Investigation of intervertebral disc degeneration using multivariate FTIR spectroscopic imaging.

Kerstin T Mader,Marco N Helder,Suzanne E L Detiger,Christine L Le Maitre,Chris Sammon,Mirte Peeters,Theo H Smit

doi:10.1039/c5fd00160a

Abstract

Traditionally tissue samples are analysed using protein or enzyme specific stains on serial sections to build up a picture of the distribution of components contained within them. In this study we investigated the potential of multivariate curve resolution-alternating least squares (MCR-ALS) to deconvolute 2nd derivative spectra of Fourier transform infrared (FTIR) microscopic images measured in transflectance mode of goat and human paraffin embedded intervertebral disc (IVD) tissue sections, to see if this methodology can provide analogous information to that provided by immunohistochemical stains and bioassays but from a single section. MCR-ALS analysis of non-degenerate and enzymatically in vivo degenerated goat IVDs reveals five matrix components displaying distribution maps matching histological stains for collagen, elastin and proteoglycan (PG), as well as immunohistochemical stains for collagen type I and II. Interestingly, two components exhibiting characteristic spectral and distribution profiles of proteoglycans were found, and relative component/tissue maps of these components (labelled PG1 and PG2) showed distinct distributions in non-degenerate versus mildly degenerate goat samples. MCR-ALS analysis of human IVD sections resulted in comparable spectral profiles to those observed in the goat samples, highlighting the inter species transferability of the presented methodology. Multivariate FTIR image analysis of a set of 43 goat IVD sections allowed the extraction of semi-quantitative information from component/tissue gradients taken across the IVD width of collagen type I, collagen type II, PG1 and PG2. Regional component/tissue parameters were calculated and significant correlations were found between histological grades of degeneration and PG parameters (PG1: p = 0.0003, PG2: p < 0.0001); glycosaminoglycan (GAG) content and PGs (PG1: p = 0.0055, PG2: p = 0.0001); and MRI T2* measurements and PGs (PG1: p = 0.0021, PG2: p < 0.0001). Additionally, component/tissue parameters for collagen type I and II showed significant correlations with total collagen content (p = 0.0204, p = 0.0127). In conclusion, the presented findings illustrate, that the described multivariate FTIR imaging approach affords the necessary chemical specificity to be considered an important tool in the study of IVD degeneration in goat and human IVDs.

Highlights

Low back pain (LBP) affects millions of people worldwide, and has been linked to degenerative changes of the intervertebral disc (IVD).[1,2] The IVD is a structurally and chemically complex cartilaginous tissue consisting of distinct regions; a so er gelatinous inner core known as the nucleus pulposus (NP) and a highly organised brous outer region known as the annulus brosus (AF).[3]
We examine the potential of multivariate curve resolution-alternating least squares (MCR-ALS) analysis to extract spatially resolved biochemical information of non-degenerated and enzymatically in vivo degenerated goat IVDs from 2nd derivative Fourier transform infrared (FTIR) microscopic imaging data collected in trans ectance mode
MCR-ALS extracted parameters for PG1 and PG2 were correlated with MRI T2*, GAG concentration and histological grades in the anterior AF (aAF), NP and posterior AF (pAF) IVD areas

Summary

Introduction

Low back pain (LBP) affects millions of people worldwide, and has been linked to degenerative changes of the intervertebral disc (IVD).[1,2] The IVD is a structurally and chemically complex cartilaginous tissue consisting of distinct regions; a so er gelatinous inner core known as the nucleus pulposus (NP) and a highly organised brous outer region known as the annulus brosus (AF).[3]. The distribution map of Factor 2 extracted from the spectral region 950–1600 cmÀ1 shows a gradual increase of intensity from the NP towards the AF typical for collagen type I distribution in IVDs and matches well with the immunohistochemical stained section (Fig. 4BB and CB).

Results

Conclusion