Here, we present a protocol to generate a rat spinal cord compression model, assess its behavioral score, and observe the compressed spinal cord region. The behavioral assessments showed decreased monitor motor disability. Hematoxylin and eosin staining and immunostaining revealed considerable neuronal apoptosis in the compressed region of the spinal cord.
As a severe progressive degenerative disease, cervical spondylotic myelopathy (CSM) has a poor prognosis and is associated with physical pain, stiffness, motor or sensory dysfunction, and a high risk of spinal cord injury and acroparalysis. Thus, therapeutic strategies that promote efficient spinal cord regeneration in this chronic and progressive disease are urgently needed. Effective and reproducible animal spinal cord compression models are required to understand the complex biological mechanism underlying CSM. Most spinal cord injury models reflect acute and structural destructive conditions, whereas animal models of CSM present a chronic compression in the spinal cord. This paper presents a protocol to generate a rat spinal cord compression model, which was further evaluated by assessing the behavioral score and observing the compressed spinal cord region. The behavioral assessments showed decreased monitor motor disability, including joint movements, stepping ability, coordination, trunk stability, and limb muscle strength. Hematoxylin and eosin (H&E) staining and immunostaining revealed considerable neuronal apoptosis in the compressed region of the spinal cord.
As a common progressive degenerative disease, CSM accounts for 5-10% of all cervical spondylosis1. If patients suffering from CSM ignore their symptoms and fail to treat them in a timely and effective manner, this could lead to severe complications, such as spinal cord injury and limb paralysis, which would deteriorate with aging, posing a substantial economic and mental burden to patients and their families2,3. The pathogenesis of CSM is complex, involving static and dynamic factors, the hypoxia-ischemia theory, endothelial cell injury, the blood spinal cord barrier destruction theory, and the inflammation and apoptosis theory4,5,6,7.
The static and dynamic mechanisms of compression on the spinal cord cause clinical symptoms. Protruding vertebral discs, deformed vertebral bodies, and calcified ligaments may cause prolonged spinal cord compression, which will gradually affect the blood-spinal cord barrier and local microvasculature in the spinal cord4,8. In turn, ischemia, inflammation, and apoptosis affect the neurons, axons, and glial cells6,9.
The experimental animal models of spinal cord injury include contusive injury, compressive injury, traction injury, photochemical-induced injury, and ischemia-reperfusion injury. Most of these models also reflect some acute and structural destructive conditions (transection or chemical toxicity). However, these animal models of CSM cannot present progressive neuronal apoptosis in the spinal cord.
This paper describes a detailed protocol to generate a rat spinal cord compression model, which was further evaluated by assessing the behavioral score and observing the compressed region of the spinal cord. This rat spinal cord compression model is a reliable animal model for further investigation of the mechanisms involved in CSM.
The following procedure was performed with approval from the Institutional Animal Care and Use Committee (IACUC), Shanghai University of Traditional Chinese Medicine. All survival surgeries were performed under sterile conditions as outlined by the NIH guidelines. Pain and risk of infections were managed with appropriate analgesics and antibiotics to ensure a successful outcome. This surgical procedure is optimized for Sprague-Dawley (SD) outbred male rats at 12 weeks of age and 400 g weight.
1. PVA-polyacrylamide hydrogel preparation
NOTE: As shown in Figure 1G, 1H, the PVA-polyacrylamide hydrogel is a water-absorbing polymer sheet. In the natural state, the gel is extremely difficult to cut into small pieces. The preparation is described as follows.
- Place a PVA-polyacrylamide hydrogel in water for 24 h to make it easier to cut after hydration.
- Use a self-made cutting tool (Figure 1H) to divide the whole hydrogel into pieces, sized 2 mm x 2 mm x 2 mm.
- Transfer these hydrogel pieces to an oven at 60 °C for 12 h for dehydration into small pieces of 1 mm x 1 mm x 1 mm as implantation materials.
2. Anesthesia and preparation
NOTE: Be sure to wear a surgical cap, disposable medical masks, and sterile surgical gloves throughout the sterile surgical process.
- Place the rat on a heating pad, and ensure that rectal temperature is maintained at 37±1 °C during anesthesia.
- Place the rat into the anesthesia chamber filled with 3% isoflurane for 3 min.
- Gently pinch the rat's limbs and toes with tweezers to test for loss of withdrawal response, indicating successful anesthetization.
- Fix the rat on the operating table in a prone position, ensuring that the limbs and head of the rat are firmly fixed.
- Fix the anesthesia mask to the face of the rat. Administer 2% isoflurane in an oxygen/air mixture via a standard rat nose mask to anesthetize the rat throughout the spinal compression surgery.
- Place a cylindrical gauze pad (size of about 30 mm x 20 mm x 60 mm) between the rat and the operating table (Figure 1A) to ensure an unobstructed airway and fully exposed surgical site throughout the surgery.
- Shave the hair around the surgical area of the rat's neck with an electric shaver.
- Apply depilating cream to remove the remaining hair and expose the skin.
- Disinfect the surgical area with iodophor.
- Cover the disinfected area with a sterile towel with a hole exposing only the surgical area on the dorsal side of the rat's neck.
3. Surgical approach
- Make a longitudinal incision in the dorsal midline with a scalpel from the second cervical spinous process to the second thoracic spinous process, after percutaneously positioning the second cervical spinous process and second thoracic spinous process.
- Blunt separate the muscles of both sides with hemostatic forceps to expose the C2-T2 lamina after cutting subcutaneous tissue and fascia layer by layer.
- Drill a hole (1 mm x 1 mm) on the cervical laminar (Figure 1B).
NOTE: To avoid excessive injury on the spinal cord, ensure that the rat’s neck is maintained in a dorsal arch state, allowing sufficient space between the cervical laminas.
- Use microsurgical forceps to grasp a piece of PVA-polyacrylamide hydrogel of the size of 1 mm x 1 mm x 1 mm and insert it into the previously drilled hole (Figure 1C, 1D).
NOTE: Transient twitching performance indicates the spinal cord compression model has been established successfully.
- Suture the muscle, fascia, subcutaneous, and skin tissues, layer by layer, using triangular needles and 5-0 suture.
- After disinfection, transfer the animals back to the cage and keep them warm.
- Subcutaneously inject buprenorphine hydrochloride analgesia (0.03 mg/kg) every 6 h for 3 days following the surgery and as needed after that.
4. Postoperative management
- Inject an equivalent of 100,000 units of penicillin intraperitoneally into the rats once a day to prevent postoperative infection and relieve pain.
- Transfer the rats to new cages that have been continuously heated with an infrared lamp to ensure adequate heat preservation postoperatively.
NOTE: Remove the heating lamp after the rat's consciousness is restored
- Maintain hygiene and ventilation of the rat's feeding cage.
- Assist the rats with eating and drinking twice a day. If necessary, administer a bladder massage to assist in urination until the urinary function is restored.
5. Behavioral assessment
- Use the Basso, Beattie, and Bresnahan (BBB) rating scale to assess postoperative behavior.
NOTE: The BBB rating scale is a gold standard (Table 1) used to evaluate spinal cord-related function in rats. It assesses rats' movement according to scores ranging from 0 (no hind limb movement was observed) to 21 (gait coordination, toe space consistency, main claw position parallel in the whole posture, consistent trunk stability, and consistent tail elevation).
6. Grip strength test
- Use an electronic grip strength meter to measure grip strength.
- Grab the lower half of the rat to suspend the rat and allow it to grab the metal rod of the front grip meter.
- When the rat grasps the metal rod, pull it away and record the grip strength.
- Measure the grip strength three times for each rat and record the highest score.
7. Inclined plate test
- Place the rat on a rubber plate with an adjustable angle.
- Gradually raise the inclined plate angle by 5° each time until the rat manages to balance and stand firm for 5 s.
- Record the maximum angle at which the rat can balance itself on the inclined plate.
- Measure the maximum angle three times for each rat and record the highest score.
8. Euthanasia, spinal cord separation, and frozen embedding
NOTE: Ensure that appropriate eye goggles and face shield/mask are worn to protect the eyes, face, and respiratory tract from paraformaldehyde and formaldehyde gas.
- Inject an equivalent of 10% chloral hydrate intraperitoneally to anesthetize the rats before opening the sternum to expose the heart.
- Insert a perfusion needle into the apex of the heart, fix it with hemostatic forceps, and slowly infuse with normal saline.
- Drill a hole on the right atrial appendage until clean normal saline flows out of the right atrium, indicating a successful infusion.
- Stop the normal saline perfusion after the liver turns white.
- Infuse with an equivalent of 10% paraformaldehyde until the rat's body becomes stiff.
- After paraformaldehyde perfusion, remove the skin, muscles, and soft tissues around the spine; separate the C2-C7 segment of the cervical spine; and immerse it into 10% paraformaldehyde for fixation overnight.
- Separate the cervical spinal cord from the spine and place it into a concentration gradient of 10%, 20%, and 30% sucrose solutions for gradual dehydration.
- Transfer the compressed spinal cord of 2 mm height along with an OCT embedding agent into a -80 °C freezer.
- After sectioning into 7-µm-thick slices and staining (H&E staining and dUTP nick end labeling (TUNEL)/neuronal nuclei (NeuN), see section 9), observe the histopathology of the spinal cord and neuronal apoptosis, respectively.
9. TUNEL/NeuN immunostaining
- Immerse the spinal cord sections in phosphate-buffered saline (PBS) for 10 min at room temperature, then block with PBS solution containing 0.3%Triton X-100 and 5% bovine serum albumin (BSA) for 1 h.
- Incubate the spinal cord sections with a rabbit polyclonal anti-NeuN antibody (diluted 1:200;) overnight at 4 °C.
- Rinse the spinal cord sections three times in PBS. Subsequently incubate with Alexa Fluor 594-conjugated secondary antibodies for 2 h at room temperature.
- Perform the one-step TUNEL apoptosis assay kit (green fluorescence) to stain the spinal cord sections' apoptotic nuclei.
Spinal cord compressive injury may lead to neuromuscular disability in limbs
As the hydrogel piece expands gradually, it persistently compresses the spinal cord region for a prolonged period, which simulates the forelimb disabilities induced by cervical spinal cord diseases8,10. In the current model, considerable ipsilateral forepaw contracture was observed in most of the rats (9/10) in the model group (Figure 2A). Further measurement and analysis of the forepaws' length and width were conducted on a piece of paper with a grid line (Figure 2B). The data revealed that the length and width of the ipsilateral forepaws in the model group were remarkably decreased one day post-surgery (P < 0.01). However, no significant difference was detected in the contralateral forepaws between the control and model groups (Figure 2C).
To evaluate the progress and neuromuscular disability in limbs, the BBB rating scale, inclined plane test, and forelimb grip test were employed for observation on days 1, 3, 7, 14, 21, and 28 after the surgery. One-way or two-way analysis with Tukey's test was performed to analyze normally distributed data. A nonparametric Mann-Whitney U-test with post hoc analysis was performed for data that were not normally distributed but contained equal variances. Data are expressed as mean ± standard deviation (SD). Differences were considered statistically significant at P < 0.05.
The results showed that the BBB scores of the rats in the model group gradually decreased on days 1 and 3 after the surgery, presenting significant functional disability during the early phase, especially on the ipsilateral side (Figure 2D, 2E, 2G). Although recovery for spinal cord compression was observed in both the model and control groups, the rats in the model group showed a tardy and incomplete recovery of the aberrant forepaw function and balancing ability compared to the control group at 4 weeks post-surgery (Figure 2E, 2G). Significant differences between the model and control groups were maintained in the inclined plane score and grip strength on day 28 post-surgery. These combined results indicate that this surgery induces progressive compression on the cervical spinal cord and causes deterioration of motor ability in rats.
Histological changes and inflammation induced by compression in the spinal cord
After separating the cervical spinal cord, a prominent indentation of 2 mm depth and 2 mm x 2 mm area could be observed on the spinal cord (Figure 3B). To assess the morphometric changes, the spinal cord sections were stained and viewed under a light microscope. The H&E staining revealed the infiltration of immune cells and a dramatic loss of neurons in the compressive region of the spinal cord (Figure 3C). In addition, the immunostaining revealed a dramatic increase in neuronal apoptosis in the spinal cord compression site in the model group (Figure 3D, 3E). Some cells or tissues have high nuclease and polymerase activity levels, which could result in nonspecific fluorescence. Hence, the tissue was immobilized immediately after it was extracted to prevent these enzymes from causing false positives. TUNEL staining is nonspecific and can be employed in the event of cell or neuron death. NeuN is a specific staining marker for neurons. As a result, merged images from TUNEL staining and NeuN staining were used to demonstrate neuronal apoptosis.
Figure 1: A schematic of the surgical procedure. (A) A gauze pad was placed under the rat to ensure that the airway of the rat was clear during the operation. (B-D) A surgical procedure of hydrogel implantation into the cervical spinal canal; the yellow arrowhead points to a tiny hole drilled on the vertebral plate of C6, and the green arrowhead indicates the dehydrated hydrogel block. (E) A schematic of the surgical procedure. (F) A three-dimensional schematic of spinal cord compression. (G) Water-absorbing property of the PVA-polyacrylamide hydrogel. (H) Preparation of the hydrogel block for spinal cord compression. Abbreviations: PVA = polyvinyl alcohol. Please click here to view a larger version of this figure.
Figure 2: Morphology of the forepaw and behavioral observations with BBB scale, grip strength test, and inclined plate test. (A) A typical feature of the ipsilateral forepaws of the control group (left) and model group (right) rats on the third day after surgery. (B) The width and length of the forepaws of the rats were measured. The transverse red arrow is from the first finger to the fourth finger, and the longitudinal red arrow is from the tip of the longest finger to the root of the palm. (C) Quantitative analysis of the length and width of the ipsilateral forepaws in the model and control groups. (D) BBB score of both the ipsilateral and contralateral sides 1, 3, 7, 14, 21, and 28 days after surgery. (E) The grip strength of both the ipsilateral and contralateral side forelimbs 1, 3, 7, 14, 21, and 28 days after surgery, assessed with the grip strength test. (F) Schematic of the inclined plate test.(G) The strength and balance of both ipsilateral side and contralateral side limbs 1, 3, 10, 20, and 28 days after surgery, assessed with the inclined plate test. Data are presented as mean ± SD. *P < 0.05 and **P < 0.01 vs. control group; n = 10/group. Abbreviation: BBB = Basso, Beattie, and Bresnahan rating scale. Please click here to view a larger version of this figure.
Figure 3: Morphological changes and inflammatory responses after prolonged cervical spinal cord compression. (A) A three-dimensional schematic of spinal cord compression. (B) An indentation of 2 mm depth and 2 mm x 2 mm-area on the spinal cord. (C) A spinal cord histological section at 28 days after compression and H&E staining. The infiltration of immune cells and a dramatic loss of neurons in the compressive region of the spinal cord. Red rectangle, ipsilateral side; green rectangle, contralateral; blue arrowheads, immune cells; yellow arrows, neurons. (D) Double staining for NeuN (red)/TUNEL (green) of sections from the spinal cord compression site in the model and control groups. Scale bars = 20 µm. (E) Quantification of NeuN and TUNEL double-positive cells. ***P < 0.001 compared to the control group; n = 10/group. Abbreviations: H & E = hematoxylin and eosin; NeuN = neuronal nuclei; TUNEL = dUTP nick end labeling. Please click here to view a larger version of this figure.
|Score||Operational definitions of categories and attributes|
|0||No observable movement of the hindlimbs|
|1||Slight (limited) movement of one or two joints, usually hip and/or knee|
|2||Extensive movement of one joint or extensive movement of one joint and slight movement of the other|
|3||Extensive movement of two joints|
|4||Slight movement of all three joints of the hindlimbs|
|5||Slight movement of two joints and extensive movement of the third joint|
|6||Extensive movement of two joints and slight movement of the third joint|
|7||Extensive movement of the three joints in the hindlimbs|
|8||Sweeping without weight-bearing or plantar support of the paw without weight-bearing|
|9||Plantar support of the paw with weight-bearing only in the support stage (i.e., when static) or occasional, frequent, or inconsistent dorsal stepping with weight-bearing and no plantar stepping|
|10||Plantar stepping with occasional weight-bearing and no forelimb-hindlimb coordination|
|11||Plantar stepping with frequent to consistent weight-bearing and occasional forelimb-hindlimb coordination|
|12||Plantar stepping with frequent to consistent weight-bearing and occasional forelimb-hindlimb coordination|
|13||Plantar stepping with frequent to consistent weight-bearing and frequent forelimb-hindlimb coordination|
|14||Plantar stepping with consistent weight support, consistent forelimb-hindlimb coordination, and predominantly rotated paw position (internally or externally) during locomotion, both at the instant of initial contact with the surface as well as before moving the toes at the end of the support stage or frequent plantar stepping, consistent forelimb-hindlimb coordination, and occasional dorsal stepping|
|15||Consistent plantar stepping, consistent forelimb-hindlimb coordination and no movement of the toes or occasional movement during forward movement of limb; predominant paw position is parallel to the body at the time of initial contact.|
|16||Consistent plantar stepping and forelimb-hindlimb coordination during gait and movement of the toes occurs frequently during forward movement of the limb; the predominant paw position is parallel to the body at the time of initial contact and curved at the instant of movement.|
|17||Consistent plantar stepping and forelimb-hindlimb coordination during gait and movement of the toes occurs frequently during forward movement of limb; the predominant paw position is parallel to the body at the time of initial contact and at the instant of movement of the toes.|
|18||Consistent plantar stepping and forelimb-hindlimb coordination during gait and movement of the toes occurs consistently during forward movement of limb; the predominant paw position is parallel to the body at the time of initial contact and curved during movement of the toes.|
|19||Consistent plantar stepping and forelimb-hindlimb coordination during gait and movement of the toes occurs consistently during forward movement of limb; the predominant paw position is parallel to the body at the instant of contact and at the time of movement of the toes, and the animal presents a downward tail some or all of the time.|
|20||Consistent plantar stepping and forelimb-hindlimb coordination during gait and movement of the toes occurs consistently during forward movement of limb; the predominant paw position is parallel to the body at the instant of contact and at the time of movement of toes, and the animal presents consistent elevation of the tail and trunk instability.|
|21||Consistent plantar stepping and coordinated gait, consistent movement of the toes; paw position is predominantly parallel to the body during the whole support stage; consistent trunk stability; consistent tail elevation|
Table 1: 21-point functional evaluation scale of Basso et al.9,11.
The goal of this surgical procedure was to generate reproducible, prolonged, neural apoptosis in the rat spinal cord. A key advantage of this model is that the expandable hydrogel implants provide a prolonged compression on the spinal cord, thereby leading to a progressive neural apoptotic response (Figure 2C), which is consistent with the pathological process of CSM. In the current study, the mortality from spinal cord injury was extremely low (~2 in 50), whereas the repeatability of this model was > 45 in 50. Incorrect size of the hydrogel pieces and vigorous implantation during the surgery might cause an acute injury to the spinal cord12,13.
An unpublished study14 found that implantation with an expansion rate of 350% resulted in temporary and acute CSM with progressive recovery for several weeks. An expansion rate of 200% caused a slow progressive paralysis in the CSM model because the implants were harder than the spinal cord. However, in this model, we were not interested in the hardness of the implanted material, only in the final size of this implantation. After 4 weeks, an indentation on the spinal cord (Figure 3A, 3B) was observed, which reflected the sustained constriction on the spinal cord, aggravated neuroinflammation, and neuronal apoptosis.
Currently, there is no consensus on the size of the implants. Several studies used absorbent sheets with a thickness of 0.5-1 mm15,16,17,18 and reported functional disability from spinal compression. Another rat spinal cord compression study19 showed that the loss of intact white matter and dramatic cord flattening were induced by severe cord compression (2.6 mm thickness), which reflected a compression strain without inflammation. Therefore, a large implant fabricated with a soft expandable material may be suitable for prolonged compression on the spinal cord.
In the current model, the size of the hydrogel pieces and drill on the vertebral plate was strictly limited to a size of 1 mm x 1 mm x 1 mm to avoid acute spinal cord injury or accidental death due to any sudden force due to oversized implants. After 48 h of hydration, the hydrogel blocks expanded to a size of 2 mm x 2 mm x 2 mm. Clinically, the aggravation of symptoms in CSM patients is related to the sudden compression of the spinal cord, which is from continuous disc herniated compression on the spinal cord and subsequent lower compensated adaptation induced by inflammation and edema4,7. This could explain why unilateral hydrogel inflammatory infiltration leads to a bilateral neurological function deficit20.
One limitation of this animal model is that rats show strong adaptation to any injury21, which facilitates quick recovery. Several studies have shown continuous improvements in neurological function over time after the compression operation15,16,17,18,21,22, whereas only a few studies have reported a deteriorating trend. In addition, most CSM patients show either a gradual recovery or deterioration in neurological function under consistent compression on the spinal cord23. As there was no significant difference in the motor function in the current model after 4 weeks, we stopped the behavioral assessment and euthanized the rats for further histological investigations. In summary, this study presents a neural apoptosis model induced by spinal cord compression in rat, a practical animal model to study the cellular and molecular mechanisms associated with CSM and spinal cord regeneration.
The authors have no conflicts of interest to disclose and state that there are no restrictions on full access to all the materials used in this study.
This study was supported by the National Key R&D Program of China (2018YFC1704300), National Natural Science Foundation of China (81930116, 81804115, 81873317, and 81704096), Shanghai Sailing Program (18YF1423800), Natural science Foundation of Shanghai (20ZR1473400). This project was also supported by the Shanghai University of Traditional Chinese Medicine (2019LK057).
|Antibiotic ointment||Prevent wound infection|
|Inhalant anesthesia equipment||Anesthesia|
|Micro ophthalmic forceps||Mingren medical equipment||Length: 11 cm, Head diameter: 0.3 mm||Clip the muscle|
|Ophthalmic forceps||Shanghai Medical Devices (Group) Co., Ltd. Surgical Instruments Factory||JD1050||Clip the skin|
|Ophthalmic scissors (10 cm)||Shanghai Medical Devices (Group) Co., Ltd. Surgical Instruments Factory||Y00030||Skin incision|
|SD male rats||Shanghai SLAC Laboratory Animal Co., Ltd||SCXK2018-0004||Animal model|
|Sterile surgical blades (22#)||Shanghai Pudong Jinhuan Medical Products Co., Ltd.||35T0707||Muscle incision|
|Small animal trimmer||Hair removal|
|Veet hair removal cream||RECKITT BENCKISER (India) Ltd||Hair removal|
|Venus shears||Mingren medical equipment||Length: 12.5 cm||Muscle incision|
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