Assessment Of Tumoral Angiogenesis And Vascular Maturity In Rat C6 Gliomas Using Perfusion CT

Partâ… Experimental Studies on Functional Response in Normal Rat Brain toHypercarbia Using Perfusion CTPURPOSE:To investigate CT perfusion value changes in normal rat brain tissue athypercarbia and analyze correlations between perfusion CT and the results ofα-SMAimmunohistochemical staining.MATERIALS AND METHODS:Total 10 male SD rats,weighting 250-300g.Perfusion CT was performed pre- and post-(15 minutes delay) inhalation of a mixture of10% CO₂ and 90% air.Perfusion CT values(CBV,CBF,PS and MTT) were measured atthe right nucleus caudatus in rat brain tissue.The rats were killed and their brains wereremoved within 24 hours after perfusion CT,the samples at nucleus caudatus in rat braintissue were examined histologically using HE and immunohistochemical staining forα-SMA.Paired t testing was used to determine differences in perfusion CT values at rightnucleus caudatus in normal rat brain pre- and post- hypercarbia.The differences in thechanging rate(%) of CBV,CBF,PS and MTT between rat right caudate nucleus and theleft caudate nucleus inhaled 10% CO₂ mixture gas were determined using paried t test.Pearson correlation coefficients were used to investigate relationships between number ofSMA positively stained vessels and changing rate(%) of CBV and CBF at right nucleuscaudatus in normal rat brain tissues.RESULTS:Normal SD rat right caudate nucleus area CBV,CBF,PS and MTT values(mean±SD) were (10.28±4.01) ml/100g,(304.95±88.77) ml/min.100g,(0.26±0.37)ml/min.100g,(1.48±0.07) s,respectively.After inhalation of a mixture of 10% CO₂ and90% air,rat right caudate nucleus area CBV,CBF values were significantly higher thanthose before hypercarbia (t=4.92,P=0.001 and t=6.75,P〈0.001).The increasing rates ofCBV,CBF were (87.14±46.45)%,(65.75±22.05)%,respectively.However,no significantdifferences were detected for changes of PS and MTT after hypercarbia (P〉0.05 for both). The changs of CBV,CBF,PS and MTT of right caudate nucleus area were not significantcompared with those of contralateral brain tissues (P〉0.05 for all).Immunohistochemicalstaining of normal SD rat brain tissues for SMA all positively expressed in smoothmuscles of vessels which present brown regular loops or tubes.The number for SMApositively stained vessels (mean±SD) were (12.7 vessels per field±3.23).Significantcorrelations were observed between the number for SMA positively stained vessels andchanging rates of CBV and CBF after hypercarbia in rat normal brain tissues (r=0.652and r=0.89,respectively; P〈0.05 for both).CONCLUSION:Perfusion CT in the change in blood PaCO₂ can reflect thehemodynamic changes in normal rat brain tissues.These results demonstrate thatchanging rates of CBV and CBF correlate well with the number of mature vessels bymeans of changing blood PaCO₂ in normal rat brain tissues.Partâ…¡Experimental Studies on Functional Response of Tumoral Vascular tohypercarbia in Rat Brain C6 Glioma Model Using Perfusion CTPURPOSE:To investigate CT perfusion paremeter changes in a C6 rat glioma model athypercarbia,analyze correlations between perfusion CT and the results ofα-SMA,Fâ…§and CD105 immunohistochemical staining and evaluate the tumoral vascular maturity usingperfusion CT.MATERIALS AND METHODS:Total 20 male SD rats,weighting 250-300g,weredivided into glioma group and control group at random.Rats in glioma group wereimplanted C6 glioma cells at right eaudate nucleus of rat brain through stereotaxicapparatus to establish a rat brain glioma model.After three weeks,perfusion CT wasperformed pre- and post-(15 minutes delay) inhalation of a mixture of 10% CO₂ and 90%air.Perfusion CT values(CBV,CBF,PS and MTT) were measured at the right nucleuscaudatus in rat brain tissue.The rats were killed and their brains were removed within 24hours after perfusion CT,the samples at nucleus caudatus in rat brain tissue wereexamined histologically using HE and immunohistochemical staining forα-SMA,Fâ…§and CD105.SMA(+)/Fâ…§-MVD(%) of each rat glioma was caculated as tumoral vascularmaturity index(VMI),and CD105-MVD/Fâ…§-MVD(%) of each rat glioma was caculated as tumoral vascular immaturity fraction(VIF).Paired t testing was used to determinedifferences in perfusion CT values of C6 gioma at right nucleus caudatus pre- and post-hypercarbia.The differences in the changing rate(%) of CBV and CBF between gliomagroup and control group inhaled 10% CO₂ mixture gas were determined usingindependent samples t test.The differences between the number of SMA positivelystaining vessels within gliomas and that of normal brain tissues in control group weredetermined using independent samples t test.Pearson correlation coefficients were usedto investigate relationships between perfusion CT parameters (CBV,CBF,PS,MTT) andimmunohistochemical parameters (Fâ…§-MVD,CD105-MVD,number of SMA positivelystained vessels),and changing rate(%) of CBV and CBF at right nucleus caudatus innormal rat brain tissues.Correlation coefficients were calculated between the changingrate(%) of CBV and CBF in hypercarbia and tumoral immunohistochemical parametersin rat gliomas by Pearson correlation analysis.RESULTS:All rat brain gliomas,three weeks after C6 glioma cells implanted,wereviewed as significantly enhanced mass on contrast enhancement CT scan.Rat gliomas atright caudate nucleus area CBV,CBF,PS and MTT values (mean±SD) were(17.35±6.73) ml/100g,(508.66±158.88) ml/min.100g,(13.92±8.96) ml/min.100g,(1.79±0.44) s,respectively.CBV,CBF and PS values in glioma group were significanthigher than those in control group (t=2.85,P=0.011; t=3.54,P=0.003; t=4.82,P〈0.001respectively).After inhalation of a mixture of 10% CO₂ and 90% air,CBV,CBF valueswithin tumors were higher than those before hypercarbia.The increasing rates of CBVand CBF within tumors were (41.21±21.28)% and (30.1±14.54)% respectively,whichwere lower than the increasing rates of CBV and CBF in normal rat brain tissues (t=2.84,P=0.014; t=4.21,P=0.001; respectively).However,no significant differences weredetected for changes of PS and MTT after hypercarbia in glioma group (P〉0.05 for both).The number for SMA positively stained vessels (mean±SD) in glioma group was (5.8vessels per field±2.2),as was statistically less than that in control group (t=5.58,P〈0.001).The number of Fâ…§-MVD and CD105-MVD in glioma group was (34.7vessels per field±7.13) and (16.6 vessels per field±4.12) respectively.VMI and vascularimmaturity fraction in glioma group was (20.46±10.31)% and (48.03±6.69)%respectively.Significant correlations were observed between the number for Fâ…§-MVD,CD105-MVD and CBV,PS in glioma group,CBF correlated well with the number for CD 105-MVD,while MTT was not significant correlation with MVD in glioma group.Nosignificant correlation was observed between perfusion CT parameters and the numberfor SMA positively stained vessels,VMI and VIF (P〉0.05 for all) in glioma group.Thechanging rates of CBV and CBF after hypercarbia in glioma group were not significantcorrelation with immunohistochemical parameters and VMI (P〉0.05 for all).CONCLUSION:Perfusion CT can reflect tumoral angiogenesis within C6 rat braingliomas.However,the changing rates of CBV and CBF in hypercarbia don't correlatewell with the number of mature vessels,VMI and VIF.Partâ…¢A Pilot Study of Antiangiogenic Therapy in Rat Brain C6 Glioma ModelUsing Perfusion CTPURPOSE:To investigate MVD changes in a C6 rat glioma model and to study thefeasibility that CT perfusion evaluates the therapeutic effect and maturity of tumorvessels atter antiangiogenic therapy.MATERIALS AND METHODS:Total 12 male SD rats,weighting 250-300g,wereimplanted C6 glioma cells at the right nucleus caudatus of rat brain through stereotaxic apparatusto establish a rat brain glioma model.Rats were divided into glioma group and treatmentgroup at random.Rats in treatment group were injected with Recombinant Human Endostatin (rhEndostatin) at a dose of 10 mg/kg/d for 7 days S.C.,and those in glioma group were injected only withthe diluent after two weeks of glioma cells implantation.Three weeks later,perfusion CT wasperformed pre- and post-(15 minutes delay) inhalation of a mixture of 10% CO₂ and 90%air.Perfusion CT parameters (CBV,CBF and PS) were measured at the right nucleuscaudatus in rat brain tissue.The rats were killed and their brains were removed within 24hours after perfusion CT,the samples at nucleus caudatus in rat brain tissue wereexamined histologically using HE and immunohistochemical staining forα-SMA,Fâ…§and CD105.SMA(+)/Fâ…§-MVD(%) of each rat glioma was caculated as tumoral vascularmaturity index(VMI).The differences in the changing rate(%) of CBV and CBF betweenglioma group and treatment group inhaled 10% CO₂ mixture gas were determined usingindependent samples t test.The differences in CT perfusion parameters,immunohistochemical parameters and VMI between glioma group and treatment group were determined using independent samples t test.Pearson correlation coefficients wereused to investigate relationships between the changing rate(%) of CBV and CBF andimmunohistochemical parameters (Fâ…§-MVD,CD105-MVD,number of SMA positivelystained vessels and VMI) after hypercarbia in treatment group.RESULTS:All rat brain gliomas,three weeks after C6 glioma cells implanted,wereviewed as significantly enhanced mass on contrast enhancement CT scan.In gliomagroup,CT parameters CBV,CBF and PS values (mean±SD) were (19.19±5.83) ml/100g,(519.31±36.35) ml/min.100g,(15.14±9.62) ml/min.100g,respectively.CBV,CBF andPS values in treatment group were (12.65±3.05)ml/100g,(468.97±109.15)ml/min,(5.08±3.83)ml/min.100g,respectively.CBV and PS values in treatment group weresignificantly lower than those in glioma group (t=2.43,P=0.043; t=2.38,P=0.039,respectively),nevertheless,no significant difference for CBF was detected betweenglioma group and treatment group (t=1.07,P=0.31).The increasing rates of CBV andCBF in treatment group were (53.81±10.29)% and (58.94±26.23)% respectively,whichwere remarkably higher than the increasing rates of CBV and CBF in glioma group [CBV:(34.08±11.65)%,CBF:(27.3±14.77)%] aider hypercarbia (t=3.11,P=0.011; t=2.58,P=0.033; respectively).The number for SMA positively stained vessels in glioma groupwas less than that in treatment group,but no significantly statistical difference was foundbetween two groups (t=1.42,P=0.187).The number of Fâ…§-MVD and CD105-MVD inglioma group was (30.17 vessels per field±8.71) and (15.5 vessels per field±8.76)respectively.The number of Fâ…§-MVD and CD 105-MVD intreatment group was (19.17vessels per field±2.64) and (5.67 vessels per field±4.18) respectively,as weresignificantly lower than those in glioma (t=2.42,P=0.036 and t=2.48,P=0.041).VMI intreatment group was significantly higher compared with that in glioma group[(46.01±17.65)% vs (20.3±11.7)%,P=0.016].The changing rates of CBV afterhypercarbia in treatment group were significant correlation with CD105-MVD and VMI(P〈0.05 for both).No significant correlation was observed between the changing rates ofCBF after hypercarbia and immunohistochemical parameters and VMI (P〉0.05 for all) intreatment group.The changing rates of CBV and CBF after hypercarbia in glioma groupwere not significant correlation with immunohistochemical parameters and VMI (P〉0.05for all).CONCLUSION:Tumoral angiogenesis in C6 rat brain glioma can be inhabited using rh Endostatin.Perfusion CT can predict the effect of antiangiogenic therapy in C6 rat braingliomas model and the changing rates of CBV in hypercarbia may reflect VMI afterantiangiogenic therapy.