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Sprague-Dawley (SD) rats were used as experimental subjects, with no restriction on gender, and aged 6-8 weeks.
1. Preoperative Preparation: Two hours before the modeling surgery, rats were intraperitoneally injected with 2×10⁵ units of penicillin sodium to achieve an anti-inflammatory effect.
2. Anesthesia, Disinfection, and Fixation: Complete the corresponding preparatory work.
3. Localization of Thoracic Vertebra T10 Segment: Palpate the rat's back with hands to first find the highest point of the thoracic vertebrae. Count 3 spinous processes upward from this point; the corresponding vertebral body position is the T10 segment of the thoracic vertebrae.
4. Dorsal Incision Operation: Make a median vertical incision on the dorsal skin corresponding to the T9-T11 segments. Subsequently, use hemostats to bluntly dissect and separate the mucosa between the skin and muscle layer at the incision site.
5. Exposure of the Surgical Field: Use auxiliary retractors to pull the skin on both sides apart, fully exposing the muscle field of view in the surgical area. Make vertical incisions on the muscles on both sides of the midline (edge of the spinous process) with ophthalmic scissors. If a small amount of bleeding occurs, clean the surgical area with cotton swabs. Next, use ophthalmic scissors to remove the spinous processes in the T9-T11 segment area and a small amount of other redundant muscle tissue, fully exposing the laminae of the T9-T11 segments.
6. Exposure of the Dorsal Spinal Cord: Remove the articular processes on both sides to form a circular exposed area with a diameter of approximately 0.5 cm on the dorsal side of the spinal cord. It should be noted that the exposed area must be larger than the diameter of the selected impact head, and no residual bone processes should remain to prevent obstruction by surrounding tissues when the weight falls freely.
7. Adjustment of Impactor Position and Height: Adjust the position of the free-fall impactor so that the impact head moves to the experimental site on the dorsal spinal cord to be impacted. Then adjust the vertical height of the impactor. Lower the impactor until the spherical surface of the impact pin just touches the spinal periosteum, and then lift it freely to an appropriate position (the impact head is lifted by 2-3 mm).
8. Placement and Localization of Weights: Insert a positioning pin at a height of 2.5 cm above the impact pin, and select an appropriate weight (10 g) to place into the metal impact tube.
9. Implementation of Impact: Quickly pull out the positioning pin, allowing the weight to hit the impact pin in a free-fall manner, thereby generating an injury-inducing energy of 25 gcf (gram-centimeters of force).
10. Postoperative Preliminary Observation: After quickly removing the free-fall impactor, carefully observe the rat's heartbeat and breathing to ensure no abnormalities.
11. Suturing and Anti-Infection Treatment: Perform suturing in the order of muscles, fascia, and skin. After suturing, disinfect the incision and its surrounding area with povidone-iodine. Sprinkle penicillin powder around the wound to prevent infection.
1. Behavioral Observation: After the impact, if the rat's body is observed to tremble, the tail shows spasmodic twitching, the hind limbs first retract and flutter rapidly and then become paralyzed, and subdurally hematoma of the spinal cord is visible to the naked eye, the model establishment is likely successful. Rats in the sham operation group only underwent spinous process and laminectomy without spinal cord injury (SCI) modeling.
2. BBB Locomotor Rating Scale: The observation duration was set to 4 minutes, and scoring was conducted within a 125 cm × 125 cm space. A three-person blind method was adopted; before scoring, the scorers were trained to fully master the scoring rules and standards. The three scorers independently observed the motor function of the rat's hind limbs at the same time, recorded and scored, and finally took the minimum value among the three scores as the functional score result. The detailed explanation of the BBB scoring criteria is as follows: For scores 0-7, focus on whether the joints move and the number of moving joints; for scores 8-14, mainly check whether the soles of the feet can touch the ground, whether movement can be performed after touching the ground, and whether the movement is coordinated; for scores 15-18, focus on observing whether the toes can grip the ground, whether the direction of the toes is consistent with the forward direction, and whether the movement of the forelimbs and hind limbs is coordinated; for scores 19-21, mainly judge whether the trunk is stable during movement and whether the tail is raised upward.
Definition of BBB Score: Place the animal in an open basin, tap the basin wall to make it crawl, and observe the movement and coordination of the animal's hips, knees, ankles during walking, as well as trunk movement. BBB Scale (Basso, Beattie & Bresnahan Locomotor Rating Scale for Rats) | |
| Scoring Criteria for (Rat Spinal Cord Injury) | |
| Score 0 | No visible hind limb movement |
| Score 1 | Minimal movement of one or two joints, usually the hip and/or knee joints |
| Score 2 | Extensive movement of one joint, or extensive movement of one joint plus minimal movement of another joint |
| Score 3 | Extensive movement of two joints |
| Score 4 | Minimal movement of all three hind limb joints |
| Score 5 | Minimal movement of two joints plus extensive movement of the third joint |
| Score 6 | Extensive movement of two joints plus minimal movement of the third joint |
| Score 7 | Extensive movement of all three hind limb joints |
| Score 8 | Palmar surface of the paw can touch the ground without weight-bearing |
| Score 9 | Sole of the foot is only in a weight-bearing position, or walks with the dorsum of the foot bearing weight occasionally/frequently/continuously, without weight-bearing walking on the sole. Weight-bearing: When the sole is in the weight-bearing position or only when the posterior trunk is lifted, the hind limb (HL) extensor muscles contract |
| 10 Points | Occasional weight-bearing movement on the palmar surface of paws; no coordinated movement of forelimbs and hindlimbs |
| 11 Points | Frequent weight-bearing movement on the palmar surface of paws, but no coordinated movement of forelimbs and hindlimbs |
| 12 Points | Frequent weight-bearing movement on the palmar surface of paws, with occasional coordinated movement of forelimbs and hindlimbs |
| 13 Points | Common weight-bearing movement on the palmar surface of paws, with frequent coordinated movement of forelimbs and hindlimbs |
| 14 Points | Continuous weight-bearing movement on the palmar surface and coordinated movement of forelimbs and hindlimbs; or common palmar surface movement, continuous coordinated movement of forelimbs and hindlimbs, with occasional movement on the dorsal surface of paws |
| 15 Points | Continuous palmar surface movement and continuous coordinated movement of forelimbs and hindlimbs; no or occasional paw-ground contact during forelimb advancement; the position of active paws is parallel to the body at initial contact |
| 16 Points | Continuous palmar surface movement and continuous coordinated movement of forelimbs and hindlimbs in gait; frequent paw-ground contact during forelimb advancement; the position of active paws is parallel to the body at initial contact, and rotation occurs after weight transfer |
| 17 Points | Continuous palmar surface movement and continuous coordinated movement of forelimbs and hindlimbs in gait; frequent paw-ground contact during forelimb advancement; the position of active paws is parallel to the body both at initial contact and after weight transfer |
| 18 Points | Continuous palmar surface movement and continuous coordinated movement of forelimbs and hindlimbs in gait; continuous paw-ground contact during forelimb advancement; the position of active paws is parallel to the body at initial contact, and rotation occurs after weight transfer |
| 19 Points | Continuous palmar surface movement and continuous coordinated movement of forelimbs and hindlimbs in gait; continuous paw-ground contact during forelimb advancement; the position of active paws is parallel to the body both at initial contact and after weight transfer. The tail droops sometimes or always |
| 20 Points | Continuous palmar surface movement, continuous coordinated gait, continuous toe-ground contact; the position of active paws is parallel to the body both at initial contact and after weight transfer; unstable trunk; tail remains raised continuously |
| 21 Points | Continuous palmar surface movement, continuous coordinated gait, continuous toe-ground contact; the position of active paws is always parallel to the body during movement; stable trunk; tail remains raised continuously |
| Note: HL (hindlimb): Hindlimb. Mild: Joint range of motion ≤ 50%. Significant: Joint range of motion ＞ 50%. The 2 joints usually refer to the hip joint and knee joint. The 3rd joint is usually the ankle joint. | |
3. Rivlin Inclined Plane Test: A modified Rivlin method was adopted. A rectangular wooden board pre-pasted with a 3mm-thick rubber pad was used as the inclined plane. The inclined plane could rotate through a hinge connected to the base plate to adjust its inclination angle. Rats were placed on the inclined plane with their heads facing forward and the long axis of their bodies perpendicular to the longitudinal axis of the inclined plane. The test started from the horizontal position (0°), and the inclined plane was rotated and raised by 5° each time to gradually increase the angle between the inclined plane and the horizontal plane. The maximum angle at which the rat could stay for 5 seconds was recorded as its functional angle value. Under normal conditions, the inclination angle of the inclined plane when a rat maintains its body position for 5 seconds is approximately 73°.
4. HE Staining: The injured segment and the cephalic-caudal segments of the spinal cord (with a total length of 1.0 cm) were carefully and completely removed, placed in a centrifuge tube pre-filled with 4% paraformaldehyde, fixed for 24 hours, then subjected to HE staining. Subsequently, the pathological changes in the spinal cord injury area and adjacent parts were observed under a light microscope.
The entire modeling cycle lasts for 3 days.
Carefully dissect and completely remove a 1.0 cm segment of spinal cord tissue, including the injured segment and the adjacent cephalic and caudal segments. Place the tissue into a centrifuge tube pre-filled with 4% paraformaldehyde for fixation for 24 hours. After HE staining, observe the pathological changes in the spinal cord injury area and adjacent regions under a light microscope.
1. Body Temperature Maintenance: The body temperature of rats is usually low after surgery. They should be placed on a body temperature maintenance device to ensure stable body temperature.
2. Urination Care: When performing urination care on rats, first use your fingers to feel for the distended bladder, then press the bottom of the bladder and gently squeeze from top to bottom to promote urine excretion. The squeezing process should be patient and gentle; rough force is strictly prohibited. If urine excretion is difficult, moderately stimulate the bladder area and external genitalia with warm water. At the same time, avoid improper mechanical stimulation of the urethral area and external genitalia to prevent urethral edema and eversion of the external genitalia due to edema, and conduct regular cleaning.
3. Precautions for BBB Scoring: Before conducting the BBB (Basso, Beattie, Bresnahan) score, it is essential to empty the rat's bladder first to avoid affecting the experimental results. During scoring, carefully and meticulously observe the walking status of the rat's limbs and keep detailed records.
4. Comprehensive Postoperative Care: After surgery, house the animals in separate cages, ensuring adequate natural light and good ventilation. Replace with clean and dry bedding every day to keep the environment dry at all times. Closely monitor the animals' mental state, dietary intake, urination and defecation status, presence of limb edema and pressure injuries, and presence of bloody secretions in the urinary system. Perform artificial urination 3 times a day, and administer penicillin via intraperitoneal injection at a dose of 100,000 units per time, twice a day. Pay attention to preventing self-mutilation behavior and intestinal obstruction in rats.
5. Model Adjustment: The severity of the model can be adjusted by modifying the number of impacts and impact height according to the specific requirements of the experiment. It is recommended to conduct a pilot experiment before the formal experiment to determine the most appropriate parameter settings.
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