⁹⁹Tc^m-MNLS’ Hypoxic Imaging Estimate The Variation About Tumor Hypoxic After Radiotherapy

Objective: Detected the feasibility of⁹⁹Tc^m-MNLS [2-（2-methyl-5-nitro-1H-imidazol-1-yl）ethyl eihydrogen phosphate，⁹⁹Tc^m–MNLS] in monitoringthe hypoxic changes of tumor after radiotherapy，and explored theadvantage in monitoring hypoxic variation compared with⁹⁹Tc^m-HL91.Methods：1. Molding the mice bearing H22liver cancerH22liver cancer cell was extracted from abdomen of mice into test tubewhich is immersed to ice water and adjusted the density to2×107/0.1ml,thenshaked and injected into the right foreleg’s subcutaneous of mice. The micefeeding in cleaning environment and waiting the diameter of tumor to1cmwith no significant difference in weight or the size were chosen to experiment.2. Labled the hypoxic imaging agent of MNLS by⁹⁹Tc^mThe solution of⁹⁹Tc^mwas injected into the bottle which was packed withMNLS，and the mixture was shaked for ten minutes，then the pure ofradioaction was determinated by Polyamide as stationary phase and10%hydrochloric acid as mobile phase when the ITLG-SG was stationary phaseand physiological salineand was mobile phase respectively on thirty minutes,one hour, two hour, three hour, four hour, six hour and eight hour in roomtemperature and37℃water bath box.3.⁹⁹Tc^m-MNLS’ biodistribution in mice and radionuclide scintigraphyimaging①The mice were random divided into seven group by the time ofscintigraphy,respective in thirty minutes, one hour, two hour, three hour, fourhour, six hour and eight hour，and every group contained six.②The0.2mCi/0.1ml of⁹⁹Tc^m-MNLS was injected into caudal vein of themice. Then，the mice were anesthetized, fastened and placed to radionuclide scintigraphy imaging.Delineated the tumor and the normal tissue,the latter wasconsidered the blackground，and calculated the ratio of T/NT.③The mice were executed after imaging and striped the tumor, stomach,heart, lung, liver, kidney, spleen, bone, small intestine and the posterior limb’smuscle which was swashed by normal saline and dried by gauze.The tissueswere measured and weighed to obtain the percentage of injected dose pergram of tissue（％ID/g）.4.⁹⁹Tc^m-MNLS radionuclide scintigraphy imaging after radiotherapy①The mice were random divided into six group by the time ofscintigraphy after radiotherapy：instant imaging(A_instant)and its black control(C_instant)，24h(A_24h)and its black control(C_24h),48h(A_48h)and its blackcontrol(C_48h)，and every group contained six.The mice about Ainstant，A_24hand A_48hwere treated by the radiotherapy about25Gy from linear acceleratorof six MV，when all the black control were not.②The0.2mCi/0.1ml of⁹⁹Tc^m-MNLS was injected into caudal vein of themice. The mice were anesthetized, fastened and placed to radionuclidescintigraphy imaging after two hours of injected. Then the ratio of T/NT werecalculated.③The mice were executed after imaging and striped the tumor intoneutral formaldehyde solution so as to immunohistochemistry. The resultswere judged by the way as following：every slice was selected five vision fromup to down and left to right at four hundred high magnification，then everywas counted two times， the average representated the vision and the averageof five vision representated the slice.5.⁹⁹Tc^m-MNLS and⁹⁹Tc^m-HL91’s radionuclide scintigraphy imagingafter radiotherapy①The mice were random divided into two group by the differentradiopharmaceuticals：⁹⁹Tc^m-HL91and⁹⁹Tc^m-MNLS， and every groupcontained six. The mice in the two groups were treated by the radiotherapyabout25Gy from linear accelerator of six MV； ②The mice of each group respectively were injected⁹⁹Tc^m-HL91or⁹⁹Tc^m-MNLS on instant and48h after radiotherapy, then anesthetized, fastenedand placed to radionuclide scintigraphy imaging after two hours of injected.③The ratio of T/NT were calculated and compared with each other.Results：1. Observed and identified the modelNinty of subcutaneously transplanted tumor models were successfullyestablished.The tumor were all located the right foreleg’s subcutaneous whichwere hard, pink, smooth and the uncleus were deepened, largened anddeformed tremendously which corresponded with the uncleus of liver cancer.2. MNLS’ labling and stabilityLabling⁹⁹Tc^m-MNLS was easy，and the stability after labled was quitegood.The labled⁹⁹Tc^m-MNLS was colorless transparent solution，and the pureof radioaction was97%on ten minutes after marked，when it is more than90%on thirty minutes, one hour, two hour, three hour, four hour, six hour andeight hour.3. Biodistribution and imaging result of⁹⁹Tc^m-MNLS①The⁹⁹Tc^m-MNLS were absorbed and cleaned up in a short time whenthe％ID/g on30min was0.243±0.026％ID/g. The absorbance of tumorswere enhanced during the two or three hours after starting imaging, keeped thebalance arounding the peak between two and four hour and reduced after fourhours.②The tumors were developed on30min，The ratios’ change of T/NTwere analogied with the absorbance of tumors.4. Imaging results of⁹⁹Tc^m-MNLS after radiotherapy①The T/NT of Ainstantwas higher than Cinstant（3.35士0.19and2.74士0.29,t=4.235，P﹤0.05）; The difference of T/NT between A24hand B24hwasinvalid，（2.65士0.27and2.98士0.16, t=0.525，P﹥0.05）; The T/NT of A48hwas lower than the one of C48h,（2.23士0.52and3.15士0.88, t=7.902，P﹤0.05）. ②The expression of HIF-1α in the group of Ainstant, A24h, A48hwererespectively85.32%士0.94%,50.62%士0.78%and21.69%士0.75%，when28.26%士0.70%，58.45%士0.98%and67.64%士0.55%were in Cinstant, C24h,C48h. The HIF-1α’s expression in Ainstantwas higher than Cinstant’s （t=3.473，P﹤0.05）; the HIF-1α’s expression in A24hand C24hhad no difference（t=2.043，P﹥0.05）, when theA48hwas lower than C48h，（t=3.258，P﹤0.05）.③The correlation between image and the content of HIF-1α:The relation between the result of⁹⁹Tc^m-MNLS’image and the expressionof HIF-1α was positive correlation. The correlation coefficient inradiotheraphy group and control group comtrastion were respectively0.493and0.756which proved that⁹⁹Tc^m-MNLS could evaluate the change abouthypoxia in tumor and curative effect of radiotherapy.5. Compared the imaging results of⁹⁹Tc^m-MNLS and⁹⁹Tc^m-HL91afterradiotherapyThe ratios of T/NT about instant and48h in⁹⁹Tc^m-MNLS and⁹⁹Tc^m-HL91were respective3.35士0.19,2.23士0.52and3.14士0.18,2.82士0.35，the difference of T/NT between⁹⁹Tc^m-MNLS and⁹⁹Tc^m-HL91was tiny（t=0.583,t=0.313，P>0.05），but99mTc-MNLS were absorbed little exceptkidney when⁹⁹Tc^m-HL91were not.Conclusion：1.⁹⁹Tc^m-MNLS could evaluate the change about hypoxia in tumor andcurative effect of radiotherapy.2.⁹⁹Tc^m-MNLS and⁹⁹Tc^m-HL91could evaluate the hypoxia but the⁹⁹Tc^m-MNLS was superior to⁹⁹Tc^m-HL91in the abdomen’ tumor.