Biophysiologic Considerations in Cryoablation: A Practical Mechanistic Molecular Review 冷冻消融的生物生理学考虑因素：实用机制分子回顾
Michael Maccini, David Sehrt, Alexandre Pompeo, Felipe A. Chicoli, Wilson R. Molina, Fernando J. Kim
Denver Health Medical Center, Division of Urology, Tony Grampsas Cancer Center, University of Colorado Health Science Center 科罗拉多大学健康科学中心，丹佛健康医疗中心，泌尿科，托尼-格兰普萨斯癌症中心
BRIEF HISTORY OF CRYOTHERAPY 冷冻疗法简史
Abstract 摘要
Cryotherapy techniques date back as far as the mid-1800s, when James Arnott demonstrated the effectiveness of salt/ice mixtures in palliation of breast, uterine, and skin cancers. Subsequent advances saw the use of liquid air and solid carbon dioxide in the treatment of various conditions, particularly benign dermatologic lesions (1). Cooper and Lee introduced the first automated cryosurgical apparatus cooled by circulating liquid nitrogen in 1961 and initially used it for treating neuromuscular disorders (2). Liquid nitrogen probes were soon being used in the treatment of benign prostatic hypertrophy and prostate cancer, though complications were quite common, resulting in the procedures falling out of favor until the 1990s, when intraoperative ultrasound techniques were developed, allowing more accurate monitoring of the freezing process (1). The advent of “third-generation” argon and helium gas probes in 2000 and preoperative computer thermal mapping techniques have allowed even more precise placement, temperature control, and further reduction in post-procedural morbidity (3). Cryosurgical techniques are currently used to treat a wide variety of conditions, but significant urologic indications include treatment of low and intermediate risk prostate cancer and renal cell carcinoma < 4<4 cm in diameter. 冷冻治疗技术最早可追溯到 19 世纪中期，当时詹姆斯-阿诺特（James Arnott）证明了盐/冰混合物在缓解乳腺癌、子宫癌和皮肤癌方面的有效性。随后，液态空气和固态二氧化碳被用于治疗各种疾病，尤其是皮肤良性病变 (1)。1961 年，Cooper 和 Lee 推出了第一台由循环液氮冷却的自动化冷冻手术设备，最初用于治疗神经肌肉疾病 (2)。液氮探针很快被用于治疗良性前列腺肥大和前列腺癌，但并发症十分常见，导致这种手术逐渐失宠，直到 20 世纪 90 年代，术中超声技术的发展使冷冻过程的监控更加精确（1）。2000 年，"第三代 "氩气和氦气探针的出现以及术前计算机热成像技术的应用，使冷冻位置和温度控制更加精确，并进一步降低了术后发病率（3）。冷冻外科技术目前可用于治疗多种疾病，但泌尿外科的重要适应症包括治疗中低危前列腺癌和直径 < 4<4 厘米的肾细胞癌。
The cryosurgical site is characterized by two zones a central zone of total coagulative necrosis, and a peripheral zone characterized by varying degrees of cellular death and injury. The mechanisms by which acute, direct cellular death occurs in the central zone are quite well-established. The two mechanisms involved include intracellular ice crystal formation resulting in mechanical trauma, and cellular dehydration with associated osmotic damage. Subsequent cell death is mediated by ischemia and apoptosis. These mechanisms of cell death are summarized in Figure-1. 冷冻手术部位有两个区域，一个是完全凝固性坏死的中心区域，另一个是细胞死亡和损伤程度不同的外围区域。中央区发生急性、直接细胞死亡的机制已相当成熟。其中涉及的两种机制包括机械性创伤导致的细胞内冰晶形成和细胞脱水及相关的渗透损伤。随后的细胞死亡由缺血和细胞凋亡介导。图 1 概述了这些细胞死亡机制。
Because water diffusion through the cellular membrane is rate-dependent, rapid cooling of tissue 由于水在细胞膜中的扩散取决于速率，因此组织的快速冷却
(near the cryoablation probe) results in intracellular ice crystal formation, as water cannot leave the cell fast enough to equilibrate the intracellular and extracellular compartments (4). Intracellular ice crystal formation results in direct mechanical trauma to the plasma membrane and organelles and is lethal (4-7). Rubinsky demonstrated dramatically increased rates of cell death at lower temperatures in ND-1 prostate cancer cells at a cooling rate of 25 degrees Celsius/ min versus both 1 degree //min/ \mathrm{min} and 5 degrees //min/ \mathrm{min} (4). (冷冻消融探针附近）会导致细胞内冰晶形成，因为水无法快速离开细胞，使细胞内和细胞外达到平衡（4）。细胞内冰晶的形成会对质膜和细胞器造成直接的机械创伤，是致命的（4-7）。Rubinsky 证实，在较低温度下，ND-1 前列腺癌细胞的细胞死亡率显著增加，冷却速度为 25 摄氏度/分钟，而 1 摄氏度 //min/ \mathrm{min} 和 5 摄氏度 //min/ \mathrm{min} 的细胞死亡率均为 25 摄氏度/分钟（4）。
Extracellular ice crystal formation occurs below -15 degrees Celsius during slow freezing (regions farther from the probe) and effectively removes water from the space surrounding cells. This creates an osmotic gradient which draws water out of the cells, stressing cell membranes and organelles and increasing intracellular electrolyte concentra- 细胞外冰晶的形成发生在零下 15 摄氏度以下的缓慢冷冻过程中（距离探针较远的区域），并有效地从细胞周围的空间中去除水分。这就形成了一种渗透梯度，将水从细胞中抽出，对细胞膜和细胞器造成压力，并增加细胞内电解质浓度。
Figure 1 - A) Closest to the cryoablation probe, rapid cooling results in intracellular ice formation, directly damaging cells and resulting in immediate cell death. B\boldsymbol{B} ) In regions farther from the probe, cooling is slower, resulting in extracellular ice formation, which creates an osmotic gradient resulting in dehydration and subsequent osmotic damage to cells, in addition to mechanical injury to membranes caused by ice crystals. C) Cryoablation also damages blood vessels, resulting in platelet activation, thrombosis, and ultimately ischemia after reperfusion. D) Lastly, cells which sustain damage - particularly damage to mitochondria (M) - not severe enough to kill them during the freezing process may undergo delayed programmed cell death (apoptosis). This may be a target for adjuvant therapies but may also be a potential mechanism for tumor resistance to cryoablation. 图 1 - A)最靠近低温消融探针，快速冷却导致细胞内冰形成，直接损伤细胞并导致细胞立即死亡。 B\boldsymbol{B} )在离探针较远的区域，冷却速度较慢，导致细胞外冰形成，形成渗透梯度，造成脱水，继而对细胞造成渗透损伤，此外冰晶还对细胞膜造成机械损伤。C) 低温消融也会损伤血管，导致血小板活化、血栓形成，最终导致再灌注后的缺血。D)最后，在冷冻过程中，细胞受到的损伤（尤其是线粒体（M）的损伤）还没有严重到足以杀死它们的程度，但它们可能会发生延迟的程序性细胞死亡（细胞凋亡）。这可能是辅助疗法的目标，但也可能是肿瘤对冷冻消融产生抗药性的潜在机制。
tions. Ice crystals outside the cell continue to grow and can cause mechanical trauma to cell membranes as well. The longer this process continues, the more likely cell death is to occur (4-7). 冰晶体在细胞外继续生长，也会对细胞膜造成机械损伤。细胞外的冰晶继续生长，也会对细胞膜造成机械损伤。这一过程持续的时间越长，细胞死亡的可能性就越大（4-7）。
Slow thawing between freezing cycles results in recrystallization and further propagation of extracellular ice crystals, disrupting tissue structure (4). Thawing eventually results in a decrease in extracellular osmolarity as ice melts, which can result in an influx of water into cells, resulting in cellular swelling and bursting (5). Repeating the freeze-thaw cycle results in markedly increased cell death rates (4). This is especially important in the treatment of tumors, as there is evidence in animal models that some tumors are more resistant to damage in single freeze-thaw cycles than normal tissue, likely due to increased fibrotic tissue. Repeated freeze-thaw cycles improved local tumor control in these studies (8). 冷冻周期之间的缓慢解冻会导致细胞外冰晶的再结晶和进一步扩散，从而破坏组织结构（4）。解冻最终会导致细胞外渗透压随着冰的融化而降低，从而导致水分涌入细胞，造成细胞肿胀和破裂（5）。重复冻融循环会导致细胞死亡率明显增加（4）。这对治疗肿瘤尤为重要，因为有动物模型证据表明，与正常组织相比，某些肿瘤在单次冻融循环中的抗损伤能力更强，这可能是由于纤维组织增加所致。在这些研究中，重复冻融循环可改善局部肿瘤控制（8）。
In the post-thaw period, ischemia induces further cell death in the central and peripheral zones. Endothelial damage to blood vessels results in platelet activation and thrombus formation as demonstrated in Rupp et al. histologic examination of changes in porcine post-cryoablation kidney tissue (9) leading to decreased perfusion. Additionally, Kimura et al. demonstrated decreased microvessel density and a positive correlation between hypoxia and necrosis in a mouse model of prostate cancer treated with cryosurgery (10). Ischemia induces regional hyperemia by the release of vasoactive mediators, resulting in an influx of inflammatory cells (neutrophils and macrophages). The ensuing “cleanup” process continues for weeks to months, with coagulation necrosis in the center of the surgery site and a band of neutrophils around the periphery (5). 在解冻后，缺血会进一步诱导中央区和外周区的细胞死亡。血管内皮损伤会导致血小板活化和血栓形成，Rupp 等人对猪冷冻术后肾组织变化的组织学检查（9）证明了这一点，从而导致灌注量减少。此外，Kimura 等人在用冷冻手术治疗前列腺癌的小鼠模型中发现，微血管密度下降，缺氧和坏死之间呈正相关（10）。缺血会通过释放血管活性介质诱发区域性充血，导致炎症细胞（中性粒细胞和巨噬细胞）大量涌入。随之而来的 "清理 "过程会持续数周至数月，手术部位中心会出现凝固性坏死，外围会出现中性粒细胞带（5）。
Recent investigations have established the role of apoptosis in peripheral zone cell death. These mechanisms are not as well understood as the immediate direct injury caused by the procedure. One hypothesis is that mitochondrial damage may activate caspase cascades, resulting in programmed cell death (5,11)(5,11). Recognition of the role of the apoptotic pathway of cell death in cryosurgery raises questions regarding the potential for resistance to cryotherapy, particularly in the treatment of prostate cancer, as it is not possible to include the entire prostate in the central necrotic zone to ensure negative margins due to the proximity of neurovascular bundles and the rectum. This means that the peripheral portions of the prostate will be in the peripheral “injury zone” of the cryosurgical lesion, where cellular death is at 最近的研究确定了细胞凋亡在外周区细胞死亡中的作用。这些机制并不像手术造成的直接损伤那样清楚。一种假设是，线粒体损伤可能会激活caspase级联反应，导致细胞程序性死亡 (5,11)(5,11) 。认识到细胞凋亡途径在冷冻手术中的作用，引发了有关冷冻疗法潜在抗药性的问题，尤其是在治疗前列腺癌时，因为靠近神经血管束和直肠，不可能将整个前列腺纳入中央坏死区，以确保阴性边缘。这意味着前列腺的外围部分将处于冷冻手术病灶的外围 "损伤区"，细胞在此死亡。
least partially dependent on apoptosis. Given that many cancers have mutations resulting in derangements of gene-regulated cell death pathways, this could be a potential risk for recurrent disease. Baust et al. examined survival rates of in vitro prostate and colorectal cancer cell lines after a single cycle of cryoablation and found an increased cell survival rate when the cells were exposed to caspase inhibitors (12). Another study by Klossner et al. demonstrated that androgen insensitive prostate cancer cell lines showed significantly increased survival rates versus androgen sensitive cell lines after treatment (13). There are, however, also encouraging indications that adjuvant therapies may increase tumor sensitivity to cryotherapy. For example, Clarke et al. demonstrated that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and cryoablation have a synergistic effect on a PC-3 cell line (14). This suggests that there may be cancers which are more amenable to cryosurgical treatment and cancers which are more likely to be resistant, and that there may be a role for adjuvant therapies. It should be noted, however, that these studies examined the effects of a single freeze-thaw cycle on the target cell populations, rather than multiple freeze-thaw cycles. 至少部分依赖于细胞凋亡。鉴于许多癌症都存在基因突变，导致基因调控的细胞死亡途径失常，这可能是疾病复发的潜在风险。Baust 等人研究了体外前列腺癌和结直肠癌细胞系在单周期低温消融后的存活率，发现当细胞暴露于 Caspase 抑制剂时，细胞存活率会增加（12）。Klossner 等人的另一项研究表明，对雄激素不敏感的前列腺癌细胞系在治疗后的存活率明显高于对雄激素敏感的细胞系（13）。不过，也有令人鼓舞的迹象表明，辅助疗法可能会增加肿瘤对冷冻疗法的敏感性。例如，Clarke 等人证实，肿瘤坏死因子相关凋亡诱导配体（TRAIL）和冷冻消融对 PC-3 细胞系有协同作用（14）。这表明，有些癌症更适合冷冻治疗，而有些癌症则更容易产生耐药性，因此辅助疗法可能会发挥作用。但应注意的是，这些研究考察的是单次冻融循环对目标细胞群的影响，而不是多次冻融循环。
DISCUSSION/CONCLUSIONS 讨论/结论
Cryosurgery has seen a resurgence in use in the past two decades, with the development of more advanced and precise equipment dramatically improving procedure safety. The mechanisms of cellular destruction and damage include both direct, immediate physical damage to cells and more delayed cell death due to local hypoxia and apoptosis. The role of apoptosis in the peripheral zone is a promising target for combination therapy, but also raises concerns due to the derangement of apoptotic pathways in many tumor cell populations, as evidenced by at least one study showing increased tumor survival after cryoablation in the presence of caspase inhibitors. If these pathways are not intact, cryoablation-mediated apoptosis may be impaired, rendering some tumors relatively resistant to cryosurgery. Further studies are needed to examine the effects of abnormal apoptotic pathways to identify potential adjuvant therapies and tumor characteris- 随着更先进、更精确的设备的发展，冷冻手术的安全性大大提高，冷冻手术在过去二十年中再次兴起。细胞破坏和损伤的机制包括对细胞直接、即时的物理损伤，以及由于局部缺氧和细胞凋亡导致的更迟缓的细胞死亡。细胞凋亡在外周区的作用是一种很有前景的综合疗法靶点，但也引起了人们的关注，因为许多肿瘤细胞群的细胞凋亡途径发生了改变，至少有一项研究表明，在有caspase抑制剂存在的情况下，冷冻消融术后肿瘤存活率增加。如果这些通路不完整，冷冻消融介导的细胞凋亡可能会受损，从而使一些肿瘤对冷冻手术产生相对抗性。需要进一步研究异常凋亡途径的影响，以确定潜在的辅助疗法和肿瘤特征。
tics which suggest the effective road to cryotherapy. In addition, using two freeze-thaw cycles in subsequent studies would more accurately simulate clinical situations. 这表明冷冻疗法是一条有效的道路。此外，在后续研究中使用两个冻融循环将更准确地模拟临床情况。
CONFLICT OF INTEREST 利益冲突
None declared. 无声明。
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Correspondence address: 通讯地址
Dr. Fernando J. Kim 费尔南多-J-金博士
Chief of Urology, DHMC DHMC 泌尿外科主任
Associate Professor of Surgery/Urology, UCHSC 加州大学旧金山分校外科/泌尿科副教授 bar(" Accepted after revision: ")\overline{\text { Accepted after revision: }}
Director of Minimally Invasive Urological Oncology 泌尿肿瘤微创科主任
Tony Grampsas Cancer Center, UCHSC 加州大学旧金山分校托尼-格兰普萨斯癌症中心
FAX: + 1303 436-6572 传真：+ 1303 436-6572
E-mail: fernando.kim@dhha.org
