新型栓塞材料聚氨酯—钡铁氧化合物微粒的生物相容性及栓塞实验
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摘要
微粒类栓塞材料是最早用于血管内栓塞的材料。它们主要是靠血流的冲击作用将栓塞微粒送入供血丰富的病变区，减慢血流速度，充盈并聚集于病变血管床，使供血动脉及其分支栓塞，以达到彻底治愈病变的作用。微粒类栓塞剂在临床上应用范围广泛，在血供丰富肿瘤的术前栓塞及动静脉畸形的栓塞治疗等方面，疗效显著。但是，自从液体性栓塞材料问世并广泛应用于临床以来，神经外科血管内治疗微粒类栓塞材料的应用及开发稍显滞后。单独应用微粒栓塞材料治疗脑血管疾病的越来越少，一般多用于和液体材料的联合使用，或是血供丰富脑肿瘤的术前栓塞。但是不能因此而否定微粒类栓塞材料在神经外科领域的应用价值。在栓塞治疗脑血管病变时，微粒材料有其独到的优势：生物相容性好；操作安全，适合粒径的微粒材料可以到达靶血管的终末支，从而达到彻底治愈病变。此点非液体类栓塞材料所能及。
本课题开发的新型BaFe_(12)O_(19)微粒及聚氨酯-BaFe_(12)O_(19)(PU-BaFe_(12)O_(19))复合型微粒，利用材料本身的吸波和结晶性能解决了不透X线的问题，且悬浮性能良好的球形微粒也避免了在栓塞治疗过程中堵塞微导管。本实验着重研究了该两种栓塞材料作为一种新开发的、具有潜在应用前景的新型血管内栓塞材料的生物相容性及动物栓塞效果。通过对该两种微粒进行生物相容性实验，来评价该两种材料的生物相容性；通过Wistar大鼠的颈动脉栓塞、新西兰兔的肾动脉栓塞及家猪颅底微血管网(RMB)的栓塞实验研究来评价该微粒栓塞材料的栓塞效果。从而为进一步的临床应用研究提供实验依据。
第一部分BaFe_(12)O_(19)和PU-BaFe_(12)O_(19)的生物相容性研究
本部分着重进行两种微粒材料的生物相容性研究，包括Ames致突变实验、小鼠骨髓细胞微核试验、体外细胞毒性实验、全身急性、亚急性及慢性毒性实验、溶血实验、出血和凝血时间测定、凝血功能实验等。
1．Ames实验：为体外致突变实验，采用鼠伤寒沙门氏菌组氨酸营养缺陷型TA_(98)、TA_(100)为指示菌株；已知致突变物敌克松(Dexon)、二氨基芴(2-AF)为阳性对照，无菌蒸馏水为阴性对照，采用平皿掺入法进行实验。实验分非活化实验(-S9)与活化实验(+S9)。测试结果以致突变比值(MR=诱变菌回落数Rt／自发回变菌落数Rc)表示。判断标准(WHO)：试验组回变菌落数超过阴性对照组2倍以上者为阳性。实验结果：BaFe_(12)O_(19)和PU-BaFe_(12)O_(19)微粒材料各浓度浸提液实验组的MR值均小于2，而Dexon及2-AF两阳性对照组的MR值均大于2，表明两种材料体外实验均无致突变作用。
2．小鼠骨髓细胞微核试验：为体内致突变实验，采用腹腔注射材料浸提液后行骨髓腔悬浮液涂片，Giemsa染色，显微镜下计数轮廓完整的嗜多染红细胞(Polychronic Cell，PCE)，统计其中含有微核的PCE数，计算各组微核出现的频率(‰)，并进行统计分析。生理盐水(阴性对照)、环磷酚胺(阳性对照)同时行平行对照实验。实验结果表明，两种材料浸提液的三个实验组的骨髓微核细胞率均在正常范围。而生理盐水阴性对照组的微核细胞率为1．93±0．56‰，环磷酰胺阳性对照组的为19．53±2．14‰。经统计分析，两种微粒材料浸提液实验组与生理盐水组对小鼠骨髓微核细胞率的影响相互之间无显著性差异(P>0．05)，而环磷酰胺阳性对照组与三个实验组、生理盐水组之间均存在显著性差异(P<0．001)。
3．体外细胞毒性实验：采用材料浸提液与小鼠成纤维细胞L929共同培养观察其对细胞的毒性作用。实验同时行培养液(阴性对照)、64g/L苯酚(阳性对照)作平行对照实验。分3个时间段即24、48、72h行MTT比色，在酶联免疫检测仪上测定各孔光吸收值(A值)，计算细胞相对增殖率(relative growth rate，RGR)。[RGR=(实验组OD值-空白OD值)/(阴性对照组OD值-空白OD值)]，并进行细胞毒性分级。结果判断标准：根据细胞毒性分级(RGR≥100％0级；75％-99％Ⅰ级：50％-74％Ⅱ级；25％-49％Ⅲ级；1％-24％Ⅳ级；0Ⅴ级)评判细胞毒性：0级和Ⅰ级被认为没有细胞毒性，Ⅱ级为轻度细胞毒性，Ⅲ级和Ⅳ级为中度细胞毒性，Ⅴ级为明显细胞毒性。实验发现，BaFe_(12)O_(19)和PU-BaFe_(12)O_(19)微粒浸提液实验组细胞生长情况与阴性对照组相似，未见明显细胞生长抑制现象。两种材料不同浓度浸提液各实验组对L929细胞的细胞毒性均为Ⅰ级，而生理盐水组为0级，苯酚阳性对照组的细胞毒性为Ⅴ级。实验表明该两种材料无细胞毒性。
4．全身急性、亚急性、慢性毒性实验：采用腹腔注射混悬液的方式观察其对小鼠的毒性作用。用无菌生理盐水将BaFe_(12)O_(19)和PU-BaFe_(12)O_(19)微粒材料分别配制成10mg／mL和5mg／mL混悬液。急性组每只腹腔注射混悬液0．5mL，亚急性组于第1、3、5d腹腔注射0．5mL混悬液，慢性组于1、7、15、30d腹腔注射0．5mL混悬液，生理盐水0．5mL同期注射作平行对照。观察小鼠一般情况，包括食欲、活动、体重、毒性反应及死亡动物数。根据毒性评价分级来评价毒性反应。按分组分别于7 d、30d、60d后处死小鼠，取脑、心、肝、脾、肺、肾、腹膜标本，行组织学观察。结果发现，全身急性、亚急性、及慢性毒性实验组动物一般情况良好，所有实验动物均无死亡、无惊厥、瘫痪、呼吸抑制、腹泻、运动减少．和体重下降等不良反应。按毒性分级，BaFe_(12)O_(19)微粒属无毒级。各标本心、肝、脾、肺、肾、脑及腹膜等组织学检查未见组织和细胞的变性及坏死，无渗出及炎细胞浸润。实验表明该两种材料无全身的毒性反应。
5．溶血实验：采用材料浸提液与红细胞悬液接触的方式检验材料有无溶血作用。抽取新鲜静脉血5ml，加入肝素抗凝，玻璃棒搅动，除去纤维蛋白原。取4ml，加入0．9％生理盐水20ml稀释，摇匀，离心，得到沉淀红细胞。将红细胞用生理盐水配制成2％的红细胞悬液，加入材料浸提液2ml，混匀后于37℃水浴，观察4h后离心，取上清液1 ml，加入0．1％Na_2CO_3溶液4 ml，在分光光度计540 nm处测定各样本吸光度，计算溶血率。实验结果：BaFe_(12)O_(19)和PU-BaFe_(12)O_(19)微粒材料浸提液和生理盐水阴性对照组均无溶血现象，双蒸水阳性对照组全部溶血。经计算得知，BaFe_(12)O_(19)和PU-BaFe_(12)O_(19)微粒实验组的溶血率分别为2．08％和2．43％，符合ISO规定的不大于5％的标准。实验表明该材料不引起溶血。
6．出血、凝血时间测定：采用小鼠腹腔注射材料混悬液7d后检测其出血及凝血时间的方式观察其影响。取10mg／ml浓度BaFe_(12)O_(19)微粒混悬液、5mg／ml浓度的PU-BaFe_(12)O_(19)混悬液，按实验分组每只小鼠腹腔注射混悬液0．5ml，对照组腹腔注射生理盐水1ml，7d后测定出血及凝血时间。实验结果行统计分析：两种材料浸提液实验组和对照组小鼠的出血、凝血时间无显著差异(p>0．05)。表明该微粒栓塞材料对小鼠的出血及凝血时间无明显影响。
7．凝血功能测定：采用材料浸提液与Wistar大鼠新鲜血液直接接触的方式检验材料对凝血功能的影响。每只大鼠尾静脉抽血1ml，分别放入20只PT抗凝管。实验组每管注入饱和微粒浸提液1ml，对照组加入1ml生理盐水。30 min后测量凝血功能全套。实验数据经统计分析后表明，两种微粒材料浸提液对血液凝血功能无显著影响，各实验组的凝血酶原时间、纤维蛋白原测定、活化部分凝血活酶时间、凝血酶时间等与对照组无显著差别，均在正常范围，说明该两种材料对大鼠的凝血功能无明显影响。
第二部分BaFe_(12)O_(19)和PU-BaFe_(12)O_(19)栓塞Wistar大鼠颈动脉分支的实验研究
目的：评价BaFe_(12)O_(19)和PU-BaFe_(12)O_(19)两种微粒栓塞材料对Wistar大鼠颈外及颈内动脉分支的栓塞效果，观察栓塞后大鼠的神经功能变化及存活情况，了解该两种材料对血管内膜及脑组织所产生的病理变化及影响。
方法：1．实验分组：BaFe_(12)O_(19)栓塞组、PU-BaFe_(12)O_(19)栓塞组和BaFe_(12)O_(19)与PU-BaFe_(12)O_(19)联合栓塞组、生理盐水对照组，每组8只大鼠。2．方法：采用套管针穿刺颈动脉法进行栓塞。用浓度为2．5mg/ml、5mg／ml的PU-BaFe_(12)O_(19)混悬液和浓度为6mg／ml、12mg/ml的BaFe_(12)O_(19)混悬液按实验分组进行栓塞，按“先低浓度后高浓度”、“先小粒径后大粒径”的原则栓塞。栓塞的靶血管为颈外动脉的各级分支和颈内动脉的各级分支。栓塞分两部，即先注射低浓度、小微粒材料，然后注射高浓度及大微粒栓塞材料，造影观察末梢血管栓塞后的闭塞情况。不要求将各个分支血管完全闭塞。术后注意观察动物的存活、生理行为及神经功能的变化情况，并于术后的第一、四、八周时行存活动物的造影复查及病理组织检查。
结果：1．术中栓塞顺利，材料推注阻力均匀，无堵管现象；2．术中可见栓塞材料注射后颈外及颈内动脉的一些末梢分支血管被成功栓塞。BaFe_(12)O_(19)和PU-BaFe_(12)O_(19)栓塞组由于注射微粒较小，术后动物存活及神经功能状况要好，而BaFe_(12)O_(19)小微粒联合PU-BaFe_(12)O_(19)较大微粒栓塞组动物术后的死亡率高，神经功能状况较差。栓塞后存活动物的血管造影复查，未发现原闭塞血管的再通现象。3．脑大体标本检查：早期可见脑表面的片状缺血区，晚期可见脑表面梗死区，皮质凹陷；病理检查见血管腔内填充栓塞材料，早期可见血管内栓塞材料周围炎性细胞聚集浸润，血管内膜无明显破坏；晚期可见异物巨细胞，栓塞剂在血管内存留良好，部分血管内膜不同程度的破坏，未见血管中层弹力板明显破坏。血管内及周围可见不同程度的炎细胞浸润。未见栓塞材料逸出血管壁外。结论：BaFe_(12)O_(19)和PU-BaFe_(12)O_(19)两种栓塞材料可以栓塞Wistar大鼠颈外及颈内动脉分支末梢血管，且栓塞作用持久，血管不易再通。组织检查发现该微粒可引起血管周围轻度的炎性反应，未见明显血管壁破坏。
第三部分BaFe_(12)O_(19)和PU-BaFe_(12)O_(19)微粒栓塞新西兰兔肾动脉的实验研究
目的：应用两种微粒材料栓塞兔肾动脉的各级分支，评价BaFe_(12)O_(19)和PU-BaFe_(12)O_(19)微粒材料的短期及长期栓塞效果，了解肾动脉栓塞后肾脏的病理变化情况。
方法：1．实验分组：BaFe_(12)O_(19)栓塞组、PU-BaFe_(12)O_(19)栓塞组和BaFe_(12)O_(19)与PU-BaFe_(12)O_(19)联合栓塞组，每组8只兔子。2．方法：采用切开股动脉法置入微导管，用浓度为2．5mg／ml、5mg／ml的PU-BaFe_(12)O_(19)混悬液和浓度为6mg／ml、12mg/ml的BaFe_(12)O_(19)混悬液按实验分组进行栓塞，采用“先低浓度后高浓度”、“先小粒径后大粒径”的栓塞方法，以使能够栓塞远端终末支目的血管床。栓塞的靶血管为肾动脉的多个二级分支或三级分支。术中成功栓塞目的血管后，及时造影复查栓塞血管的显影情况，必要时追加栓塞剂量，直至完全栓塞。动物存活的中间时段行彩色多普勒超声检查，观察栓塞侧肾动脉的血流情况。术后两周、四周、八周、三个月时行栓塞侧肾动脉造影复查，并处死动物取肾脏标本，观察大体形态及行组织学检查。
结果：1．术中栓塞顺利，材料推注阻力均匀，无堵管现象；2．术中均可成功完全栓塞目的靶血管，PU-BaFe_(12)O_(19)栓塞组、BaFe_(12)O_(19)和PU-BaFe_(12)O_(19)联合栓塞组栓塞的血管造影复查，均未发现栓塞后的血管出现再通现象，无侧枝循环出现，再通率为0％。；而单纯BaFe_(12)O_(19)组在栓塞的18条肾动脉分支血管中有两条复发再通，再通率为11％。3．肾脏大体标本检查可见早期肾脏表面的片状梗死区，晚期肾脏萎缩、变小，皮质凹陷；病理检查见血管腔内填充栓塞材料，早期肾间质内轻度炎性反应，血管壁无明显破坏，晚期肾组织结构稀疏，小球小管结构大多消失，肾组织呈弥漫性微粒样变性，间质小血管管壁透明变性，血管周围可见炎性细胞浸润。
结论：PU-BaFe_(12)O_(19)单独应用或与BaFe_(12)O_(19)联合应用对新西兰兔肾动脉的栓塞效果较好，而单独应用BaFe_(12)O_(19)微粒材料效果稍差，有一定的复发再通可能。组织学检查提示该两种材料对血管的影响作用较轻。
第四部分家猪脑AVM不同流量模型的建立及栓塞实验
目的：1．利用家猪先天颅底微血管网(RMB)通过显微血管吻合建立高、中、低三种不同流量的脑AVM动物模型；2．使用该两种微粒材料栓塞三种不同流量的脑AVM动物模型，探讨其栓塞不同流量脑AVM的技术可行性、栓塞有效性及安全性；3．通过栓塞后的病理检测评价该材料对血管及组织的影响。
方法：1．动物模型的建立：8只动物随机分成2只低流量组、3只中流量组及3只高流量组。低流量模型：利用猪天然颅底RMB，无手术干预；中流量模型：单纯结扎右侧颈总动脉，造成右侧颈总动脉结扎远端以上由对侧咽升动脉经双侧RMB供血，人为增大了双侧RMB的流速及流量；高流量模型：行右侧颈总动脉远端与右颈内静脉近端行端-侧或端端吻合，形成以左侧咽升动脉为供血动脉，双侧RMB为畸形团，右颈总动脉→右颈内静脉为回流静脉的脑AVM模型。2．脑AVM模型栓塞：两种BaFe_(12)O_(19)和PU-BaFe_(12)O_(19)微粒材料分别和联合对三个流量组的动物模型行栓塞实验。各组动物采用股动脉切开插管法，将微导管置于左侧咽升动脉内，注射栓塞材料栓塞RMB。造影证实完全栓塞后，实验完毕。中高流量组动物栓塞后第1～3天处死，取RMB和脑组织作病理学检查。低流量组动物2周及2个月后复查造影。处死前再次造影，检查RMB是否显影。动物处死后取双侧RMB及部分周围脑组织行组织病理学检查。
结果：1．动物模型：显微吻合血管操作顺利，成功建立中、高流量脑AVM模型，高流量组1只动物模型出现吻合口狭窄。2．全部8只动物均顺利完栓塞RMB，复查造影未见RMB显影。栓塞均在未更换导管情况下完成栓塞，未发现堵塞导管现象。急性期病理检查见栓塞材料在RMB内弥散铸型良好，栓塞的微粒材料周围可见炎性细胞浸润，血管内膜破坏不明显；慢性期可见血管内仍被栓塞微粒材料及夹杂的血栓所闭塞。栓塞物周围可见异物巨细胞。栓塞剂在血管内存留良好，血管壁不同程度的变薄及破坏，血管内及周围可见不同程度的异物巨细胞及炎细胞浸润。所有标本未发现微粒材料外逸现象。
结论：1．通过结扎一侧颈总动脉，以及行颈部动静脉血管吻合，改变血流供应及回流通路，能够建立不同流量的脑AVM模型。2．微粒材料BaFe_(12)O_(19)和PU-BaFe_(12)O_(19)能够有效栓塞不同流量的AVM模型，操作方便、安全性高，有望成为一种新型的栓塞脑AVM的微粒性栓塞材料。
Granule materials were first used in endovascular embolization for curing diseases. With the affluxion of flood, the materials reached and gathered together in vessel beds, slowing down the blood stream and blocked the nutrition artery and other diseased branches. Granule materials embolization is widely used in clinic especially for pre-operation of tumors and endovascular treatment of cerebral arteriovenous malformations (AVM). However, with the appearance and application of liquid materials, the development, of Granule materials appears slowly relatively. Even though they are rarely used alone, and usually used together with liquid embolic materials. However, granule materials shouldn't be ignored in clinic for so many advantages, such as ideal biocompatibility, safe and easily control in operation and suitable diameter particles can be used to cure various diseased vessals.
This innovative BaFe_(12)O_(19) particles can be seen in X-ray, having spherical shape and good suspension quality, thus they wouldn't block the catheter easily as some liquid materials. The author carried out some experiments to examine the biocompatibility and embolization effect of BaFe_(12)O_(19) and polyurethane-BaFe_(12)O_(19) (PU-BaFe_(12)O_(19)) particles as a new kind of embolic materials. Embolization experiments were evaluated in carotid artery of Wista rats, renal artery of New Zealand rabbits and rete mirabile (RMB) of pigs, providing some positive evidences for further clinical studies.
Part 1: Study on Biocompatibility of BaFe_(12)O_(19) andPU-BaFe_(12)O_(19) Particles
The experiment of biocompatibility for BaFe_(12)O_(19) and PU-BaFe_(12)O_(19) particles was evaluated including Ames test, bone marrow micronucleus test, acute and subacute systemic toxicity test, hemolysis test, hemopexis test, bleeding and clotting time measurement test.
1. Ames test is a vitro mutagenesis test. The indicator strains used in this test were Salmonella typhimurium TA_(98) and TA_(100) with or without S9 The positive control objects were mutagens of Dexon and diaminofluorene (2-AF), while the negative control was sterile distilled water. The results was evaluated by mutation rate (MR=mutagenesis colony number/ spontaneous colony number). Standards (WHO): the test is positive if MR>2. The result showed that MR value of all concentration of leaching liquors of BaFe_(12)O_(19) and polyurethane-BaFe_(12)O_(19) was smaller than 2 while bigger than 2 for both Dexon and 2-AF groups. This results Indicates these two experimental materials have no mutagenesis effection.
2. Bone marrow micronucleus test in mice is a vivo mutagenesis test. After intraperitoneal injection of leaching liquor for 30h, cavum ossis suspension was taken and smeared, then the number of polychronic cells (PCE) containing micronucleus was counted and the frequency calculated (‰). The positive and negative control was cyclophosphamide and saline, tested in parallel controlled trial. The results showed that negative results in three groups of leaching liquor of both materials. The frequency of micronucleated cell for normal saline group was 1.93±0.56‰while for cyclophosphamide was 19.53±2.14‰. By statistical analysis of the data, this results indicates that three experiment groups had no significant difference compare to normal saline group(P>0.05) , and the cyclophosphamide group showed significant difference compare to the experiment and normal saline groups(P<0.001).
3. Vitro cytotoxicity test. L929 (fibroblast of mice) cells were cultivated with leaching liquor so as to evaluate its toxicity. The negative control was culture fluid while the positive was Phenol (64g/L), and the three groups were tested in parallel controlled trial. The samples were tested by MTT assay and optical density values (OD) were measured at 24h, 48h, 72h respectively. The OD value and relative growth rate (RGR) was calculated and the cytotoxicity was evaluated. Standars: 0 Ord (RGR=100%) I Ord (RGR 75%-99%) II Ord (RGR 50%-74%) IIIOrd (RGR 25%-49%) IVOrd (RGR 1%-24%) V Ord (RGR=0); 0& I (no cytotoxicity) II (slight cytotoxicity) III&IV(middling cytotoxicity) V (manifest cytotoxicity). The results showed that L929 cells growth condition in experiment groups similarly to negative group, no obvious growth inhibition. Cytotoxicity of different concentration of leaching liquor of both experiment materials to the L929 cells was I Ord, the NS group 0 Ord and the Phenol was V Ord. So we can draw a conclusion that both materials have no cytotoxic effect.
4. Acute, subacute and chronic systemic toxicity test. NIH rats were Intraperitoneal injection of experiment materials suspension so as to observe the toxicity in mice. With sterile saline BaFe_(12)O_(19) and PU-BaFe_(12)O_(19) particulate materials were prepared in 10 mg / ml and 5 mg / ml suspension. Each mouse of every group underwent intraperitoneal injection of 0.5 ml suspension while the control group injection of NS 0.5mL during the same period in parallel control. The general condition of mice was observed including appetite, activity, weight change, toxic reactions and even the number of dead animals. According to different groups, the mice were sacrificed in differently period respectively. Their brain, heart, liver, spleen, lung, kidney, pancreas and peritoneum were taken respectively prepared for histology analysis. The results showed that general condition of all experimental animals were well and no one death, no convulsions, no paralysis, no respiratory depression, no diarrhea, no activity reduce or weight loss. In all specimens there was no cell degeneration or necrosis, no exudation and infiltration of inflammatory cells. The experiments showed the two materials had no systemic toxicity.
5. Hemolysis test. To determine whether the materials have hemolysis effect by means of adding the leaching liquor into the erythrocyte suspensions. 5ml of fresh blood were collected, heparin added, glass rod to be used stirred to remove fibrinogen. Take 4ml of above blood, add 20ml NS to dilute, shake even, centrifuge and make RBC sediment. RBC was diluted into 2% suspension by adding NS, and add 2ml leaching liquor into it, shake even, bathe in 37℃water and centrifuge observed. After 4h, 1 ml of the supernatant was taken, 4 ml of 0.1% Na2CO3 solution were added, and then optical density was measured on spectrophotometer at 540 nm, then calculated the hemolysis rate. The results showed hemolysis rate of BaFe_(12)O_(19) and PU- BaFe_(12)O_(19) particulate were 2.08% and 2.43% respectively, it achieve the standard of not exceeding 5% according to ISO. This experiment indicates that the materials do not cause hemolysis.
6. Bleeding and clotting time test. To observe the impact on the bleeding and clotting time after intraperitoneal injection of materials suspension. Materials suspension was intraperitoneal injected into each experimental mouse, and 1 ml NS for each mouse in control group. Bleeding and clotting time was detected after 7d. The results showed that there was no significant difference (p>0.05) between experimental groups and control group, indicating these two kinds of materials have no obvious impact on mice bleeding and clotting time.
7. Blood hemopexis function test. The leaching liquor and blood of Wistar rat were directly added together to determine whether the materials have impact on blood hemopexis function. 1ml of each rat tail vein blood was respectively added to 20 anticoagulant tubes. Every experiment tube was added with 1 ml of leaching liquor while the control group 1 ml of NS. Blood coagulation time was measured after 30 mins. Statistical analysis of experimental data showed that there was no significant difference in prothrombin time, Fibrinogen assay, activated partial thromboplastin time and thrombin time between experimental group and control group.
Part 2 Experiment Study on Embolization of Carotid Artery Brancheswith BaFe_(12)O_(19) and PU-BaFe_(12)O_(19) Particles in Wistar Rats
Objective: To evaluate the embolization effect on carotid artery branches with BaFe_(12)O_(19) and PU-BaFe_(12)O_(19) particles in Wistar rats, to observe the changes in neural function and survival status, and to get a better understanding of the impact on pathology on vascular endothelium and brain tissues.
Method: 1. Experiment groups: (1) BaFe_(12)O_(19) embolization group; (2) PU-BaFe_(12)O_(19) embolization group; (3) BaFe_(12)O_(19) and PU-BaFe_(12)O_(19) co-embolization group; (4) NS control group, 8 rats for each group. 2. Methods: Puncture trocars were used to puncture the carotid artery then finish embolization. With the concentration of 2.5mg/ml, 5mg/ml of the PU-BaFe_(12)O_(19) suspension and 6mg/ml, 12mg/ml of BaFe_(12)O_(19), distal branches of ECA and ICA were embolized successfully according to the principle of "low concentration first" "small size particles first". Embolization consisted of two steps. Low concentration of small particles was first injected, and then the high concentrations of bigger particles were injected subsequently. Angiographic was made to observe the occlusion situation of the artery branches. Not every branch to be embolized imperatively. To keep observation on the survival, behavior and physiological changes in nerve function of animals, and to recheck angiographic of carotid arteries of survival animals, and then make macroscopic and microscopic examination at 1w, 4w, 8w after the operation.
Results: 1. The endovascular operation was successful performed and the particle injection resistance was well-distributed without blocking the catheters; 2. Some branches of ECA and ICA were embolized successfully in the operation, but the survival and neurological condition were better for using little particle of BaFe_(12)O_(19) and PU-BaFe_(12)O_(19), while much worse for using BaFe_(12)O_(19) and PU-BaFe_(12)O_(19) co-embolized. DSA for survived animals showed there weren't recanalization of previous occluded vessels. 3. Pathological detection showed some sporadic lamellar ischemia regions on brain surface observed in early phase, while infarction and introcession of cortex formed in late phase. Histological examination showed vessels were stuffed with these embolic materials, no obvious damage to the wall of vessels, and inflammatory cells gathered around the Embolization particles in early phase, while forein-body giant cells were observed in late phase. The embolic agents stayed well in vessels, some vascular walls thinningz, and varying degrees of damage, varying degrees of inflammatory cells was found around blood vessels.
Conclusion: BaFe_(12)O_(19) and PU-BaFe_(12)O_(19) as embolic materials can be used to embolize branches of ECA and ICA in Wistar rats, and the embolization effect is duration without artery recanalization, only mild vessal endomembrane inflammatory reaction in pathological examination.
Part 3 Experiment Study on Embolization of Renal Artery withBaFe_(12)O_(19) and PU-BaFe_(12)O_(19) Particles in NewZealand Rabbit
Objective: To evaluate the long-term and short-term Embolization effects of rabbit renal artery embolization with BaFe_(12)O_(19) and PU- BaFe_(12)O_(19) particles and to investigate the pathological changes in kidney afer Embolization.
Method: 1. According to the kinds of particles, 24 rabbits were divided averagely into three groups: Experimental groups: BaFe_(12)O_(19) embolization group, BaFe_(12)O_(19) embolization group and BaFe_(12)O_(19) and BaFe_(12)O_(19) co-embolization group, 8 rabbits for each group. 2. Methods: Use the operation of slitting femoral artery to cannulate 4F introducer sheath, and the micro-catheter was inserted into the renal artery of one side. With the concentration of 2.5mg/ml, 5mg/ml of the PU-BaFe_(12)O_(19) suspension and 6mg/ml, 12mg/ml of BaFe_(12)O_(19), the distal terminal branches of renal artery were embolized, according to the principle of "low concentration first", "small size particles first". The target vessels were second grade renal artery branches or the third grade artery branches. Angiography was taken immediately after embolized to review the target vessels whether or not visualization, if necessary, additional dose injected until embolization completely. Color Doppler ultrasound examination was taken at the middle period of survived animals to observe the blood stream of the embolized renal artery. Angiographic was rechecked for the embolized artery afer operation of 1w, 2w, 4w, 8w respectively, then the animals were killed, kidney specimens to be observed and for histological examination.
Results: 1. The endovascular operation was successful and the injection resistance was smooth without blocking the tubes; 2. The target vessels were successfully embolized and DSA of PU-BaFe_(12)O_(19) embolization group, BaFe_(12)O_(19) and BaFe_(12)O_(19) co-embolization showed no collateral circulation, no recanalization. But 2 of 18 renal artery branches underwent recanalization in the simple BaFe_(12)O_(19) group, the recanalization rate was 11%. 3. Kidney specimen examination showed flake infarction on the surface of early renal, atrophy in late renal and cortical depression; pathological examination showed vessels stuffed with embolization agents, mild inflammatory reaction in early renal interstitial, no destruction to vessel wall. The pathological specimen of late renal showed that the structure was sparse, the structure of glomerulus and renal tubules mostly disappeared and kidney showed diffuse granulation degeneration, mesenchymal small vessels wall hyalinized, perivascular infiltration of inflammatory cells visible.
Conclusion: PU-BaFe_(12)O_(19) alone or combined with BaFe_(12)O_(19) can be used to embolize the New Zealand rabbit renal artery effectively, while a separate application of BaFe_(12)O_(19) particles alone were lower effect than above mentioned. Some arteries have the possibility of recanalization and the toxic histopathologic for vessal response to the material is mild.
Part4 Modeling and Embolization for Different Flows ofArteriovenous Malformation (AVM) of Brain in Swine
Objective: 1. To establish three flows of brain AVM animal models with high, medium and low flows by microvascular anastomosis on the base of pig's rete mirabile (RMB). 2. To research the technical feasibility, effectiveness and safety of embolization by using these two particle materials to embolize AVM of animal model. 3. To evaluate the impact of the experiment materials on vessels and brain tissues by making macroscopic and microscopic examination.
Method: 1. Animal models: 8 animals were divided into three groups randomly-2 for low-flow group, 3 for medial-flow group and 3 for high-flow group. The normal RMB is used as low-flow of AVM model. The medial flow model is established by ligation of common carotid artery of one side, which increases the blood flow through bilateral RMB. The high flow model is built on by end-side or end-end vascular anastomosis between right carotid artery proximal end and right jugular vein distal end, which increases greatly the blood flow of RMB because the direct arteriovenous fistula. In high flow model, left and right ascending pharyngeal (AP) artery acted as afferent artery and draining vein respectively, the bilateral RMB as AVM nidus. 2. Embolization of cerebral AVM model: BaFe_(12)O_(19), PU-BaFe_(12)O_(19) and mixture of the two materials embolized the three groups respectively. All animals adopted femoral artery incision intubation, micro-catheter being placed in the left ascending pharyngeal artery where injection of embolic materials can embolize RMB entirely. Animals in the high and medial-flow groups were sacrificed on 1 -3d. while animals in low flow group were taken angiography after 2 weeks and 2 months respectively, then the swine were sacrificed and their bilateral RMB including some surrounding brain tissues were taken for macroscopic and microscopic examination.
Results: 1. Animal models: The vascular anastomosis operation was successful and medial-flow, high-flow cerebral AVM models were established ideally, only one animal in high-flow group undergoing anastomotic stenosis. 2. All RMB of experimental animals were embolized successfully and angiography rechecked showed no visualization of RMB. No catheter blocked and therefore the catheter wasn't replaced in the embolization process. Acute pathological examination showed that the embolic material was well dispersed and located in the mold RMB. Inflammatory cells Infiltrated and no obvious damage to the endothelium. Chronic pathological examination showed the vessels still blocked by experiment material with foreign body giant cells surround, some vessel walls underwent varying degrees of thinning and damage. All specimens showed no leak of embolic material.
Conclusions: 1. Different cerebral AVM animal models can be formed by one carotid artery ligation as well as the anastomosis of carotid artery and jugular vein changing the blood supply and pathway. 2. Granular materials of BaFe_(12)O_(19) and PU-BaFe_(12)O_(19) can be embolized AVM models of different flow and have many advantages such as operation convenient, safe embolization and so on. So BaFe_(12)O_(19) maybe become a new type of embolization material for cerebral AVM and has great potential as a therapeutic embolic agent.
本课题开发的新型BaFe_(12)O_(19)微粒及聚氨酯-BaFe_(12)O_(19)(PU-BaFe_(12)O_(19))复合型微粒，利用材料本身的吸波和结晶性能解决了不透X线的问题，且悬浮性能良好的球形微粒也避免了在栓塞治疗过程中堵塞微导管。本实验着重研究了该两种栓塞材料作为一种新开发的、具有潜在应用前景的新型血管内栓塞材料的生物相容性及动物栓塞效果。通过对该两种微粒进行生物相容性实验，来评价该两种材料的生物相容性；通过Wistar大鼠的颈动脉栓塞、新西兰兔的肾动脉栓塞及家猪颅底微血管网(RMB)的栓塞实验研究来评价该微粒栓塞材料的栓塞效果。从而为进一步的临床应用研究提供实验依据。
第一部分BaFe_(12)O_(19)和PU-BaFe_(12)O_(19)的生物相容性研究
本部分着重进行两种微粒材料的生物相容性研究，包括Ames致突变实验、小鼠骨髓细胞微核试验、体外细胞毒性实验、全身急性、亚急性及慢性毒性实验、溶血实验、出血和凝血时间测定、凝血功能实验等。
1．Ames实验：为体外致突变实验，采用鼠伤寒沙门氏菌组氨酸营养缺陷型TA_(98)、TA_(100)为指示菌株；已知致突变物敌克松(Dexon)、二氨基芴(2-AF)为阳性对照，无菌蒸馏水为阴性对照，采用平皿掺入法进行实验。实验分非活化实验(-S9)与活化实验(+S9)。测试结果以致突变比值(MR=诱变菌回落数Rt／自发回变菌落数Rc)表示。判断标准(WHO)：试验组回变菌落数超过阴性对照组2倍以上者为阳性。实验结果：BaFe_(12)O_(19)和PU-BaFe_(12)O_(19)微粒材料各浓度浸提液实验组的MR值均小于2，而Dexon及2-AF两阳性对照组的MR值均大于2，表明两种材料体外实验均无致突变作用。
2．小鼠骨髓细胞微核试验：为体内致突变实验，采用腹腔注射材料浸提液后行骨髓腔悬浮液涂片，Giemsa染色，显微镜下计数轮廓完整的嗜多染红细胞(Polychronic Cell，PCE)，统计其中含有微核的PCE数，计算各组微核出现的频率(‰)，并进行统计分析。生理盐水(阴性对照)、环磷酚胺(阳性对照)同时行平行对照实验。实验结果表明，两种材料浸提液的三个实验组的骨髓微核细胞率均在正常范围。而生理盐水阴性对照组的微核细胞率为1．93±0．56‰，环磷酰胺阳性对照组的为19．53±2．14‰。经统计分析，两种微粒材料浸提液实验组与生理盐水组对小鼠骨髓微核细胞率的影响相互之间无显著性差异(P>0．05)，而环磷酰胺阳性对照组与三个实验组、生理盐水组之间均存在显著性差异(P<0．001)。
3．体外细胞毒性实验：采用材料浸提液与小鼠成纤维细胞L929共同培养观察其对细胞的毒性作用。实验同时行培养液(阴性对照)、64g/L苯酚(阳性对照)作平行对照实验。分3个时间段即24、48、72h行MTT比色，在酶联免疫检测仪上测定各孔光吸收值(A值)，计算细胞相对增殖率(relative growth rate，RGR)。[RGR=(实验组OD值-空白OD值)/(阴性对照组OD值-空白OD值)]，并进行细胞毒性分级。结果判断标准：根据细胞毒性分级(RGR≥100％0级；75％-99％Ⅰ级：50％-74％Ⅱ级；25％-49％Ⅲ级；1％-24％Ⅳ级；0Ⅴ级)评判细胞毒性：0级和Ⅰ级被认为没有细胞毒性，Ⅱ级为轻度细胞毒性，Ⅲ级和Ⅳ级为中度细胞毒性，Ⅴ级为明显细胞毒性。实验发现，BaFe_(12)O_(19)和PU-BaFe_(12)O_(19)微粒浸提液实验组细胞生长情况与阴性对照组相似，未见明显细胞生长抑制现象。两种材料不同浓度浸提液各实验组对L929细胞的细胞毒性均为Ⅰ级，而生理盐水组为0级，苯酚阳性对照组的细胞毒性为Ⅴ级。实验表明该两种材料无细胞毒性。
4．全身急性、亚急性、慢性毒性实验：采用腹腔注射混悬液的方式观察其对小鼠的毒性作用。用无菌生理盐水将BaFe_(12)O_(19)和PU-BaFe_(12)O_(19)微粒材料分别配制成10mg／mL和5mg／mL混悬液。急性组每只腹腔注射混悬液0．5mL，亚急性组于第1、3、5d腹腔注射0．5mL混悬液，慢性组于1、7、15、30d腹腔注射0．5mL混悬液，生理盐水0．5mL同期注射作平行对照。观察小鼠一般情况，包括食欲、活动、体重、毒性反应及死亡动物数。根据毒性评价分级来评价毒性反应。按分组分别于7 d、30d、60d后处死小鼠，取脑、心、肝、脾、肺、肾、腹膜标本，行组织学观察。结果发现，全身急性、亚急性、及慢性毒性实验组动物一般情况良好，所有实验动物均无死亡、无惊厥、瘫痪、呼吸抑制、腹泻、运动减少．和体重下降等不良反应。按毒性分级，BaFe_(12)O_(19)微粒属无毒级。各标本心、肝、脾、肺、肾、脑及腹膜等组织学检查未见组织和细胞的变性及坏死，无渗出及炎细胞浸润。实验表明该两种材料无全身的毒性反应。
5．溶血实验：采用材料浸提液与红细胞悬液接触的方式检验材料有无溶血作用。抽取新鲜静脉血5ml，加入肝素抗凝，玻璃棒搅动，除去纤维蛋白原。取4ml，加入0．9％生理盐水20ml稀释，摇匀，离心，得到沉淀红细胞。将红细胞用生理盐水配制成2％的红细胞悬液，加入材料浸提液2ml，混匀后于37℃水浴，观察4h后离心，取上清液1 ml，加入0．1％Na_2CO_3溶液4 ml，在分光光度计540 nm处测定各样本吸光度，计算溶血率。实验结果：BaFe_(12)O_(19)和PU-BaFe_(12)O_(19)微粒材料浸提液和生理盐水阴性对照组均无溶血现象，双蒸水阳性对照组全部溶血。经计算得知，BaFe_(12)O_(19)和PU-BaFe_(12)O_(19)微粒实验组的溶血率分别为2．08％和2．43％，符合ISO规定的不大于5％的标准。实验表明该材料不引起溶血。
6．出血、凝血时间测定：采用小鼠腹腔注射材料混悬液7d后检测其出血及凝血时间的方式观察其影响。取10mg／ml浓度BaFe_(12)O_(19)微粒混悬液、5mg／ml浓度的PU-BaFe_(12)O_(19)混悬液，按实验分组每只小鼠腹腔注射混悬液0．5ml，对照组腹腔注射生理盐水1ml，7d后测定出血及凝血时间。实验结果行统计分析：两种材料浸提液实验组和对照组小鼠的出血、凝血时间无显著差异(p>0．05)。表明该微粒栓塞材料对小鼠的出血及凝血时间无明显影响。
7．凝血功能测定：采用材料浸提液与Wistar大鼠新鲜血液直接接触的方式检验材料对凝血功能的影响。每只大鼠尾静脉抽血1ml，分别放入20只PT抗凝管。实验组每管注入饱和微粒浸提液1ml，对照组加入1ml生理盐水。30 min后测量凝血功能全套。实验数据经统计分析后表明，两种微粒材料浸提液对血液凝血功能无显著影响，各实验组的凝血酶原时间、纤维蛋白原测定、活化部分凝血活酶时间、凝血酶时间等与对照组无显著差别，均在正常范围，说明该两种材料对大鼠的凝血功能无明显影响。
第二部分BaFe_(12)O_(19)和PU-BaFe_(12)O_(19)栓塞Wistar大鼠颈动脉分支的实验研究
目的：评价BaFe_(12)O_(19)和PU-BaFe_(12)O_(19)两种微粒栓塞材料对Wistar大鼠颈外及颈内动脉分支的栓塞效果，观察栓塞后大鼠的神经功能变化及存活情况，了解该两种材料对血管内膜及脑组织所产生的病理变化及影响。
方法：1．实验分组：BaFe_(12)O_(19)栓塞组、PU-BaFe_(12)O_(19)栓塞组和BaFe_(12)O_(19)与PU-BaFe_(12)O_(19)联合栓塞组、生理盐水对照组，每组8只大鼠。2．方法：采用套管针穿刺颈动脉法进行栓塞。用浓度为2．5mg/ml、5mg／ml的PU-BaFe_(12)O_(19)混悬液和浓度为6mg／ml、12mg/ml的BaFe_(12)O_(19)混悬液按实验分组进行栓塞，按“先低浓度后高浓度”、“先小粒径后大粒径”的原则栓塞。栓塞的靶血管为颈外动脉的各级分支和颈内动脉的各级分支。栓塞分两部，即先注射低浓度、小微粒材料，然后注射高浓度及大微粒栓塞材料，造影观察末梢血管栓塞后的闭塞情况。不要求将各个分支血管完全闭塞。术后注意观察动物的存活、生理行为及神经功能的变化情况，并于术后的第一、四、八周时行存活动物的造影复查及病理组织检查。
结果：1．术中栓塞顺利，材料推注阻力均匀，无堵管现象；2．术中可见栓塞材料注射后颈外及颈内动脉的一些末梢分支血管被成功栓塞。BaFe_(12)O_(19)和PU-BaFe_(12)O_(19)栓塞组由于注射微粒较小，术后动物存活及神经功能状况要好，而BaFe_(12)O_(19)小微粒联合PU-BaFe_(12)O_(19)较大微粒栓塞组动物术后的死亡率高，神经功能状况较差。栓塞后存活动物的血管造影复查，未发现原闭塞血管的再通现象。3．脑大体标本检查：早期可见脑表面的片状缺血区，晚期可见脑表面梗死区，皮质凹陷；病理检查见血管腔内填充栓塞材料，早期可见血管内栓塞材料周围炎性细胞聚集浸润，血管内膜无明显破坏；晚期可见异物巨细胞，栓塞剂在血管内存留良好，部分血管内膜不同程度的破坏，未见血管中层弹力板明显破坏。血管内及周围可见不同程度的炎细胞浸润。未见栓塞材料逸出血管壁外。结论：BaFe_(12)O_(19)和PU-BaFe_(12)O_(19)两种栓塞材料可以栓塞Wistar大鼠颈外及颈内动脉分支末梢血管，且栓塞作用持久，血管不易再通。组织检查发现该微粒可引起血管周围轻度的炎性反应，未见明显血管壁破坏。
第三部分BaFe_(12)O_(19)和PU-BaFe_(12)O_(19)微粒栓塞新西兰兔肾动脉的实验研究
目的：应用两种微粒材料栓塞兔肾动脉的各级分支，评价BaFe_(12)O_(19)和PU-BaFe_(12)O_(19)微粒材料的短期及长期栓塞效果，了解肾动脉栓塞后肾脏的病理变化情况。
方法：1．实验分组：BaFe_(12)O_(19)栓塞组、PU-BaFe_(12)O_(19)栓塞组和BaFe_(12)O_(19)与PU-BaFe_(12)O_(19)联合栓塞组，每组8只兔子。2．方法：采用切开股动脉法置入微导管，用浓度为2．5mg／ml、5mg／ml的PU-BaFe_(12)O_(19)混悬液和浓度为6mg／ml、12mg/ml的BaFe_(12)O_(19)混悬液按实验分组进行栓塞，采用“先低浓度后高浓度”、“先小粒径后大粒径”的栓塞方法，以使能够栓塞远端终末支目的血管床。栓塞的靶血管为肾动脉的多个二级分支或三级分支。术中成功栓塞目的血管后，及时造影复查栓塞血管的显影情况，必要时追加栓塞剂量，直至完全栓塞。动物存活的中间时段行彩色多普勒超声检查，观察栓塞侧肾动脉的血流情况。术后两周、四周、八周、三个月时行栓塞侧肾动脉造影复查，并处死动物取肾脏标本，观察大体形态及行组织学检查。
结果：1．术中栓塞顺利，材料推注阻力均匀，无堵管现象；2．术中均可成功完全栓塞目的靶血管，PU-BaFe_(12)O_(19)栓塞组、BaFe_(12)O_(19)和PU-BaFe_(12)O_(19)联合栓塞组栓塞的血管造影复查，均未发现栓塞后的血管出现再通现象，无侧枝循环出现，再通率为0％。；而单纯BaFe_(12)O_(19)组在栓塞的18条肾动脉分支血管中有两条复发再通，再通率为11％。3．肾脏大体标本检查可见早期肾脏表面的片状梗死区，晚期肾脏萎缩、变小，皮质凹陷；病理检查见血管腔内填充栓塞材料，早期肾间质内轻度炎性反应，血管壁无明显破坏，晚期肾组织结构稀疏，小球小管结构大多消失，肾组织呈弥漫性微粒样变性，间质小血管管壁透明变性，血管周围可见炎性细胞浸润。
结论：PU-BaFe_(12)O_(19)单独应用或与BaFe_(12)O_(19)联合应用对新西兰兔肾动脉的栓塞效果较好，而单独应用BaFe_(12)O_(19)微粒材料效果稍差，有一定的复发再通可能。组织学检查提示该两种材料对血管的影响作用较轻。
第四部分家猪脑AVM不同流量模型的建立及栓塞实验
目的：1．利用家猪先天颅底微血管网(RMB)通过显微血管吻合建立高、中、低三种不同流量的脑AVM动物模型；2．使用该两种微粒材料栓塞三种不同流量的脑AVM动物模型，探讨其栓塞不同流量脑AVM的技术可行性、栓塞有效性及安全性；3．通过栓塞后的病理检测评价该材料对血管及组织的影响。
方法：1．动物模型的建立：8只动物随机分成2只低流量组、3只中流量组及3只高流量组。低流量模型：利用猪天然颅底RMB，无手术干预；中流量模型：单纯结扎右侧颈总动脉，造成右侧颈总动脉结扎远端以上由对侧咽升动脉经双侧RMB供血，人为增大了双侧RMB的流速及流量；高流量模型：行右侧颈总动脉远端与右颈内静脉近端行端-侧或端端吻合，形成以左侧咽升动脉为供血动脉，双侧RMB为畸形团，右颈总动脉→右颈内静脉为回流静脉的脑AVM模型。2．脑AVM模型栓塞：两种BaFe_(12)O_(19)和PU-BaFe_(12)O_(19)微粒材料分别和联合对三个流量组的动物模型行栓塞实验。各组动物采用股动脉切开插管法，将微导管置于左侧咽升动脉内，注射栓塞材料栓塞RMB。造影证实完全栓塞后，实验完毕。中高流量组动物栓塞后第1～3天处死，取RMB和脑组织作病理学检查。低流量组动物2周及2个月后复查造影。处死前再次造影，检查RMB是否显影。动物处死后取双侧RMB及部分周围脑组织行组织病理学检查。
结果：1．动物模型：显微吻合血管操作顺利，成功建立中、高流量脑AVM模型，高流量组1只动物模型出现吻合口狭窄。2．全部8只动物均顺利完栓塞RMB，复查造影未见RMB显影。栓塞均在未更换导管情况下完成栓塞，未发现堵塞导管现象。急性期病理检查见栓塞材料在RMB内弥散铸型良好，栓塞的微粒材料周围可见炎性细胞浸润，血管内膜破坏不明显；慢性期可见血管内仍被栓塞微粒材料及夹杂的血栓所闭塞。栓塞物周围可见异物巨细胞。栓塞剂在血管内存留良好，血管壁不同程度的变薄及破坏，血管内及周围可见不同程度的异物巨细胞及炎细胞浸润。所有标本未发现微粒材料外逸现象。
结论：1．通过结扎一侧颈总动脉，以及行颈部动静脉血管吻合，改变血流供应及回流通路，能够建立不同流量的脑AVM模型。2．微粒材料BaFe_(12)O_(19)和PU-BaFe_(12)O_(19)能够有效栓塞不同流量的AVM模型，操作方便、安全性高，有望成为一种新型的栓塞脑AVM的微粒性栓塞材料。
Granule materials were first used in endovascular embolization for curing diseases. With the affluxion of flood, the materials reached and gathered together in vessel beds, slowing down the blood stream and blocked the nutrition artery and other diseased branches. Granule materials embolization is widely used in clinic especially for pre-operation of tumors and endovascular treatment of cerebral arteriovenous malformations (AVM). However, with the appearance and application of liquid materials, the development, of Granule materials appears slowly relatively. Even though they are rarely used alone, and usually used together with liquid embolic materials. However, granule materials shouldn't be ignored in clinic for so many advantages, such as ideal biocompatibility, safe and easily control in operation and suitable diameter particles can be used to cure various diseased vessals.
This innovative BaFe_(12)O_(19) particles can be seen in X-ray, having spherical shape and good suspension quality, thus they wouldn't block the catheter easily as some liquid materials. The author carried out some experiments to examine the biocompatibility and embolization effect of BaFe_(12)O_(19) and polyurethane-BaFe_(12)O_(19) (PU-BaFe_(12)O_(19)) particles as a new kind of embolic materials. Embolization experiments were evaluated in carotid artery of Wista rats, renal artery of New Zealand rabbits and rete mirabile (RMB) of pigs, providing some positive evidences for further clinical studies.
Part 1: Study on Biocompatibility of BaFe_(12)O_(19) andPU-BaFe_(12)O_(19) Particles
The experiment of biocompatibility for BaFe_(12)O_(19) and PU-BaFe_(12)O_(19) particles was evaluated including Ames test, bone marrow micronucleus test, acute and subacute systemic toxicity test, hemolysis test, hemopexis test, bleeding and clotting time measurement test.
1. Ames test is a vitro mutagenesis test. The indicator strains used in this test were Salmonella typhimurium TA_(98) and TA_(100) with or without S9 The positive control objects were mutagens of Dexon and diaminofluorene (2-AF), while the negative control was sterile distilled water. The results was evaluated by mutation rate (MR=mutagenesis colony number/ spontaneous colony number). Standards (WHO): the test is positive if MR>2. The result showed that MR value of all concentration of leaching liquors of BaFe_(12)O_(19) and polyurethane-BaFe_(12)O_(19) was smaller than 2 while bigger than 2 for both Dexon and 2-AF groups. This results Indicates these two experimental materials have no mutagenesis effection.
2. Bone marrow micronucleus test in mice is a vivo mutagenesis test. After intraperitoneal injection of leaching liquor for 30h, cavum ossis suspension was taken and smeared, then the number of polychronic cells (PCE) containing micronucleus was counted and the frequency calculated (‰). The positive and negative control was cyclophosphamide and saline, tested in parallel controlled trial. The results showed that negative results in three groups of leaching liquor of both materials. The frequency of micronucleated cell for normal saline group was 1.93±0.56‰while for cyclophosphamide was 19.53±2.14‰. By statistical analysis of the data, this results indicates that three experiment groups had no significant difference compare to normal saline group(P>0.05) , and the cyclophosphamide group showed significant difference compare to the experiment and normal saline groups(P<0.001).
3. Vitro cytotoxicity test. L929 (fibroblast of mice) cells were cultivated with leaching liquor so as to evaluate its toxicity. The negative control was culture fluid while the positive was Phenol (64g/L), and the three groups were tested in parallel controlled trial. The samples were tested by MTT assay and optical density values (OD) were measured at 24h, 48h, 72h respectively. The OD value and relative growth rate (RGR) was calculated and the cytotoxicity was evaluated. Standars: 0 Ord (RGR=100%) I Ord (RGR 75%-99%) II Ord (RGR 50%-74%) IIIOrd (RGR 25%-49%) IVOrd (RGR 1%-24%) V Ord (RGR=0); 0& I (no cytotoxicity) II (slight cytotoxicity) III&IV(middling cytotoxicity) V (manifest cytotoxicity). The results showed that L929 cells growth condition in experiment groups similarly to negative group, no obvious growth inhibition. Cytotoxicity of different concentration of leaching liquor of both experiment materials to the L929 cells was I Ord, the NS group 0 Ord and the Phenol was V Ord. So we can draw a conclusion that both materials have no cytotoxic effect.
4. Acute, subacute and chronic systemic toxicity test. NIH rats were Intraperitoneal injection of experiment materials suspension so as to observe the toxicity in mice. With sterile saline BaFe_(12)O_(19) and PU-BaFe_(12)O_(19) particulate materials were prepared in 10 mg / ml and 5 mg / ml suspension. Each mouse of every group underwent intraperitoneal injection of 0.5 ml suspension while the control group injection of NS 0.5mL during the same period in parallel control. The general condition of mice was observed including appetite, activity, weight change, toxic reactions and even the number of dead animals. According to different groups, the mice were sacrificed in differently period respectively. Their brain, heart, liver, spleen, lung, kidney, pancreas and peritoneum were taken respectively prepared for histology analysis. The results showed that general condition of all experimental animals were well and no one death, no convulsions, no paralysis, no respiratory depression, no diarrhea, no activity reduce or weight loss. In all specimens there was no cell degeneration or necrosis, no exudation and infiltration of inflammatory cells. The experiments showed the two materials had no systemic toxicity.
5. Hemolysis test. To determine whether the materials have hemolysis effect by means of adding the leaching liquor into the erythrocyte suspensions. 5ml of fresh blood were collected, heparin added, glass rod to be used stirred to remove fibrinogen. Take 4ml of above blood, add 20ml NS to dilute, shake even, centrifuge and make RBC sediment. RBC was diluted into 2% suspension by adding NS, and add 2ml leaching liquor into it, shake even, bathe in 37℃water and centrifuge observed. After 4h, 1 ml of the supernatant was taken, 4 ml of 0.1% Na2CO3 solution were added, and then optical density was measured on spectrophotometer at 540 nm, then calculated the hemolysis rate. The results showed hemolysis rate of BaFe_(12)O_(19) and PU- BaFe_(12)O_(19) particulate were 2.08% and 2.43% respectively, it achieve the standard of not exceeding 5% according to ISO. This experiment indicates that the materials do not cause hemolysis.
6. Bleeding and clotting time test. To observe the impact on the bleeding and clotting time after intraperitoneal injection of materials suspension. Materials suspension was intraperitoneal injected into each experimental mouse, and 1 ml NS for each mouse in control group. Bleeding and clotting time was detected after 7d. The results showed that there was no significant difference (p>0.05) between experimental groups and control group, indicating these two kinds of materials have no obvious impact on mice bleeding and clotting time.
7. Blood hemopexis function test. The leaching liquor and blood of Wistar rat were directly added together to determine whether the materials have impact on blood hemopexis function. 1ml of each rat tail vein blood was respectively added to 20 anticoagulant tubes. Every experiment tube was added with 1 ml of leaching liquor while the control group 1 ml of NS. Blood coagulation time was measured after 30 mins. Statistical analysis of experimental data showed that there was no significant difference in prothrombin time, Fibrinogen assay, activated partial thromboplastin time and thrombin time between experimental group and control group.
Part 2 Experiment Study on Embolization of Carotid Artery Brancheswith BaFe_(12)O_(19) and PU-BaFe_(12)O_(19) Particles in Wistar Rats
Objective: To evaluate the embolization effect on carotid artery branches with BaFe_(12)O_(19) and PU-BaFe_(12)O_(19) particles in Wistar rats, to observe the changes in neural function and survival status, and to get a better understanding of the impact on pathology on vascular endothelium and brain tissues.
Method: 1. Experiment groups: (1) BaFe_(12)O_(19) embolization group; (2) PU-BaFe_(12)O_(19) embolization group; (3) BaFe_(12)O_(19) and PU-BaFe_(12)O_(19) co-embolization group; (4) NS control group, 8 rats for each group. 2. Methods: Puncture trocars were used to puncture the carotid artery then finish embolization. With the concentration of 2.5mg/ml, 5mg/ml of the PU-BaFe_(12)O_(19) suspension and 6mg/ml, 12mg/ml of BaFe_(12)O_(19), distal branches of ECA and ICA were embolized successfully according to the principle of "low concentration first" "small size particles first". Embolization consisted of two steps. Low concentration of small particles was first injected, and then the high concentrations of bigger particles were injected subsequently. Angiographic was made to observe the occlusion situation of the artery branches. Not every branch to be embolized imperatively. To keep observation on the survival, behavior and physiological changes in nerve function of animals, and to recheck angiographic of carotid arteries of survival animals, and then make macroscopic and microscopic examination at 1w, 4w, 8w after the operation.
Results: 1. The endovascular operation was successful performed and the particle injection resistance was well-distributed without blocking the catheters; 2. Some branches of ECA and ICA were embolized successfully in the operation, but the survival and neurological condition were better for using little particle of BaFe_(12)O_(19) and PU-BaFe_(12)O_(19), while much worse for using BaFe_(12)O_(19) and PU-BaFe_(12)O_(19) co-embolized. DSA for survived animals showed there weren't recanalization of previous occluded vessels. 3. Pathological detection showed some sporadic lamellar ischemia regions on brain surface observed in early phase, while infarction and introcession of cortex formed in late phase. Histological examination showed vessels were stuffed with these embolic materials, no obvious damage to the wall of vessels, and inflammatory cells gathered around the Embolization particles in early phase, while forein-body giant cells were observed in late phase. The embolic agents stayed well in vessels, some vascular walls thinningz, and varying degrees of damage, varying degrees of inflammatory cells was found around blood vessels.
Conclusion: BaFe_(12)O_(19) and PU-BaFe_(12)O_(19) as embolic materials can be used to embolize branches of ECA and ICA in Wistar rats, and the embolization effect is duration without artery recanalization, only mild vessal endomembrane inflammatory reaction in pathological examination.
Part 3 Experiment Study on Embolization of Renal Artery withBaFe_(12)O_(19) and PU-BaFe_(12)O_(19) Particles in NewZealand Rabbit
Objective: To evaluate the long-term and short-term Embolization effects of rabbit renal artery embolization with BaFe_(12)O_(19) and PU- BaFe_(12)O_(19) particles and to investigate the pathological changes in kidney afer Embolization.
Method: 1. According to the kinds of particles, 24 rabbits were divided averagely into three groups: Experimental groups: BaFe_(12)O_(19) embolization group, BaFe_(12)O_(19) embolization group and BaFe_(12)O_(19) and BaFe_(12)O_(19) co-embolization group, 8 rabbits for each group. 2. Methods: Use the operation of slitting femoral artery to cannulate 4F introducer sheath, and the micro-catheter was inserted into the renal artery of one side. With the concentration of 2.5mg/ml, 5mg/ml of the PU-BaFe_(12)O_(19) suspension and 6mg/ml, 12mg/ml of BaFe_(12)O_(19), the distal terminal branches of renal artery were embolized, according to the principle of "low concentration first", "small size particles first". The target vessels were second grade renal artery branches or the third grade artery branches. Angiography was taken immediately after embolized to review the target vessels whether or not visualization, if necessary, additional dose injected until embolization completely. Color Doppler ultrasound examination was taken at the middle period of survived animals to observe the blood stream of the embolized renal artery. Angiographic was rechecked for the embolized artery afer operation of 1w, 2w, 4w, 8w respectively, then the animals were killed, kidney specimens to be observed and for histological examination.
Results: 1. The endovascular operation was successful and the injection resistance was smooth without blocking the tubes; 2. The target vessels were successfully embolized and DSA of PU-BaFe_(12)O_(19) embolization group, BaFe_(12)O_(19) and BaFe_(12)O_(19) co-embolization showed no collateral circulation, no recanalization. But 2 of 18 renal artery branches underwent recanalization in the simple BaFe_(12)O_(19) group, the recanalization rate was 11%. 3. Kidney specimen examination showed flake infarction on the surface of early renal, atrophy in late renal and cortical depression; pathological examination showed vessels stuffed with embolization agents, mild inflammatory reaction in early renal interstitial, no destruction to vessel wall. The pathological specimen of late renal showed that the structure was sparse, the structure of glomerulus and renal tubules mostly disappeared and kidney showed diffuse granulation degeneration, mesenchymal small vessels wall hyalinized, perivascular infiltration of inflammatory cells visible.
Conclusion: PU-BaFe_(12)O_(19) alone or combined with BaFe_(12)O_(19) can be used to embolize the New Zealand rabbit renal artery effectively, while a separate application of BaFe_(12)O_(19) particles alone were lower effect than above mentioned. Some arteries have the possibility of recanalization and the toxic histopathologic for vessal response to the material is mild.
Part4 Modeling and Embolization for Different Flows ofArteriovenous Malformation (AVM) of Brain in Swine
Objective: 1. To establish three flows of brain AVM animal models with high, medium and low flows by microvascular anastomosis on the base of pig's rete mirabile (RMB). 2. To research the technical feasibility, effectiveness and safety of embolization by using these two particle materials to embolize AVM of animal model. 3. To evaluate the impact of the experiment materials on vessels and brain tissues by making macroscopic and microscopic examination.
Method: 1. Animal models: 8 animals were divided into three groups randomly-2 for low-flow group, 3 for medial-flow group and 3 for high-flow group. The normal RMB is used as low-flow of AVM model. The medial flow model is established by ligation of common carotid artery of one side, which increases the blood flow through bilateral RMB. The high flow model is built on by end-side or end-end vascular anastomosis between right carotid artery proximal end and right jugular vein distal end, which increases greatly the blood flow of RMB because the direct arteriovenous fistula. In high flow model, left and right ascending pharyngeal (AP) artery acted as afferent artery and draining vein respectively, the bilateral RMB as AVM nidus. 2. Embolization of cerebral AVM model: BaFe_(12)O_(19), PU-BaFe_(12)O_(19) and mixture of the two materials embolized the three groups respectively. All animals adopted femoral artery incision intubation, micro-catheter being placed in the left ascending pharyngeal artery where injection of embolic materials can embolize RMB entirely. Animals in the high and medial-flow groups were sacrificed on 1 -3d. while animals in low flow group were taken angiography after 2 weeks and 2 months respectively, then the swine were sacrificed and their bilateral RMB including some surrounding brain tissues were taken for macroscopic and microscopic examination.
Results: 1. Animal models: The vascular anastomosis operation was successful and medial-flow, high-flow cerebral AVM models were established ideally, only one animal in high-flow group undergoing anastomotic stenosis. 2. All RMB of experimental animals were embolized successfully and angiography rechecked showed no visualization of RMB. No catheter blocked and therefore the catheter wasn't replaced in the embolization process. Acute pathological examination showed that the embolic material was well dispersed and located in the mold RMB. Inflammatory cells Infiltrated and no obvious damage to the endothelium. Chronic pathological examination showed the vessels still blocked by experiment material with foreign body giant cells surround, some vessel walls underwent varying degrees of thinning and damage. All specimens showed no leak of embolic material.
Conclusions: 1. Different cerebral AVM animal models can be formed by one carotid artery ligation as well as the anastomosis of carotid artery and jugular vein changing the blood supply and pathway. 2. Granular materials of BaFe_(12)O_(19) and PU-BaFe_(12)O_(19) can be embolized AVM models of different flow and have many advantages such as operation convenient, safe embolization and so on. So BaFe_(12)O_(19) maybe become a new type of embolization material for cerebral AVM and has great potential as a therapeutic embolic agent.
引文
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4. Azzam A. Khankan, Keigo Osuga, Shinichi Hori, et al. Embolic effects of superabsorbent polymer microspheres in rabbit renal model: comparison with Tris-acryl gelatin microspheres and polyvinyl alcohol. Radiation Medicine, 2004, 22(6): 384-390.
5. Kwak BK, Shim HJ, Han SM, et al. Chitin-based Embolic Materials in the Renal Artery of Rabbits: Pathologic Evaluation of an Absorbable Particulate Agent. Radiology, 2005, 236(1):151-158.
6. LeeJ I, Kwak BK, Shim HJ, et al. Embolization effects of chitosan microspheres on rabbit kidney. Chung-Ang Journal of Medicine, 2003; 28(1):55-64.
7. Yamamoto A, Imai S, Kobatake M, et al. Evaluation of tris-acryl gelatin microsphere embolization with monochromatic X Rays: comparison with polyvinyl alcohol particles. J Vasc Interv Radiol, 2006,17(11): 1797-802.
8. Gobin YP, Vinuela F, Vinters HV, et al. Embolization with radiopaque microbeads of polyacrylonitrile hydrogel: evaluation in swine. Radiology, 2000, 214(1): 113-9.
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10. Spies JB, Allison S, Flick P, et al. Spherical polyvinyl alcohol versus tris-acryl gelatin microspheres for uterine artery embolization for leiomyomas: results of a limited randomized comparative study. J Vasc Interv Radiol, 2005 ,16(11):1431-7.
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14. Ohta S, Nitta N, Takahashi M, et al. Degradable gelatin microspheres as an embolic agent: an experimental study in a rabbit renal model. Korean J Radiol, 2007, 8(5):418-428.
15. Derdeyn CP, Graves VB, Salamat MS, et al. Collagen-coated acrylic microspheres for embolotherapy: in vivo and in vitro characteristics. Am J Neuroradiol, 1997,18(4): 647-653.
16. Beaujeux R, Laurent A, Wassef M, et al. Trisacryl gelatin microspheres for therapeutic embolization, II: preliminary clinical evaluation in tumors and arteriovenous malformations. AJNR, 1996,17(3):541-548.
17. Kai Y, Hamada J-i, Morioka M, et al. The utility of the microcrystalline cellulose sphere as a particulate embolic agent: an experimental study. AJNR, 2000, 21(6): 1160-1163.
18. van Es RJ, Nijsen JF, Dullens HF, et al. Tumour embolization of the Vx2 rabbit head and neck cancer model with Dextran hydrogel and Holmium-poly(L-lactic acid) microspheres: a radionuclide and histological pilot study. J Craniomaxillofac Surg. 2001, 29(5):289-97.
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2. Kai Y, Hamada J-i, Morioka M, et al. The utility of the microcrystalline cellulose sphere as a particulate embolic agent: an experimental study. AJNR, 2000, 21(6): 1160-1163.
3. Turjman F, Massound TF, Vinters HV, et al. Collagen microbeads: experimental evaluation of an embolic agent in the rete mirabile of swine. AJNR Am J Neuroradiol, 1995,16:1031-1036.
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5. Yamamoto A, Imai S, Kobatake M, et al. Evaluation of tris-acryl gelatin microsphere embolization with monochromatic X Rays: comparison with polyvinyl alcohol particles. J Vasc Interv Radiol, 2006,17(11):1797-802.
6. Gobin YP, Vinuela F, Vinters HV, et al. Embolization with radiopaque microbeads of polyacrylonitrile hydrogel: evaluation in swine. Radiology, 2000,214(1):113-9.
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1. Brocheriou I, Capron F. Arteriovenous malformation of the brain: histological study and micrometric measurement of abnormal vessels. J Neuroradiol, 2004, 31(5):359-61.
2. Turjman F, Massound TF, Vinters HV, et al. Collagen microbeads: experimental??evaluation of an embolic agent in the rete mirabile of swine. AJNR Am J Neuroradiol, 1995,16:1031-1036.
3. Osuga K, Hori S, Kitayoshi H, Embolization of high flow arteriovenous malformations: experience with use of super absorbent polymer microspheres. J Vasc Interv Radiol, 2002,13(11):1125-1133.
4. Azzam A. Khankan, Keigo Osuga, Shinichi Hori, et al. Embolic effects of superabsorbent polymer microspheres in rabbit renal model: comparison with Tris-acryl gelatin microspheres and polyvinyl alcohol. Radiation Medicine, 2004, 22(6): 384-390.
5. Kwak BK, Shim HJ, Han SM, et al. Chitin-based Embolic Materials in the Renal Artery of Rabbits: Pathologic Evaluation of an Absorbable Particulate Agent. Radiology, 2005, 236(1):151-158.
6. LeeJ I, Kwak BK, Shim HJ, et al. Embolization effects of chitosan microspheres on rabbit kidney. Chung-Ang Journal of Medicine, 2003; 28(1):55-64.
7. Yamamoto A, Imai S, Kobatake M, et al. Evaluation of tris-acryl gelatin microsphere embolization with monochromatic X Rays: comparison with polyvinyl alcohol particles. J Vasc Interv Radiol, 2006,17(11): 1797-802.
8. Gobin YP, Vinuela F, Vinters HV, et al. Embolization with radiopaque microbeads of polyacrylonitrile hydrogel: evaluation in swine. Radiology, 2000, 214(1): 113-9.
9. Matsumaru Y, Hyodo A, Nose T, et al. Embolic materials for endovascular treatment of cerebral lesions. J Biomater Sci Polym Ed, 1997, 8(7):555-569.
10. Spies JB, Allison S, Flick P, et al. Spherical polyvinyl alcohol versus tris-acryl gelatin microspheres for uterine artery embolization for leiomyomas: results of a limited randomized comparative study. J Vasc Interv Radiol, 2005 ,16(11):1431-7.
11．马廉亭，吴佐泉，秦尚振等．经微导管注入真丝微粒和线段治疗脑血管AVM．中华外科杂志，1991，19(8)：56．
12. Khankan A A, Osuga K, Hori S, et al. Embolic effects on superabsorbent polymer microspheres in rabbit renal model: comparison with trisacryl gelatin microspheres and polyvinyl alcohol. Radiat Med, 2004, 22(6):384-390.
13. Yamamoto A, Imai S, Kobatake M, et al. Evaluation of tris-acryl gelatin microsphere embolization with monochromatic X Rays: comparison with polyvinyl alcohol particles. J Vasc Interv Radiol, 2006,17(11): 1797-1802.
14. Ohta S, Nitta N, Takahashi M, et al. Degradable gelatin microspheres as an embolic agent: an experimental study in a rabbit renal model. Korean J Radiol, 2007, 8(5):418-428.
15. Derdeyn CP, Graves VB, Salamat MS, et al. Collagen-coated acrylic microspheres for embolotherapy: in vivo and in vitro characteristics. Am J Neuroradiol, 1997,18(4): 647-653.
16. Beaujeux R, Laurent A, Wassef M, et al. Trisacryl gelatin microspheres for therapeutic embolization, II: preliminary clinical evaluation in tumors and arteriovenous malformations. AJNR, 1996,17(3):541-548.
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