| Part Ⅰ. Comparison between Acetazolamide Challenge and10%Carbon Dioxide using Perfusion CT in Rat C6GliomaPURPOSE:Perfusion computed tomography (PCT) was used to investigate cerebral and C6glioma perfusion changes induced by acetazolamide (ACZ) and a mixture of10%carbon dioxide and90%air challenges. Correlations were investigated between PCT with ACZ and the results of Vascular endothelial growth factor (VEGF) and FⅧ-microvessel density (MVD).MATERIALS AND METHODS:Dynamic PCT was performed on32male Sprague-Dawley rats (age3month, weight250-300g. selected randomly) including20rats with rat C6glioma and12rats served as controls. Ten rats with orthotopically implanted C6gliomas and6normal rats underwent PCT at rest and15minutes after1mg/kg intravenous ACZ. Another ten rats with C6gliomas and six normal rats underwent PCT at rest and15minutes after inhalation of a mixture of10%carbon dioxide and90%air which lasted for15minutes. The interval between PCT with challenge and at rest was at least1hour. All examinations were performed with a256-slice scanner. PCT was performed at glioma area and the right caudate nucleus in controls. The raw data were processed using Philips CT brain perfusion software which could provide time-density curve (TDC) curves and colour maps. Carbon dioxide partial pressure (PaCO2) and potential of hydrogen (pH) were recorded. At the end of the experiment, each animal was sacrificed in24hours. Their brains were resected and fixed, which were cut at the level of the right caudate nucleus. A coronal5μm thick slice of brain tissue from each rat was cut and stained according to the standard hematoxylin and eosin (HE) staining protocol. The samples were examined using immunohistochemical staining for VEGF and FⅧ-MVD. The statistical analysis was performed using the SPSS17.0package (SPSS Inc., Chicago, IL. USA). ANOVA test was used to compare the difference among different groups, after confirmation of normal distribution of the data. In the multiple comparison procedure. P<0.01was considered significant after Bonferroni correction. Independent-samples t tests were used to compare the difference between perfusion parameters changes with ACZ challenge and perfusion parameters changes with10%carbon dioxide. Pearson correlation coefficients were used to investigate relationships between cerebral blood flow (CBF) percentage changes, cerebral blood volume (CBV) percentage changes, permeability and the results of VEGF and FⅧ-MVD. P less than0.05was considered significant.RESULTS:In the end of this experiment, those C6rats were dissected and proven to be gliomas. The tumor periphery had higher CBF, CBV, and permeability values than tumor central parenchyma (P<0.01). Tumor central parenchyma had higher CBF, CBV, and permeability values than controls (P<0.01). Twelve normal rats (6rats with ACZ stimuli,6rats with10%carbon dioxide stimuli) had increased CBF and CBV values after challenge (P<0.01). In controls, CBF percentage change apparently differed using ACZ stimuli (117.42%) compared to10%carbon dioxide (65.86%)(P<0.01). As for CBV percentage change, there was no significant difference between ACZ stimuli (107.51%) and10%carbon dioxide (92.95%)(P=0.02). In parenchyma of rat C6glioma, CBF percentage change was different using ACZ stimuli (72.73%) compared to10%carbon dioxide (28.47%)(P<0.01). CBV percentage change was37.85%with ACZ and24.69%with10%carbon dioxide. There was no significant difference in CBF, CBV and permeability percentage change between contralateral normal tissue and controls, beween peritumoral edema tissue and controls, and between tumor central parenchyma and the periphery. Significant correlations were observed between VEGF and FⅧ-MVD and blood volume percentage change (P<0.01). Blood flow percentage change correlated well with VEGF.CONCLUSION:PCT with ACZ is suitable for evaluation of blood vessels in C6gliomas instead of inhalation of10%carbon dioxide without the side effects induced by10%carbon dioxide. CBV percentage is correlated change with the FⅧ-MVD and VEGF in gliomas. CBF percentage change is correlated with the VEGF in gliomas. Part II. Cerebral Perfusion CT with Acetazolamide Challenge in C6GliomasPURPOSE:PCT with ACZ challenge was used to investigate perfusion changes of C6gliomas at10days,14days and18days. Correlations were investigated between PCT with ACZ and the results of CD105-MVD, VEGF, vascular maturity index (VMI) and Ki67.MATERIALS AND METHODS:Dynamic PCT was performed on32male Sprague-Dawley rats (age3month, weight250-300g, selected randomly) including8rats with rat C6glioma10days,8rats with rat C6glioma14days,8rats with rat C6glioma18days and8rats served as controls. Intravenous catheter for contrast medium administration pre-and post-challenge was put in the vena dorsalis penis of rats.24rats with orthotopically implanted C6gliomas and6normal rats underwent PCT at rest and15minutes after1mg/kg intravenous ACZ. The interval between PCT with challenge and at rest was at least1hour. All examinations were performed with a256-sIice scanner. PCT was performed at glioma area and the right caudate nucleus in controls. The raw data were processed using Philips CT brain perfusion software which could provide TDC curves and colour maps. PaCO2and pH were recorded. At the end of the experiment, each animal was sacrificed in24hours. Their brains were resected and fixed, which were cut at the level of the right caudate nucleus. A coronal5um thick slice of brain tissue from each rat was cut and stained according to the standard HE staining protocol. The samples were examined using immunohistochemical staining for CD105-MVD, FⅧ-MVD, VEGF, alpha-smooth muscle actin (a-SMA)-MVD and Ki67. VMI was calculated as follows:VMI (%)=α-SMA-MVD/FⅧ-MVD X100%. The statistical analysis was performed using the SPSS17.0package. Paired-samples t tests were used to compare the difference between perfusion parameters pre-and post-ACZ challenge. ANOVA test was used to compare the difference among different groups, after confirmation of normal distribution of the data. In the multiple comparison procedure, P<0.01was considered significant after Bonferroni correction. Pearson correlation coefficients were used to investigate relationships between CBF percentage changes, CBV percentage changes, permeability, CBF, CBV and the results of CD105-MVD, VEGF, VMI and Ki67. P less than0.05was considered significant. RESULTS:Gliomas of4groups were all proved to have elevated PaCO2and decreased pH after ACZ challenge (P<0.01). Significant CBF. CBV difference was observed before and after ACZ challenge. Rat glioma in14days and18days group had higher CBF than controls (P<0.01). Rat glioma inl0days,14days and18days group had higher CBV than controls (P<0.01). CD105-MVD of Rat C6glioma is10.64±6.13. CD105-MVD of controls was0.84±0.28. Significant correlations were observed between CD105-MVD. VEGF, VMI, Ki67and CBV percentage change, CBF percentage change, permeability. CBV and CBF (P<0.01).CONCLUSION:There was difference between10days,14days.18days gliomas and controls. PCT with ACZ challenge could provide useful parameters, such as CBF percentage change, CBV percentage change, permeability, CBF. and CBV, which could correlate with glioma angiogenesis. Part Ⅲ. Evaluation of human glioma angiogenesis using Perfusion CTPURPOSE:To evaluate the feasibility of human glioma perfusion CT and analyse the relationship between perfusion parameters and human glioma angiogenesis.MATERIALS AND METHODS:From January2010to February2012,55subjects underwent perfusion256-CT imaging. Among them, the histopathological results of40patients were gliomas. Perfusion CT imaging was performed in15healthy volunteers served as controls. The raw data were processed using CT perfusion software. Perfusion parameters including CBF, CBV, MTT and permeability surface-area product. The statistical analysis was performed using the SPSS17.0package. ANOVA test was used to compare the difference among controls, low grade glioma group and high grade glioma. In the multiple comparison procedure. P<0.01was considered significant after Bonferroni correction. Paired-samples t tests were used to compare the difference between perfusion parameters of peripheral and solid tissue. Pearson correlation coefficients were used to investigate relationships between CBF. CBV. permeability surface-area product and the results of CD105-MVD, Hypoxia-inducible factor-1alpha (HIF-1α), VMI. CD34-MVD and a-SMA-MVD. P less than0.05was considered significant.RESULTS:Twenty-five patients were proved to be high grade gliomas, and fifteen patients were proved to be low grade gliomas. Significant CBF, CBV difference was observed between high grade gliomas and low grade gliomas (P<0.01). Cutoff value of permeability surface-area product was2.88ml/min/100g, the sensitivity and specificity of which were96%and93%. Cutoff value of CBV was3.91ml/100g. the sensitivity and specificity of which were92%and93%. Cutoff value of CBF was38.90ml/100g/min. the sensitivity and specificity of which were92%and86%. Significant CBF, CBV difference was observed between low grade gliomas and controls (P<0.01). The periphery had higher CBF, CBV and permeability surface-area product than the solid tissue in high grade gliomas (P<0.01). There was no significant difference in MTT (P=0.14). Significant correlations were observed between CD105-MVD, HIF-la, VMI, CD34-MVD. a-SMA-MVD and CBV, CBF, permeability surface-area product (P<0.01).CONCLUSION:PCT could provide useful parameters, such as CBF, CBV and permeability surface-area product, which could correlate with glioma angiogenesis. |
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