Development of a Novel Internal Fixation Model for Rat Radial Fractures: Fracture Healing Assessment and Dorsal Root Ganglion Isolation

Summary

The study innovatively combines sutures and titanium needles for stable internal fixation in a rat radial fracture model. Fracture healing was assessed via micro-computed tomography (micro-CT), and primary dorsal root ganglion (DRG) neurons were isolated and cultured for further investigation.

Abstract

The rat radial fracture model is of significant importance in orthopedic research, particularly for studying fracture-healing mechanisms, drug screening, and evaluating biological materials. Despite certain limitations, its role in scientific research remains irreplaceable. Since the radius of rats is relatively thin, the steel plates and screws currently used in clinical practice are not suitable for the treatment of rat radial fractures. To address this limitation, this study innovatively employs suture and titanium needle fixation, an internal fixation model for radial fractures that provides relatively stable fixation and establishes a robust animal model for investigating internal fixation of such fractures. In vivo imaging was performed using a live-animal micro-CT system to detect radial fracture healing in Sprague-Dawley (SD) rats. The extraction and application of the rat DRG are important steps in neuroscience research, widely used in fields such as neuroscience, pathology, and cell biology. This protocol is suitable for the primary culture of dorsal root ganglion neurons from rats with radial fractures and can be used to study mechanisms of nerve injury and repair after fractures, such as neuronal apoptosis and neurotransmitter release.

Introduction

Radial fractures are among the most common clinical fractures, with a substantial global incidence, and rank as a leading cause of long-bone fractures in the extremities^1,2,3. Population studies reveal a bimodal age distribution for radial fractures, typically peaking in both pediatric and elderly populations⁴. Delayed treatment often results in forearm rotational dysfunction, severely impairing activities of daily living (ADLs)^5,6. Controversies persist in radial fracture management, primarily centered on fracture classification systems and treatment modality selection^7,8. Clinical evidence suggests that open reduction internal fixation (ORIF) significantly improves elbow or wrist joint range of motion (ROM) and strength recovery⁹; however, this approach is associated with higher rates of postoperative infections and internal fixation-related complications^10,11.

The DRG serves as a critical anatomical and functional bridge for sensory information transmission between the peripheral and central nervous systems (CNS). Beyond its primary role in collecting and relaying peripheral sensory signals (e.g., touch, temperature), the DRG actively regulates abnormal sensations such as pain, making it a key structure underlying both normal sensory function and clinical pain management^12,13,14. Pathological changes such as injury, inflammation, or neurodegeneration in the DRG can trigger sensory abnormalities (e.g., numbness, tingling) or chronic pain, positioning it as a focal point of research in neuroscience and pain medicine.

Herein, the 8-week-old male SD rats weighing approximately 200 g were selected to construct (1) an internal fixation model for radial transverse fractures in rats and subsequent post-healing assessment, and (2) the isolation and in vitro culture of DRGs from rats with an internal fixation model for radial fractures. This protocol is designed to facilitate reproducible experimentation in studies exploring fracture healing mechanisms, DRG-mediated sensory changes, and potential therapeutic targets for post-fracture pain or dysfunction.

Protocol

All animal experiments were approved by the Animal Ethics Committee of Nanjing University of Chinese Medicine (No. 202508A046). All rats used in the following assays were maintained in specific pathogen-free (SPF) conditions. A schematic representing the protocol is shown in Figure 1.

1. Establishment of an internal fixation model for radial fractures in rats

NOTE: Rats were acclimated to a quiet environment with soft, diffused lighting prior to surgery, shielding them from noise and harsh illumination. During handling, the rats' abdomen and chest were supported with gentle manual restraint, avoiding tail grasping, a practice known to induce pain and distress and to reduce discomfort associated with struggling.

1. Prepare the surgical instruments as shown in Figure 2. The surgical instruments include a scalpel blade, a scalpel handle, operating scissors, a needle holder, hemostatic forceps, and toothed tissue forceps. The male Sprague-Dawley (SD) rat was approximately 8 weeks old and weighed about 200 g.
2. Anesthetize the rat using an isoflurane gas anesthesia machine. For induction, use 4% isoflurane and maintain ventilatory anesthesia (2% isoflurane) using a nose cone throughout the surgical procedure. Maintain the operating room temperature at approximately 28 °C.
   NOTE: Determine the success of anesthesia by checking vital signs (such as respiration and heart rate), corneal reflex (no response indicates effective anesthesia), blink reflex (absence), and toe contraction response (no retraction after stimulation).
3. Place the rat in the lateral position, expose the right forelimb, and shave off the hair using a hair clipper. Drape a sterile sheet and wrap a tourniquet around the proximal right forelimb to reduce surgical bleeding.
4. Prepare the skin by alternating alcohol and betadine scrubs three times.
5. Make a 1.5-cm longitudinal incision on the dorsal radial side of the right forearm, as shown in Figure 3. Separate the muscle spaces and fully expose the right forelimb radius.
   NOTE: The radius is exposed by bluntly separating the muscles along the muscle interspace. Only the dorsal muscles of the radius are exposed without completely stripping the surrounding muscles.
6. Use operating scissors to cut the radius to create a transverse fracture. The scissors are oriented at a 90-degree angle to the radius (Figure 4).
7. Take a 0.8 mm-diameter, 10 mm-long titanium needle and insert it into the radius fracture along its longitudinal axis (Figure 5). Fix the radius and the titanium needle with suture (4-0 [1.5 metric] silk braided non-absorbable), as shown in Figure 6.
8. Tie the suture with three consecutive knots, and secure the titanium needle to the radius via four interrupted sutures.
   NOTE: The proximal and distal ends of the fracture are fixed twice, respectively.
9. Suture (4-0 [1.5 metric] silk braided non-absorbable) the skin, cut off the excess suture, and close the wound. Disinfect the skin with Betadine scrubs.
10. Cover the wound with sterile gauze and secure it with bandages. Place the rat in a cage to regain consciousness until recovery.
11. Postoperative pain management:
    1. If the animal demonstrates signs of pain, is not eating, and/or hesitates to walk on their hindlimbs, consult with veterinary personnel and administer additional analgesics.
       NOTE: Cefuroxime sodium is used in the treatment of bacterial infections. Tramadol hydrochloride is used to administer analgesics.

2. Micro CT imaging

1. Anesthetize the rat using an isoflurane gas (induction dose 4%) anesthesia machine.
2. Place the rat in a prone position within the scanning chamber, adjust limb and head positioning to center the target scanning area in the field of view, and maintain continuous ventilation and anesthesia (2% isoflurane) throughout the procedure.
3. Push the fixed animal bed into the scanning chamber and close the chamber door.
4. Set the micro-CT scanning parameters as follows. Scan mode: standard; exposure time: 2 min; field of view: 72 mm; voxel size: 144 µm; voltage: 90 kV; current: 88 µA; filter: 3D-soft.
   NOTE: The scan data is automatically transmitted to the software, and a three-dimensional CT image is generated.
5. Separate the target tissues.
   1. Analyze the selected bone tissue area from the original micro-CT images. Slide the transfer function line to separate the bone from the muscle, avoiding too much muscle tissue included. Perform the high-resolution reconstruction on the segmented bone tissue area to generate a 3D model.
6. Export the processed images.
   1. After processing the images, locate and click the end button to finalize the current workflow.
   2. A pop-up window will appear, first select still to confirm you want to export a static image (not a dynamic sequence).
   3. Choose jpeg as the output file format from the available options.
   4. Select large size to ensure the exported image retains high resolution suitable for analysis or documentation.
   5. Click OK to complete the export process.

3. Isolation and culture of DRG in rats with radial fractures

NOTE: Euthanasia is administered with 4% isoflurane inhalation anesthesia, followed by rapid cervical dislocation to euthanize the animals. The DRGs are extracted 20 days after the radial fracture model establishment.

1. Sever the spine, remove the C1-C7 vertebral bodies. Cut the cervical spine along the anterior and posterior midlines, and divide it into left and right halves. If necessary, cut off the spinous processes to facilitate the operation.
2. Gently clean the spinal cord tissue and nerve fibers inside the intervertebral foramina. Rinse the intervertebral foramina with a syringe filled with PBS to remove the blood to expose the lateral recesses.
   NOTE: The DRG is located inside the lateral recesses, presenting as a light-yellow nodule, with nerve fibers connected at both ends.
3. Use toothed tissue forceps to pick up the DRG, gently lift it, and then use operating scissors to cut the nerve fibers at both ends.
4. Transfer the extracted DRGs to a 1.5 mL centrifuge tube with extraction medium; place on ice.
   NOTE: Prepare the extraction medium by adding 2% fetal bovine serum (FBS) to the α-Minimum Essential Medium (α-MEM). The DRGs are incubated in the extraction medium for about 30 min before proceeding to the next step.
5. Slowly aspirate and discard the extraction medium. Add 1 mL of the mixed digestive enzyme working solution. Digest in a 37 °C water bath for 60-70 min.
   NOTE: To prepare the mixed digestive enzyme working solution, dissolve 100 mg of type I collagenase and 100 mg of dispase in 1 mL of Phosphate Buffered Saline (PBS). When using, dilute it to a working solution of 5 mg/mL and store it on ice.
6. Coat a Petri dish by pipetting an appropriate volume of the fibronectin working solution, then incubate at 37 °C for 1 h.
   NOTE: Human plasma fibronectin working solution (Fibronectin working solution) is prepared by diluting the 1 mg/mL Fibronectin stock solution with ddH₂O to a 0.02 mg/mL Fibronectin working solution.
7. Post-digestion, aspirate and discard the mixed digestive enzyme working solution. Add 1 mL of complete medium, and gently pipette the DRG tissue several times.
   NOTE: To prepare complete medium, mix 45 mL of MEM-α medium, 4.5 mL of FBS, and 0.5 mL of Penicillin-Streptomycin (PS) solution, and sterilize by filtration through a 0.22 µm sterilizing-grade microporous membrane.
8. Collect filtrate, centrifuge at 75 × g for 8.5 min at 25 °C.
9. After centrifugation, aspirate and discard the supernatant in the centrifuge tube, and add 1 mL of complete medium to resuspend the precipitate.
10. Take out the Petri dish. Discard the Fibronectin working solution and wash with PBS twice.
11. Inoculate the cells into the coated Petri dish, add 1 mL of complete medium, and incubate in a constant-temperature incubator at 37 °C with 5% CO₂ for 8-12 h.
12. After 8-12 h, replace the complete medium with replacement medium. Then, replace half of the volume with the replacement medium every 2-3 days.
    NOTE: To prepare replacement medium, mix 44.5 mL of MEM-α medium, 4.5 mL of FBS, 0.5 mL of Penicillin-Streptomycin (PS) solution, and 0.5 mL Deoxyuridine. Sterilize by filtration through a 0.22 µm sterilizing-grade microporous membrane.

Results

The micro-CT images confirmed the successful establishment of radial fracture rat models in Figure 7 and the internal fixation model for rat radial fractures in Figure 8. The micro-CT images were compared between taking on the day of constructing the internal fixation model of fracture and twenty days after the model establishment. Twenty days post-modeling, micro-CT analysis of SD rats in the fracture model group revealed a favorable prognosis. Images showed a ...

Discussion

Radial fracture is one of the most common long bone fractures in clinical practice. Its healing process involves complex interactions between bone tissue repair and neural regulation (such as the regulation of osteoblasts/osteoclasts by neuropeptides and neurotrophic factors released from the dorsal root ganglion)^15,16,17,18. Rats are a classic model for studying the mechanism of fracture heali...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
5-Fluoro-2' deoxyuridine	sigma	F0503
Cefuroxime sodium	Shandong Runze Pharmaceutical Co., Ltd., China	Guoyao Zhunzi H20205058
DAPI Staining Solution	Biyotime	C1005
Dispersase	Shanghai yuanye Bio-Technology Co., Ltd.	S25046
Fetal bovine serum (FBS)	Thermo	16000-044
hemostatic forceps	Shanghai Medical Instrument (Group) Co.,Ltd.	J31070
Human plasma fibronectin (Human Fibronectin)	sigma	F0895
Isoflurane	RWD	R510-22-10
Live animal micro-CT imaging system	PerkinElmer in the USA	Quantum GX
MEM-ALPHA Culture medium	BI	C3060
needle holder	Shanghai Medical Instrument (Group) Co.,Ltd.	200622
operating scissors	Shanghai Medical Instrument (Group) Co.,Ltd.	J21130
penicillin	Solarbio	140051251
Penicillin-streptomycin solution	NovaCells	N-PSS125
povidone-iodine	Meilunbio	MB1313800g
scalpel blade	Shanghai Medical Instrument (Group) Co.,Ltd.	RYY070
scalpel handle	Shanghai Medical Instrument (Group) Co.,Ltd.	J11010
suture	Johnson & Johnson MEDICAL (CHINA) Ltd.	SA84G
suture needle	Shanghai XIAOYU MEDICAL EQUIPMENTS.	Δ3/8 ·3×6
titanium needle	Tianjin Yutong Medical Device	2-0X250
toothed tissue forceps	Shanghai Medical Instrument (Group) Co.,Ltd.	J41055
Tramadol hydrochloride	Jiangsu Jiuxu Pharmaceutical Co., Ltd., China	CYHB2201997
Tubulin beta-III	sigma	CBL412X
Type I collagenase	Thermo	17100-017
Zeiss Axio Vert A1 Inverted Fluorescence Microscope	Zeiss	Axio Vert A1