Therapeutic Effect Of Intranasal Administration With Temozolomide In Glioma Rat Model In Vivo

PartⅠ: Establishment of the rat C6glioma model and analysis of therelated factorsObjective: To establishment rat glioma model, C6glioma cells werestereotactically implanted into the right caudate nucleus of male Wistar rats.General presentation, suivival time and pathological mechanism were observed.The purpose was to explore the accurate procedure in establishing the rat gliomamodel for the further research.Methods:135male Wistar rats weighted200-300g were used to establish themodels. C6glima cells were stereotaxically implanted into the right caudatenucleus in each of135Wistar rats.(the number of cells was1-2×106/10μl).Onpostoperative day(POD)5, MR scan were used to examine the glima.Results:5Wistar rats were dead in surgery,8Wistar rats on POD1and2. Tumorgrowth is visible in98rats with MRI scans. Tumor formed at a rate of80.3%insurvived rats. Each rat fur lose luster after inoculation, activity decreased, theintake of food and water to reduce, and diarrhea.2weeks after surgery, the ratsstarted to lose weight, hemiplegia, nasal hemorrhage, congestion and othersymptoms of the eyes. Significantly worse after3weeks, according to thephenomenon such as fasted and denied water. After tumor implantation began todeath over the second weekend, to POD60days all the rats were died.Conclusion: The established method of C6glioma model is stable with a maturemethod of culture, tumor rate is higher, can satisfy the experimental research ofglioma. To improve the rate of planting tumor, cell line selection and training isthe key. The original generation of glioblastoma cells were chosed to guaranteethe cell antigenicity. The logarithmic phase cells were more conducive to cellgrowth in cell culture of glioma model. Grasp the dosage of animal anesthesia, avoid death caused by excessive anesthesia. Strict aseptic operation was used toavoid infection. Part II: Therapeutic effect of intranasal administration withtemozolomide in glioma rat model in vivoObjective: Preparation for the temozolomide(TMZ) intranasal brain targetingdrug combinations. Study the therapeutic effect of intranasal administration withtemozolomide on rat C6glioma in vivo.Methods:72Wistar rat models bearing brain glioma C6cells were selected.72rats were randomly divided into six groups which were treated with physiologicalsaline solution, TMZ by intravenous injection, gavage and low-dose,medium-dose and high-dose intranasal pathway. The tumor size, survival time andpathological changes were observed in each group. MR were used to examine thetumor volume.Results: Clear MRI imaging were captured by MR scans. The increase in tumorvolume in group1to group6was66.25±9.18mm3,35.08±4.16mm3,37.17±5.36mm3,11.17±1.28mm3,18.0±2.39mm3，and11.33±2.49mm3.ANOVAanalysisshowed that TMZ administration could significantly decrease the tumor volume inall groups (p=0.001), and intravenous group had similar effect with gavage group(p=0.861), and there was no significant differences among intranasal groups(p=0.067). The increase in tumor volume was significantly reduced in low-doseintranasal group compared with intravenous group (p=0.043) or gavage group(p=0.047), suggesting that intranasal delivery was more effective in inhibiting theglioma growth. The median survival time of the control, intravenous, gavage,low-dose, medium-dose, and high-dose intranasal groups was21.5days,18.5days,21.5days,37.5days,45.5days, and30.0days, respectively. The mediansurvival time of low-dose intranasal treatment group was significantly longer thanthat of control, intravenous, and gavage group (low-dose for eg. P=0.001, P=0.02, P=0.02), suggesting that intranasal targeted therapy could prolong the survivaltime. However, the median survival time showed no significant difference amonglow-dose, medium-dose, and high dose intranasal groups (P=0.642). HEdemonstrate that all the rat glioma models were successfully established. PCNAexpression in glioma tissues of intranasal group was significantly lower comparedwith those in other group. Apoptosis rate of glioma cells in intranasal group wasmarkedly higher compared with those in other group.Conclusion: TMZ transport by intranasal pathway could improve the efficacy inmalignant glioma chemotherapy, it has a potential value in clinic therapy ofglioma. Part III: Quantitative dynamic contrast enhanced MR scaned on C6glioma rats treated with TMZ brain targeting drugObjective: To discuss the feasibility and value of magnetic resonance perfusionimaging of dynamic quantitative study in glioma through contrasting theparameters before and after the treatment of TMZ brain targeting drug. Confirmfor the therapy effect of TMZ brain targeting drugs.Methods:10Wistar rat models were selected to establish a glioma model. MRscans on POD5, scan sequence including T1map and dynamic enhanced T1WI.One rat was failed to growth a tumor.2rats were randomly picked out from theother9rats and were sacrificed to get brain specimens. From POD6, the other ratshad been treated for5days with TMZ brain targeting drugs with a dosage of4mg/kg/d. On POD11, MR scan were performed again with the same sequence asthe first time.2rats were randomly picked out from the other rats and weresacrificed to get brain specimens. Image data were processed by Image J, Ktransand Vpwere calculated, statistical analysis were performed. The specimens wereused to perform HE staining and VEGF immunohistochemical test.Results: The ktransvalue are0.103±0.058/min and0.042±0.031/min before andafter TMZ therapy. ktranswere significantly decreased after therapy of TMZ braintargeting drugs(P=0.027). Vpvalue are0.210±0.126/min，0.136±0.058/min beforeand after TMZ therapy, the change is not significant(p=0.117). HE stainingshowed cell density decreased after treatment， positive rate of VEGF alsodecreased。Conclusion：TMZ brain targeted drugs can inhibit tumor angiogenesis， ktransdecreased after treatment， magnetic resonance perfusion imaging of dynamicquantitative research in glioma is feasible. The validity of the TMZ brain targetingdrug treatment for C6rat glioma were further confirmed by pathological andimmunohistochemical manifestations and ktransvalues.