MRI Study On Vascular Endothelia Growth Factor Antisense Gene Regulation For Leptomeningeal Metastasis

Part I MRI study on a rabbit model of leptomeningeal metastasiscorrelated with pathologyObjective â‘  To establish a leptomeningeal metastasis rabbit model. (2)To evaluated MRI characteristics correlated with pathology with the modeland to estabish the MRI standard of diagnose in the leptomeningealmetastais.Methods 30 New Zealand rabbits were divided into 2 groups.Group A: 24 rabbits were inoculated with 0. 2m1(1×10⁵/ml) VX2 tumor cells into subarachnoid space through cisterna magna.Group B: 6 rabbits were inoculated with 0. 2ml physiological saline only into subarachnoid space through cisterna magna.MRI examinations were performed at different times after inoculation on a SIGNA^? 1.5 Tesla scanner (General Electric Medical Systems, Waukesha, WI) . The knee coil was used for imaging. Routine scans used SE sequence for T1WI (TR/TE=350/15ms) FSE sequence for T2WI (TR/TE=3000/90ms) and FLAIR (TR/TE /TI 8800/127.5/2200). rabbits of Group A were done triple-dose (0.3 mmol/kg) Gd-DTPA enhanced MR T1WI scanning/enhanced fast-FLAIR and enhanced fat-suppressed T1WI scanning respectively. Pathological study was made under microscope by HE and MRI characteristics and the correlated pathological observations were summarized. Results1. MR features1) Group A:4 in 24 rabbits has been found the nodus with a diameter of 5. 8±2. 3mm in plain MR scanning. All tumors appeared hypointensity or isointensity on T1W images, and isointensity or hyperintensity on T2W images. 12 in 24 rabbits has been found the nodus on FLAIR MRI. All tumors appeared linear, nodular or mixed enhancement . 15 in 24 rabbits has been found the positive signs on post-enhanced T1WI MRI. 18 in 24 rabbits has been found the positive signs on post-enhanced FLAIR MRI. 21 in 24 rabbits has been found the positive signs on triple-doseimages. 2) Group B:The MRI findings are normal except that one rabbit had a linearenhancement on 6^th and 9^th day. 2. Pathology1) Specimen: The pathological findings were normal on 3^rd day. After 6 days, the linear and nodular thickened leptomeniges were found in Group A. The pathological findings were normal in Group B.2) Microscopic: In Group A, tumors infiltrated alone with the leptomeninges and the superficial part of the brain and spinal cord were affected. The pathological findings were normal in Group B.3.Statistic results1) The tumor morbility in Group A are much more than that in Group R(P <0.05).2) GD-DTPA enhanced MR scanning (21/24) can find more leptomeningeal metastasis than plain MR scanning (4/24) .3) Quantitative analysis demonstrated an increase in relative signal intensity (C/N) when compared triple-dose studies (14.37 ± 4.93) with routine-dose studies ( 7.28 ± 3.21 ) (P＜0.05). More abnormal leptomeningeal lesions were found on triple-dose images.4) Quantitative analysis demonstrated an increase in relative signal intensity (C/N) when compared enhanced FLAIR(12. 51 ± 3. 05) with FLAIR(6.35 ± 2.55) (P＜0.01). More leptomeningeal lesions were found on enhanced FLAIR MRI.Conclusion â‘  The animal model is able to provide a good platform for medical imaging study. â‘¡ MRI with Gd-DTPA enhancement is highly sensitive for detecting the leptomeingeal metastasis. Triple-dose Gd-DTPA enhanced MRI , FLAIR , enhance FLAIR and enhanced fat - suppressed TIWI are all useful to improve the detection of leptomeningeal disorders. Part II Dynamic contrast enchanced MRI of leptomeningeal metastasis and its relationship with VEGF expressionObjective â‘  To evaluate the VEGF expression level in the metastasis, plasma and cerebrospinal fluid (CSF) of the animal models . â‘¡ To analyse the correlation of DCE-MRI with VEGF expression and assess the value of DCE-MRI in tumor biology. Methods30 New Zealand rabbits were divided into 2 groups.Group A: 24 rabbits were inoculated with 0. 2ml (1 × 10⁵/ml) VX2 tumor cells into subarachnoid space.Group B: 6 rabbits were inoculated with 0. 2ml physiological saline only into subarachnoid space.DCE-MRI (TR10ms/TE2ms/Flip angle20⁰) was performed on different days. MER and SLE were calculated.Before DCE-MRI examination of the rabbit, the VEGF level of plasma and CSF were evaluated by ELISA method. After DCE-MRI examination, the rabbits was sacrificed randomly and the corresponding tumor specimen was harvested. HE staining, VEGF immunohistochemical staining were carried out. VEGF expression level in the specimen was evaluated by means of HIS hemi-quantification.Results â‘ VEGF positive staining was localized in tumor cytoplasms and cell membranes, and some epithelial cells also had a positive expression. VEGF immunohistochemical score bore a positive correlation with SLE(P<0.01) by rank correlation statistical method. VEGF immunohistochemical score bore a negative correlation with SLE(P<0. 05) by rank correlation statistical method.â‘¡The VEGF level in the aplasma and CSF in the Group A are higher than that in the Group B(P<0.01), and they are different in the different time(P<0.05). The VEGF level in the plasma are higher than that in the CSF in the Group A(P<0. 05).The VEGF level in the plasma bore a positive correlation with that in the CSF (P<0. 05) by rank correlation statistical method. The VEGF level in the plasma bore a positive correlation with SLE (P<0. 05) by rank correlation statistical method. The VEGF level in the CSF bore a positive correlation with SLE (P<0. 05) by rank correlation statistical method. Conclusions â‘ The VEGF level in the plasma and CSF in the leptomenigeal metastasis are higher than that in the normal tissue. â‘¡DCE-MRI had a goodcorrelation with VEGF expression level of leptomenigeal metastasis.â‘¢DCE-MRI is a good mothed to assese the tumor angiogenesis and the effectof antiangiogenisis therapy.PART III MRI study on antisense VEGF cDNA regulation for leptomeningeal metastasisObjective â‘ Using molecular biology technique to construct eukaryoticexpression vector of rabbit VEGF antisense cDNA. â‘¡Using MRI to monitorthe regulation for leptomeningeal metastasis. And discuss the possibilityin prevention and cure of leptomeningeal metastasis by antiangiogenesistherapy.Methodsâ‘  Construction of eukaryotic expression vector of rabbit VEGFantisense cDNA:Total RNA was isolated from rabbit kidney tissue with the trizol method.VEGF cDNA was amplifyed by RT-PCR from total RNA and was sequenced. PCRproduct was digest and was inserted into polylinker site of plasmidpcDNA3.1. By using restricion enzyme Nco I and Hind III digestion andDNA sequence, we successfully constructed rabbit antisence VEGF₁₆₅eukaryotic expression vector pcDNA3. 1-ASVEGF.(2) MRI study on antisense VEGF cDNA regulation for leptomeningealmetastasis50 New Zealand white rabbits were divided into5 groups, with 10 in groupA for 150μg pcDNA3. 1-ASVEGF:Liposome and VX2 cells in the same time; 10in group B for 150μg pcDNA3. 1-ASVEGF:Liposome injected intrathecally onthe 7^th day; 10 in group C for 300μg pcDNA3. 1-ASVEGF:Liposome injectedintrathecally on the 7^th day; 10 in group D for only VX2 cells; 10 in groupE for Liposome and VX2 cells. MRI examinations were performed at differenttimes after inoculation on a SIGNA^TM1.5 Tesla scanner (General ElectricMedical Systems, Waukesha, WI) . The knee coil was used for imaging.Routine scans used SE sequence for T1WI (TR/TE=350/15ms) and FSE sequence for T2WI (TR/TE=3000/90ms) . DCE-MRI were acquired using a 2D spoiled gradient echo pulse sequence (TR/TE/Flip angle=9. 7ms/l. 7ms/20° ) and SLE of time-signal curve was analysed. Before DCE-MRI examination of the rabbit, the VEGF level of plasma and CSF were evaluated by ELISA method. Pathological study was made under microscope by HE and immunohistochemical stains.Results â‘ We successfully constructed eukaryotic expression vector of rabbit VEGF antisense cDNA (pcDNA3.(?)-ASVEGF). â‘¡SLE in group A is lower than that in group B, C, D and E after 6 days inculating the VX2 tumor cells. SLE in group A B and C are lower than that in group D and E after 9 days inculating the VX2 tumor cells. â‘¢The numbers and size of foci showed difference between group A, B, C and group D, E (P <0. 01). â‘£The average survival time of animals in group A , B, C are longer than that in groupD and E (P <0.01). ⑤Immunohistochemistry assay showed that there was difference of VEGF expression between groups(P < 0.05). VEGF immunohistochemical score bore a positive correlation with SLE(P<0. 01) by rank correlation statistical method. â‘¥The VEGF level in the aplasma and CSF in the EG are higher than that in the CG(P<0.01). The VEGF level in the aplasma bore a positive correlation with SLE (P<0. 05) by rank correlation statistical method. The VEGF level in the CSF bore a positive correlation with SLE (P<0.05) by rank correlation statistical method. Conclusion â‘ Antisense VEGF cDNA may play an inhibition role on the early stage of leptomeningeal metastases. â‘¡Antisense VEGF cDNA can inhibit the VEGF expression of leptomeningeal foci on molecule level. ? MRI can accurately show the leptomeningeal metastases , it can be used in early diagnosis and monitoring therapeutic effectiveness on leptomeningeal metastasis.