Comparing Transperitoneal Retroperitoneal Access and Percutaneous Puncture for Lumbar Disc Degeneration Modeling in Rabbits

Summary

This protocol compared percutaneous and trans-retroperitoneal punctures in a rabbit intervertebral disc degeneration (IVDD) model. Both methods induced IVDD; however, the trans-retroperitoneal approach resulted in more extensive changes and lower mortality.

Abstract

This study compares the efficacy of two methods for inducing intervertebral disc degeneration (IVDD) in rabbits: percutaneous and trans-retroperitoneal puncture of the annulus fibrosus. Fifteen healthy male New Zealand White rabbits were randomly assigned to three groups: sham, percutaneous puncture, and trans-retroperitoneal puncture. A comprehensive assessment, including mortality rates, morphological and histological evaluations, radiological imaging, and biomarker analysis, was conducted to ensure an accurate and detailed comparison between the two methods. The results demonstrate that both puncture techniques successfully induced IVDD in the rabbit model. However, the trans-retroperitoneal approach resulted in more pronounced degenerative changes in the intervertebral discs while maintaining a significantly lower mortality rate compared to the percutaneous method. These findings highlight the advantages of the trans-retroperitoneal approach in IVDD modeling. This study provides valuable insights into the establishment of IVDD models and lays a foundation for future investigations into effective treatment strategies for low back pain, ultimately improving patient outcomes.

Introduction

Over the past few decades, low back pain (LBP) has emerged as the most significant musculoskeletal disorder affecting quality of life¹. LBP has become an increasingly important public health concern, imposing a substantial economic burden on society due to lost labor and additional medical expenses^2,3. In the United States alone, the direct and indirect costs associated with LBP exceed $100 billion annually, including medical expenditures, income losses, and labor losses⁴. LBP is often caused by intervertebral disc degeneration (IVDD)^5,6,7,8. Given the high prevalence and economic impact of LBP, accurately modeling IVDD is crucial for exploring treatment strategies.

To understand the pathophysiology of IVDD and evaluate treatment strategies, various preclinical in vivo animal models have been developed and utilized⁹. Multiple methods have been employed in these models to induce disc degeneration, including surgical or chemical disc injury, non-invasive mechanical stress, genetic modification, and natural occurrence¹⁰. Among these methods, surgical injury accounts for up to 64.9% of IVDD induction, with needle puncture being the primary surgical technique¹¹. The needle puncture model is characterized by its ease of establishment and minimal damage to experimental animals. Common needle puncture approaches include open retroperitoneal access to the lumbar disc space and percutaneous posterolateral puncture. The depth of insertion can be determined using radiographic monitoring or needle length. Notably, the percutaneous approach may reduce iatrogenic tissue damage compared to open surgical methods, while retroperitoneal access provides the benefit of direct visualization-features that have not been quantitatively compared in prior literature. While studies have investigated the effects of using needles of different diameters¹² and puncturing different discs¹⁰ on IVDD induction, comparative studies focusing on different needle puncture approaches remain limited. The selected rabbit model offers particular utility for researchers requiring cost-effective longitudinal studies with frequent imaging assessments, given its anatomical similarity to human discs and its advantages over rodent models in terms of size and structure¹³.

In this study, rabbit models of lumbar IVDD were established using two methods: open retroperitoneal access to puncture the lumbar disc space and percutaneous posterolateral puncture. A comprehensive set of outcome measures, including morphological, histological, and radiological changes, was analyzed.

Protocol

The animal experimental procedures strictly adhered to the Guide for the Care and Use of Laboratory Animals issued by the National Institutes of Health and were approved by the Experimental Animal Ethics Committee of Chengdu University of Traditional Chinese Medicine (Ethics Approval Number: 2021-23). Fifteen healthy, 4-month-old, clean-grade New Zealand White rabbits (2.25 kg ± 0.25 kg) were used, including seven males and eight females. The animals were housed in an environment with a room temperature of 23 °C ± 3 °C and a humidity of approximately 60% ± 10% for one week of adaptation, with free access to water and food. Prior to the experiment, the 15 rabbits were randomly assigned to one of three groups: the sham group (Group A), the percutaneous annulus fibrosus puncture group (Group B), and the trans-retroperitoneal space annulus fibrosus puncture group (Group C), with five rabbits in each group. The details of the reagents and equipment used in this study are listed in the Table of Materials.

1. Establishment of rabbit IVDD model via percutaneous annulus fibrosus puncture

NOTE: The rabbit IVDD model was established using the percutaneous annulus fibrosus puncture method. The procedure followed the puncture modeling method described by Luo TD et al.¹⁴ and was performed under X-ray guidance (Figure 1).

1. Prepare the rabbit.
   1. Fast the rabbits for 24 h before surgery, ensuring access to water.
   2. Administer anesthesia via intravenous injection of 3% pentobarbital sodium (1.3 mL/kg) into the ear vein (following institutionally approved protocols).
   3. Confirm successful anesthesia by checking for immobility, relaxed muscles, lack of corneal reflex, and absence of pain response.
2. Position and mark the rabbit.
   1. Fix the rabbit in a prone position on a fixation board.
   2. Shave and prepare the surgical area, then palpate bone landmarks.
   3. Palpate the bony landmarks on the rabbit's lumbar back. Locate the lowest rib on the rabbit, which typically corresponds to the vertebra just above the L1 spinous process.
   4. Identify the spinous process immediately below this vertebra to determine the L1 spinous process.
   5. Locate the highest points of the iliac crests, approximately level with the L6 vertebra.
   6. Trace down from the L1 spinous process to sequentially identify each spinous process down to L7.
   7. Use a marking pen to mark the L1 spinous process clearly on the rabbit's back.
   8. Move to the next spinous process and mark it as L2.
   9. Continue marking each subsequent spinous process as L3, L4, L5, L6, and L7. Ensure that each mark is distinct and in sequential order for clear identification.
3. Locate and mark the puncture site.
   1. Palpate the transverse processes and locate the midpoint between the distal ends of L5 and L6.
   2. Mark this point and prepare to insert the puncture needle approximately 1 cm above it.
4. Insert the puncture needle.
   1. Hold the puncture needle horizontally and insert it towards the ground, breaking the skin.
   2. Advance the needle to reach the L4 vertebral body and verify the correct positioning under X-ray guidance.
   3. Tilt the needle slightly cephalic at an angle of approximately 20° toward the L4-5 intervertebral disc. Puncture the disc and confirm the accuracy of the puncture under X-ray examination.
5. Perform disc punctures.
   1. Precisely puncture the annulus fibrosus, using X-ray guidance if necessary.
   2. Repeat the puncturing process for the L2-3 and L3-4 intervertebral discs, puncturing each once.
   3. Maintain a puncturing depth of approximately 5 mm with a dwell time of 5 s for each disc.
6. Post-procedure care
   1. Disinfect and bandage the puncture site.
   2. Inject penicillin intramuscularly into the gluteus maximus at a dosage of 40,000 U per rabbit daily for 3 days.
      NOTE: Adjust the approach if the needle encounters hard tissue. Use X-ray guidance for precise puncturing. Monitor the rabbit's recovery and provide appropriate care.

2. Establishment of rabbit IVDD model via trans-retroperitoneal space annulus fibrosus puncture

NOTE: The rabbit IVDD model was established using the trans-retroperitoneal space annulus fibrosus puncture method¹² (Figure 2).

1. Fast the rabbits for 24 h before surgery, allowing access to water.
2. Anesthetize the rabbit by intravenously injecting 3% pentobarbital sodium (1.3 mL/kg) into the ear vein (following institutionally approved protocols).
3. Ensure that the rabbit is immobile, with relaxed muscles, no corneal reflex, and no pain response to pressure to confirm successful anesthesia.
4. Fix the rabbit in a prone position on a fixation board.
5. Shave and prepare the surgical area.
6. Palpate bone landmarks, marking the L1-L7 lumbar spinous processes on the rabbit's lumbar back with a marking pen.
7. Re-palpate the transverse processes of the rabbit to determine the surgical incision location.
8. Place a sterile drape and disinfect the local skin to ensure aseptic conditions.
9. Use a posterior retroperitoneal approach to dissect the fascia and muscles layer by layer, exposing the lateral aspect of the lumbar intervertebral disc.
10. Puncture the annulus fibrosus with a puncture needle to a depth of approximately 5 mm and a dwell time of 5 s.
11. Sequentially puncture the L3-4, L4-5, and L5-6 intervertebral discs, ensuring that each disc is punctured only once.
12. Suture the tissues layer by layer using a 0.25 mm diameter suture thread.
13. Disinfect and bandage the puncture site after modeling.
14. Inject penicillin intramuscularly into the gluteus maximus of the rabbit daily at a dosage of 40,000 U per rabbit for three consecutive days.
    NOTE: Use penicillin with a specification of 800,000 units/vial and approval number Veterinary Drug 140051251.

3. Selection of IVDD models and outcome evaluation

1. Mortality and general condition assessment of rabbits
   1. Observe rabbits weekly to determine survival and record general condition, including mental status, activity patterns, food and water intake, as well as fecal and urinary output.
   2. Record observations accurately and note any changes in condition.
2. Weight monitoring of rabbits
   1. Record the body weight of rabbits before and after model establishment, as well as prior to tissue collection.
   2. Ensure accurate weight recording and note any significant changes.
3. Radiological assessment
   1. Obtain sagittal 1.5T T2-weighted magnetic resonance imaging of the entire lumbar vertebral explant sequence of each white rabbit before and 4 weeks after model establishment.
   2. Observe the extent of intervertebral disc degeneration.
   3. Perform a quantitative assessment of intervertebral disc degeneration using the modified Pfirrmann grading system proposed by Griffith et al.¹⁵. Have three independent blinded radiologists evaluate T2-weighted MRI sequences according to established criteria: disc height, nucleus pulposus signal intensity, and annulus fibrosus integrity.
   4. Determine final grades through consensus when discrepancies exceed one grade level. Conduct all assessments using standardized DICOM viewing software with calibrated display settings.
4. Histopathological evaluation and scoring
   1. Euthanize the rabbits 4 weeks after modeling using an intravenous overdose of pentobarbital sodium (following institutionally approved protocols), then rapidly harvest the L2-L3, L3-L4, and L4-L5 intervertebral discs on ice¹⁵.
   2. Fix the L2-L3 discs in 4% paraformaldehyde and store the remaining samples at -80 °C.
   3. Immerse the fixed discs in a decalcifying solution, such as 10% EDTA, ensuring complete submersion. Change the decalcifying solution every 2-3 days to maintain effectiveness.
   4. Monitor the decalcification process regularly until full decalcification is achieved, which may take several days to a week, depending on disc size and thickness.
   5. Rinse the decalcified discs thoroughly with running water to remove any traces of the decalcifying solution.
   6. Dehydrate the discs by immersing them in a series of graded ethanol solutions, starting with 70% ethanol and gradually increasing to 100% ethanol. Conduct each dehydration step for 1-2 h per grade.
   7. Infiltrate the dehydrated discs with paraffin wax (melting point 56-58 °C) for a minimum of 2 h, ensuring complete infiltration.
   8. Embed the infiltrated discs in a wax block, positioning them for sectioning. Allow the wax block to cool and solidify completely.
   9. Section the embedded discs into thin, uniform slices (5-10 µm) using a microtome. Mount the sections onto glass slides for further analysis, such as histological staining or immunohistochemistry^12,13.
   10. Perform hematoxylin and eosin (HE) staining, capture images under an optical microscope, and assign HE staining scores using the IVD histopathological grading scale¹².
5. TUNEL assay
   1. Dewax and rehydrate intervertebral disc tissue sections, then perform antigen retrieval and membrane permeabilization.
   2. Add a mixture of reagent 1 (TdT) and reagent 2 (dUTP) at a ratio of 1:9 and incubate in a humidified chamber.
   3. Wash sections with PBS buffer, apply DAPI stain, and incubate in the dark at room temperature for 10 min.
   4. Capture images using a fully automated panoramic scanner and processing software.
6. Cytokine detection
   1. Euthanize the experimental rabbits (step 3.4.1) and collect blood samples from the abdominal aorta.
   2. Centrifuge rabbit blood samples at 2000 × g for 10 min at 25 °C to separate serum from blood cells. Carefully collect the supernatant (serum), ensuring no cellular debris is included.
   3. Follow the instructions provided in the ELISA kit to detect the expression of TGF-β in serum samples.
   4. Prepare reagents and standards as directed in the ELISA kit.
   5. Add serum samples to the appropriate wells in the ELISA plate.
   6. Incubate the plate at the recommended temperature and duration according to the kit instructions.
   7. Wash the plate as instructed to remove unbound reagents.
   8. Add the detection antibody and other necessary reagents, following the kit's protocol.
   9. Incubate the plate again for the specified time and temperature.
   10. Wash the plate thoroughly to remove excess reagents.
   11. Add the substrate solution to the wells and incubate for the recommended period to allow for color development.
   12. Measure the optical density (OD) using a spectrophotometer at the wavelength specified in the kit instructions.
   13. Calculate sample concentrations by substituting OD values into the provided equation or using the standard curve generated from the known concentrations of the standards.

4. Statistical analysis

1. Perform statistical analysis using commercially available software.
2. Express continuous variables as mean ± standard deviation.
3. Use one-way ANOVA to test differences between groups.
4. Apply LSD tests for pairwise comparisons.
5. Use repeated measures ANOVA for analyzing repeated measures data.
6. Perform Spearman correlation analysis to assess the correlation between variables.
7. Set the significance level at α = 0.05 and consider P values less than 0.05 as statistically significant.

Results

The surgical procedures were performed without complications. One rabbit from Group B (percutaneous puncture group) died after the procedure. All other animals resumed normal feeding and activity patterns postoperatively and survived throughout the experimental period. No prolonged bleeding or infection was observed at the surgical sites.

Mortality and general condition assessment
The mortality rate was 0% for both Group A and Group C, while it was 20% for Group B (

Discussion

The findings of this study indicate that both percutaneous and trans-retroperitoneal puncture approaches are effective in inducing intervertebral disc degeneration (IVDD) in rabbit models. Notably, based on a comprehensive evaluation of general condition, mortality, histopathological assessment, TUNEL assay, and serum TGF-β levels, the trans-retroperitoneal puncture model resulted in more extensive degenerative changes in the intervertebral discs while maintaining a lower mortality rate.

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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.3 T Veterinary Maenetic Resonance lmaging(MRI)	NINGBO CHUANSHANJIA	CSJ-MR
Alcohol medical	LIRCON	20230107
Benzylpenicillin potassium	Jiangxi Keda Animal Pharmaceutical	140051251
Haemostatic forceps	SHINVA	20211239
Injection syringe	CONPUVON	20153151307
Knife blades	Hons Medincal	20210615
Medical absorbent cotton ball	Cofoe	20210006
Medical suture needle	Shanghai Xiaoyi Medical Devices	20192020430
Medullo-puncture needle	Yangzhou Jiangzhou Medical Devices	20190902	Used to puncture lumbar disc
Physiological saline	NeilMed	C1210504D2
Povidone iodine solution	Sichuan IJIS Medical Technology	20221209
Quasi-microbalance	Explorer
Rabbit dissection operating table	Zhenhua Biomedical	ZH-BXT-3Z
Shaver	AUX
Statistical analysis softeare	IBM	SPSS
Sterile gauze	Cofoe	20202140675
Surgical gloves	DR.LERSH	20172140028
Surgical knife	Hons Medinca	20210019
Surgical tweezers	SHINVA	20210233
USB-C data transmission line	KINI
White light photography microscope	Nikon	Eclipse Ci-L