Confocal Microscope Raman Spectroscopy Analysis Of The Diabetic Mice Keratopathy

【Objective】To invetagate the differential expression of biochemical substances(water,nucleic acid,amino acid,protein,glucose,collagen,lipid,etc.)in corneal tissue of diabetic and normal mice by confocal Raman spectroscopy(HORIBA Xplo RA PLUS)technology,revealing possible mechanism of diabetic keratopathy.【Methods】SPF male DBA / 1 mice(6-8 weeks old)were divided into two groups according to the random number table method: one group is the diabetic mouse group(experimental group);the citrate buffer group was the negative control group.After 1 week of normal rearing,mice in the experimental group were induced to form a type 1 diabetes model by tail vein injection with streptozocin(STZ).2weeks later,the whole cornea was removed as sample.The confocal microscope Raman spectrometer was used to detect and analyze the differential expression of biochemical substances in the corneal tissue of these two groups.【Results】1.After injecting STZ via tail vein,mice in the experimental group gradually developed clinical symptoms of "three highs and one low" typical diabetes such as polyphagia,polydipsia,polyuria and weight loss.Two weeks later,their hair glossiness,lassitude,and mobility were observed.And the body weight was significantly reduced compared to the normal group.The blood glucose measured by the tail vein were all over than 16.7 mmol / L.Testing cornea with 1% rose bengal staining,and it was found that the diabetic mouse began to have a slight dotted corneal epithelium staining at 1 week after modeling,and a little dotted staining at 2 weeks,which was more than that at 1 week.2.The results of Confocal Raman Spectroscopy: 2 weeks after modeling,cornea was taken as sample.The average cornea Raman spectrum of diabetic mice and normal mice had similarities in position,morphology and intensity.But some specific Raman spectra varies greatly.The cornea of diabetic mice has higher intensity than normal mouse at 708cm-1,813cm-1,995cm-1,1443cm-1,1655cm-1 Raman peaks.These peaks are assigned to methionine,collagen,ribose,lipid and protein,amideⅠband(collagen);and at 846cm-1,930cm-1,1028cm-1,1239cm-1,the Raman peaks of diabetic mice were lower than normal.These peaks belong to the a-glucose,proline,methoxy,and amide III bands.Principal component analysis(PCA)was used to distinguish the corneal Raman spectra of normal mice and diabetic mice.Combined with PCA-LDA,the diagnostic sensitivity and specificity of corneal Raman spectroscopy to distinguish normal mice and diabetic mice were respectively 58% and 89%,and the accuracy of differentiation was 74%.The receiver operating characteristic curve(ROC)drawn by SPSS,the AUC = 0.745(AUC value is generally between 0.5 ～ 1.0,the larger the value,the better the classification effect of the model),indicating that this model ’s classification effect is exceptional.【Conclusion】In the hyperglycemic environment,the content of methionine,collagen,ribose,lipid and protein,amideⅠband(collagen)were increased in the corneal tissues of early diabetic keratopathy,while the content of a-glucose,proline,methoxy,amide III band were decreased relatively.The changes of above substances are consistent with the corneal damage mechanism caused by diabetes.