A Mouse Model of Lumbar Spine Instability

Summary

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

Abstract

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–L₅ spinous processes exposure, detachment of paraspinous muscles, resection of processes and ligaments, and skin closure. L₄–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.

Introduction

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 studies^12,13,14,15.

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 IDD^12,18.

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 process^20,21.

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.

Protocol

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

1. Instrument sterilization: Steam-sterilize surgical instruments in an autoclave (121 °C for 15 min) prior to the surgery. Pack instruments in a metal container and maintain them until they are used in the surgery.
2. 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. Add a hotpad underneath surgical pad for thermal support.
3. Animal preparation
   1. 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.
   2. Apply chlortetracycline hydrochloride eye ointment to prevent corneal dryness during the surgery.
   3. 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.
   4. 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.
   5. 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.

2. Exposure of the lumbar third to lumbar fifth (L₃–L₅) spinous processes

1. 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.
2. Rinse the skin using 75% alcohol. Perform a 3–4 cm midline skin incision over the lumber region from the mid-thoracic region to the hip using a scalpel blade to expose the fascia.
3. 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 “V” shapes. The last “V” tip connects to the first sacral (S₁) fascia and the first “V” tip corresponds to the L₃ spinous process (Figure 2B).
4. 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.
5. Separate the muscle layers using two ophthalmic forceps to expose L₃ to L₅ spinous processes and supraspinous ligaments.

3. Resection of L₃–L₅ spinous processes along with the ligaments

1. Separate individual spinous processes by cutting off interspinous ligaments using Venus shears (Figure 2D).
2. Resect the L₃–L₅ spinous processes along with the interspinous ligaments with Venus shears (Figure 2E).
3. Suture the skin incision with sterile silk braided (suture size 5.0) without reattachment of the paravertebral muscles.
4. Apply Chlortetracycline Hydrochloride Eye Ointment to the surgical site.
5. Administer Buprenorphine-SR (25 uL per gram of mouse weight) immediately after LSI surgery for analgesia.
6. 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.
7. 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.
8. Carry out sham operations only by the detachment of the posterior paravertebral muscles from the L₃–L₅ vertebrae.

Results

The LSI mouse model is applied in the studies of IDD mechanism, IDD treatment, endplate (EP) degeneration such as sclerosis, and sensory innervation in EP^20,21,22,23. 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...

Discussion

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...

Materials

List of materials used in this article

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