Evaluation Of Radiobiological Effects Of Carbon Ion Irradiation In Rat Glioma Based On Spectral CT

Objective:To investigat the effects of different doses of carbon-ion irradiation(¹²C⁶⁺IR)on C6 cells and rat model of C6 glioma,which include cell proliferation,activity,invasiveness,¹²C⁶⁺IR dose tolerance,as well as analyses of repeatability and imaging in vivo,and further provide a theoretical basis for heavy ion-beam radiation as a potential treatment strategy for glioma.Methods:Annexin V/PI double staining flow cytometry was used to assessapoptosis ratio(in C6 cells after¹²C⁶⁺IR),western blot of¹²C⁶⁺IR-exposed cells was used to assess expression of MAPK and Akt signal transduction pathway associated protein.Based on the analysis of the spectrum CT multi-parameter quantitativeanalysis of the clinical tumor cases,the quantitative parameters,which can reflect the tumor proliferation and invasion activity,were initially screened for the detection of C6 glioma in rats.In comparison with C6 brain glioma CT,CT perfusion andpathological results,the CT multi-parameter quantitative test model of rat brainglioma was constructed.Then we used this model to detect C6 glioma in rats before,and 7 and 14 days after exposure to¹²C⁶⁺IR.We also observed tumor-burdened rats for their survival days,changes in eating habits and physical disorders.Rats identified as close to death were sacrificed by heart perfusion under anesthesia,followed byformalin preservation of their whole brains in wax blocks,which were sectioned and immunostained for?-SMA,VEGF and CD34.Results:The percentage of surviving tumor cell in C6 cells after ¹²C⁶⁺IR?at 0,1.0,2.0,and 4.0 Gy?was 0.1833±0.3512%,6.2800±0.3606%,15.4867±0.0971%,20.5833±0.1604%respectively.Differences in colony formation rates in¹²C⁶⁺IR-exposed C6 cells?relative to intreated cells?were 0.9328±0.0394 at 0.5 Gy,0.8243±0.1209 at 1.0 Gy,and 0.6816±0.1553 at 2.0 Gy respectively?P<0.01?.Differences in ratio of BCL2/BAXin¹²C⁶⁺IR-exposed C6 cells was 0.7619±0.0360at 0.5 Gy,0.7517±0.0250 at 1.0 Gy,and 0.1181±0.0034 at 2.0 Gy respectively?P<0.01?.At 10 days after implantation of logarithmic phase C6 cells at right basal ganglia region of male Wistar rats,application of a stereotactic apparatus with enhanced CT examination showed 86.7%?13/15?success rate in establishing gliomas,and the tumor diameter was 1.8-3.8 mm?Median diameter:2.27mm?.32 cases of GS and GST spectral CT multi-phase enhanced showed that the 40-70 keV energy CT value,energy spectrum curve and iodine level of GS was lower than GST,the differences were significant?P<0.05?.In 13 tumor-burdened rats,CT perfusion showed that the blood flow and blood volume in central of tumor was higher than that in the surrounding area 12 days after C6 cell implantation,tumor surface vascular permeability was higher than that in the central area,the differences were significant?P<0.05?.Spectral CT at 65 keV showed the optimal contrast-to-noise ratio,and the slope of spectral curve,65 keV CT value,and iodine concentration in tumor were significantly higher than that in normal brain tissue?P<0.01?and were positively related to the CT perfusion parameters of CBF,CBV?P<0.01?,and ki67 expression(P<0.01.Results of 3.0 T MRI contrast scan T₁WI clearly showed lesion contours,T₂WI clearly showed the tumor's peripheral edema and liquefaction;however,quantitative detection was difficult to carry out due to the lack of objective quantitative analysis indicators.Tumor-burdened rats exposed to 0,2.0 or 4.0 Gy of¹²C⁶⁺IR,the 2.0 Gy group showed that blood perfusion is slightly reduced on solid area,but abnormal tumor angiogenesis,perfusion,and invasiveness was still strong.7 days after intervention,tumors were slightly increased,and their iodine concentrations and CT values increased gradually.The 4.0 Gy group showed solid tumor area shrinking,with internal liquefied necrotic area in tumors further expanded.In solid tumor area,the 65keV CT value,iodine concentration and spectrual curve slope showed a declining trend.Moreover,?-SMA staining showed that tumor vascular structure was incomplete,with decreased VEGF expression,CD34 expression and mature tumor blood vessel count.One rat survived on 49 days,median survival of the 4.0 Gy group?n=3?was 37 days,significantly higher than that of the 0 Gy group?median survival:27 days,maximum survival:32 days?.Conclusion:¹²C⁶⁺IR decreases protein expression in the MAPK and AKT signaling pathways,thus inducing the apoptosis and inhibiting their proliferation activity of glioma cells.The 65keV CT value,iodine concentration and spectral curve slope can be used to detect the malignancy of the tumor.Heavy ion irradiation can effectively protect normal brain tissue while inhibit tumor growth and invasion,and may be used to treat refractory and recurrent glioblastoma.