A Safe, Effective, and Economical Protocol for Rabbit Vertebral Screw Insertion

Summary

This article describes a safe, effective, and cost-efficient step-by-step protocol for posterior lumbar screw fixation in New Zealand White rabbits, establishing a standardized approach for vertebral screw insertion in this animal model.

Abstract

Posterior lumbar screw fixation is the most common surgical method for lumbar disc herniation, but patients often face multiple complications postoperatively. The occurrence of screw track loosening can lead to fusion failure and even life-threatening screw track extrusion. However, there is currently a lack of animal models specifically targeting changes in the screw track following lumbar screw fixation. This study developed a vertebral screw insertion method based on New Zealand white rabbits, designed to minimize the risk of postoperative screw penetration into the spinal canal, thus avoiding spinal cord injury. In this protocol, the intersection point of the horizontal line from the upper edge of the transverse process and the junction of the pedicle is recommended as the entry point. The insertion angle should be approximately 45°-55° outward and 20°-40° downward. Additionally, based on screw placement under direct vision and the use of commercial instruments,this method is simpler and more cost-effective compared to existing large animal modeling techniques. It provides an animal model for subsequent basic research on screw track loosening and the microenvironment surrounding the screw track.

Introduction

Lumbar Disc Herniation (LDH) is a common cause of chronic lower back pain and radiating leg pain, particularly in young and middle-aged individuals^1,2,3. The condition usually results from a tear in the annulus fibrosus of the intervertebral disc, leading to the herniation of the nucleus pulposus and compression of the nerve roots^4,5. While many patients benefit from conservative treatments like rest and lifestyle modification, medication therapy, physical therapy, rehabilitation exercises, wearing a brace, and nerve block or epidural injection therapy⁶, surgery is necessary for others, especially those with severe symptoms or ineffective conservative management^7,8,9.

Posterior Lumbar Screw Fixation (PLF) is a standard spinal procedure, especially suitable for patients with spinal instability, nerve root compression, or degenerative changes³. Screw loosening occurs when the fixation strength between the inserted screws and the bone is lost, preventing the screws from stabilizing the vertebrae^10,11. Loosening can range from slight movement to complete detachment and is typically associated with recurring symptoms, worsening back pain, or spinal instability¹². Studies report that screw loosening occurs in 5% to 20% of PLF cases¹⁰, depending on factors like hospital technical level, patient age, and postoperative activities. The specific mechanisms underlying screw loosening remain unclear. Currently, appropriate animal models for such investigations are limited. This study details the development of a surgical protocol for posterior lumbar screw fixation in New Zealand White rabbits, which may provide a foundational model for future research into the mechanisms of screw loosening.

In basic research, selecting appropriate animal models is crucial for determining the credibility and results of studies. In lumbar disc degeneration research, mice and rats have limited applicability due to their relatively small intervertebral discs¹³. Other animals, like goats, pigs, and dogs, have limitations related to anesthesia and lack broad applicability¹⁴, due to the costs of animal purchase, breeding, and breeding facilities. The New Zealand white rabbit, a medium-sized animal, is widely used in disc research due to its relatively large vertebrae, sufficient operating space, and simple anesthesia via ear vein injection without the need for a ventilator¹⁵. Given these factors, the New Zealand white rabbit is considered an ideal model. Currently, no animal model for PLF exists, so a surgical method for rabbit lumbar spine surgery has been developed based on prior experience.

Protocol

The animal experimental protocol was reviewed and approved by the Ethics Committee of Laboratory Animal Welfare and Ethics of the Institute of Radiological Medicine, Chinese Academy of Medical Sciences (Ethics Number: IRM/2-IACUC-2404-005). All handling procedures for the animals complied with the relevant national regulations on animal welfare and ethics.

NOTE: In this study, New Zealand white rabbits were used for experimental investigation. The rabbits were of common grade, weighing approximately 2.5-3.0 kg, aged 3-5 months, and female. The rabbits were housed in a standardized ordinary-grade rabbit room.

1. Anesthesia

1. Disinfect the operating room with ultraviolet light for 30 min using an ultraviolet lamp.
2. Anesthetize the rabbit by administering 3% pentobarbital sodium (30 mg/kg) in sterile saline through the marginal ear vein.
   NOTE: Inject 2/3 of 3% pentobarbital sodium solution and observe the rabbit's pupil response and pupillary reflex. Ensure that the pupils constrict and the pupillary reflex weakens. If there is no weakening after 5 min, consider injecting the remaining 1/3. If the reflex weakens, inject 2 mL of 0.9% saline.

2. Surgical procedure

1. Once anesthesia is adequate,observe the direct corneal reflex and ensure that the corneal reflex is diminished, indicating successful anesthesia. Position the rabbit in the prone position for surgical site preparation. Prior to the surgery, disinfect the incision site of the rabbit twice with iodine tincture, and place a sterile surgical drape during the procedure (Figure 1A).
   NOTE: Ensure that all surgical instruments are sterilized by autoclaving.
2. Make a 5 cm midline skin incision in the lumbar region (Figure 1B) and sequentially cut through the skin and superficial fascia with a scalpel.
3. Using the extensibility between the skin and subcutaneous tissue, carefully separate and fully expose the subcutaneous tissue, clearly revealing the root of the spinous process  Figure 1C).
4. Using hemostatic forceps, bluntly dissect the transverse process and pedicle of the surgical segment, avoiding adjacent facet joint segments (Figure 1D). Use a small retractor to fully expose the surgical area (Figure 1E).
5. Identify the entry point for the needle at the intersection of the horizontal line from the upper edge of the transverse process and the junction of the pedicle (Figure 1F). Drill a small hole into the cortical bone at an angle of approximately 45°-55° outward and 20°-40° downward using a surgical drill (Figure 1G).
6. Insert the pedicle screws (3.5 mm × 10.0 mm) one by one (Figure 1H).
7. Install an appropriately sized longitudinal connecting rod and tighten with a cap screw (Figure 1I to Figure 1J).
8. Reconfirm that the screws are securely fixed within the vertebral body and that the fixation rod is properly locked. Verify the positioning of the internal fixation through intraoperative direct visualization. Ensure there is no significant bleeding, irrigate the surgical area with saline, and perform continuous suturing of the muscles and fascia using a 4-0 absorbable suture.
9. Use a 7-0 suture for intermittent vertical mattress sutures for the skin incision, followed by iodine-based antiseptic and coverage with sterile gauze, securing it in place. The surgery is completed (Figure 1K).

3. Postoperative care

1. Once consciousness is regained, check the motor ability of the hind limbs to assess the success of the surgery.
   NOTE: Confirm that after the rabbit regains consciousness from anesthesia, its limb motor function is intact, it can move normally, and there are no signs of hemiplegia or paraplegia.
2. For the first 3 days post-surgery, administer 1.2 × 10⁶ U/kg of penicillin via intramuscular injection daily to prevent wound infection.

4. Morphological examination

1. At the end of week 12 post-surgery, induce air embolism via the marginal ear vein to euthanize the animal. Afterward, use a posterior lumbar approach to expose the paraspinal soft tissues and perform a lumbar discectomy. After collecting the specimens and confirming the surgical segment, store them temporarily in 4% paraformaldehyde for tissue fixation. Then, perform Micro-CT and X-ray scans.
   1. Remove the spinal specimens from 4% paraformaldehyde and wipe off the surface fixation liquid. Then, subject the specimens to Micro-CT scanning. Set the scanning parameters as follows: voltage 60 kV, current 200 µA, and resolution 8 µm. Perform post-processing of the regions of interest (ROIs) in the scanned files using specialized image analysis and visualization software (see Table of Materials). Perform X-ray imaging simultaneously with the Micro-CT scanning.

Results

Motor function assessment after awakening
Immediately after the rabbit regained consciousness from anesthesia, the motor function of its hind limbs was assessed. It was found that the insertion of the lumbar screw fixation system did not affect the rabbit's motor function (See Supplementary video 1), indicating that the spinal cord was not injured during the procedure.

Micro-CT and X-ray evaluation
To investigate the exact position of ...

Discussion

The most severe complication of posterior lumbar screw fixation (PLF) is the potential for spinal cord injury caused by the screw penetrating the spinal canal during insertion¹⁰. The most critical aspect of this method is the exposure of the transverse process and pedicle, as well as the angle at which the screws are inserted. Adjusting the insertion angle can prevent the screw from entering the spinal canal and causing spinal cord injury. Additionally, due to the anatomical characteristics of the...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
CTAn	BURKER		used in anaylsis
Datviewer	BURKER		used in anaylsis
Micro-CT	BURKER		used in anaylsis
New Zeland Rabbit Pentobarbital	Tianjin Yuda Laboratory Animal Breeding Co., Ltd. Beijing Jiehui Biotechnology Co. MCE		used in this research
pedicle screws	Hebei Ruihe Medical Equipment Co., Ltd	532003510	used in Surgical Procedure
penicillin	MCE	HY-17591R	used in Postoperative Care
Pentobarbital	Beijing Jiehui Biotechnology Co.		used in Anesthesia
rods	Hebei Ruihe Medical Equipment Co., Ltd	532003510	used in Surgical Procedure
surgical instruments and sutures	Hebei Ruihe Medical Equipment Co., Ltd	532003510	used in Surgical Procedure