Effect of percutaneous nucleoplasty with coblation on phospholipase A2 activity in the intervertebral disks of an animal model of intervertebral disk degeneration: a randomized controlled trial

This study was approved by the Animal Experimental Ethics Committee of Beijing Army General Hospital. A total of 46 New Zealand white rabbits (weight, 2.0–2.5 kg; age, 2.0–2.5 months) were purchased from Vital River Laboratories (Beijing, China). They were randomly assigned to an animal model group (n = 38) and a control group (n = 8). A surgical procedure was performed in the animal model group to establish the animal model of intervertebral disk degeneration. Two rabbits died of infection after the surgery. Thirty-six animal models were successfully established and then randomly divided into two groups: the coblation group (n = 18) and coblation control group (n = 18). At each time point, six rabbits in the coblation and coblation control groups were euthanized for the analysis of PLA2 activity (Figure 1). Food and drinking water were available ad libitum.

All rabbits were anesthetized with sodium pentobarbital (35 mg/kg, intraperitoneal injection). Magnetic resonance imaging (MRI) (Signa TwinSpeed 1.5 T Excite II; GE Healthcare, USA) and X-ray were performed to confirm the preoperative status of the involved disks prior to surgery. The animal model of intervertebral disk degeneration was induced as previously described [8]. A C-arm with image intensification (BV300; Philips Electronics, Eindhoven, The Netherlands) was used to identify the location of L4–5 and L5–6. A posterolateral incision was made in the iliac crest, and the L4–5 and L5–6 disks were exposed. A 16-gauge puncture needle was used to induce an injury to the disk to a depth of 5 mm. Muscles and skin were closed with 3-0 silk suture. Four weeks postoperatively, the animal model group underwent MRI and X-ray examinations to assess the degeneration of the disks, and 36 animal models were successfully established. Two animals died of iatrogenic infection. The MR images of the disk was assessed by an observer blinded to the study using modified Thompson classification based on changes in the degree and area of signal intensity from grades 1 to 4 (1 = normal, 2 = minimal decrease in signal intensity but obvious narrowing of high-signal, 3 = moderate decrease in signal intensity, 4 = severe decrease in signal intensity). The animal model with grade 2 or grade 3 was suitable for the study.

Nucleoplasty (Coblation®; ArthroCare Spine, USA) was performed in the coblation group as soon as we identified the successful establishment of the animal model of intervertebral disk degeneration, while no procedure was performed in the coblation control group. Each rabbit was tranquilized as described above. The operative field was prepared in a sterile fashion using betadine. L4–5 and L5–6 were identified with the C-arm while using Kirschner wires as markers. The procedure was performed as follows. The skin was stabbed by a 16-gauge hypodermic needle in preparation for puncture. Under fluoroscopic guidance, the access needle was inserted at an angle of 40° from the mid-back, targeting the tip of the stylet to the center of the nucleus in both the coronal and sagittal planes. The exact positioning of the stylet was then confirmed using anteroposterior and lateral views. The stylet was withdrawn from the needle, and a DC SpineWand was inserted under fluoroscopic guidance. The SpineWand was then connected to the cable. The controller was set at power level 1. The COAG pedal on the foot controller was depressed for 0.5 s. The ABLATION pedal on the foot controller was depressed for 3 to 5 s while the flange was rotated 180° in a back-and-forth motion. At the same time, the effective length was confirmed to extend from the inner part of the annulus fibrosus to that of the opposite side. The depth of penetration was controlled to extend not more than 5 mm from the needle tip. After the coblation zone had been created, the SpineWand was withdrawn from the needle and the needle was then withdrawn from the rabbit. Throughout all procedures, care was taken to avoid disturbing the peripheral tissues around the vertebrae. Following the surgeries, the rabbits were permitted free cage activity (4,000 cm²), food, and water. Gentamicin was given intramuscularly for three consecutive days.

The PLA2 activity assessment (titrimetric analysis) was performed as previously published [9]. All reagents were purchased from Beijing Chemical Preparation Company (Beijing, China). The substrate buffer solution comprised phosphatidylcholine (3.75 mmol/L), glycine (0.1 mmol/L), boric acid (3.57 mmol/L), and sodium deoxycholate (6.03 mmol/L). Before use, the solution was placed in a water bath at 60°C for 30 min, and the pH was adjusted to 8.50 with sodium hydroxide solution (1 mol/L). The components of the PLA2 diluent were similar to those of the substrate buffer solution, but phosphatidylcholine was not included. Dilute hydrochloric acid (0.005 mmol/L) was also prepared for titration and determination of the amount of base.

The rabbits in the coblation and coblation control groups were followed serially by both MRI and X-ray at different time points: real time, 1 week, and 4 weeks after nucleoplasty. At each of these time points, six rabbits were randomly euthanized for analysis of disk specimens. A posterior longitudinal skin incision was made along the lumbar spine in a sterile fashion. Both the L4–5 and L5–6 motion segments were dissected from the lumbar spine. The L4–5 and L5–6 intervertebral disks were then carefully separated with a scalpel. Each disk was cut into 1-mm³ pieces. After being weighed, the disk tissue was mixed with PLA2 diluent (4 ml/g) and centrifuged at 4,000 r/min for 10 min. The supernatant was mixed with 0.4 ml of PLA2 diluent, centrifuged at 8,000 r/min for 20 min, and collected and stored at 4°C.

First, each sample was placed in a 60°C thermostatic water bath for 30 min. Next, 0.4-ml samples and different reagents according to Table 1 were added to two tubes (tubes A and B), then placed in a water bath at 37°C for 60 min. Tube A was added with 0.2 ml calcium chloride (0.2 ml), and ethylenediaminetetraacetic acid (EDTA) (1.1 ml) was added to tube B. The pH in tube A was carefully adjusted to match that in tube B by titration with dilute hydrochloric acid. A sensitivity pH meter (sensitivity, 0.001) (inoLab pH 740; WTW Co., Weilheim, Germany) was used to detect the endpoint of the reaction. The PLA2 activity was considered to indicate the consumption of dilute hydrochloric acid.

(V: volume of hydrochloric acid solution consumed; N: concentration of hydrochloric acid solution; t: time of titration).

All data were statistically analyzed with SPSS 16.0 (SPSS, Inc., Chicago, IL, USA). The data are presented as mean ± standard deviation (SD). The PLA2 activity between the coblation control group and control group was compared using Student’s t-test. One-way analysis of variance or Fisher’s least significant difference test was used to compare the differences in the PLA2 activities at different time points in the coblation group. A P value of <0.05 was considered statistically significant.