Percutaneous lumbar annular puncture: A rat model to study intervertebral disc degeneration and pain-related behavior

Abstract

Background: Previous animal models of intervertebral disc degeneration (IDD) rely on open surgical approaches, which confound the degenerative response and pain behaviors due to injury to surrounding tissues during the surgical approach. To overcome these challenges, we developed a minimally invasive percutaneous puncture procedure to induce IDD in a rat model.

Methods: Ten Fischer 344 male rats underwent percutaneous annular puncture of lumbar intervertebral discs (IVDs) at L2-3, L3-4, and L4-5. Ten unpunctured rats were used as controls. Magnetic resonance imagings (MRIs), serum biomarkers, and behavioral tests were performed at baseline and 6, 12, and 18 weeks post puncture. Rats were sacrificed at 18 weeks and disc histology, immunohistochemistry, and glycosaminoglycan (GAG) assays were performed.

Results: Punctured IVDs exhibited significant reductions in MRI signal intensity and disc volume. Disc histology, immunohistochemistry, and GAG assay results were consistent with features of IDD. IVD-punctured rats demonstrated significant changes in pain-related behaviors, including total distance moved, twitching frequency, and rearing duration.

Conclusions: This is the first reported study of the successful establishment of a reproducible rodent model of a percutaneous lumbar annular puncture resulting in discogenic pain. This model will be useful to test therapeutics and elucidate the basic mechanisms of IDD and discogenic pain.

Keywords: intervertebral disc degeneration; low back pain; percutaneous annular puncture; rat model.

FIGURE 1

Overview of experimental design illustrating the time course of different assays (A) and types of post‐sacrifice outcome measures performed on each disc (B)

FIGURE 2

Percutaneous lumbar annular puncture procedure (A) and comparison of MRI imaging of rat and human anatomy at the lumbar region (B). (A.1) Starting point identified 3 cm left of the midline and 1.5 cm rostral to a line connecting the iliac crests. (A.2) AP (left) and lateral (right) fluoroscopic images confirming needle puncture into intervertebral disc space. Following fluoroscopic confirmation of proper needle placement, the needle tip is (A.3) rotated 360° around its longitudinal access, then swung 60 degrees in a (A.4) cranial‐caudal and (A.5) anterior–posterior direction. (B) Axial T2 MRI imaging of a rat at the level of the L3 vertebra (B.1) and a human at the level of the L2 vertebra (B.2) demonstrating the location of the kidneys and aorta in relationship to the spine. The described path of the needle puncture is indicated by the dotted line. AP, anteroposterior; MRI, magnetic resonance imaging

FIGURE 3

T2 MRI of rat spines. (A) The process used for quantifying disc signal intensity. Individual pixels of each NP were highlighted (middle), and signal intensity was quantified using DSI Studio software. This process was repeated for each “phantom” marker isointense to water (right), and average disc intensity values were standardized to the nearest average phantom intensity in order to account for spatial intensity variability based on the location of the disc in relation to the MRI coil. (B) Representative mid‐sagittal T2 MRI images from a control and experimental animal at baseline and 6, 12, and 18 weeks post puncture. A decrease in disc signal intensity in the punctured discs (arrows) is noted. Mean disc signal intensities after normalization to mean L6/S1 value are shown in the control and experimental groups at each timepoint. Asterisks represent a significant difference (p < 0.05) in mean intensity at indicated timepoint when compared to baseline for a given disc. There were no significant differences between groups at any timepoint in the unpunctured discs (L1/L2 and L5/L6), or baseline in the punctured discs (L2/L3, L3/L4, and L4/L5). There was a significant difference between groups at all postoperative timepoints in the punctured discs (p < 0.001). Error bars represent one standard error. (C) Sham control rats exhibited no MRI evidence of IDD. Representative mid‐sagittal T2 MRI images from a control and sham surgical animal at baseline, 12, and 24 weeks post surgery. L2–L3, L3–L4, and L4–L5 discs corresponding to the levels of punctured discs in experimental rats are noted (arrows). Graphs represent the mean disc signal intensities after normalization to the mean L6/S1 value in the control and experimental groups at each timepoint. There were no significant differences between groups at any timepoint in the lumbar discs of control and sham rats. Error bars represent one standard error. IDD, intervertebral disc degeneration; MRI, magnetic resonance imaging; NP, nucleus pulposus

FIGURE 4

Rat behaviors assessed by Open Field Assay. Representative behavioral parameters from two separate sets of experiments comparing (A) unstabbed control and experimental groups, and (B) unstabbed control and sham groups. Asterisks represent significant differences (p < 0.05) within groups when compared to baseline (indicated by the asterisks above and below the plots) and between groups at the specified timepoint (indicated by the asterisks between the plots). Error bars represent one standard error

FIGURE 5

Serum concentrations of NPY and RANTES in control and experimental groups (A) and between control and sham groups (B). Asterisks represent significant differences (p < 0.05) within groups when compared to baseline (indicated by the asterisks above and below the plots) and between groups at the specified timepoint (indicated by the asterisks between the plots). Error bars represent one standard error. NPY, neuropeptide Y; RANTES, Regulated on Activation Normal T Cell Expressed and Presumably Secreted

FIGURE 6

Effects of annular puncture on rat disc gross morphology and histological features. (A) Comparison of gross morphology in a control (left) and experimental (right) L2/L3 disc. Punctured discs exhibited loss of hydration and structural organization. Representative 6‐μm axial slices of control (left column) and experimental (right column) discs following H&E staining. Images shown at 20× (top row, black scale bar = 500 mm), 40× (middle row, black scale bar = 200 mm), and 100× (bottom row, black scale bar = 60 mm) magnification. Experimental discs show distinct changes in disc architecture, including the loss of a clear NP/AF boundary (black arrows), serpentine AF lamellae (yellow arrowheads), and large fissures/clefts in the NP (red arrowheads). AF, annulus fibrosus; NP, nucleus pulposus

FIGURE 7

Immunohistochemistry. (A) IHC staining for aggrecan (reddish brown) in control (left) and experimental (right) L2/L3 discs. Images shown at 40× (top, black scale bar = 600 mm) and 100× (bottom, black scale bar = 200 mm) magnification. (B) IHC staining for MMP‐13 (reddish brown) in control (left) and experimental (right) L2/L3 discs. Representative discs from four individual animals, two control (left) and two experimental (right), are shown in this Figure. (C) Quantification of IHC staining revealed a smaller percentage of NP area positive for aggrecan (left) and a greater percentage of NP area positive for MMP‐13 (right) in the punctured discs. Error bars represent one standard error. IHC, immunohistochemistry; NP, nucleus pulposus

FIGURE 8

GAG content in control and experimental animal NP (left) and AF (right), as standardized to DNA content (top) and tissue mass (bottom). DNA content is also shown standardized to tissue mass (middle). Error bars represent one standard error. AF, annulus fibrosus; GAG, glycosaminoglycan; NP, nucleus pulposus