Study Of Nucleus Pulposus From Patients With Lumbar Disc Herniation Using Raman Spectroscopy

Objectives:Lumbar disc herniation is considered the major factor associated with low back pain,which is a global health problem affecting quality of life,and which has imposed an enormous economic burden on society.In China,lumbar disc herniation has an incidence rate of 4.26%,affecting 1.9%to 7.6%of males and 2.2%to 5.0%females.Previous studies have shown that both type II collagen and proteoglycan,are the most important components of extracellular matrices within the nucleus pulposus,and decreased content of these two proteins is the major characteristic of Intervertebral disc degeneration.Raman spectroscopy is an emerging optical technique based on inelastic scattering of light.It is widely used in the fields of medicine and life sciences because of its advantages of being label-free,noninvasive,and real-time.Raman spectroscopy can detect the structure and composition of proteins,lipids,DNA,and other biological macromolecules in biological samples,providing abundant molecular information at the microscopic level.The aim of this study was to use Raman spectroscopy detection with different degree of degeneration of nucleus pulposus in patients with lumbar disc prolapse extracellular matrix biochemical component content and the change of microstructure,which could help for the further study of the lumbar disc degeneration mechanisms.Methods:According to the inclusion and exclusion criteria,a total of 30 nucleus pulposus samples from 29 patients with surgically treated lumbar disc herniation were included in this study from November 2015 to June 2018.All intraoperative after removal of nucleus pulposus sample liquid nitrogen frozen quickly,then kept-80?.All samples were thoroughly washed in physiological saline solution,then embedded with optimal cutting temperature?OCT?medium.All frozen tissue blocks were cryo-sectioned to a thickness of 30?m,mounted on glass coverslips,and stored at-20°C.Part?:All Magnetic Resonance Imaging data of the lumbar spine?L1/L2 to L5/S1?were collected on a Siemens Magnetom Trio 3.0 T Scanner.The T2 signal intensity value of the nucleus pulposus was measured by Siemens post-processing software on Sagittal T2-weighted images.The Pfirrmann grading system,based on T2 weighted MRI images,was used to classify the degenerative discs.In this study,Pfirrmann grades??II were defined as a non-degenerated group;grades III,IV,and V were defined as degenerated groups.Part?:All spectroscopic assessments in this study were made by means of a Laser Micro-Raman Spectrometer?Thermo Scientific DXR Raman Microscope?at room temperature.Spectral data were processed and analyzed by OMINIC software,and further analyzed the content and structure of proteoglycan and collagen in nucleus pulposus.Results:Part?:1.A total of 30 nucleus pulposus samples from 29 patients were included in the study.The mean age was 47.5±15.53 years?range 21–75 years?in male patients?n=16?,and 50.4±15.4 years?range 18–67 years?in female patients?n=13?.Pfirrmann grade II:n=5;grade III:n=9;grade IV:n=9;and grade V:n=7;no discs were graded as Pfirrmann grade I.2.Correlation analysis between nucleus pulposus T2 signal intensity and Pfirrmann grading showed that with the increase of the degree of intervertebral disc degeneration,the T2 signal intensity of nucleus pulposus decreased?r=-0.789,P<0.000?,and the T2 signal intensity of nucleus pulposus in the degeneration group was significantly lower than that in the non-degeneration group?P<0.001?.Part?:1.The relative content of proteoglycan was inversely significantly correlated with the Pfirrmann grades?r=-0.703,P<0.000?,the relative content of proteoglycan(I₁₀₆₄/I₁₀₀₄)in the degenerated group was significantly lower than the non-degenerated groupwith aggravation of the intervertebral disc?P<0.01?.2.The content of Amide-III was positive significantly correlated with the Pfirrmann grades?r=0.556,P<0.001?,and reached a maximum at grade IV;the content of Amide-I was positive significantly correlated with the Pfrrmann grades?r=0.707,P<0.000?,and reached a maximum at grade IV.3.The collagenization degree of the NP?Amide-III/Proteoglycan?in the degeneration group was higher than that in the non-degenerated group with increasing degeneration based on Pfirrmann grades?P<0.05?.The collagenization degree of the NP?Amide-I/Proteoglycan?in grade IV and grade V were significantly increased compared with grade II?P<0.001?.4.The collagen quality parameter(I₁₂₄₅/I₁₂₇₀,Amide-III)was not significantly different between the degenerated group and the non-degenerated group with aggravation of intervertebral discs?P>0.05?.The collagen quality parameter(I₁₆₇₀/I₁₆₄₀,Amide-I)in grade III and grade V were higher than that in grade II?P<0.05?.Conclusions:1.With the increase of degree of intervertebral disc degeneration,the nucleus pulposus T2 signal intensity value is reduced,Pfirrmann grading and negatively correlated with nucleus pulposus T2 signal intensity value.2.Raman spectroscopy can identify changes in the content and structure of the proteoglycan and collagen in the extracellular matrix of the nucleus pulposus at the microscopic level.3.Compared with the non-degenerative group,the relative content of extracellular matrix proteoglycan in nucleus pulposus(I₁₀₆₄/I₁₀₀₄)was significantly decreased in the degenerative group,while the content of collagen was significantly increased,and the collagenization degree of the nucleus pulposus in the extracellular matrix of nucleus pulposus was increased.4.The increase of collagen mass parameters in nucleus pulposus(I₁₆₇₀/I₁₆₄₀)suggests that secondary structure of collagen protein in nucleus pulposus is out of order,which may be evidence of structure abnormalities in nucleus pulposus.