Assessment Of Angiogenesis And Anti-angiogenesis Therapy In The Rat Brain Gliomas With Contrast Agent Steady-State Susceptibility MR Perfusion Technology: Pre-clinical Study

Glioma is the most common primary malignant cerebral tumors in clinical,characterizing rapid growth, strong invasiveness and high vascularization. In the pastseveral decades, the traditional therapy of malignant gliomas consisting of surgicalremoval and postoperative radiation has undergone extensive improvement from atechnical viewpoint. However, clinical outcome of these patients still remains poor.Angiogenesis is the sprouting of capillaries from preexisting blood vessels by theproliferation, differentiation and migration of endothelial cells. Angiogenesis isindispensable for the growth and metastasis of glioma, characterized by a higher vesseldensity in some regions of the tumor and by vessels of larger lumen than in normaltissues. Recently, researchers pay more attentions to the effect of anti-angiogenesisdrugs on glioma. MR perfusion imaging can reflect the biological characteristics ofglioma, often be used to evaluate vascularization of tumor. Recently, Steady-Statesusceptibility contrast-enhanced MR perfusion imaging give rise to the strong interestsof researchers, it can measure tumor blood volume (BV) and vessel size index (VSI) toobtain the information of structure and function of tumor microcirculation, which is thetarget point of anti-angiogenesis therapy.The creation of this study is in that we combined the leading mechanism of theformation and therapy of glioma with imaging study, elucidate the formation,development and therapetic reaction of gliomas in molecular imaging levels, and correlate with histopathology in order to further learn the imaging parameter.PurposeBoth normal rat brian and rat cerebral glioma models were taken with theSteady-State susceptibility contrast-enhanced MR perfusion imaging. To explore thefeasibility of this imaging technique in the evaluation of microcirculation structure andfunctional change. To observe the microvessel structure and functional change duringthe period of the formations and developments of the rat cerebral gliomas. To observethe microvessel morphology and function change of tumor when the bearing-tumor ratswere given by human recombination endostatin therapy.Material and Method1. The preparation of rat C6 cerebral gliomaAll experiments were carried out on 77 female Wistar rats. The rats wereanesthetized and placed on a stereotactic headholder. A middle scalp incision was made.The cell suspension(10⁶ cell in lul) was slowly injected using a Hamilton syringethrough a burr in right caudate nucleus (lmrn posterior to suture, 3-4mm lateral to themidline, 5.5mm depth form the dura).2. Anti-angiogenesis therapy of the bearing-tumor ratsWe randomly divided the rats with the bearing-tumor 14 days into 2 groups (studygroup and contrast group ), we injected i.p. human-recombination endostatin10mg/kg/d into the study group animals and the same dose saline into contrast groupevery day, for 7 days.3. MR perfusion scanMR perfusion imagings were performed to evaluate angiogenesis of the six-grouprats( as following :the normal rats, the rats with implanted tumor 1w, 2w, 3w, and therats with bearing-tumor 14 days before and after anti-angiogenesis therapy). Theimaging sequence including: SE , GE and DWI before injecting SPIO and SE, GEsequence after injecting SPIO, and then Gd-DTPA T1WI enhance scanning. 4. Parameter calculationThe set of region of interest (ROI) : We choose the right cortex, caudate nucleus,callosum in the normal rat ; tumor parenchyma and corresponding normal cerebralparenchyma in the opposite side in 1 week group rats; peripheral tumor area, centraltumor area and normal parenchyma in the opposite side in other rats in other groups.The calculation ofΔR₂^* andΔR2:ΔR₂^* (ΔR₂)=(-1/TE)'[ln(SI_pre/SI_post)]BV=3/(4Ï€)·[ΔR₂^*/(D/r·Δx·B0)]VSI_MRI(um)=0.424 (D/r·Δx·B0)^1/2(ΔR₂^*/ΔR₂)^3/25. The examination of histopathologyAfter MR examination , we endocardically perfused heparin-saline, then 4%paraforrnaldehyde on the anaesthesia rats. We decapitated to get the rat brainimmediately after endocadiacal purfusion . After the brains were fixed, they wereparaffin-embedded, sectioned, and examined histologically using both hematoxylin andeosin(HE) and immunohistochemical staining for mono-anti CD34 to mark endothelialcells.To observe the tumor morphous under light microscope, and use CD34immunohistochemical method to measure the radius of microvessel.6. Statistical analysisResults were expressed as the mean±SD. SPSSll.5 for Windows was used for allstatistics analysis such as one-way ANOVA, non-match T-test and correlation ofSpearman. If p＜0.05, there is a statistic difference . If p＜0.01, there is a obviousstatistic difference.Results1.The growth condition of rats with C6 brain gliomasIn the 2nd week after tumour cells implanted, some rats had slight intracranialhypertension manifestation, such as the activity and body weight slightly decrease. In the 3rd week, the symptom aggravated, foraging and foodintake decrease, some havedyscrasia, and periorbitalis hemorrhage .The therapy group had not hemiparalysis,intermittence convulsive seizure, dyscrasia, only decrease in activity, foragingfoodintake and body weight.2.The imaging results and histologic correlation of normal rats andrats with C6 brain gliomas(1).There was an obvious positive correlation between the contrast concentrationchange and theΔR₂^* andΔR₂ of 3 ROIs in normal rats .The study showed whenusing 12mg/kg contrast agent dose in MR imaging, BV and VSI_MRI we measured weremore closed to the histologic measurent results.(2). BV and VSI_MRI of peripheral tumor areas were 5.93±1.75% and 10.34±3.21um,central tumor areas were 4.64±1.92% and 14.57±7.13um, there was statisticaldifference compared with the normal cerebral tissue in the opposite side (P＜0.05) .Compared with histologic measurement results, the correlation coef-ficiency were 0.884and 0.825 (P＜0.01) respectively.(3).With bearing-tumor week increasing, BV of central tumor area had anincrease-decrease curve-like change, which related to the host blood vessel regressionin tumor central area, the decrease of blood vessel dentisy and obvious necrosishistopathologically. BV of peripheral tumor areas were increasing, because of activeblood angiogenesis in this area.(4).Gone with the time, the VSI_MRI increase gradually, especially in the centraltumor area, there was a statistical difference between the peripheral and central areaat the same time. There was obviously correlation between the VSI_MRI and VSl_histo(P＜0.01 ) .(5).After the therapy of human recombination endostatin, there was obviousdifferences between the tumor BV of therapy group and contrast group (P＜0.05) . BVand VSI_MRI of both peripheral and central areas of tumor decreased, particularly in the peripheral area (P＜0.01) , because endostatin inhibited vascularization in theperipheral tumor areas. The decrease of BV in the central tumor area did not havestatistical difference, and its mechanism was complicated.Conclusion1. There was obvious linear relationship between the contrast agent concentrationchange andΔR_z^*andΔR₂ of 3 ROIs in normal rats. It shows it was rational to useMonte Carlo model in this technique.2. As a blood-pool contrast agent, SPIO had a relative long plasma half-time,whose plasma blood concentration can remain constant for a long time, so it can beused to steady-state susceptibility contrast-enhanced MR perfusion imaging. Becauseits hydration diameter was more than 30nm, SPIO was hard to leakage the extracellularextravessel space through the destructive blood-brain barrier (BBB), which made surethe accurateness of measurement.3. The BV and VSI_MRI we measured can exactly reflect the morphology andfunctional information of microcirculation of tumors during the growth anddevelopment of gliomas.4. As a "broad-spectrum" anti-angiogenesis drug, hr-endostatincan can inhibit ratC6 glioma angiogenesis and improve the life quality of rats.