Basic Research And Clinical Application Of Photoacoustic Imaging In Photodynamic Therapy Of Port Wine Stains

Background and objectivePort wine stains(PWS)is a congenital microvascular malformation.Photodynamic therapy(PDT)is one of the main treatment methods.PDT can activate photosensitizer mainly by light irradiation of corresponding wavelength,produce a large number of active singlet oxygen in the part of absorbing photosensitizer,cause oxidation reaction of adjacent tissues,destroy cells,and cause cell death.Its curative effect is significant.However,PDT,as a new therapeutic method,is still in the early stage of clinical application,and its specific molecular mechanism for PWS treatment has not been fully defined.At the same time,,there are two clinical problems in the treatment of PWS.Firstly,visual method is mainly used to diagnose PWS,which has strong subjectivity and large error.Secondly,the vascular changes cannot be monitored in real time during the treatment process,and the disease progress and prognosis evaluation lack of quantitative indicators.Photoacoustic imaging(PAI),as a new micro imaging technology,has been gradually used in the treatment of PWS in recent years.Through functional imaging of blood vessels and detection of the ratio of red blood cell area fraction,it provides quantitative parameters for the diagnosis and prognosis of PWS.In this study,the molecular mechanism of PDT in the treatment of PWS was discussed through in vivo and in vitro as well as in clinical case analysis.The effect of PAI on PDT was analyzed to provide theoretical guidance and technical support for the application of PAI to PDT in PWS.Research method1.Screening of differential metabolites in PDT treatment of PWS and study on the mechanism of vascular injuryIn vivo experiment,25 samples of chicken sideburns were selected and 5 in each group.The intervention methods of each group were as follows:con group(blank control,saline injection,no laser irradiation),G1(low concentration and low power group,photosensitizer 5mg,laser power density of 90 W/cm2),G1-1(low concentration and high power group,photosensitizer 5mg,laser power density of 100 W/cm2),G2-1(high concentration and low power group,photosensitizer 10mg,laser power density of 90 W/cm2),G2(high concentration and high power group,photosensitizer 10mg,laser power density of 100 W/cm2),using LC-MS metabolomics to screen differential metabolites.In vitro experiments,human endothelial cell lines(HUVEC)were selected to simulate vascular endothelial cells,and were divided into Con group(control group with no treatment),Intervention group(using Daidzein as intervention),and Inhibitor group(using inhibitor to inhibit Daidzein effect)to observe the changes of 24,48 and 72 hours respectively.The changes of IC50,ROS,SOD,MDA,CO A,RE?,PPAR?,VEGF and Endostatin were detected by MTT,ELISA,RT-qPCR and Western blot.2.PAI detection and application in PDT treatment of PWS animal modelThe sideburns of chicken rich in blood vessels were taken as the experimental object.Through the blood vessel localization,photoacoustic signal collection and imaging parameter adjustment,to explore the best image location,imaging area and image analysis scheme for observing the blood vessels of chicken sideburns.Observe the changes of the treatment area on day 0,2,4,7,and 14,determine the morphological changes by HE staining,and analyze the changes of red blood cell area fraction ratio(RAR)in each time period by PAI.3.Clinical effect of PAI in PDT for PWSIn clinical study,227 patients who received PDT were analyzed to explore the difference of cure rate,side effects and patient satisfaction between PDT group(diagnosis by visual inspection and adjustment of plan)and PDT+PAI group(diagnosis and adjustment of plan by PAI Technology).Research results1.Screening of differential metabolites in PDT treatment of PWSThrough differential expression analysis,daidzein 7-o-beta-d-glucoside and daidzein were screened out.Through pathway analysis of differential metabolites,citrate cycle(TCA cycle)was selected as the main expression pathway.The half effect inhibitory concentration of 1 M Daidzein measured by MTT on HUVEC varied with time.It was 30.58±1.667? mol/L,2.935±0.548 ?mol/L and 0.459±0.258 ?mol/L at 24 hours,48 hours and 72 hours respectively.The content of oxidative stress factors was determined by flow cytometry and ELISA.When daidzein was added,the content of oxidative factors decreased and the content of antioxidant factors increased,while when inhibitor was added,the index of oxidative stress increased.RT-qPCR and Western blot showed that the gene and protein expression of Er ?,PPAR ?,VEGF and endostatin varied with time.2.PAI detection and application in PDT treatment of PWS animal modelObserving the change of treatment area,we found that the sideburns of Con group were ruddy and even in color.After PDT treatment,the blood vessels of G1 and G2 groups were damaged in varying degrees.After 14 days,only the center of G1 group was light white,and the surrounding area was light red.The treatment area of G2 group was also red,but the remaining white treatment area was relatively large.The photoacoustic images also showed that the vessels of Con group were arranged orderly,and there was no significant difference in the images of 5 time periods.In the intervention group,the vascular arrangement was more disordered,the photoacoustic signal was weaker,and the red signal was mostly scattered in the shape of a circle and mixed with the black signal.According to the analysis of PAI images,the average rar value of the five time periods in the con group was 72.08%,and the whole data fluctuation was small;in the G1 group,the RAR value showed a downward trend within 7 days after PDT treatment,and rose on the 14th day,from the average distribution of the RAR value;the RAR value in G2 group decreased significantly within 2 days after treatment,and rose slightly on the 4th day.3.Clinical effect of PAI in PDT for PWSCase analysis showed that,there was no significant difference between the two groups ni Fail effect but the number of effective people in PAI+PDT group increased.Combined with the data of subgroup analysis,the therapeutic effect of head and face was significantly different.The location and detection of PAI+PDT group was more accurate than that of PAI group,and the RAR value was less fluctuated in the group without PAI.The incidence of side effects in PAI+PDT group was 6%lower than that in PDT group.Research conclusion1.The effect of PDT treatment to PWS in sideburns was related to the concentration of photosensitizer and power density.Daidzein and its homologues play an important role in this process.The protective effect of Daidzein on vascular injury is mainly through reducing oxidative stress factors and inhibiting vascular proliferation,which may be related to interventing the gene and protein expression of ER?,PPAR ?,VEGF,endo statin.2.PAI is available for detecting the injury of sideburns blood vessels under PDT.It could be quantificationally evaluated the treatment effect by analyzing RAR value.The GAN based deep learning model may optimize the processing and analysis of PAI image.3.It provides more accurate location and clinical observation for PWS treatment with the help of PAI,which improves the effect of PWS treatment.