Lumbar intervertebral disc puncture under C-arm fluoroscopy: a new rat model of lumbar intervertebral disc degeneration

Abstract

To establish a minimally invasive rat model of lumbar intervertebral disc degeneration (IDD) to better understand the pathophysiology of the human condition. The annulus fibrosus of lumbar level 4-5 (L4-5) and L5-6 discs were punctured by 27-gauge needles using the posterior approach under C-arm fluoroscopic guidance. Magnetic resonance imaging (MRI), histological examination by hematoxylin and eosin (H&E) staining, and reverse transcription polymerase chain reaction (RT-PCR) were performed at baseline and 2, 4, and 8 weeks after disc puncture surgery to determine the degree of degeneration. All sixty discs (thirty rats) were punctured successfully. Only two of thirty rats subjected to the procedure exhibited immediate neurological symptoms. The MRI results indicated a gradual increase in Pfirrmann grade from 4 to 8 weeks post-surgery (P<0.05), and H&E staining demonstrated a parallel increase in histological grade (P<0.05). Expression levels of aggrecan, type II collagen (Col2), and Sox9 mRNAs, which encode disc components, decreased gradually post-surgery. In contrast, mRNA expression of type I collagen (Col1), an indicator of fibrosis, increased (P<0.05). The procedure of annular puncture using a 27-gauge needle under C-arm fluoroscopic guidance had a high success rate. Histological, MRI, and RT-PCR results revealed that the rat model of disc degeneration is a progressive pathological process that is similar to human IDD.

Fig. 1.

Intraoperative fluoroscopy images from a representative rat Both the anteroposterior radiograph (a) and lateral radiograph (b) showed the tips of the needles were located in the center of the discs. An enlarged image (c) from 1b indicated needles locations. The numbers (1–6) indicated the lumbar vertebrae.

Fig. 2.

Sagittal T2-weighted images by MRI at 4 observed time points Pre-operative image (a) showed the homogeneous structures of the L4–5 and L5–6 discs. A hyperintense signal was still seen at 2 weeks post-operation (b), but the area of high intensity decreased in L4–5 disc (arrow). At 4 weeks post-operation (c), T2-weighted image by MRI showed an intermediate gray signal in the L4–5 and L5–6 discs (arrows). At 8 weeks post-operation (d), signal intensity from both L4–5 and L5–6 discs was significantly reduced. A ‘black disc’ (L4–5, arrow) was observed in several T2-weighted images at this time. The numbers (1–6) indicated the lumbar vertebrae.

Fig. 3.

Changes in the Pfirrmann classification grade after annular puncture The degree of disc degeneration was assessed from T2-weighted images acquired before surgery (pre-op; n=16) and at 2 (post-op 2 w; n=16), 4 (post-op 4 w; n=16), and 8 (post-op 8 w; n=16) weeks post-operation using the Pfirrmann classification [20]. Grade ranged from I (normal) to V (advanced degeneration). **P<0.01; ***P<0.001

Fig. 4.

Histological changes after annular puncture H&E-stained sagittal sections before (a & b) and 2 weeks (c & d) after surgery showed intact annulus fibrosus and cell-enriched nucleus pulposus. From 4 (e & f) to 8 weeks (g & h) post-surgery, the number of chondrocyte-like cells in nucleus pulposus was reduced gradually, and cracks (arrows) among the layers of collagen fibers of the annulus fibrosus appeared.

Fig. 5.

Changing mRNA expression levels of aggrecan, type II collagen (Col2), Sox9, type I collagen (Col1), and β-actin RT-PCR was used to examine the expression of genes related to disc degeneration before surgery (A) and 2 (B), 4 (C), and 8 weeks (D) post-operation. Gene expression was normalized to β-actin.