Label-Free Spectral Imaging Unveils Biochemical Mechanisms of Low-Level Laser Therapy on Spinal Cord Injury

Abstract

Background/Aims: Low-level laser therapy (LLLT) leads to complex photochemical responses during the healing process of spinal cord injury (SCI). Confocal Raman Microspectral Imaging (in combination with multivariate analysis) was adopted to illustrate the underlying biochemical mechanisms of LLLT treatment on a SCI rat model. Methods: Using transversal tissue sections, the Raman spectra can identify areas neighboring the injury site, glial scar, cavity, and unharmed white matter, as well as their correlated cellular alterations, such as demyelination and up-regulation of chondroitin sulfate proteoglycans (CSPGs). Multivariate data analysis methods are used to depict the underlying therapeutic effects by highlighting the detailed content and distribution variations of the biochemical constituents. Results: It is confirmed that photon-tissue interactions might lead to a decay of the inhibitory response to remyelination by suppressing CSPG expression, as also morphologically demonstrated by reduced glial scar and cavity areas. An inter-group comparison semi-quantitatively confirms changes in lipids, phosphatidic acid, CSPGs, and cholesterol during SCI and its LLLT treatment, paving the way for in vitro and in vivo understanding of the biochemical changes accompanying pathobiological SCI events. Conclusion: The achieved results in this work not only have once again proved the well-known cellular mechanisms of SCI, but further illustrate the underlying biochemical variability during LLLT treatment, which provide a sound basis for developing real-time Raman methodologies to monitor the efficacy of the SCI LLLT treatment.

Raman spectroscopy combined with multivariate analysis to study the biochemical mechanism of lung cancer microwave ablation

“…The acquired spectral data were pre-processed using WITec Project 4 (WITec GmbH, Germany) following our previously generated protocols [31][32][33], including a nine-order polynomial fit to subtract the spectral background and a five-order Savitzky-Golay smoothing. Each pre-processed spectrum was then normalized by the integrated area under the curve in the range from 600 to 1800 cm −1 , and from 2800 to 3000 cm −1 to eliminate interference from unrelated factors and provide a superior comparison of the spectral features of different tissues.…”

“…The equipment used for Raman spectroscopy has been described in detail in previous studies [31][32][33]. Briefly, the WITec Alpha500 confocal micro-Raman spectroscopy system (WITec GmbH, Germany) was used for single spectrum acquisition with a 633 nm He-Ne laser (35 mW, Research Electro-Optics, Inc., USA) as the excitation source.…”

“…The main characteristic peaks observed in both the Pre-MWA group and Post-MWA groups at the following locations (with their respective tentative biochemical assignments shown in parentheses) [38,[40][41][42][43][44][45][46][47]: 669 cm −1 (T, G of nucleic acids); 750 cm −1 (symmetric breathing of tryptophan); 1002 cm −1 (C-C symmetric ring breathing of phenylalanine); 1123 cm −1 (C-N stretching of proteins); 1248 cm −1 (β-sheet conformation of collagen and proline); 1316 cm −1 (CH 2 CH 3 bending of collagen); 1448 cm −1 (CH 2 CH 3 bending of collagen, CH 2 bending of phospholipids); 1552 cm −1 (C = C stretching of tryptophan); 1608 cm −1 (C = C stretching of phenylalanine); 2878 cm −1 (CH 2 asymmetric stretch of lipids and proteins); and 2927 cm −1 (CH 2 anti-symmetric stretching of lipids). However, the characteristic peaks in healthy lung tissues were observed at the following locations: 860 cm −1 (C = O stretching of phosphatidic acid) [32]; 933 cm −1 (C-C stretching skeletal of collagen backbone, hydroxyproline, proline) [42]; 1002 cm −1 , 1248 cm −1 , and 1454 cm −1 (overlapping asymmetric CH 3 bending and CH 2 scissoring of collagen and phospholipids) [48]; 1552 cm −1 , and 1665 cm −1 (amide I band of collagen) [42]; and 2878 cm −1 and 2940 cm −1 (C-H vibrations in lipids and proteins) [49]. Furthermore, the normalized intensity of the nucleic acid peak at 669 cm −1 , tryptophan at 750 and 1552 cm −1 , phenylalanine at 1002 and 1608 cm −1 , collagen at 1248 cm −1 , and the lipids peaks at 1448, 2878 and 2927 cm −1 in the Post-MWA group were significantly higher than those in the Pre-MWA group.…”

Non-destructive diagnostic testing of cardiac myxoma by serum confocal Raman microspectroscopy combined with multivariate analysis

“…After the spectral pre-processing, the acquired spectra were normalized by the area-under-curve method to avoid the effects of the experimental apparatus, such as sample inhomogeneity and excitation light intensity drift. 22…”

“…Aer the spectral pre-processing, the acquired spectra were normalized by the area-under-curve method to avoid the effects of the experimental apparatus, such as sample inhomogeneity and excitation light intensity dri. 22 SVM, developed by Vapnik and Burges, is considered superior to traditional linear methods due to its ability to represent nonlinear features in data. 23,24 As a result, SVM is attracting increasing interest for the classication of spectral data.…”