbar motion segment due to the weakness of the surrounding supportive tissue such as ligaments and muscles. It is also commonly seen post spinal fusion operation19. SI is considered as the main cause of IDD. Therefore, we aim to develop a SI mice model (focused on lumbar spine) that mimics the human IDD process20,21.
In the protocol, we introduced the procedure of establishing lumbar spinal instability (LSI) mouse model by the resection of lumbar third (L3) to lumbar fifth (L5) spinous processes along with the supraspinous and interspinous ligaments (Figure 1A,B). The animal model develops IDD as early as 1-week post-surgery as shown by hypertrophy and porosity in endplates (EPs). IVD volume starts to decrease 2 weeks post-surgery through 16 weeks along with increased IVD score, which indicates the degree of IDD. We believe the detailed and visualized procedure is useful for researchers to establish the LSI mouse model in their laboratory and apply to IDD research as needed.

<table><tbody><tr><td>Chlortetracycline Hydrochloride Eye Ointment</td><td>Shanghai General Pharmaceutical Co., Ltd.</td><td>H31021931</td><td>Prevent eye dry, Prevent wound infection</td></tr><tr><td>C57BL/6J male mice</td><td>Tian-jiang Pharmaceuticals Company (Jiangsu, CN)</td><td>SCXK2018-0004</td><td>Animal model</td></tr><tr><td>Disposable medical towel</td><td>Henan Huayu Medical Devices Co., Ltd.</td><td>20160090</td><td>Platform for surgical operation</td></tr><tr><td>Inhalant anesthesia equipment</td><td>MIDMARK</td><td>Matrx 3000</td><td>Anesthesia</td></tr><tr><td>Isoflurane</td><td>Shenzhen RWD Life Technology Co., Ltd.</td><td>1903715</td><td>Anesthesia</td></tr><tr><td>Lidocaine hydrochloride</td><td>Shandong Hualu Pharmaceutical Co., Ltd.</td><td>H37022839</td><td>Pain relief</td></tr><tr><td>Medical suture needle</td><td>Shanghai Pudong Jinhuan Medical Products Co., Ltd.</td><td>20S0401J</td><td>Suture skin</td></tr><tr><td>Ophthalmic forceps</td><td>Shanghai Medical Devices (Group) Co., Ltd. Surgical Instruments Factory</td><td>JD1050</td><td>Clip the skin</td></tr><tr><td>Ophthalmic scissors(10cm)</td><td>Shanghai Medical Devices (Group) Co., Ltd. Surgical Instruments Factory</td><td>Y00030</td><td>Skin incision</td></tr><tr><td>silk braided</td><td>Shanghai Pudong Jinhuan Medical Products Co., Ltd.</td><td>11V0820</td><td>Suture skin</td></tr><tr><td>Small animal trimmer</td><td>Shanghai Feike Electric Co., Ltd.</td><td>FC5910</td><td>Hair removal</td></tr><tr><td>Sterile surgical blades(12#)</td><td>Shanghai Pudong Jinhuan Medical Products Co., Ltd.</td><td>35T0707</td><td>Muscle incision</td></tr><tr><td>Veet hair removal cream</td><td>RECKITT BENCKISER (India) Ltd</td><td>NA</td><td>Hair removal</td></tr><tr><td>Venus shears</td><td>Mingren medical equipment</td><td>Length:12.5cm</td><td>Clip the muscle and spinous process</td></tr></tbody></table>

a mouse model of lumbar spine instability

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.

The LSI mouse model is applied in the studies of IDD mechanism, IDD treatment, endplate (EP) degeneration such as sclerosis, and sensory innervation in EP20,21,22,23. The LSI mouse develops IDD and EP degenerative changes, as identified, by decreased IVD volume and height, increased EP volume, and increased IVD and EP scores.
The dissected and fixed lower thoracic an...

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 ...

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Research

JoVE Journal

Biology

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Learn a reproducible surgical mouse model for lumbar intervertebral disc degeneration using C57BL/6 mice. This method enables 3D histomorphometric analysis of disc volume, height changes, and vertebral bone loss for orthopedic research.

A Mouse Model of Lumbar Spine Instability

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