Background Necrotizing enterocolitis (NEC) is one of the leading causes of death in premature infants. To determine the factors present in the disease that lead to increased morbidity and mortality, manipulation of variables that are shown to have a positive response has been tested using various animal models. Testing and manipulation of these variables are unwarranted in humans due to regulatory health standards. Methods The purpose of this review is to provide an update to previous summaries that determine the significance of animal models in studying the mechanisms of NEC. A large variety of animal models including rats, mice, rabbits, piglets, nonhuman primates, and quails have been described in literature. We reviewed the reported animal models of NEC and examined the pros and cons of the various models as well as the scientific question addressed. Results The animals used in these experiments were subject to gavage feeding, hypoxia, hypothermia, oxygen perfusion, and other methods to induce the disease state. Each of these models has been utilized to show the effects of NEC on the premature, undeveloped gut in animals to find a correlation to the disease state present in humans. We found specific advantages and disadvantages for each model. Conclusions Recent advances in our understanding of NEC and the ongoing therapeutic strategy developments underscore the importance of animal models for this disease. 背景 坏死性小肠结肠炎 （NEC） 是早产儿死亡的主要原因之一。为了确定疾病中存在的导致发病率和死亡率增加的因素，已经使用各种动物模型测试了对显示具有阳性反应的变量的操作。由于监管健康标准，在人类中测试和操纵这些变量是没有必要的。方法 本综述的目的是对以前的总结进行更新，以确定动物模型在研究 NEC 机制中的重要性。文献中描述了各种各样的动物模型，包括大鼠、小鼠、兔子、小猪、非人灵长类动物和鹌鹑。我们回顾了已报道的 NEC 动物模型，并研究了各种模型的优缺点以及所解决的科学问题。结果 这些实验中使用的动物采用管饲喂养、缺氧、低温、氧气灌注和其他方法诱导疾病状态。这些模型中的每一个都被用来显示 NEC 对动物过早、未发育的肠道的影响，以发现与人类存在的疾病状态的相关性。我们发现了每种型号的具体优点和缺点。结论 我们对 NEC 的理解和正在进行的治疗策略发展的最新进展强调了动物模型对这种疾病的重要性。
INTRODUCTION 介绍
Necrotizing enterocolitis (NEC) is a devastating disease affecting premature infants. Approximately 10%10 \% of infants born weighing less than 1500 g will develop the disease, with mortality rates for affected infants being as high as 20%-30%.^(1)20 \%-30 \% .^{1} Mechanisms leading to the development of NEC are not completely understood, which makes the study of this disease difficult. ^(2){ }^{2} Although the exact pathophysiology of NEC is not fully understood, most experts agree that its etiology is multifactorial. It is felt that impaired gastrointestinal (GI) motility, intestinal barrier dysfunction, decreased digestive ability, poor circulatory regulation, intestinal microbial overgrowth, and immature immune defenses predispose 坏死性小肠结肠炎 （NEC） 是一种影响早产儿的毁灭性疾病。大约 10%10 \% 出生体重低于 1500 克的婴儿会患上这种疾病，受影响婴儿的死亡率高达 20%-30%.^(1)20 \%-30 \% .^{1} 导致 NEC 发展的机制尚不完全清楚，这使得对这种疾病的研究变得困难。 ^(2){ }^{2} 尽管 NEC 的确切病理生理学尚不完全清楚，但大多数专家都认为其病因是多因素的。人们认为胃肠道 （GI） 运动受损、肠道屏障功能障碍、消化能力下降、循环调节不良、肠道微生物过度生长和免疫防御不成熟是易感因素
premature newborns to intestinal injury leading to NEC. ^(34){ }^{34} 早产儿肠道损伤导致 NEC。 ^(34){ }^{34}
Naturally, development of robust animal models to aid in the study of this disease process is of critical importance. Traditionally, gavage formula fed/hypoxia/hypothermia models have been used for induction of NEC in mice, rats, and piglets. Asphyxia has been shown to hold a crucial role in the development of NEC because formula and bacterial exposure without asphyxia typically do not result in the phenotypical injury pattern seen in NEC. ^(2){ }^{2} Another significant variable that has been manipulated in various models is the removal of breast milk. It is well known that breast milk contains large amounts of immunoglobulin A (IgA), smaller amounts of Immunoglobulin G (IgG), active lymphocytes and macrophages, and specific antibodies against many types of microorganisms. ^(3){ }^{3} Barlow et al ^(4){ }^{4} theorized that without the passive immunity and intestinal flora control of breast milk, the enteric mucous barrier will be destroyed, leading to a pathologic cascade characterizing NEC. ^(4){ }^{4} 自然，开发健壮的动物模型来帮助研究这种疾病过程至关重要。传统上，灌胃配方饲喂/缺氧/低温模型已用于诱导小鼠、大鼠和仔猪的 NEC。窒息已被证明在 NEC 的发展中起着至关重要的作用，因为没有窒息的配方奶粉和细菌暴露通常不会导致 NEC 中出现的表型损伤模式。 ^(2){ }^{2} 在各种模型中操纵的另一个重要变量是母乳的去除。众所周知，母乳中含有大量的免疫球蛋白 A （IgA）、少量的免疫球蛋白 G （IgG）、活性淋巴细胞和巨噬细胞，以及针对多种微生物的特异性抗体。 ^(3){ }^{3} Barlow 等人的 ^(4){ }^{4} 理论认为，如果没有母乳的被动免疫和肠道菌群控制，肠道粘液屏障将被破坏，导致 NEC 的病理级联反应。 ^(4){ }^{4}
To induce intestinal damage and microbial dysbiosis, pathogenic bacteria can be administered to disrupt the intestinal epithelial barrier leading to an inflammatory response and translocation of pathogens. ^(5){ }^{5} Although this explains the presence of intestinal epithelial disruption that characterizes NEC, it does not explain the presence of intestinal necrosis that is seen in patients with this disease, nor can this explanation readily reconcile the observation that most patients who develop NEC do not actually have an antecedent ischemic event. ^(6){ }^{6} 为了诱导肠道损伤和微生物生态失调，可以施用病原菌来破坏肠道上皮屏障，导致炎症反应和病原体易位。 ^(5){ }^{5} 虽然这解释了 NEC 特征性肠上皮破坏的存在，但它并不能解释在患有这种疾病的患者中观察到的肠坏死的存在，这种解释也不能轻易地调和大多数发生 NEC 的患者实际上没有既往缺血事件的观察结果。 ^(6){ }^{6}
The clinical NEC seen in different animal models resembles NEC presented in human patients. Robust animal models closely resembling human disease are critical to test biochemical pathways, cell receptors, enteral nourishment, mechanisms of direct bowel injury, and studies that cannot ethically be conducted in humans. Animal models of NEC utilizing rats, mice, nonhuman 在不同动物模型中看到的临床 NEC 类似于人类患者中出现的 NEC。与人类疾病非常相似的稳健动物模型对于测试生化途径、细胞受体、肠内营养、直接肠损伤机制以及无法在人类中进行的伦理研究至关重要。利用大鼠、小鼠、非人 NEC 的动物模型
Figure 1 Pregnant time-dated Sprague-Dawley rats were delivered by C-section under CO_(2)\mathrm{CO}_{2} anesthesia on day 21.5 of gestation. Newborn rats were placed in an incubator and fed via gavage with formula containing 15 g Similac 60/40 in 75 mL Esbilac, providing 836.8kJ//kg836.8 \mathrm{~kJ} / \mathrm{kg} per day. Feeds were started at 0.1 mL every 4 hours beginning 2 hours after birth and advanced as tolerated up to a maximum of 0.4 mL per feeding by the fourth day of life. Animals were exposed to a single dose of intragastric LPS ( 2mg//kg2 \mathrm{mg} / \mathrm{kg} ) 8 hours after birth, and were stressed by exposure to hypoxia ( 100%100 \% nitrogen for 1 min ) followed by hypothermia ( 4^(@)C4^{\circ} \mathrm{C} for 10 min ) two times per day beginning immediately after birth until the end of the experiment on day 5 (IRB protocol # 8180041, Loma Linda university health). LPS, lipopolysaccharide. 图 1 妊娠时间 Sprague-Dawley 大鼠在妊娠第 21.5 天在麻醉下 CO_(2)\mathrm{CO}_{2} 通过剖腹产分娩。将新生大鼠置于培养箱中，通过强饲法喂养，配方奶粉中含有 15 g Similac 60/40 在 75 mL Esbilac 中，每天提供 836.8kJ//kg836.8 \mathrm{~kJ} / \mathrm{kg} 。从出生后 2 小时开始，开始喂养每 4 小时 0.1 mL，并在出生后第 4 天根据耐受程度提高到每次喂养最多 0.4 mL。动物在出生后 8 小时暴露于单剂量的胃内 LPS （ 2mg//kg2 \mathrm{mg} / \mathrm{kg} ），并通过暴露于缺氧（ 100%100 \% 氮气 1 分钟）然后低温（ 4^(@)C4^{\circ} \mathrm{C} 10 分钟）每天两次受到压力，从出生后立即开始直到第 5 天实验结束（IRB 方案 # 8180041，Loma Linda 大学健康）。LPS，脂多糖。
primates, and piglets have been used to study the pathogenesis of NEC. 灵长类动物和仔猪已被用于研究 NEC 的发病机制。
The advantages and disadvantages to use animal models are to be discussed in further detail. Because of the differences in size, genetic manipulation, cost of use, and preterm viability, each animal model can be examined to highlight the beneficial aspects of how NEC can be modeled. 使用动物模型的优缺点将进一步详细讨论。由于大小、遗传操作、使用成本和早产活力的差异，可以检查每种动物模型，以突出如何建模 NEC 的有益方面。
ANIMAL MODELS 动物模型
Rat model 大鼠模型
The rat model has been used most commonly for NEC analysis due to the similarity to human preterm infants. NEC mainly affects premature and low birthweight neonates and is characterized by ischemic necrosis of the bowel wall frequently leading to perforation and death. ^(3){ }^{3} As such, neonatal rats can be useful models for comparison due to their level of bowel immaturity at day 21 of gestation. One of the earliest studies to use rats concluded that the gut flora and absence of breast milk were suggested to play important roles in NEC pathogenesis. ^(7){ }^{7} In Barlow’s model, a formula was developed which simulated rat breast milk and could be mixed with a bacterial contaminant. In this model, the pups are removed immediately after Cesarian section and are placed in an incubator, with avoidance of breast feeding. ^(8){ }^{8} The stressors used to induce a NEC like state in the animal models includes hypoxia, hypothermia, addition of a lipopolysaccharide 由于大鼠模型与人类早产儿相似，因此最常用于 NEC 分析。NEC 主要影响早产儿和低出生体重新生儿，其特征是肠壁缺血性坏死，经常导致穿孔和死亡。 ^(3){ }^{3} 因此，由于新生大鼠在妊娠第 21 天的肠道不成熟水平，它们可以作为有用的比较模型。最早使用大鼠的研究之一得出结论，肠道菌群和母乳的缺乏被认为在 NEC 发病机制中起重要作用。 ^(7){ }^{7} 在 Barlow 的模型中，开发了一种模拟大鼠母乳的配方奶粉，可以与细菌污染物混合。在该模型中，幼崽在剖腹产后立即取出并放置在培养箱中，避免母乳喂养。 ^(8){ }^{8} 在动物模型中用于诱导 NEC 样状态的压力源包括缺氧、体温过低、添加脂多糖
(LPS) and hyperosmolar formula during the course of feeding throughout a week long experiment (figure 1). In this model, a formula is given four times per day with an increasing caloric intake each day of the week. ^(8){ }^{8} At the end of the protocol, or when rat pups have clinical signs of NEC, intestines are harvested and histologically graded for intestinal injury 0-40-4 with lessions greater than 2 being considered as NEC (figure 2). （LPS） 和高渗配方奶粉在为期一周的实验中饲喂（图 1）。在这个模型中，每天给予 4 次配方奶粉，每周每天增加热量摄入。 ^(8){ }^{8} 在方案结束时，或者当大鼠幼崽具有 NEC 的临床体征时，收获肠道并针对肠道损伤 0-40-4 进行组织学分级，小于 2 的减少被视为 NEC（图 2）。
Several laboratories have modified this formula feeding/hypoxia/hypothermia rat NEC model using additional elements to help induce NEC. One model has administered commensal bacteria found in stool, whereas others have tried different types of bacteria. ^(7){ }^{7} Some researchers introduced Cronobacter sakazakii (CS), formerly known as Enterobacter sakazakii, a bacteria that was reported to be associated with NEC, to the formulafeeding/hypoxia rat NEC model. ^(7){ }^{7} 几个实验室已经修改了这种公式喂养/缺氧/低温大鼠 NEC 模型，使用额外的元素来帮助诱导 NEC。一种模型使用了粪便中发现的共生细菌，而其他模型则尝试了不同类型的细菌。 ^(7){ }^{7} 一些研究人员将坂崎克罗诺杆菌 （CS），以前称为坂崎肠杆菌，一种据报道与 NEC 相关的细菌，引入配方奶喂养/缺氧大鼠 NEC 模型。 ^(7){ }^{7}
Rat models are an attractive option when studying NEC due to their low cost, preterm viability post-Cesarian section, and resilience to common stressors used to induce the disease. ^(9){ }^{9} One disadvantage seen is the lack of commercial antibodies produced to target selected receptors involved in NEC. Another disadvantage is that rats are highly tolerant to different types of bacteria and it can be difficult to achieve success when attempting to induce disease. Last but not least, another major drawback is the lack of genetic diversity found in rats compared with other species of rodents. ^(7){ }^{7} 在研究 NEC 时，大鼠模型是一个有吸引力的选择，因为它们成本低、剖宫产后早产存活率以及对用于诱发疾病的常见压力源的恢复力。 ^(9){ }^{9} 一个缺点是缺乏针对 NEC 中涉及的选定受体产生的商业抗体。另一个缺点是大鼠对不同类型的细菌高度耐受，在尝试诱导疾病时可能很难取得成功。最后但并非最不重要的一点是，与其他种类的啮齿动物相比，大鼠缺乏遗传多样性。 ^(7){ }^{7}
Using this model, many promising preventative and therapeutic interventions have been tested such as administration of probiotics, growth factors, stem cells, human milk oligosaccharides, tumor necrosis factor (TNF) blockers, and various antioxidants. ^(9){ }^{9} To date, we found more than 400 papers that have used the rat model with or without modifications from the original description. 使用该模型，已经测试了许多有前途的预防和治疗干预措施，例如益生菌、生长因子、干细胞、母乳低聚糖、肿瘤坏死因子 （TNF） 阻滞剂和各种抗氧化剂的给药。 ^(9){ }^{9} 迄今为止，我们发现了 400 多篇论文使用了大鼠模型，对原始描述进行了或没有修改。
Mouse model 小鼠模型
One of the major challenges and limitations of various animal models is that humans have an inherently different pattern of gene expression and therefore immune response to the development of NEC. In addition to this limitation, mice are also different due to their size compared with newborn rats or piglets. However, despite these limitation, mouse models have produced greater insights into the significance of prematurity in animals who develop NEC. ^(7){ }^{7} 各种动物模型的主要挑战和局限性之一是，人类具有本质上不同的基因表达模式，因此对 NEC 发展的免疫反应也不同。除了这个限制之外，与新生大鼠或仔猪相比，小鼠的大小也不同。然而，尽管存在这些限制，小鼠模型还是对早产对 NEC 动物的意义有了更深入的了解。 ^(7){ }^{7}
Humanized mouse models have initiated an area of interest for modeling NEC through genetic manipulation. Humanized mice can be defined as animals that are genetically manipulated to express human genes. ^(10){ }^{10} This model also relies on the use of hyperosmolar formula gavage feeds, hypoxia, and hypothermia. The addition of LPS to increase the incidence of NEC is variable across the published literature. 人源化小鼠模型开创了通过基因操作对 NEC 进行建模的感兴趣领域。人源化小鼠可以定义为经过基因操纵以表达人类基因的动物。 ^(10){ }^{10} 该模型还依赖于使用高渗配方管饲喂养、缺氧和低温。在已发表的文献中，添加 LPS 以增加 NEC 的发病率是可变的。
The most useful advantage of the mouse model comes from the ability to monitor and manipulate genetic variability and the use of transgenic animals to compare knockout (KO) mice to wild-type. The advantage of a 小鼠模型最有用的优势来自于监测和操纵遗传变异的能力，以及使用转基因动物将敲除 （KO） 小鼠与野生型小鼠进行比较。的优点
Histological grading: 组织学分级：
0 normal 0 正常
1 epithelial cell lifting or separation 1 上皮细胞提升或分离
2 necrosis to mid villus level 2 坏死至中绒毛水平
3 necrosis of entire villus 3 整个绒毛坏死
4 transmural necrosis 4 透壁坏死
Histological grading:
0 normal
1 epithelial cell lifting or separation
2 necrosis to mid villus level
3 necrosis of entire villus
4 transmural necrosis| Histological grading: |
| :--- |
| 0 normal |
| 1 epithelial cell lifting or separation |
| 2 necrosis to mid villus level |
| 3 necrosis of entire villus |
| 4 transmural necrosis |
Figure 2 Histologic injury score in rat pups subjected to experimental NEC. Shown are representative H&E-stained sections showing Grade 0, normal intestine, Grade 1, epithelial cell lifting or separation, Grade 2, sloughing of epithelial cells to the mid villous level, Grade 3, necrosis of the entire villous, and Grade 4, transmural necrosis. Magnification xx40\times 40. H&E, hemotoxylin and eosin; NEC, necrotizing enterocolitis. 图 2 实验性 NEC 大鼠幼崽的组织学损伤评分。显示的是代表性的 H&E 染色切片，显示 0 级、正常肠道、1 级、上皮细胞提升或分离、2 级、上皮细胞脱落至绒毛中层、3 级、整个绒毛坏死和 4 级透壁坏死。放大倍率 xx40\times 40 .H&E、血毒素和伊红;NEC，坏死性小肠结肠炎。
humanized mouse model is the interrogation of human tissue while examining the progression of the disease. ^(7){ }^{7} With this ability, the complex biological processes can be appreciated better in an attempt to model human neonates. For example, KO and transgenic (TG) models have been used to test the role of different growth factors in the prevention and treatment of NEC such as epidermal growth factor (EGF) and heparin-binding EGF-like growth factor (HB-EGF). ^(1112){ }^{1112} 人源化小鼠模型是在检查疾病进展的同时对人体组织进行询问。 ^(7){ }^{7} 有了这种能力，可以更好地理解复杂的生物过程，以尝试模拟人类新生儿。例如，KO 和转基因 （TG） 模型已被用于测试不同生长因子在预防和治疗 NEC 中的作用，例如表皮生长因子 （EGF） 和肝素结合 EGF 样生长因子 （HB-EGF）。 ^(1112){ }^{1112}
HB-EGF TG mice were designed to specifically overexpress the human HB- EGF precursor (proHB-EGF) in the intestine. ^(11){ }^{11} Using this model, the investigators were able to demonstrate that decreased endogenous HB-EGF expression predisposed the intestines to the development of NEC and the opposite being true for HB-EGF TG mice that overexpressed HB-EGF. ^(11){ }^{11} HB-EGF TG 小鼠被设计为在肠道中特异性过表达人 HB-EGF 前体 （proHB-EGF）。 ^(11){ }^{11} 使用该模型，研究人员能够证明内源性 HB-EGF 表达降低使肠道易发生 NEC，而过表达 HB-EGF 的 HB-EGF TG 小鼠则相反。 ^(11){ }^{11}
The advantages of this model lie in the ability to easily breed mice, the ability to genetically alter lines, and the cost-benefit of using a smaller animal model compared with the other animal models. On the other hand, the smaller size of the pups at birth predisposes them to a increased rate of complications. The mouse pups are more difficult to handle and gavage feed with formula through an orogastric catheter compared with rats. 该模型的优势在于能够轻松繁殖小鼠、能够改变品系进行基因改变，以及与其他动物模型相比，使用较小的动物模型具有成本效益。另一方面，幼崽出生时体型较小，使它们容易发生并发症。与大鼠相比，小鼠幼崽更难处理，并通过口胃导管用配方奶管饲饲。
Piglet model 仔猪模型
Compared with rodents, piglets share a high degree of anatomical, developmental, nutritional, and physiological similarity to the GI tract of humans. ^(7){ }^{7} The piglet is therefore a good model for studies looking at pathogenesis of NEC, evaluation of specific feeding regimens, preclinical drug studies for prevention and potential 与啮齿动物相比，仔猪在解剖学、发育、营养学和生理学上与人类的胃肠道具有高度相似性。 ^(7){ }^{7} 因此，仔猪是研究 NEC 发病机制、评估特定饲喂方案、预防和潜力的临床前药物研究的良好模型
therapy, as well as for the development of radiological diagnostic methods. ^(7){ }^{7} During the neonatal period, protein deposition is very rapid, and owing to similarities of postnatal nutrition and intestinal development to humans, the piglet can be viewed as an accelerated model of postnatal growth and development. ^(13){ }^{13} In addition, the piglet model is also appropriate for modeling other biological systems that may have an impact on the development of NEC such as liver function and metabolism due to its similarity with human systems. ^(13){ }^{13} 治疗，以及放射学诊断方法的发展。 ^(7){ }^{7} 在新生儿期，蛋白质沉积非常迅速，由于出生后营养和肠道发育与人类相似，仔猪可以被视为出生后生长发育的加速模型。 ^(13){ }^{13} 此外，仔猪模型还适用于模拟其他可能对 NEC 发育产生影响的生物系统，例如肝功能和新陈代谢，因为它与人类系统相似。 ^(13){ }^{13}
Generally, with small variations, the model involves piglets that are delivered by C-section at 90%90 \% term. Following birth, preterm piglets go through a period of natural hypoxia/hypothermia, and then are formula fed to induce injury. ^(14)A{ }^{14} \mathrm{~A} variation uses total parental nutrition at birth followed by a transition to enteral formula feeds on the second day of life. ^(91516){ }^{91516} Many investigators have used variations of this model as outlined in table 1. 一般来说，该模型涉及足月剖腹产的 90%90 \% 仔猪，差异很小。出生后，早产仔猪会经历一段时间的自然缺氧/体温过低，然后用配方奶喂养以诱导损伤。 ^(14)A{ }^{14} \mathrm{~A} 变异使用出生时父母的全部营养，然后在出生后的第二天过渡到肠内配方奶喂养。 ^(91516){ }^{91516} 许多研究人员使用了表 1 中概述的该模型的变体。
Cohen et al ^(17){ }^{17} described a model in which newborn piglets were subjected to a hypoxic insult for 30 min and hypothermic stress (core temperature reduced to 35^(@)C35^{\circ} \mathrm{C} for 30 min ). After 3-4 days, approximately half of the animals developed histological changes that resembled NEC. Other investigators induced NEC through a combination of cow-based formula and ischemia/ reperfusion such as the model described by Crissinger et al^(18)a l^{18} or by means of administered isosmolar acidified casein solution into intestinal segments of 1day-old piglets. ^(19){ }^{19} Cohen 等人 ^(17){ }^{17} 描述了一个模型，其中新生仔猪受到低氧损伤 30 分钟和低温应激（核心温度降低到 35^(@)C35^{\circ} \mathrm{C} 30 分钟）。3-4 天后，大约一半的动物出现类似于 NEC 的组织学变化。其他研究人员通过基于奶牛的配方奶粉和缺血/再灌注的组合（例如 Crissinger 等 al^(18)a l^{18} 人描述的模型）或通过将异摩尔酸化酪蛋白溶液注入 1 日龄仔猪的肠道段来诱导 NEC。 ^(19){ }^{19}
All these reported variations of the piglet model serve to underline the importance of this model to study the pathogenesis of NEC. 所有这些报道的仔猪模型变体都强调了该模型对研究 NEC 发病机制的重要性。
