Vibrational Spectroscopic Imaging and Multiphoton Microscopy of Spinal Cord Injury

Abstract

Spinal cord injury triggers a series of complex biochemical alterations of nervous tissue. Up to now, such cellular events could not be studied without conventional tissue staining. The development of optical, label-free imaging techniques could provide powerful monitoring tools with the potential to be applied in vivo. In this work, we assess the ability of vibrational spectroscopy to generate contrast at molecular level between normal and altered regions in a rat model of spinal cord injury. Using tissue sections, we demonstrate that Fourier transform infrared (FT-IR) spectroscopy and spontaneous Raman spectroscopy are able to identify the lesion, the surrounding scar, and unharmed normal tissue, delivering insight into the biochemical events induced by the injury and allowing mapping of tissue degeneration. The FT-IR and Raman spectroscopic imaging provides the basis for fast multimodal nonlinear optical microscopy (coherent anti-Stokes Raman scattering, endogenous two-photon fluorescence, and second harmonic generation). The latter proves to be a fast tool for imaging of the lesion on unstained tissue samples, based on the alteration in lipid content, extracellular matrix composition, and microglia/macrophages distribution pattern. The results establish these technologies in the field of regeneration in central nervous system, with the long-term goal to extend them to intravital use, where fast and nonharmful imaging is required.

Interpreting the biochemical specificity of mouse spinal cord by confocal raman microspectral imaging

“…Furthermore, a prominent spectral feature in white matter is that its relative lipid band intensity is higher than that of gray matter due to more myelinated axons constitutions. 9 The other spectral feature of white matter at 875 cm À1 is the symmetric and antisymmetric stretch vibrations of the choline group N þ (CH 3 Þ 3 in phosphatidylcholine, which is essential for fat metabolism and functions as a methyl donor.…”

“…The 913 and 1137 cm À1 Raman peaks are attributed to vibrations of the saccharides, as well as vibrations of C-H and C-OH bonds. [7][8][9] Besides providing protection and lubrication for the spinal cord, saccharides are the skeleton of nerve cells in gray matter. 18 Strong vibrations are also evident in regions assigned to protein (1250 and 1328 cm À1 Þ, namely, to the vibrations of amide III and C-H bonds.…”

“…8 Galli et al assessed the ability of Fourier transform infrared spectroscopy and spontaneous RS to generate contrast data between normal and altered regions on the molecular level in an SCI rat model. 9 They also applied RS for the characterization of morphochemical properties of surgically induced SCI in the rat that received an implant of soft unfunctionalized alginate hydrogel. 10 Besides that, coherent anti-Stokes RS was also used for visualization of myelin sheets in normal spinal cord and for study of myelin sheet swelling and myelin sheet retraction and repair after SCI.…”

“…10 Besides that, coherent anti-Stokes RS was also used for visualization of myelin sheets in normal spinal cord and for study of myelin sheet swelling and myelin sheet retraction and repair after SCI. 9,11 In this work, a spectral assessment of spinal cord tissue was achieved on longitudinal tissue sections from healthy mice. The acquired spectral information addressed biochemical characteristics of white and gray matter, as well as some speci¯c substance variances between them.…”

Accumulating advantages, reducing limitations: Multimodal nonlinear imaging in biomedical sciences – The synergy of multiple contrast mechanisms

“…Especially the differentiation of benign and malignant brain tissue [42,91,93,[140][141][142], is of utmost importance, which is why much work has focused on the accurate detection of morphologic changes associated with malignant transformation. But also the structure of nerve fibers, the spinal cord and myelin related disorders and injuries have been investigated [143][144][145][146][147].…”