Blood analysis by Raman spectroscopy for laser stimulation on mouse prefrontal cortex

Shu-Yang Li; Yu-Dan Xia; Jing Tian; Chunlei Shan; Heng Zhang; Na Chen; Shu-Peng Liu

doi:10.4103/2773-2398.365027

RESEARCH ARTICLE

Year : 2022 | Volume : 1 | Issue : 4 | Page : 173-178

Blood analysis by Raman spectroscopy for laser stimulation on mouse prefrontal cortex

Shu-Yang Li¹, Yu-Dan Xia¹, Jing Tian², Chunlei Shan³, Heng Zhang¹, Na Chen¹, Shu-Peng Liu¹
¹ Key Laboratory of Specialty Fiber Optics and Optical Access Networks, Shanghai Institute for Advanced Communication and Data Science, Shanghai University, Shanghai, China
² School of Electron and Computer, Southeast University Chengxian College, Nanjing, Jiangsu Province, China
³ Department of Rehabilitation Medicine, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine; School of Rehabilitation Science, Shanghai University of Traditional Chinese Medicine, Shanghai, China

Date of Submission	06-Jun-2022
Date of Decision	25-Aug-2022
Date of Acceptance	04-Nov-2022
Date of Web Publication	30-Dec-2022

Correspondence Address:
Shu-Peng Liu
Key Laboratory of Specialty Fiber Optics and Optical Access Networks, Shanghai Institute for Advanced Communication and Data Science, Shanghai University, Shanghai, China
China

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2773-2398.365027

Abstract

Low-level laser therapy, a noninvasive physical therapy, is applied to a wide range of conditions and has many effects including anti-inflammatory, analgesic, and anti-allergic effects. Some reports show that low-level laser therapy improves memory for patients. In this study, we explored the effect of laser stimulation on the prefrontal cortex of Alzheimer’s disease model mice. Ten 4-month-old APP/PS1 double-transgenic Alzheimer’s disease model mice were selected for prefrontal cortex stimulation by an 808-nm laser for 40 minutes every day. The peak intensities of blood Raman spectroscopy at 675, 747, 1124 (P < 0.05), 1223 (P < 0.05), 1305, 1340, 1372, 1540, and 1637 cm^-1 were different between the laser stimulation group and the control group. The results indicated that laser stimulation of the mouse prefrontal cortex may induce some changes in blood components, such as porphyrins and glucose. Laser stimulation could play a role in the neurophysiological activity, thereby triggering the changes in blood components that could be detected by Raman spectroscopy.

Keywords: Alzheimer’s disease; APP/PS1 mice; laser; prefrontal cortex; Raman spectroscopy

How to cite this article:
Li SY, Xia YD, Tian J, Shan C, Zhang H, Chen N, Liu SP. Blood analysis by Raman spectroscopy for laser stimulation on mouse prefrontal cortex. Brain Netw Modulation 2022;1:173-8

How to cite this URL:
Li SY, Xia YD, Tian J, Shan C, Zhang H, Chen N, Liu SP. Blood analysis by Raman spectroscopy for laser stimulation on mouse prefrontal cortex. Brain Netw Modulation [serial online] 2022 [cited 2023 Jun 7];1:173-8. Available from: http://www.bnmjournal.com/text.asp?2022/1/4/173/365027

Funding: This study was supported by Shanghai Clinical Research Center for Rehabilitation Medicine (No. 21MC1930200) and the National Natural Science Foundation of China (Nos. 62175142 and 61875118).

Introduction

Near-infrared light promotes a wide range of biological effects including enhanced energy production, gene expression, immune regulation, improved blood circulation, and cell death prevention (Rojas et al., 2008). Low-level laser therapy (LLLT) utilizes weak laser light to produce photochemical and biological effects that enhance the metabolic capacity of neurons and stimulate neurogenesis and synaptogenesis. With the increasing applications of LLLT in neurological and psychotherapeutic treatment (Tyan et al., 2012), it is expected to become a new physical therapy for dementia.

Alzheimer’s disease is a chronic neurodegenerative disease with progressive cognitive impairment, and there are currently no clear biomarkers to identify cognitive decline. However, blood metabolites have gradually been recognized as promising biomarkers for the diagnosis of early dementia (Livingston et al., 2017; Ma et al., 2020). Studies have shown that blood glucose (Chatterjee et al., 2016), phosphatidylcholine (Proitsi et al., 2017), hypertension (Li et al., 2010), hypertriglyceridemia, phospholipids, and cholesterol metabolites (Reitz and Mayeux, 2014) all play an important role in cognitive impairment (Chatterjee et al., 2016; Livingston et al., 2017; Frison et al., 2021).

Raman spectroscopy, known as “fingerprint spectroscopy” of substances, provides vibrational information about biomolecules such as nucleic acids, glucose, proteins, and lipids. Raman spectroscopy comprehensively detects the overall spectral markers, and can be used to obtain comprehensive physiological and metabolic information (Lochocki et al., 2020). To explore the metabolic effect of LLLT on the prefrontal cortex of Alzheimer’s disease model APP/PS1 mice, Raman spectroscopy was used to analyze the blood components of the stimulated mice in this study. Laser stimulation of the mouse prefrontal cortex may lead to neurophysiological activity, and we hypothesize that this may cause changes in blood components.

Materials and methods

Design

The experiment was designed and reported according to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines (Percie du Sert et al., 2020). The animal study was approved by the Ethics Committee of Shanghai University (approval No. ECSHU 2021-106; Date: March 5, 2021). Twenty specific pathogen-free 4-month-old APP/PS1 double-transgenic Alzheimer’s disease mice (APP/PS1, Cat# 14001A), all male, weighing 25 ± 5 g, were purchased from Beijing Weishang Lituo Technology Co., Ltd. (Beijing, China; license No. SCXK (Jing) 2018-0010). The mice were randomly divided into a control group (APP/PS1 group) and a laser group (APP/PS1_laser group), with 10 mice in each group. The surrounding skin and hair were removed and disinfected with 75% alcohol. The mice in the APP/PS1_laser group were fixed with a mouse holder, and then were stimulated in the anterior frontal area (Covington et al., 2010; Selimbeyoglu et al., 2017) with the laser (808 nm, 20 mW), as shown in [Figure 1] . To gradually acclimatize the mice to the stimuli, the stimulation duration was increased by 10 minutes per day until 40 minutes per day. The mice in the APP/PS1 group were fixed in the immobilizer without any operations. The immobilizer and laser used in this study were disinfected with 75% alcohol, and the experiment lasted for 30 days. After the stimulation, approximately 0.15 mL of blood was collected from the tail vein, and Raman spectroscopy was performed for blood analysis.

Figure 1: The stimulation schematic with infrared laser device.

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Spectral data collection

Raman spectroscopy was performed immediately after the mouse blood samples were collected. The Horiba HR Evolution LabRAM laser confocal Raman microscope (Horiba, Japan) was used in the experiment. The experimental conditions and parameters were set as follows: laser wavelength, 532 nm; laser power, 3.3 mW; wavenumber resolution, 1 cm^-1; photon collection time, 10 seconds; and scanning range, 400-1800 cm^-1. The instrument was calibrated using the 520 cm^-1 peak position of the standard silicon sample before each measurement. The laser was focused on the upper surface of the blood sample [Figure 2] through an L50× telephoto objective (N.A. 0.5), and Raman spectra were randomly collected at 10 different locations for each blood sample to minimize measurement errors.

Figure 2: Spectrum measurement of blood samples using confocal Raman spectroscope.
Note: CCD: Charge coupled device.

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Spectral preprocessing

For preprocessing ofthe Raman spectral data, the asymmetric least squares approach was used to smooth the original data, to subtract the baseline (parameters were set as: asymmetry factor, 0.001; threshold, 0.05; smoothing factor, 4; iteration, 10 times), and to reduce the interference of the fluorescence background. Then the spectral data were normalized to the interval [0, 1], and the average spectrum was calculated for each group, as shown in [Figure 3].

Figure 3: Raman spectra after baseline removal and normalization.
Note: The average spectra curves A and B correspond to the average Raman spectra of the APP/PS1 and APP/PS1_laser groups, respectively, and curve C represents the difference between the two groups. The red and blue shading represent the spectral standard deviation ranges for each curve. a.u.: Absorbance unit.

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Statistical analysis

All data were expressed as mean ± standard error of the mean (SEM) using the software Excel (Office 2019, Microsoft Corporation, Redmond, WA, USA), the paired sample t-test was used to assess whether the difference between APP/PS1 and APP/PS1_laser groups was significant. P < 0.05 was considered statistically significant.

Results

Peak assignment of blood Raman spectrum

Raman spectroscopy is a kind of fingerprint spectrum, and the peak intensity in Raman spectroscopy is related to the number of molecules (Yu et al., 2006). Raman shifts of the APP/PS1_laser and the APP/PS1 groups are marked in [Figure 4]. The corresponding blood components were determined according to the possible peaks assigned for components reported in previous studies (Frank et al., 1995; Huang et al., 2003; Binoy et al., 2004; Ruiz-Chica et al., 2004; Stone et al., 2004; Krafft et al., 2005; Lau et al., 2005; Katainen et al., 2007; Szabó et al., 2007; Bergholt et al., 2011; Tfaili et al., 2012; Silveira et al., 2014; Ditta et al., 2019; Naseer et al., 2019) [Table 1]. The effect of laser stimulation on the mouse prefrontal cortex was evaluated by analyzing the height changes of peaks. Raman spectra of the blood samples in the two groups of mice had height differences in the peaks at 675, 747, 1081, 1124, 1223, 1305, 1340, 1372, and 1540 cm^-1. The different contents of blood components, such as glucose, lipids, and porphyrin, suggest that laser stimulation could cause certain physiological effects.

Figure 4: Average Raman spectra of APP/PS1 and APP/PS1_laser groups.
Note: a.u.: Absorbance unit.

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Table 1: The possible biological substances corresponding to the peak positions of the Raman spectrum

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Effects of laser stimulation on glucose in the blood of APP/PS1 mice

The peaks at 1081, 1124, and 1340 cm^-1, which correspond to the characteristic peaks of glucose, were analyzed [Figure 5], and [Table 1]. The average intensities of these characteristic peaks in the APP/PS1_laser group were lower than those in the APP/PS1 group, especially the peak intensity at 1124 cm^-1 (P < 0.05), suggesting that laser stimulation of the prefrontal cortex reduced blood glucose levels in the APP/PS1 mice.

Figure 5: Raman spectra of characteristic peak intensities in the blood samples of APP/PS1 and APP/PS1_laser groups.
Note: Data are expressed as Mean ± SEM, n = 10. *P < 0.05, vs. APP/PS1 group. a.u.: Absorbance unit.

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Effects of laser stimulation on lipids in the blood of APP/PS1 mice

The typical feature of Raman spectra of lipids is related to hydrocarbon chains, and the lipid-related Raman peaks are usually at 1081, 1124, 1223, 1305, 1340, and 1372 cm^-1 [Figure 5] and [Table 1]. The Raman spectral intensities at 1081, 1305, 1340, and 1372 cm^-1 are mainly attributed to lipids such as saturated and unsaturated fatty acids, triacylglycerols, cholesterol, and phospholipids. As shown in [Figure 5], the intensities of the APP/PS 1_laser group at 1081 (P > 0.05), 1124 (P < 0.05), 1223 (P < 0.05), 1305 (P > 0.05), 1340 (P > 0.05), and 1372 (P > 0.05) cm^-1 were lower than those of the APP/PS1 group, indicating low-power laser stimulation may reduce blood lipids and promote physiological metabolism in APP/PS1 mice.

Effects of laser stimulation on respiratory metabolites in the blood of APP/PS1 mice

Peak positions at 675, 1124, and 1372 cm-1 reflect the content of porphyrin in the blood [Figure 5] and [Table 1]. The peak intensities of porphyrins (675 (P > 0.05), 1124 (P < 0.05), and 1372 cm^-1 (P > 0.05)) in the blood of the APP/PS1_laser group were lower than those in the APP/PS1 group. Porphyrin plays an important role in O₂/CO₂ exchange during respiration (Katainen et al., 2007). Therefore, porphyrin levels in the blood reflect the capacity of blood to transport O₂ and CO₂. We speculate that the reduced porphyrin content in blood suggests that laser stimulation of the prefrontal cortex may contribute to reducing the O₂/ CO₂ exchange rate in blood and improving the physiological metabolic capacity of the APP/PS1 mice.

Discussion

LLLT is a novel method of noninvasive neural stimulation that can safely penetrate biological tissues and reach the cerebral cortex (Ilic et al., 2006). LLLT promotes cellular survival in times of reduced energy substrate through interactions with cytochrome oxidase to enhance oxidative phosphorylation (Leung et al., 2002; Mochizuki-Oda et al., 2002). Raman spectroscopy is commonly used in chemical research because vibrational information is specific to the chemical bonds and symmetry of molecules by which the molecule can be identified, and is widely used in biomedicine (Huang et al., 2011).

In this paper, Raman spectroscopy was used to analyze the mouse blood after laser stimulation to explore the effect of laser stimulation of the prefrontal cortex on blood metabolism. The average intensities of peaks at 1081, 1124, and 1340 cm^-1 in the APP/PS1_laser group were lower than those of the APP/PS1 group. We speculate that low-power laser stimulation may reduce the blood glucose level in APP/PS1 mice. The peak intensities 1081, 1305, 1340, and 1372 cm^-1 of the Raman spectra were attributed to lipids such as saturated and unsaturated fatty acids, triacylglycerols, cholesterol, and phospholipids. The characteristic peak intensities at 1124 cm^-1 and 1223 cm^-1 in the APP/PS1_laser group were lower than those in the APP/PS1 group, suggesting that low-power laser stimulation may promote a reduction in blood lipids and promote mouse physiological metabolism. In addition, the peak positions 675, 747, 1124, 1372, and 1540 cm^-1 reflect erythrocyte porphyrin content, which plays an important role in blood O₂/CO₂ exchange, and the characteristic peak intensities 675, 1124, and 1372 cm^-1 of the APP/PS1_laser group were lower than those in the APP/PS1 group. We speculate that low-power laser stimulation may improve the oxygen tolerance of mice, suggesting that low-power laser stimulation promotes respiration and metabolism in APP/PS1 mice. In addition, blood samples were only collected from male mice in this study; therefore, laser stimulation may have different effects between male and female mice. In conclusion, the results indicate that LLLT may promote glucose and lipid metabolism in the blood of APP/PS1 mice, and may improve the respiratory and metabolic capacity of APP/PS1 mice, which may be due to laser stimulation-induced changes in neurophysiological activity. Raman spectroscopy is a fast and reliable tool for detecting changes in blood components. However, its quantitative analysis requires validation by high-performance liquid chromatography/mass spectroscopy.

Acknowledgments

We appreciate the great help from the Yonghua Ji’s Lab, Shanghai University.

Author contributions

SYL, CS, NC, and SPL performed the concepts; SYL, YDX, CS, and NC performed the design; SYL, JT, and SPL contributed to literature search; YDX defined the intellectual content; SYL, YDX, HZ, and SPL performed the experimental studies; YDX, JT acquired the data; SYL and JT analyzed the data; SYL, HZ, SPL draft the manuscript; NC and SPL reviewed the manuscript. All authors approved the final manuscript.

Conflicts of interest

The authors declare that they have no conflict of interest.

Editor note: Chunlei Shan is an Editorial Board member of Brain Network and Modulation. He was blinded from reviewing or making decisions on the manuscript. The article was subject to the journal’s standard procedures, with peer review handled independently of this Editorial Board member and his research group.

Open access statement

This is an open access journal, and articles are distributed under the terms of the Creative Commons AttributionNonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.[32]

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