姜凯,陈继业,刘洋,刘江,刘爱军,董家鸿,黄志强
外科手术切除是肝细胞癌(hepatocellu lar carcinoma,HCC)的首选治疗方法,但目前手术切除率仅为20%~40%,对于不能手术切除的HCC,多采用以局部治疗为主的综合疗法。射频消融(radiofrequency ablation,RFA)是肝癌局部治疗的有效方法之一。研究表明,射频消融对小HCC的疗效和手术相当[1-2],但对于肝内位于大血管旁的肿瘤,尤其是位于第一肝门处时,因热沉降效应(heat-sink effect,HSE)的作用,很难达到完全消融,容易残存肿瘤组织并导致术后复发和转移[2-4]。为达到对肿瘤的一次性完全射频消融,对抗热沉降效应,必须提高射频消融的能量与作用时间,但过多的热量可能导致肝功能严重损害[5]。目前,对热沉降效应流失热量所引起的河流效应(river-flow effect,RFE)尚缺乏认识,关于河流效应对毗邻肿瘤相应门静脉流域肝细胞热损伤的病理学改变也尚未见报道。本研究通过建立活体猪肝射频消融模型,观察河流效应对相应门脉流域肝细胞造成的病理学改变,为临床HCC的一次性完全射频消融治疗提供依据。
1.1 实验动物及术前麻醉 选取健康广西巴马小型猪6头,雌雄不限,体重15~20kg,术前12h禁食水。予以10mg/kg氯胺酮、1mg硫酸阿托品肌内注射进行麻醉前诱导,用l%硫喷妥钠0.5m l/kg麻醉,气管插管后连接呼吸机,颈动脉插管监测心率、血压,颈静脉插管建立输液通道。
1.2 动物模型建立 实验动物采用平卧位,右肋缘下J形切口逐层切开入腹,实验中对肝脏行3个位点的单束电极(3cm-active tip cooled,Valleylab,USA)射频消融。射频消融过程中,在肝叶、肝胃、肝肠之间放置冷盐水纱布保护,防止对其他组织、器官的影响和破坏。为避免射频消融导致胆囊损伤,常规切除胆囊。术中超声(Philips CX50多功能超声仪,超声探头L5-12,美国Philips公司)探查肝脏,辨别供应相应肝段的门静脉。在超声引导下紧贴肝段血管(距离<5mm)插入单束电极,注意避免电极头端直接进入胆管或血管。连接射频消融系统(Cool-tipTMRF ablation system,Valleylab,USA),用冷却注射用水循环使RF电极尖端温度保持低于20℃。每个位点持续消融6min。消融结束后将实验动物进行放血处死,完整取出肝脏,沿射频消融毗邻区域门静脉纵向轴线切开,测量射频消融范围后,立即用甲醛固定肝脏组织。
1.3 病理学检查 沿射频消融区域门静脉血管纵轴方向,由近而远,垂直纵轴,横向对肝段组织进行连续取材,每块组织厚度0.5cm,石蜡包埋、切片,HE染色,显微镜下观察射频消融后相应门静脉流域肝细胞的病理学改变。
在超声引导下,冷循环射频消融电极均成功精确穿刺到目标部位,所有动物术中麻醉满意,射频消融过程无动物死亡。
2.1 大体标本观察 射频消融毁损区域形态相似,呈椭圆型,毁损最大宽径为2.2±1.1cm。直径>3mm的血管内未见血栓形成,射频消融区域沿门静脉纵向轴线的切面色泽较周围肝脏组织变暗,由近而远逐渐减轻(图1)。
图1 肝脏大体标本观察Fig.1 Observation of gross specimen of liverA. During RFA in gross specimen, the color of distal region (white arrow) along relevant portal vein was darker than that of approximate region (black arrow) away from ablated point; B. No thrombus was seen in axial section plane (white thin arrow) along portal vein (>3mm);the ablated region shaped ellipse (green arrow) and distal tissue (hollow arrow) was darker than approximate part (white bold arrow) obviously
2.2 显微镜病理学观察 射频消融区域血管及胆管正常结构消失,肝组织细胞大片溶解坏死;距射频消融部位较近区域肝窦广泛充血,明显扩张,汇管区及肝窦大量炎性细胞浸润,肝细胞固缩变性,灶性坏死;距消融部位较远的门脉流域肝小叶中细胞肿胀明显,部分小叶血窦扩张,汇管区少量炎性细胞浸润,程度较近端区域轻,但仍呈现较为明显的肿胀、空泡样改变。消融区域周边肝段组织、肝细胞结构形态正常,未见明显异常改变(图2)。
射频消融具有安全、微创、重复性好、疗效确切等优点,被广泛运用到肝脏等实体肿瘤的治疗中,在肿瘤微创治疗中发挥着越来越重要的作用[1,3]。Lu等[6]的回顾性分析显示,105例原发或继发肝脏肿瘤行射频消融治疗后的局部复发率为15%,其中位于大血管旁的肿瘤复发率为48%,远离血管周围的肿瘤复发率为7%,进一步统计分析发现,肿瘤位于大血管(>3mm)周围是影响射频消融治疗效果的独立相关因素[7-8]。目前认为导致这一结果最主要的原因是肿瘤周围大量血流形成的热沉降效应,使得肿瘤血管一侧的温度上升缓慢,从而导致肿瘤组织周边有残余[2-4]。为减少热沉降效应对射频消融治疗效果的影响,有学者提出了增加射频消融时间,或者射频消融治疗前暂时阻断动脉或门脉血流等方法,结果显示,这些方法均可提高射频消融的治疗效果[4,8-10]。
对射频消融过程中因热沉降效应流失的过多热量对相应门静脉流域肝脏组织产生的损伤,我们称之为河流效应,强调的是为达到肿瘤一次性完全射频消融,过多的热量对肝段或肝叶区域细胞的损伤,即如同河流流过需要灌溉的区域一样,热量随门静脉或肝动脉流入区域性肝段或肝叶,对流过区域的肝细胞造成热损伤,最终影响肝脏功能。热量随肝静脉流入全身也使机体温度升高,机体代谢与体液消耗增加。
图2 肝组织显微镜病理学观察Fig.2 Pathological sections of liver tissueA. Scene of liver tissue close to ablated point (HE staining, ×200): appearance of dilated sinusoid, abundant of inflammatory cells aggregation in portal area (black arrow) and necrotic hepatocytes were seen more obviously extending from the central vein of hepatic lobule (solid arrow);B. Scene of liver tissue distal to ablated point (HE staining, ×200): partial dilated sinusoid in hepatic lobule (white arrow), swollen and vacuolar degenerated hepatocytes and partial normal structured hepatic lobule (black arrow) were seen; C. Scene of liver tissue close to ablated point (HE staining, ×400): necrotic solid hepatocytes (white arrow), dilated and congestive sinusoid (black arrow) were seen obviously; D. Scene of liver tissue distal to ablated point (HE staining, ×400): swollen hepatocytes, small amount of congestive and slightly dilated sinusoid (white arrow), and aggregation of small amount of inflammatory cells in portal area (black arrow) were seen
本活体动物实验结果显示,直径>3mm血管无血栓形成,射频消融难以使之闭塞,沿射频消融区域门静脉血管纵轴方向肝组织颜色变暗。镜下病理学观察发现,距离射频消融部位较近区域肝窦广泛充血,肝细胞广泛固缩变性,灶性坏死,炎性细胞大量出现,说明流失的热量对肝细胞的影响明显;远端区域肝细胞肿胀,较距消融部位较近区域轻,但仍呈较明显的损伤改变。这一结果证实射频消融过程中热沉降效应流失的热量具有河流效应,可以导致相应门脉流域的肝脏细胞出现明显的热损伤,这一损伤沿门脉血管供应范围分布,随距离增加损伤程度逐渐减弱。同时,本研究还显示,射频消融区域肝段外临近的肝段细胞组织未见明显热损伤,进一步证实损伤沿血管流向分布,并不会传导到其他临近的肝段组织,对其他区域肝脏细胞功能的影响较小。
对射频消融过程中流失热量的阐述,热沉降效应与河流效应的临床意义是对立的:前者强调的是热量流失对血管及其周围组织的保护使肿瘤组织残留而不能达到完全射频消融;后者强调的是为达到肿瘤一次性完全射频消融,过多的热量对肝段或肝叶区域细胞的损伤。在热量与时间恒定的情况下,随热沉降效应增强,河流效应随之增加;当热沉降效应达到极限时,温度越高(热量越大),时间越长,河流效应作用越大。对完全处于肝脏内部的肿瘤进行射频消融治疗,在全肝门阻断、血流完全停止时,热沉降效应与河流效应的肝内作用消失。
根据本实验的病理学结果,为达到肿瘤的一次性完全射频消融,我们推测河流效应可能具有的实际临床意义如下。鉴于肝脏门静脉系统解剖学的分布特点,越接近第一肝门的肿瘤,行射频消融时河流效应对肝脏的影响范围越大,越接近肝脏表面对肝脏的影响范围越小;在同样的热量条件下,流过的面积越小,肝细胞损伤程度越重,但范围小;流过的面积越大,对肝细胞的损害程度相对小,但累及范围增大,影响总体的肝脏功能。因此,越接近肝脏表面,血管越细(直径<3mm),热沉降效应的作用越小,河流效应局部热量聚集越多,越适合肿瘤一次性完全消融(图3)。总之,射频消融过程中河流效应对相应门脉流域肝细胞具有热损伤作用,损伤沿该门脉血管供应范围分布,向远处程度逐渐减弱。
图3 河流效应(RFE)作用示意图Fig.3 Schema of river-flow effectThe heat extend more easily to grander surface of liver through portal vein or hepatic artery as the ablation for tumor closer to hepatic hilar (central circle, HA+PV: hepaticartery+portal vein); upper half of the largest circle(the whole liver) indicates right or left half liver, solid medium circle (segment) and minimum solid circle (sub-segment) mean pedicle of portal system, and proportion extended to surface of liver(white arrow) indicates hepatic lobe, segment and subsegment (3, 2, 1)
[1] Bhardwaj N, Strickland AD, Ahmad F, et a l. Liver ablation techniques: a review[J]. Surg Endesc, 2010, 24(2): 254-265.
[2] Ng KK, Poon RT, Lam CM, et al. Efficacy and safety of radiofrequency ab lation fo r perivascu lar hepatocellu lar carcinoma without hepatic inflow occlusion[J]. Br J Surg, 2006,93(4): 440-447.
[3] Kotoh K, Morizono S, Kohjima M, et al. Evaluation of liver parenchymal pressure and portal endothelium damage during radiofrequency ablation in an in vivo porcine model[J]. Liver Int, 2005, 25(6): 1217-1223.
[4] Thanos L, Mylona S, Galani P, et al. Overcom ing the heat-sink phenomenon: successful radiofrequency thermal ablation of liver tumors in contact with blood vessels[J]. Diagn Interv Radiol,2008, 14(1): 51-56
[5] Kai J, M ing S, Yang L, et al. Complete radiofrequency ablation of hepatocellular carcinoma adjacent to the main bile duct and blood vessels between the first and the second hepatic portal[J].Cell Biochem Biophys, 2012 [Epub ahead of print].
[6] Lu DS, Ram an SS, Limanond P, et al. In fluence of large peritumoral vessels on outcome of radiofrequency ablation of liver tumors[J]. J Vasc Interv Radiol, 2003, 14(10): 1267-1274.
[7] Kurokohchi K, Watanabe S, Masaki T, et al. Combination therapy of percutaneous ethanol injection and radiofrequency ablation against hepatocellular carcinomas difficult to treat[J]. Int J Oncol, 2002, 21(3): 611-615.
[8] Hou YB, Chen MH, Yan K, et al. Ad juvant percutaneous radiofrequency ablation of feeding artery of hepatocellular carcinoma before treatment[J]. World J Gastroenterol, 2009,15(21): 2638-2643.
[9] Ishiko T, Beppu T, Sugiyama S, et al. Radiofrequency ablation with hand-assisted laparoscopic surgery for the treatment of hepatocellular carcinoma in the caudate lobe[J]. Surg Laparosc Endosc Percutan Tech, 2008, 18(3): 272-276.
[10] Rossi S, Garbagnati F, De Francesco I, et al. Relationship between the shape and size of radiofreguency induced thermal leisions and hepatic vascularization[J]. Tumori, 1999, 85(2):128-132.