| Research BackgroundThe concept of optical molecular imaging was first proposed by Weissleder in 1999 (Weissleder R. Molecular imaging:exploring the next frontier [J]. Radiology, 1999,212(3):609-14.), it refers to use the method of imaging in living conditions reflecting the change at cellular and molecular level.Relative to the test in vitro, its advantage are lies of dynamic, real-time and non-invasive observation in the same body. Optical imaging has the advantages of high sensitivity, relatively simple imaging process, no radiation, and small investment. Compared with other medical imaging, molecular imaging method has high specificity, high sensitivity and image resolution, which can truly achieve clinical diagnosis at the molecular level, to provide anatomical structure, the disease occurrence and development of information based on molecular level. As it can be shown at the cell or subcellular level and identify different physiological and pathological processes in vivo, relative to the previous image technology aimed at understanding disease process at the level of morphology and structure, molecular imaging techniques focusing on biochemistry and intracellular way to reveal the occurrence and the development process of diseases (Blow N. In vivo molecular imaging:the inside job[J]. Nature Methods, 2009,6(6):465-469.).As the development of molecular biology, the progress of computer technology, and the clinical medical disciplines offer medical precise quantitative calculation model, which greatly promote the development of the medical practice, but there are still many experiment can’t going on in vivo.So, small animals test is still the important means of gene disease study of disease pathogenesis, clinical manifestation to better understand the corresponding physiological and pathological information, especially the malignant diseases, which provide a reliable basis for disease diagnosis and the development of new medical means. As aboved said, small animal imaging in vivo can provide biological tissue for internal genetic disease in a relatively short time and in high resolution image and track the change of the diseased tissue from the perspective of molecular and genetic studies of physiology and pathology aspects, such as disease development details, especially for malignant diseases such as tumor pathological tissues via imaging studies in vivo, which would greatly promote the human understanding of such diseases and help health care workers to study the effective method for the treatment of such diseases (Baker M. Whole-animal imaging: The whole picture [J]. Nature,2010,463(7283):977-980.).The first part of this subject using the optical molecular imaging technology to build subcutaneous and in situ the animal liver model for the research of liver cancer pathogenesis, drug efficacy evaluation, early surgical treatment and set up a good animal model in a noninvasive, real-time, continuous and visually observe liver tumor biological behavior in vivo with the help of IVIS200 molecular imaging system (Xenogen company) belonged to the institute of automation, Chinese academy of sciences key laboratory.Conventional imagings are usually used to evaluate the effect of the treatment methods of tumor diseases, such as by looking at a certain time after the tumor size shrink to observe curative effect. While clinical imaging diagnosis of disease is based on the pathological morphology of human body function change, lags far behind in molecular, cellular, tissue lesions.Optical molecular imaging technology could detect disease in a way of molecular pathological changes, and not just the end-stage disease of anatomical changes, such as:chemotherapy only observe the drug targets if there is any change, some of the key in the process of drug molecular markers have change, can have concluded that the treatment effect, this kind of extremely sensitive evaluation method has great application value in curative effect evaluation, so, molecular imaging can be more accurate than conventional image earlier to find lesions, qualitative and the pathological changes, in this way, clinical doctors can observe the treatment effect in the occurrence of diseasesearly (Zhang Q, Du Y, Xue Z, et al. Comprehensive Evaluation of the Anti-Angiogenic and Anti-Neoplastic Effects of Endostar on Liver Cancer through Optical Molecular Imaging. PLoS One, 2014,9(1):8555-9.)。 In recent years, apigenin have been widely applied in anti-tumor research because of its proven anti-inflammatory, anti-tumor and the ability of scavenging free radicals, it is a kind of naturally occurring flavonoids, have potential antitumor activity. Several studies have showed that apigenin has obvious inhibitory effect for gastric cancer, colon cancer, lung cancer, uterine sarcoma (Shukla S, Gupta S. Apigenin:a promising molecule for cancer prevention. Pharm Res 2010, 27(6):962-78.). In the second part of this subject we combined constructed liver subcutaneous and in situ animal model with the advanced optical molecular imaging technology to investigate and verify the apigenin inhibition of liver cancer in order to provide the beneficial reference for finding a new treatment of liver cancer.Small animal optical imaging technology is mainly by means of bioluminescent and fluorescence imaging, which is mainly used in:(1) the use of fluorescein specific or non-specific distribution in the body to study the fluorescein metabolic processes in the body, this study used ICG fluorescence characteristics to achieve monitoring the metabolic processes of ICG in the liver;(2) application of the tag fluorescent in the body of distribution and dynamic change process;(3) using different kinds of luciferase cells or DNA, the genetic markers on targets and expression of gene, according to biological reaction process to study cell and its level of the following biological process;(4) application and the related drug markup distribution metabolic changes of fluorescence report groups in the body to understand the effect of drugs and pathological changes.At present, a lot of the technology used in the observation of biological medicine targeted drug metabolism and function, the expression of genes and the reaction process, tracer different kinds of stem cells, monitoring and understanding of the inflammatory reaction process of organ transplantation model of monitoring, primary tumor, metastasis found that antitumor drug screening, the reproductive growth and control of bacterial virus (Weissleder R, Pittet MJ. Imaging in the era of molecular oncology. Nature,2008,452(7187):580-9.).This topic in the third part used fluorescence imaging combined with classic liver detection technology to study the MRP2 function of ICG hepatic metabolism, and proved MRP2 is one of channel of ICG in the liver, provided preliminary results for optical imaging technology used in the study of the liver of liver diseases and physiological mechanism in vivo, it can be believed that this imaging technique will become a new research method in the liver pathophysiology research.In medical practice, the resection tumor range of surgery usually based primarily on to determine the organization’s color, texture, shape and resection, so it have close relationship with the doctor’s clinical experience.If it can provide an objective in intraoperative tumor boundary or specific organization boundary information, doctors will improve operation success rate, reduce the surgical trauma and medical costs, avoid operation accidents and promote patients recover.In 2013 in the journal nature medicine said that the tumor boundary information of access will provide important value for clinical surgery.Compared with the subjective visual inspection and palpation during the operation, imaging method could give an effective and objective evaluation of the tumor and other lesions organization residual, helping doctors to achieve the purpose of intraoperative accurate diagnosis and treatment (Nguyen QT, Tsien RY. Fluorescence-guided surgery with live molecular navigation-a new cutting edge. NatRev Cancer,2013,13(9):653-62.).Therefore, in view of the intraoperative application of imaging equipment has to meet the following criteria:no radiation, the procedure of high letter back than real-time imaging, convenient operation. At present, the radionuclide imaging or computed tomography (CT) and other medical imaging equipments can’t move to surgery, and the imaging equipment in the process of operation for doctors and patients have a certain amount of radiation, so non-intrusive and real-time imaging need to be further explored easy to operate. Optical molecular imaging surgery navigation system developed by automation research institute of Chinese academy of sciences to meet the above criteria. Compared to the other intraoperative imaging system, optical molecular imaging surgery navigation system not only keep the characteristics of simple operation, also can make sure that the letter back with higher than under real-time imaging, compared to single camera imaging system at the same time, the system can also provide real-time anatomical structures in the image, this kind of high resolution, low noise and high sensitivity of the image provides an important guarantee to doctors clinical precise positioning operation.In recent years, with the rapid development of optical molecular imaging techniques, indocyanine green with produce near-infrared fluorescence characteristics of sentinel lymph node biopsy for breast cancer by mining dye, preliminary studies show that intradermal indocyanine green fluorescence can find the sentinel lymph node, and has the effect of real-time, dynamic, higher detection rate and low false negative rate compared with the traditional method (Chi C, Ye J, Ding H, et al. Use of Indocyanine Green for Detecting the Sentinel Lymph Node in Breast Cancer Patients: From Preclinical Evaluation to Clinical Validation. PLoS One,2013, 8(12):8273-9.).This topic in the fourth part explored the use of ICG， as a new type of nir fluorescent dyes and in optical molecular imaging guided surgery navigation system used in the feasibility of liver tumor resection, around the hepatocellular carcinoma in situ tumor, subcutaneous tumor and metastatic tumor were carried out by previous animal experiments and clinical verification, the experimental results can be concluded that optical molecular imaging surgery navigation system can quickly pinpoint liver tumor position, gives objective tumor boundary information, at the same time can also shows the location of the tumor metastases.According to statistical results, through the optical molecular imaging surgical navigation technology treatment of mice, survival time significantly higher than that of conventional surgical excision method to the naked eye, it also suggests that the precise treatment can effectively improve the survival rate, improve prognosis effect, we hope in the near future, the surgery navigation method based on molecular imaging can be widely used in clinical intraoperative precise positioning tumor resection.Part I. Optical molecular imaging technology aided construction of animal models of liver cancerObjective1. With the aid of optical molecular imaging technology to construct subcutaneous and orthotopic transplantation animal models of liver cancer.2. Set the animal models for early diagnosis and treatment of subsequent liver cancer drug curative effect evaluation.Methods1. HepG2 GFP and HepG2 fLuc markers of hepatocellular carcinoma cells subcutaneous hepatocellular carcinoma model in nude miceTake 20 only 4 to 6 weeks, male BALB/c nude mice respectively to extract containing 1.0x107/ml HepG2 GFP and HepG2 fLuc cell concentration cell suspension including 100μL of axillary subcutaneous inoculation in nude mice, HepG2-GFP cells to right armpit, HepG2-fLuc cells to the left armpit, each 10, to establish subcutaneous portability models of liver cancer.2. HepG2-fLuc hepatocellular carcinoma cells orthotopic liver cancer model in nude miceAseptic conditions, anesthesia nude mice by 1cm under the costal margin oblique incision on the right side, along the liver lobe membrane slowly into the tumor cells, step by step closed abdomen, the same method to establish 10 orthotopic liver cancer models.3. After completion of the above two inoculation tumor cells methods, tumor fluorescence intensity and tumor volume of nude mice were recorded daily with the aid of IVIS 200 molecular imaging system and three-dimensional reconstruction measurement, and then calculate the volume/fluorescence/time growth curve drawing, statistical tumor formation rate and incubation period, the growth and metastasis of the tumor. When tumor-burdened rat died, observe the general shape of the tumor tissue, infiltrating growth situation, and anatomical tumor-burdened rats to observe the distant metastasis.4. Statistical processingFor continuous variables adopt mean±standard deviation, according to the tumor volume/fluorescent growth in general statistical description and correlation analysis, assuming that P< 0.05 for the difference is statistically significant.All statistical analysis using IBM SPSS 19.0 statistical software for data analysis, Prism5.0 graphing software drew diagrams.Results1. The subcutaneous tumor model group:all 20 nude mice were formted into the tumor and the tumor formation rate was 100%. There had no distant metastasis lesions in all nude mice.2. The orthotopic tumor model group:in the process of tumor cells plantation, three mice were died due to over anesthesia (the excessive additional three nude mice lacking were supplementeded) and the tumor formation rate was 90%,3 of them found distance viscera metastatic lesions, four of them appeared abdominal metastatic lesions, so tumor metastasis rate was 70%, the complications such as infections did not appear in all nude mice.3. The quantitative fluorescence intensity and excitation fluorescence intensity analysis results:(1) BLI and FMI image data were got at least 12h after vaccination.Tumor cell growth and the fluorescence intensity are present grow trend after attenuation, after about 4d, tumor fluorescent to achieve the strength of the inoculation on the same day, then index growth trends.(2) through to the tumor size, tumor bioluminescence tumor and quantitative fluorescence quantitative calculation result of correlation analysis found that tumor size and tumor biological fluorescence intensity is highly correlation, tumor size and the fluorescence intensity is highly correlation.Conclusions:1. Subcutaneous and orthotopic transplantation animal models of liver cancer have characteristics with easy operation, high tumor formation rate, short tumor growth cycle and easy to control. So, they are the ideal tool to study of liver cancer biology characteristics and screening of anticancer drugs.2. Tumor bioluminescence intensity and fluorescence intensity reflects the number of living cells within the tumor.3. Optical molecular imaging techniques can realize real-time, dynamic, continuous and noninvasive, visually observed liver tumor biological behavior of nude mice in vivo.Part Ⅱ Application of molecular imaging technology in evaluating the inhibiting effect of apigenin in vivo on hepatocellular carcinoma Purpose:The aim of the study was to use optical imaging techniques to find the inhibition effect of apigenin for liver cancer so that to provide the beneficial reference to discover new drug treatment of liver cancer.Method:1. Study of subcutaneous tumor inhibition 20 Balb/c nude mice were randomly divided into 2 groups:the control group (normal saline group) and the experimental group (apigenin group),10 mice for each group with HepG2 fLuc and HepG2 GFP labeled cell suspension inoculation in nude mice in upper limb left armpit and right armpit.2. Study of orthotopic tumor inhibition 20 Balb/c nude mice were randomly divided into 2 groups:the control group (normal saline group) and the experimental group (apigenin group),10 mice for each group with HepG2-fLuc markers of cell suspension inoculation to the liver capsule:3. After the tumor cells were implanted the experimental group and control group for the subcutaneous tumor-bearing mice were given intraperitoneal injections with apigenin and an equal amount of 0.9% saline for the rest of days. According to the experimental group give corresponding different drugs treatment.(1) in the control group:every 3 d abdominal injection capacity of sterile saline solution;(2) experimental group:apigenin 50mg/kg, intraperitoneal injection, injection every 3 d, a total of 7 times.4. After all tumor-burdened rat died, stripping tumors, observe general morphology and tumor weight, will each tumor tissue is divided into two parts, part of the fixed tumor tissues including the metastasis niduses were excised, processed by standard methods, embedded in paraffin, sectioned and stained with H&E, the rest of the tumor tissue-80℃ cryopreserved.5. Each group comparison and test indexThe bioluminescent and fluorescent signals of the subcutaneous tumor were obtained daily and their intensity and distribution of tumors were observed and compared between two groups. The body weight and general physical status of the animal were recorded daily. The tumor volume was measured by caliper every two days. At the endpoint of the study, the tumors were removed and weighed.6. Statistical analysis:Results of the quantitative studies were expressed as mean ± SD. Means were compared using the independent-samples T test and by using the SPSS Statistical Software Package, version 19.0 (SPSS, Inc), and p value< 0.05 were considered statistically significant. Prism5.0 graphing software drew diagrams.Results:1. General situationsTumors growed slowly before 10 days,, tumors began to accelerate growed between 21 to 30 days after inoculation, nude mice’s state of mind than before become sluggish, decreased activity, unresponsive, angular, etc.30 days later, tumors growed more quickly. There was no unexpected death during the whole experiment process.2 Antitumor effects of apigenin(1) Subcutaneous tumor inhibitionAfter treatment began to measure tumors fluorescence intensity value and draw tumors and fluorescence intensity growth curve.Draw the experimental group and control group two groups of tumor (including HepG2 fLuc-and HepG2-GFP subcutaneous tumor model of two kinds of cell marker) tumor size and the fluorescence intensity at each time point growth curve after 20 days, which can be concluded that BLI and FMI signal strength value of experimental group was significantly lower than the control group at each time point, and always maintain the inhibition. In 45 days after inoculation of tumor inhibitory efffect was most significant, In tumor weight, tumor size and the fluorescence intensity, apigenin showed the subcutaneous tumor inhibitory effect of nude mice. After nude mice died, stripping tumors, observe its general form:nodules tumor, membrane integrity, rich blood supply, section are pale, no surrounding tissue infiltration focal necrosis and hemorrhage.①. Groups overall analysis:A. In terms of BLI subcutaneous tumor volume, it can be seen that the experimental group in 20d,25d,30d,35d,40d,6 points were lower than the control group, the experimental group compared with control group, F= 579.544, P= 0.000, the difference between the two groups have statistical significance.B. In BLI subcutaneous fluorescence intensity, it can be seen that the experimental group in 20d,25d,30d,35d,40d,6 points were lower than the control group, the experimental group compared with control group, F= 293.045, P ± 0.000, the difference between the two groups have statistical significance.C. In FMI subcutaneous tumor volume, it can be seen that the experimental group in 20d,25d,30d,3d,40d,6 points were lower than the control group, the experimental group compared with control group, F= 109.346, P= 0.000, the difference between the two groups have statistical significance.D. In FMI subcutaneous tumor fluorescence intensity, it can be seen that the experimental group in 20d,25d,30d,35d,40d,6 points were lower than the control group, the experimental group compared with control group, F= 422.243, P= 0.000, the difference between the two groups have statistical significance.②. Single time point analysis:A. In HepG2-fLuc subcutaneous tumor model, the experimental group and control group on day 45 after inoculation tumor with weight, tumor size and the intensity of fluorescence BLI comparison, the experimental group in cancer tumor weight (0.519±0.076g vs 0.859±0.114g,t=7.862, P=0.000), tumor volume (400.80±37.154 mm3 vs 624.30±39.797mm3, t=12.981, P=0.000) and BLI intensity (4543000.00±401221.745 photons/cm2 vs 6994000.00±395592.383 photons/cm2, t=13.756, P=0.000) were significantly lower than that in control group, the differences between the two groups have statistical significance.B. in HepG2-GFP marker of subcutaneous tumor model, the experimental group and control group in the tumor cells to 45 days after inoculation tumor weight, tumor size and the FMI fluorescence intensity comparison, the experimental group in cancer tumor weight (0.447±0.939g vs 0.756±0.117g, t=27.779, P=0.000), tumor volume (373.50±31.160 mm3 vs 564.20±42.512mm3, t=11.441,P=0.000) and FMI fluorescence intensity (3.51xl09±2.456×108 photons/cm2 vs 4.47×109±1.677×108 photons/cm2,t=10.206, P=0.000) was significantly lower than control group, the differences between the two groups have statistical significance.C. in respect of weight (19.700±2.540g vs 19.830±2.239g,t=0.121,P=0.905) experimental group and the control group there was no statistically significant difference in both groups.(2). Orthotopic tumor inhibition:①. The growth curve of the tumor volume and the quantitative light intensity based on HepG2-fLuc cells line. The results showed that the orthotopic tumor BLI signal of the experiment group was significantly inhibited since day 5. In situ tumor volume, it can be seen that the experimental group at four time points were lower than the control group, the experimental group compared with control group, F= 293.045, P= 0.000, the difference between the two groups was statistically significant.In terms of BLI fluorescence intensity, it can be seen that the experimental group at four time points were lower than the control group, the experimental group compared with control group, F= 1342.145, P= 0.000, differences between the two groups had statistical significance.Above all, we could see that apigenin has obvious inhibitory effect on liver cancer.②. Comparisons of the orthotopic tumor models between the experimental group and control group in tumor volueme, BLI light intensity and mouse weight. Experimental group in tumor volume (29.10±5.405 mm3 vs 30.20±6.663 mm3, t= 0.405, P= 0.690) and fluorescence intensity (349500.00±10394.977 photons/cm2 vs 349700.00±16193.620 photons/cm2,々= 0.033, P= 0.974) has no statistically significant differences between the two groups.35 days after inoculation, the experimental group in tumor volume (161.80±16.212 mm3 vs 328.90±9.949 mm3, t= 27.779, P= 0.000) and fluorescence intensity (865600.00±21613.782 photons/cm2 vs 1249000.00±53841.331 photons/cm2,t 20.897, P= 0.000) was significantly lower than the control group, while in terms of weight (22.800±2.732g vs.18.740±1.585g,t= 4.065, P= 0.001) in experimental group was higher than the control group, the differences between the two groups was statistically significant.Conclusions1. Apigenin could be expected as a new drug to treat hepatocellular carcinoma.2. Optical molecular imaging technology enabled the non-invasive and reliable assessment of anti-tumor drug efficacy on liver cancer.Part III Using molecular imaging technology to evaluate the role of multidrug resistance-associated protein 2 on the metabolism of indocyanine green in liver Purpose:1. To identify the role of multidrug resistance-associated protein 2 (MRP2) on the metabolism of indocyanine green (ICG) in vivo by using a novel small animal molecular imaging technology.2. Explore the validation of optical molecular imaging techniques in study of liver pathophysiological mechanisms.Method:1. Laboratory reagents Injected indocyanine green and MRP2 inhibitor MK-571.2. The experimental apparatus IVIS 200 noninvasive quantitative molecular imaging system3. The subjects and groups Take 4 to 8 weeks) 20 BALB/c mice were randomly divided into 2 groups:the control group A: ICG; Experimental group B:ICG+ MK-571,10 in each group.4. The animal in vivo imaging recording methodThe mice were fasted overnight prior to the experiment to prevent food from interfering with the bioluminescence results. The mouse was first anesthetized with 2% isoflurane through a respiratory mask which was attached to the mouse rack and received injection of ICG (0.5 mg/kg) and MK-571 (4 mg/kg) before fluorescence image was started. The mice were kept in lie supine and affixed on the mouse rack. The fluorescence image was scanned from 10min to 90min (nine time points in total) after injection of ICG and MK-571 from mice tail intravenous on the IVIS Spectrum in vivo imaging system.In order to study the ICG best dose in mice experiment,15 BALB/c mice were randomly divided into 3 groups (a, b, c),5 in each group, three different doses of ICG (O.lmg/kg,0.5mg/kg, 1.0mg/kg) were injected from mouse tail intravenous respectively, then ICG fluorescence intensity values were measured after 5min, 10min, 20min,40min,60min,80min 6 points, it can be seen from the figures in group b than in group a can reach higher peak (group a maximum of 80; group b maximum peak of 200), the changing trend was more significant than that of group c at various time points (b group of maximum peak was 200, minimum value was 19, group c maximum peak of 230, the minimum value of 132). From the results we could obtain the optimal 0.5mg/kg dosage of ICG in the study.5. The ICG fluorescence quantitative analysis of luminous intensity in the liverThe average fluorescence intensity and distribution of each image was measured on a Caliper Life Sciences IVIS Spectrum in vivo imaging system. The average photon emission of each sample was measured after subtraction of cosmic radiation using a region of interest (ROI) centered on the vial using Living Image software, and the radiance determined from the ROI was decay-corrected. The ICG fluorescence intensity and distribution in the liver were observed and compared.6. Statistical processingFor statistical analysis, data were expressed as mean ± standard deviation (SD). Analysis of variance was used by using the IBM SPSS 19.0 statistical software, andp value< 0.05 were considered statistically significant. Prism5.0 graphing software drew diagrams.Results:1. Fluorescence imagingFluorescence displayed immediately when ICG was injected into the liver. As time goes on, intensity and distribution of fluorescence increased to the peak gradually and got more and more small finally. The whole process of observation was in accordance with known metabolism activities of ICG in the human liver, which showed that using small animal molecular imaging technology to observe the metabolism of indocyanine green was feasible and reliable.2. Comparison of the mean fluorescence intensity of liver of two groupsIn the observation of each time point, ICG fluorescence intensity of two groups had a certain difference:the mean fluorescence intensity and distribution of group A was lower than that of group B at all nine time points. In order to clearly show the differences between two groups, with ICG fluorescence intensity at each observation point minus the fluorescence intensity of injection at the very beginning to get the average fluorescence intensity/time curve drawing, which clearly showed the trend of the difference between two groups. Comparison of the average fluorescence intensity on 9 observation points showed that there was statistically significant difference between two groups (p< 0.05).The results show that the MK-571 could postpone the metabolism time of ICG in liver.Conclusion:1. The biliary excretion of ICG was delayed by reduced MRP2 inhibitor MK 571 greatly, indicating that MRP2 is essential for its biliary excretion.2. The molecular imaging technology can continuously, real-time and no damage to display the intrahepatic metabolism process of ICG in liver in vivoPart IV Using a novel optical molecular imaging surgical navigation system (SNS) for management of liver tumor to assess tumor margins and decrease residual cancersPurpose:The aim of this study was to use a novel optical molecular imaging surgical navigation system (SNS) for management of liver tumor to assess tumor margins and decrease residual cancers.The early experiments on animalsMethod:1. Laboratory reagents Indocyanine green for injection.2. The experimental apparatus IVIS 200 noninvasive quantitative molecular imaging system Optical molecular imaging surgical navigation system3. The subjects and groupingAll male nude mice aged 4 to 6 weeks without thymus were bought from the milit ary acad emy of medical sciences, Beijing) by the military academy of medical sciences, the permission of animals used for experimental mice.After mice were in anesthesia on small animal operation bed (HARVARD, A Thirty mice for the preclinical setting and 5 patients with hepatocellular carcinoma (HCC) for the clinical evaluation were studied in our experiments.30 BALB/c nude mice which were randomly divided into the routine tumor resection group (group A) and the precise tumor resection group guided by SNS (group B).4.The experimental method Surgery navigation methods compared with conventional methods subcutaneous tumor excision of nude miceFor our experiment, we used the IVIS Spectrum imaging system (PerkinElmer Life Sciences, Hopkinton, MA, USA) to follow the growth of tumors by measuring the average bioluminescence intensity and assessing tumor volumes using 3D-reconstruction via Living Image 4.4 software. Mice were first anesthetized with 2% isoflurane through a respiratory mask which was attached to the mouse rack. D-luciferin (Caliper Life Sciences, Hopkinton, MA) dissolved in PBS (1.5mg luciferin/100ul PBS) was injected intraperitoneally at a dose of 150mg luciferin/kg, and serial images were acquired with an exposure time of 30sec, an f/stop of 1, and pixel binning at 8～12 minutes to come the peak bioluminescence. Mice were kept in lie supine and affixed on the mouse rack. The bioluminescence imaging (BLI) was scanned on the IVIS Spectrum imaging system in vivo. IVIS is calibrated to enable absolute quantitation of the bioluminescent signal and longitudinal studies can be performed over many months and over several orders of signal magnitude without compromising the quantitative result. Measurement data are displayed in the table together with all experimental parameters relating to the image capture which can be saved or exported for analysis.3D-Modality also enable classify the volumetric data by specifying color and opacity values for different intensity ranges so that researchers can view or hide certain parts of the data as needed.At the day of 45 after inoculation, a midline incision was made in the skin above the tumor and the tumor was surgically removed in group A (n= 15). The tumors in group B was surgically excised guided by SNS (n= 15). Following tumor removal, SNS was used again to detect the residual cancers. The resulting resection cavity was copiously irrigated and the skin closed with 7-0 Vicryl suture (JAJ, New Brunswick, NJ). After operation, animals were kept in a warm cage, observed for 1～2h, and subsequently returned to the animal room after full recovery from the anesthesia was documented. All experimental protocols were approved by the Animal Care and Use Committees of Zhu Jiang Hospital, Southern Medical University. All efforts were made to minimize suffering. The tumor remnant rate and the survival time after operation were compared between two groups.5. Histopathologic validation The tumor tissues including the metastasis niduses were excised, processed by standard methods, embedded in paraffin, sectioned and stained with H&E.6. Statistical analysisResults of the quantitative studies were expressed as mean ± SD. Survival analysis and Chi-square test (the Fisher exact test was used when conditions for the Chi-square test were not fulfilled) were used to determine the significance of differences. A p value< 0.05 was considered statistically significant. |
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