| ObjectivesThe goal of this study was to analyze the feasibility of embolization of cerebralarteriovenous malformation by thermosensitive chitosan(CH)/β-glycerophosphate disodiumsalt (β-GP) hydrogels through in vivo/in vitro experiments.Methods1. To study the effects that thermosensitive chitosan(CH)/β-glycero-phosphate disodiumsalt(β-GP) solution-sol/gel system has on the gelation time and mechanical properties afterdifferent contrast agents (CA) were added at37℃in order to determine contrast agent releasefrom hydrogel.2. Nine swines were selected as experimental animals. Cerebral arterio-venous malformationmodel of swines was built with Rete Mirabile（REM）of swines used as nidus, one commoncarotid artery and internal jugular vein anastomosed using microsurgical technique, andexternal carotid artery ligated. The swines were put into three groups with three in one group,and arteriography was operated to common carotid arteries of swines in acute phase(1-3daysafter operation),subacute phase(1-3weeks after operation) and chronic phase(over3weeksafter operation). This is to evaluate whether the model has been successfully built througharteriography.3. Chitosan(CH)/β-glycerophosphate disodium salt (β-GP) with addition of the appropriatecontrast agent(CA) was used to conduct embolization experiment of cerebral arteriovenousmalformation model of swines.3.1To prepare2%w/v chitosan and12%w/v β-glycerophosphate disodium salt solution, andto mix it with contrast agent20%v/v Visipaque with their volume ratio of1:1:1at23℃. Toprepare fresh when it will be used.To determine gelation time with inverted tube test.3.2To embolize bilateral rete mirabile of swines with chitosan/β-glycerophosphatedisodium salt/Visipaque solution and sol/gel tran-sition system.3.2.1Eighteen swines were divided into two groups: Low flow experimental group：One group was nine swines directly embolism on one side of the REM; High flow experimentalgroup: The other group was nine swines embolism on two sides of the REM aftersuccessfully-built cerebral arteriovenous malformation models with common carotid arteryand internal jugular vein anastomosis. To conduct femoral artery puncture with Seldingertechnique, put6F catheter sheath inside femoral artery, insert5F angiography catheterselectively into the other common carotid artery of the anastomosis of blood vessel, injectVisipaque contrast agent using high pressure injector to perform angiography (injection rate4ml/s, dosage6ml) to understand the basics of AP, REM and connected arteries. Change6Fguiding catheter with the head end close to AP, conduct digital subtraction angiography(dosage6ml, rate4ml/min, pressure300mmHg). And then place a Tracker18microcatheter/Seeker14microguidewire combina-tion through guiding catheter superselectively into thestart area of REM via AP. Next, after angiography, conduct superselective angiograms usingnonionic isotonic Visipaque (dosage2ml, rate1ml/min, pressure180mmHg). Injectchitosan/β-glycerophosphate disodium salt/Visipaque solution and sol/gel transition systemthrough a microcatheter (injection rate0.2ml-0.5ml/min, dosage0.3-0.5ml, injection time1.5-3min). In order to observe the degree of embolization, conduct DSA with the injection ofcontrast agent after fluoroscopic observation of the embolization of embolic agent and theflow direction with hand push contrast agent “smoking”during the injection of embolic agent.Continue to inject embolic agent if REM fails to be occluded completely until REM does notdevelop through microcatheter angiography. Use Tracker10microcatheter to enter RA andAA superselectively to conduct superselective angiograms. Inject chitosan/β-glycero-phosphate disodium salt/Visipaque solution and sol/gel transition system(injection rate0.2ml-0.3ml/min, dosage0.1-0.2ml, injection time1.5-3min) until REM does not developthrough guiding catheter angiography. After embolotherapy, slowly withdraw themicrocatheter., press the puncture site for15minutes, and apply sterile pressure dressing.3.2.2Low flow experimental animals and high flow experimental group were separately putinto3groups, with3swines in each group. They were re-examined with angiography1week,2weeks and8weeks after embolization respectively to evaluate the existence ofrevascularization. 3.2.3After re-examinations with angiography1week,2weeks and8weeks afterembolization, the swines in each group were executed with over-dose narcotic amobarbital（100mg/kg), and then were subsequently conducted REM histological examination.4. Statistical analysisExperimental data are reported as means±SD. Data were analyzed using one wayanalysis of chi square tests with a minimum confidence level (p <0.05) for statisticalsignificance.Results1. chitosan (CH)/β--glycerophosphate disodium salt (β-GP) hydrogels have goodbiocompatibility, thermosensitivity and high viscosity. The addition of contrast agent ofcertain concentration did not impede the formation of thermosensitive chitosan hydrogels.2. The chitosan (CH)/β--glycerophosphate disodium salt (β-GP) solution has high viscosityat23℃, while it is good for the control of injection rate when it forms hydrogels at37℃.3. Same volume mixed solution of94%degree of deacetylation (DD) and2%w/v chitosan,12%w/v β--glycerophosphate disodium salt and20%v/v nonionic isotonic Visipaque hasmoderate gelation time at37℃(120.00±1.82s), which is good for embolism injection.4. The cerebral arteriovenous malformation model was successfully built throughmicrosurgical formation end-to-end anastomosis arteriovenous fistula between the rightcommon carotid artery and the internal jugular vein. With angiography, it could be seen thatblood flew through three feeding arteries, i.e, ascending pharyngeal artery AP, ramusanastomoticus RA and arteria anastomotica AA. Nidus (bilateral retia mirabilia), throughdraining vein, i.e., right ascending pharyngeal artery and right common carotid artery, goesinto the right internal jugular vein via the anastomotic stoma. It proved that this model wasacute and subacute phase animal model, which can be experimented for new embolismmaterials.5. The chitosan (CH)/β--glycerophosphate disodium salt (β-GP) hydrogel embolized thecerebral arteriovenous malformation model of swines, and REM was completely embolized.Eighteen swines of embolism was successful except one swine cause convulsions, stoppage of breathing and death in interventional procedure. Due to the embolization of REM and externalcarotid artery at the same time, one swine was found to develop ipsilateral facial paralysis,masticatory muscle weakness, little intake of food and emaciation.6. Angiography was conducted again after1week,2weeks and8weeks of embolization, andrevascularization was not seen.7. Histological examination. REM sample was generally found to be gray, tough and easy tobe peeled. Light microscopic examination (HE staining):3days after embolization bloodvessel lumens were filled with hydrogels, the vascular intima remain intact and the necrosis ofit was not seen. No neutrophils in blood vessel lumens or obvious inflammatory reactionswere found.2weeks after embolization scattered macrophages were found through REMlight microscopic exam, blood vessel lumens were still filled with hydrogels, thrombusformed, vascular intima did not drop, slight inflammatory change was found within bloodvessel lumens, scattered lymph cells were discovered in adventitia of blood vessel, thenecrosis of vascular walls was not seen.8weeks after embolization arterioles were still filledwith hydrogels through REM light microscopic exam, slight inflammatory reactions werefound, no necrosis or dropping of vascular intima was found, that is to say inflammatorymacrophages swallowed hydrogels, the vascular intima remain intact, and medial membranestructure remain normal.ConclusionThermosensitive chitosan(CH)/β--glycerophosphate disodium salt (β-GP) hydrogels canembolize cerebral arteriovenous malformation model of swines, and have the potential ofbecoming new embolic materials. |
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