Molecular characterization of human peripheral nerves using desorption electrospray ionization mass spectrometry imaging

Abstract

Desorption electrospray ionization mass spectrometry imaging (DESI-MSI) is a molecular imaging method that can be used to elucidate the small-molecule composition of tissues and map their spatial information using two-dimensional ion images. This technique has been used to investigate the molecular profiles of variety of tissues, including within the central nervous system, specifically the brain and spinal cord. To our knowledge, this technique has yet to be applied to tissues of the peripheral nervous system (PNS). Data generated from such analyses are expected to advance the characterization of these structures. The study aimed to: (i) establish whether DESI-MSI can discriminate the molecular characteristics of peripheral nerves and distinguish them from surrounding tissues and (ii) assess whether different peripheral nerve subtypes are characterized by unique molecular profiles. Four different nerves for which are known to carry various nerve fiber types were harvested from a fresh cadaveric donor: mixed, motor and sensory (sciatic and femoral); cutaneous, sensory (sural); and autonomic (vagus). Tissue samples were harvested to include the nerve bundles in addition to surrounding connective tissue. Samples were flash-frozen, embedded in optimal cutting temperature compound in cross-section, and sectioned at 14 μm. Following DESI-MSI analysis, identical tissue sections were stained with hematoxylin and eosin. In this proof-of-concept study, a combination of multivariate and univariate statistical methods was used to evaluate molecular differences between the nerve and adjacent tissue and between nerve subtypes. The acquired mass spectral profiles of the peripheral nerve samples presented trends in ion abundances that seemed to be characteristic of nerve tissue and spatially corresponded to the associated histology of the tissue sections. Principal component analysis (PCA) supported the separation of the samples into distinct nerve and adjacent tissue classes. This classification was further supported by the K-means clustering analysis, which showed separation of the nerve and background ions. Differences in ion expression were confirmed using ANOVA which identified statistically significant differences in ion expression between the nerve subtypes. The PCA plot suggested some separation of the nerve subtypes into four classes which corresponded with the nerve types. This was supported by the K-means clustering. Some overlap in classes was noted in these two clustering analyses. This study provides emerging evidence that DESI-MSI is an effective tool for metabolomic profiling of peripheral nerves. Our results suggest that peripheral nerves have molecular profiles that are distinct from the surrounding connective tissues and that DESI-MSI may be able to discriminate between nerve subtypes. DESI-MSI of peripheral nerves may be a valuable technique that could be used to improve our understanding of peripheral nerve anatomy and physiology. The ability to utilize ambient mass spectrometry techniques in real time could also provide an unprecedented advantage for surgical decision making, including in nerve-sparing procedures in the future.

Keywords: DESI-MSI; anatomy; innervation; mass spectrometry imaging; peripheral nerve.

FIGURE 1

Workflow of peripheral nerve tissue analysis using DESI‐MSI. Samples of four nerves (vagus, sciatic, femoral, and sural) were carefully dissected from a fresh cadaveric donor. Nerve samples were flash frozen, embedded in optimal cutting temperature, and sectioned. Tissue sections were analyzed using DESI‐MSI and subsequently stained with H&E. Statistical analysis was performed using MetaboAnalyst 5.0. Ion images were compared to the associated H&E micrographs to confirm separation between nerve and surrounding tissue. Image created using BioRender.com. DESI‐MSI, desorption electrospray ionization mass spectrometry imaging; H&E, hematoxylin and eosin.

FIGURE 2

DESI‐MSI of a peripheral nerve bundle. (a) H&E image of a sciatic nerve sample. Histological analysis was used to delineate the nerve bundles (outlined in black dashed lines) for comparison to ion images. (b) DESI‐MS ion image of the sciatic nerve. Glycerophospholipid m/z 700.5 shown in red was abundant in the nerve bundles and m/z 281.2 (oleic acid), shown in green, was abundant in the adjacent tissue. Comparison of the DESI‐MS ion image to the associated H&E micrograph shown in image (a) illustrates good histologic correlation. (c) Two‐dimensional PCA plot of DESI‐MSI spectra for nerve (green) and adjacent tissue (red) illustrates clustering between classes. (d) Separation of the two classes was confirmed with K‐means clustering. The spectra obtained from the K‐means clustering analysis further illustrate putative differences in molecular composition between the two classes, with an approximate m/z scale shown. DESI‐MSI, desorption electrospray ionization mass spectrometry imaging; H&E, hematoxylin and eosin; PCA, principal component analysis.

FIGURE 3

Ion images of selected lipids and metabolites distributed across nerve samples and compared to the associated histological images. Heat map images are shown for selected ions characteristic of nerves (m/z 700.5286 and m/z 728.5587) and adjacent tissue (m/z 281.2463). Differences in the relative abundance of these ions can be noted across the various nerve types with yellow representing the most intense signal and blue representing the least intense signal. Images with m/z 281.2463 and m/z 700.5286 are shown overlayed in green and red in the final column indicating good separation of nerve and background tissue across nerve samples. See Table 1 for details on ion identification.

FIGURE 4

(a) Volcano plot illustrating significant differences in ion composition between the nerve and adjacent tissues. (b, c) Associated boxplots showing differences in ion distribution between the nerve (green) and adjacent tissues (red) for ions m/z 700.5 and m/z 281.2.

FIGURE 5

DESI‐MSI characterization of nerve subtypes. (a) Differences in ion expression were investigated using ANOVA, which identified statistically significant differences in ion expression between the nerve samples. Associated boxplot of m/z 700.528 shows differences in the relative abundance of this ion across the nerve subtypes. Two‐dimensional PCA plot shown in (b) and K‐means clustering analysis (c) shows some separation of the nerve subtypes into different classes. The accompanying spectra for each cluster illustrate overall patterns of ion expression within similar m/z ranges, though differences in relative abundance across nerve types can observed. DESI‐MSI, desorption electrospray ionization mass spectrometry imaging; PCA, principal component analysis.