Lumbar endplate microfracture injury induces Modic-like changes, intervertebral disc degeneration and spinal cord sensitization - an in vivo rat model

Abstract

Background context: Endplate (EP) injury plays critical roles in painful IVD degeneration since Modic changes (MCs) are highly associated with pain. Models of EP microfracture that progress to painful conditions are needed to better understand pathophysiological mechanisms and screen therapeutics.

Purpose: Establish in vivo rat lumbar EP microfracture model and assess crosstalk between IVD, vertebra and spinal cord.

Study design/setting: In vivo rat EP microfracture injury model with characterization of IVD degeneration, vertebral remodeling, spinal cord substance P (SubP), and pain-related behaviors.

Methods: EP-injury was induced in 5 month-old male Sprague-Dawley rats L4-5 and L5-6 IVDs by puncturing through the cephalad vertebral body and EP into the NP of the IVDs followed by intradiscal injections of TNFα (n=7) or PBS (n=6), compared with Sham (surgery without EP-injury, n=6). The EP-injury model was assessed for IVD height, histological degeneration, pain-like behaviors (hindpaw von Frey and forepaw grip test), lumbar spine MRI and μCT, and spinal cord SubP.

Results: Surgically-induced EP microfracture with PBS and TNFα injection induced IVD degeneration with decreased IVD height and MRI T2 signal, vertebral remodeling, and secondary damage to cartilage EP adjacent to the injury. Both EP injury groups showed MC-like changes around defects with hypointensity on T1-weighted and hyperintensity on T2-weighted MRI, suggestive of MC type 1. EP injuries caused significantly decreased paw withdrawal threshold, reduced axial grip, and increased spinal cord SubP, suggesting axial spinal discomfort and mechanical hypersensitivity and with spinal cord sensitization.

Conclusions: Surgically-induced EP microfracture can cause crosstalk between IVD, vertebra, and spinal cord with chronic pain-like conditions.

Clinical significance: This rat EP microfracture model was validated to induce broad spinal degenerative changes that may be useful to improve understanding of MC-like changes and for therapeutic screening.

Keywords: Endplate microfracture; In vivo rat model; Intervertebral disc degeneration; Modic changes; Pain; Spinal cord sensitization; Spine.

Figure 1:

EP microfracture in-vivo model and study design. A) Schematic of procedure with anterior approach. Experimental groups included Sham (n=6); EP injury + PBS injection (n=6) and EP injury + TNFα injection (n=7). Syringes were inserted through a tight-fitting K-wire channel with syringe depth and bevel position confirmed intraoperatively with a C-Arm to be in the center of NP region to enable injectates to be absorbed by the IVD and marrow and minimize the chance of injectate leakage. B) Output variables after t=56 days (8 weeks). C) Timeline of behavioral measurements.

Figure 2:

Behavioral testing demonstrates axial sensitivity and hindpaw mechanical hypersensitivity. A) Paw withdrawal threshold after von Frey test; B) Normalized Peak force and C) normalized mean force after grip test over time # EP+PBS compared to Sham with p < 0.05, * EP+TNFα compared to Sham with p < 0.05.

Figure 3:

EP injury causes IVD height loss. A) Faxitron imaging. B) Relative disc height was quantified by tracing the cephalad and caudal vertebral endplates and using a spline program to measure disc heights (yellow and red shape). The relative disc height was calculated by normalizing to pre-operative values at the same level for the same animal. C) Relative disc height was reduced with injury. **and **** indicate significant differences with p<0.01 and p<0.0001 respectively.

Figure 4:

EP caused IVD degeneration. A) Histology with thin sections stained with Safranin-O/Fast green and thick ground and polished sections stained with Toluidine Blue. Red arrows indicating EP defect. B) IVD degeneration grading using Scoring System [49]. **, *** and **** indicate significant differences with p<0.01, p<0.001 and p<0.0001 respectively.

Figure 5:

MRI analyses show significant injury following EP injury for both PBS and TNF α. A) Hypointensity on T1w and T2w MRI (blue arrows) and increased T2 relaxation time (sec) (red arrows) are visible around EP defects. B) Mean T2 relaxation time of NP decreased with injury. C) There was no difference in mean T2 relaxation time of the whole IVD among the 3 groups highlighting that this is a localized injury. D) Mean T2 relaxation time at the EP looking at a larger area (left) and more focused small ROI focused on the microfracture site (right) in which a significant difference between sham and EP=PBS was found. * and ** indicate significant differences between groups with p<0.05 and p<0.01, respectively.

Figure 6:

μCT analyses show vertebral disruption and remodeling at the injury site that is not impacted adjacent or at the far field. A) EP defects are highly visible on μCT, and mid-coronal plane of injured lumbar spinal regions vertebrae show trabecular bone remodeling (white arrow) around EP defect (green arrow). B) Quantitative μCT analyses show extensive injuries at the injury site in the area of the endplate. Bone changes were minimal adjacent to the injury site and not observed in the far field. * indicate significant difference between groups with p<0.05.

Figure 7:

Spinal Cord Substance P was increased from EP injury. A) Immunofluorescence images of spinal cord (SC) with outlined gray matter showing Neurons (red) and presence of substance P (green) in different groups with focus on dorsal horns. B) hematoxylin and eosin staining of spinal cord with outline of grey matter. C) quantification of SubP in dorsal horn. ** indicate significant difference between groups with p<0.01.

Figure 8:

Correlations of NP T2 with IVDD, Spinal cord (SC) SubP, and pain-related behavioral measurements of von Frey and peak grip force.