Behavioral Compensations and Neuronal Remodeling in a Rodent Model of Chronic Intervertebral Disc Degeneration

Low back pain is associated with degeneration of the intervertebral disc, but specific mechanisms of pain generation in this pathology remain unknown. Sensory afferent nerve fiber growth into the intervertebral disc after injury-induced inflammation may contribute to discogenic pain. We describe a clinically relevant behavioral phenotype in a rodent model of chronic intervertebral disc degeneration which provides a means to map sensory neuron changes to a single affected lumbar intervertebral disc. Unilateral disc puncture of one lumbar intervertebral disc revealed a bilateral behavioral phenotype characterized by gait changes and decreased activity. Moreover, neurons extracted from the dorsal root ganglia in animals with intervertebral disc injury demonstrated altered TRPV1 activation in vitro independent of exogenous NGF administration. Finally, neuronal nuclear hypertrophy and elevated expression of p75NTR provide evidence of active adaptation of innervating sensory neurons in chronic intervertebral disc degeneration. Therefore, this model and findings provide the template for future studies to establish specific mechanisms of nociceptive pain in chronic intervertebral disc degeneration.


Mechanical Sensitivity
Paw withdrawal from von Frey filaments (Stoelting, Wood Dale, IL) was tested in a pilot study to determine the threshold force for 50% paw withdrawal (50% withdrawal threshold) via Chaplan's up-down testing method 1 . During testing, animals were placed in an elevated wirebottomed cage and each filament was placed against the paw for 6 seconds with enough applied force to bend the filament. Quick paw withdrawal or retreating from the stimulus with licking of the affected paw were considered positive responses. Data was normalized to the naïve group baseline.

Site-Specific Pressure Sensitivity
Local site pressure sensitivity of the low back was assessed using a small animal algometer (SMALGO algometer, Bioseb, Vitrolles, 13845 France). This device consists of a force gauge with a 5 mm diameter tip that was pressed directly on the skin over the dorsal aspect of the L5-L6 IVD at midline while the animal was held by an investigator. The force was increased until the animal produced an escape reaction or audible vocalization 12 to determine the applied pressure threshold.

Burrowing
Methods were adapted from Deacon 2 . Briefly, 10 cm diameter white PVC pipe was sectioned into 32 cm lengths and one end of each length was closed with a fitted black foam PVC sheet.
The open end of each tube was raised 6 cm by two 8 cm bolts, each placed 2.5 cm inward from the opening and spaced 7 cm apart. Each tube was filled with 2500 g of pea-shingle gravel (diameter <1 cm, Rock City Sand & Gravel, Red Hook, NY) and placed into a rat cage (26.0 cm x 47.6 cm x 20.3 cm). Each animal was placed into an individual burrowing cage and allowed to burrow freely for 60 minutes during the dark portion of a 12 hour light-dark cycle, and without the presence of investigators or other personnel. The behavioral testing room was illuminated by red light. The weight of displaced gravel was calculated and normalized to the amount displaced at baseline. Animals were included in the burrowing assessment analysis if they displaced at least 10% of the total gravel (250 g) at the baseline timepoint (naïve/LDP n=4, sham n=5).

Lateral Bending Maze
Pain tolerance on lateral bending was assessed using a custom-built maze with 4 alternating left and right turns, which each require extreme lateral bending of the spine for completion. At each timepoint, rats were placed at one end of the maze and allowed 2 minutes to move freely. The time each rat needed to complete 2, 4, and 8 progressive turns through the maze was recorded, as well as the total number of turns completed. Animals were included in the lateral bending maze analysis if they completed at least 8 turns within 2 minutes at the baseline timepoint (naïve n=3, sham n=6, LDP n=8).

Quantitative Analysis of DRG Immunostaining Using ImageJ Software
Neurons on the digital images of immunolabeled DRGs were identified based on the PI staining ( Supplementary Fig. S4a) and manually segmented into ROIs ( Supplementary Fig. S4b). ROIs were thresholded using the isodata algorithm to output a binary image ( Supplementary Fig. S4c, ImageJ), and the percentage of pixels above the threshold and average intensity for all thresholded pixels (white; Supplementary Fig. S4c) determined the area fraction of positive stain in each neuron (areacytoplasm) and mean intensity of labeling (Icytoplasm) for the antibody of interest (TrkA, p75NTR, TRPV1). 4 Background regions were defined as all pixels outside all ROIs for a single field of view.
Huang's fuzzy thresholding method 3 was applied to the image background to identify the average intensity of background fluorescence (Ibackground, Supplementary Fig. S4d).

Statistical Analyses for Histology Grading and Correlations to Behavioral Changes
Histologic images were graded to consensus by two investigators blinded to treatment group and other identifiers. Grading criteria were modified from Tam and co-workers (Supplementary Table S1) 4 . Briefly, each section was evaluated for grade of degeneration on a 0-4 scale (0: nondegenerate, 4: degenerate). The most degenerate grade in each group of sections was assigned as the grade for the entire IVD. The Wilcoxon rank-sum test was used to determine differences in the grade of degeneration between sham and LDP treatment groups (α<0.05; GraphPad).
Correlation analysis was performed for behavioral changes against grade of IVD degeneration at the 20 week timepoint. This was done using data from each animal that completed the open field and treadmill gait assessments and the corresponding grade of degeneration for each IVD, which were ranked (GraphPad) and then correlated across all groups (CORREL function, Microsoft Excel). The Spearman's rank-order correlation coefficient (rs) was tested for significance against the null hypothesis that rs=0 (α<0.05). For these datasets and a two-tailed analysis, significance was declared if rs<-0.35 or >0.35, or rs<-0.39 or >0.39, respectively (Supplementary Table S2). Supplementary Fig. S1. Surgical procedure to visualize and puncture the L5-L6 IVD. A 3 cm dorsal midline skin incision (a) was made was made, exposing the subcutaneous connective tissue (b). This tissue was dissected away from the skin, and in this manner the skin of the left side of the animal was freed (B, red arrow). Then the animal was carefully rotated onto its right side (B, black arrows) and the interface of the paraspinal muscles and peritoneal cavity was palpated (c). A left-sided dorsolateral incision was made through the exposed abdominal muscles at this interface (d), and blunt dissection was performed to isolate the retroperitoneal space from the peritoneal cavity, and the transverse processes were identified (e). Then the transverse processes were followed anteriorly and the animal was carefully rotated dorsally (e, black arrows) to allow access to the anterolateral aspect of the spine (f). The psoas muscle was separated to expose the disc space. The L5-L6 IVD was then exposed via blunt dissection (g) and punctured to a depth of 2 mm with a 27 gauge needle plus injection of 0.3 mL air (10x) (h). Background regions were identified using Huang's fuzzy thresholding method (d).