Molecular pain markers correlate with pH-sensitive MRI signal in a pig model of disc degeneration

Abstract

Intervertebral disc (IVD) degeneration is a leading cause of chronic low back pain that affects millions of people every year. Yet identification of the specific IVD causing this pain is based on qualitative visual interpretation rather than objective findings. One possible approach to diagnosing pain-associated IVD could be to identify acidic IVDs, as decreased pH within an IVD has been postulated to mediate discogenic pain. We hypothesized that quantitative chemical exchange saturation transfer (qCEST) MRI could detect pH changes in IVDs, and thence be used to diagnose pathologically painful IVDs objectively and noninvasively. To test this hypothesis, a surgical model of IVD degeneration in Yucatan minipigs was used. Direct measurement of pH inside the degenerated IVDs revealed a significant drop in pH after degeneration, which correlated with a significant increase in the qCEST signal. Gene analysis of harvested degenerated IVDs revealed significant upregulation of pain-, nerve- and inflammatory-related markers after IVD degeneration. A strong positive correlation was observed between the expression of pain markers and the increase in the qCEST signal. Collectively, these findings suggest that this approach might be used to identify which IVD is causing low back pain, thereby providing valuable guidance for pain and surgical management.

Conflict of interest statement

G.P., Z.G., and D.G. were shareholders in TavorStem Therapeutics, which provided no funding for this study.

Figures

Figure 1

IVD degeneration timeline. Annular injuries were created in four IVD levels in minipigs to induce disc degeneration (depicted in red). Following this procedure, the animals were randomly divided into 3 groups (3 animals in each group) and imaged at 2, 6, and 10 weeks. At each time point, the animals in one group were sacrificed and the pH within their injured IVDs was measured. The IVDs were harvested for gene expression, histological, and immunohistofluoresence analyses.

Figure 2

IVD degeneration following intradiscal puncture. (A) The progress of IVD degeneration at 2, 6, and 10 weeks following intradiscal puncture, as monitored by T2-weighted sagittal MRI. White arrows denote the degenerated IVDs. Quantification of (B) T2, (C) T1, and (D) T1ρ mappings of degenerated IVDs compared to healthy controls at 2, 6, and 10 weeks after puncture (n = 12 per experimental group; *p < 0.05, ****p < 0.0001). (E) Hematoxylin and eosin staining of representative degenerated IVDs 2, 6, and 10 weeks after intradiscal puncture; microphotographs are shown at both low (upper subfigures; scale bars = 1 mm) and high (lower subfigures; scale bars = 100 μm) magnifications.

Figure 3

pH and qCEST changes following IVD degeneration. (A) Representative axial anatomical images of IVDs and their corresponding qCEST heat maps. (B) pH and (C) qCEST measurements within the degenerating IVDs at 2, 6, and 10 weeks after intradiscal puncture. (D) Correlation between the qCEST signal, represented by the exchange rate between the solute pool and the water pool (ksw), and the pH measured within the IVD following animal sacrifice (n = 12 per experimental group; *p < 0.05, **p < 0.01, ****p = 0.0001; qCEST = quantitative chemical exchange saturation transfer).

Figure 4

Upregulation of pain and inflammatory markers in degenerating IVDs. Quantitative RT-PCR analyses of (A–C) pain-related genes (CGRP, BDKRB1 and COMT) and (D,E) inflammation-related genes (IL-6 and BDNF) harvested from healthy and degenerated IVDs 2, 6, and 10 weeks after intradiscal puncture (n = 3 per group; *p < 0.05, **p < 0.01; RQ, relative quantification, CGRP, calcitonin gene-related peptide, BDKRB1, bradykinin receptor B1, COMT, catechol-Ο-methyltransferase, BDNF, brain-derived neurotrophic factor).

Figure 5

Immunofluorescence of pain-related marker upregulation. Microphotographs showing IVD tissue samples after immunostaining against COMT, CGRP, and BDKRB1, and counterstaining with DAPI; the samples were examined 2, 6, and 10 weeks after intradiscal puncture. Merged panels of the various stains are presented in the right column (NP = nucleus pulposus, CGRP = calcitonin gene-related peptide, BDKRB1 = bradykinin receptor B1, COMT = catechol-O-methyltransferase).

Figure 6

Linear correlation between qCEST and biomarkers in degenerating IVDs. Correlation plots between qCEST signal and expression levels of (A) CGRP, (B) BDKRB1, (C) COMT, (D) IL-6, and (E) BDNF extracted from degenerated and healthy annulus fibrosus and nucleus pulposus (RQ, relative quantification).