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Induction of Colitis and Colitis-associated Colorectal Cancer (CAC)
结肠炎及其相关结肠直肠癌(CAC)的诱发   

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Abstract

Inflammatory bowel disease (IBD) including Crohn’s disease and ulcerative colitis are characterized by chronic, progressive and relapsing inflammatory disorders. Existing evidence indicate that IBD is associated with a higher risk of developing CAC, which is directly related to the duration and extent of colitis. Thus, animal models have been developed to understand the biology of colitis and CAC. The most commonly used model of colitis is to treat with dextran sodium sulfate (DSS). DSS given in the drinking water is toxic to the colonic epithelial lining and induces bloody diarrhea, ulceration and inflammation, similar to colitis in IBD patients. To study CAC, DSS treatment is combined with a single intraperitoneal injection of the DNA alkylation reagent Azoxymethane (AOM).

Keywords: inflammation(炎症), tumor(瘤), intestine(肠), mouse(小鼠), carcinogen (致癌物)

Materials and Reagents

  1. 8-10 weeks old mice (in-house bred mice mostly C57BL/6 background, both male and female; we also used mice with mixed C57BL/6-129v background to induce colon tumors)
  2. Azoxymethane (AOM) (Sigma-Aldrich, catalog number: A5486 )
  3. DSS salt (molecular weight 36,000-50,000 Da) (MP Biomedicals, catalog number: 0126011090-500 g)
  4. Sterile 1x phosphate-buffered saline (PBS)
  5. 10% formalin solution (Sigma-Aldrich, catalog number: HT-501128)
  6. Autoclaved drinking water

Equipment

  1. 1 ml and 5 ml syringes (ENFA, catalog numbers: JS1 and JS3)
  2. 10 cm Petri dish, tissue culture grade (BD Biosciences, Falcon®, catalog number: 353006)
  3. Needles (25 G for AOM injection and 21 G to wash the colon)
  4. Digital caliper (Sylvac, catalog number: S_Cal WORK 910.0502.10)
  5. Dissection equipment (forceps, tweezers, scissors)
  6. Balance
  7. Tissue processor (Sakura, catalog number: VIP6-E2)

Procedure

  1. DSS- induced colitis
    1. On day 1, weigh the mice and distribute them into control group (untreated) and experimental group (DSS treatment). We usually mix mice with different genotypes in the same cage so that they are all subject to similar conditions.
    2. Prepare the 2% DSS solution (wt/vol) by dissolving DSS salt in autoclaved drinking water. Prepare enough DSS for 5 days. We do not change DSS during the treatment.
    3. Fill the drinking bottles of the cages with DSS solution.
    4. Weigh the mice every other day. Body weight loss indicates that the DSS treatment is working. Mice that lose more than 20% body weight at any time point should be sacrificed following guidelines for animal care. If the majority of mice are losing more than 20% body weight, DSS concentration should be reduced to ease mortality.
    5. On day 6, replace the DSS containing bottles by autoclaved water. In our experience, 5-days treatment with DSS suffices to induce acute colitis in C57BL/6 background mice, but the outcome is highly dependent not only on the mouse line but also on the housing facility and the batch of DSS, so it must be tested in each case.
    6. To study acute colitis, mice can be killed by cervical dislocation at day 7, one day after termination of the DSS treatment. In our experimental conditions, we see maximum colitis score in histological analysis at day 7. Afterwards, mice start to recover from the DSS induced damage. If the aim of the study is to determine recovery from DSS-induced damage, further time points should be analyzed, for example at days 10 and 13. In this case, mice should be weighted until the endpoint.

  2. AOM/DSS-induced CAC
    1. On day 1, weigh the mice and distribute them into control group (untreated) and experimental group (AOM/DSS treatment).
    2. Prepare AOM at the concentration of 1 mg/ml in sterile PBS. AOM is a cancer-causing reagent, thus appropriate precaution and protective measures should be taken to handle it.
    3. Inject mice intraperitoneally with AOM solution using 1 ml syringe. Final concentration is 10 mg/kg body weight of mice, i.e. 100 μl AOM solution of 1 mg/ml concentration per 10 g mouse body weight.
    4. Weigh the mice every other day as in step A4.
    5. On day 6, prepare the 2% DSS solution (wt/vol) and treat mice for 5 days as in step A. Continue weighing the mice until they recover the body weight of day 1 of DSS treatment.
    6. On day 11, replace DSS containing bottles with autoclaved drinking water and leave for 14 days.
    7. On days 26-31 and days 46-51, repeat DSS treatment as above.
    8. On day 100, evaluate tumor development.

  3. Sample collection
    1. Sacrifice mice by cervical dislocation at the endpoint of the experiment, for example at day 7 for colitis analysis and at day 100 for tumor analysis.
    2. Open the mice using sterile forceps and scissors to carefully remove the entire colon and place it in a 10 cm Petri dish with 15 ml cold sterile PBS, which should be enough to cover the entire colon.
    3. Carefully hold the distal part of the colon with the help of forceps and then flush to remove feces with 5-10 ml cold sterile PBS using a 21 G needle attached to a 5 ml syringe. After flushing the feces, put the cleaned colon in a new Petri dish.
    4. Carefully open the colon longitudinally and rinse again with cold PBS to remove remaining feces.

  4. Evaluation of colitis and tumor development
    1. For the colitis experiment, fix the longitudinally open colon as “swiss-rolls” in 10% formalin solution at room temperature overnight and then embed in paraffin using standard protocols. Briefly, colon tissue is processed in an automatic tissue processor for dehydration with ascending series of ethanol (70% ethanol, 2 incubations in 96% ethanol, 3 incubations in absolute ethanol, 1 h each), then cleaned with xylene (3 incubations of 1 h each), followed by 3 incubations in paraffin wax at 61 ºC and finally embedded in a paraffin block. To evaluate colitis, paraffin sections should be stained with H&E and analyzed by an experienced pathologist in blinded fashion to score for epithelial damage and inflammation. An additional parameter for the severity of colitis is the percentage of body weight loss during DSS treatment, which is calculated by comparing the body weight at different times with that of day 1 and can be represented in a graph. In our hands, using 2% DSS and mostly C57BL/6 background, WT mice lose 5-8% of body weight while mice lacking p38α in intestinal epithelial cells may lose up to 15% body weight, which correlates with a higher colitis score (inflammation and epithelial damage) evaluated in H&E-stained colon sections.
    2. For the AOM/DSS experiment, count the macroscopic tumors in the longitudinally open colon. In our in-house bred mice with mostly C57BL/6 background, tumor development is observed in 100% of the WT animals (Gupta et al., 2014). Tumor size (average diameter) is measured using a digital caliper and tumor load per mouse is calculated by summing up the sizes of all the tumors in a given mouse. After counting tumor numbers and load, longitudinally opened colons can be photographed (Figure 1) before fixing them as ”swiss-rolls” in 10% formalin solution and then embedding in paraffin. H&E stained paraffin sections (Figure 2) should be analyzed for tumor grading and immune cells infiltration by an experience pathologist.

Representative data


Figure 1. Example of a longitudinally open colon at day 100 of AOM/DSS-treatment showing colon tumors (arrows). Tumors are counted macroscopically and tumor sizes are determined using a digital caliper.


Figure 2. Example of H&E-stained colon sections at day 100 of AOM/DSS-treatment showing colon tumors (arrows) and the adjacent normal colon tissue

Notes

This protocol is highly dependent on the mouse genetic background, the animal housing conditions and the AOM and DSS batches. The working concentration of DSS should be determined empirically in pilot experiments. We have used the AOM/DSS protocol to induce colon tumors in mostly C57BL/6 background and mostly FVB background as well as in mixed C57BL/6-129v mice. In all cases, tumors were observed in 100% of the mice analyzed at day 100 of AOM/DSS treatment. If no tumors are detected at this time point, AOM concentration and/or DSS concentration should be increased. For the colitis experiment, between 2% and 5% DSS for 5 days should be enough to induce epithelial damage. Alternatively, the DSS treatment can be extended if little or no damage is observed at day 7.

Acknowledgments

Our work is supported by the Fundación BBVA and by grants from the Spanish MICINN (BFU2010-17850) and the European Commission FP7 (INFLA-CARE 223151 and ERC 294665). This protocol is a modification of the protocol published by Neufert et al. (2007).

References

  1. Gupta, J., del Barco Barrantes, I., Igea, A., Sakellariou, S., Pateras, I. S., Gorgoulis, V. G. and Nebreda, A. R. (2014). Dual function of p38alpha MAPK in colon cancer: suppression of colitis-associated tumor initiation but requirement for cancer cell survival. Cancer Cell 25(4): 484-500.
  2. Neufert, C., Becker, C. and Neurath, M. F. (2007). An inducible mouse model of colon carcinogenesis for the analysis of sporadic and inflammation-driven tumor progression. Nat Protoc 2(8): 1998-2004.

简介

包括克罗恩病和溃疡性结肠炎的炎性肠病(IBD)的特征在于慢性,进行性和复发性炎症性疾病。 现有证据表明IBD与发展CAC的较高风险相关,其与结肠炎的持续时间和程度直接相关。 因此,已经开发了动物模型来理解结肠炎和CAC的生物学。 最常用的结肠炎模型是用葡聚糖硫酸钠(DSS)治疗。 在饮用水中给予的DSS对结肠上皮衬里有毒并且诱导血性腹泻,溃疡和炎症,类似于IBD患者的结肠炎。 为了研究CAC,将DSS治疗与单次腹膜内注射DNA烷基化试剂偶氮甲烷(AOM)组合。

关键字:炎症, 瘤, 肠, 小鼠, 致癌物

材料和试剂

  1. 8-10周龄小鼠(内部繁殖的小鼠大多数为C57BL/6背景,雄性和雌性;我们也使用具有混合的C57BL/6-129v背景的小鼠诱导结肠肿瘤)
  2. 氧化甲烷(AOM)(Sigma-Aldrich,目录号:A5486)
  3. DSS盐(分子量36,000-50,000Da)(MP Biomedicals,目录号:0126011090-500g)
  4. 无菌1×磷酸盐缓冲盐水(PBS)
  5. 10%福尔马林溶液(Sigma-Aldrich,目录号:HT-501128)
  6. 高压蒸馏水

设备

  1. 1ml和5ml注射器(ENFA,目录号:JS1和JS3)
  2. 10cm培养皿,组织培养级(BD Biosciences,Falcon ,目录号:353006)
  3. 针(25 G用于AOM注射,21 G用于冲洗结肠)
  4. 数字卡尺(Sylvac,目录号:S_Cal WORK 910.0502.10)
  5. 解剖设备(镊子,镊子,剪刀)
  6. 余额
  7. 组织处理器(Sakura,目录号:VIP6-E2)

程序

  1. DSS诱导的结肠炎
    1. 在第1天,称重小鼠并将其分配到对照组中 (未处理)和实验组(DSS处理)。 我们通常混合鼠标 在不同的基因型在同一个笼子,使他们都是主题 到类似的条件。
    2. 通过制备2%DSS溶液(wt/vol) 将DSS盐溶解在高压灭菌的饮用水中。 准备足够的DSS   5天。 我们在治疗期间不更换DSS
    3. 用DSS溶液填充饮料瓶的瓶子。
    4. 每隔一天称重小鼠。 体重减轻表明   DSS治疗正在工作。 体重减轻超过20%的小鼠 任何时间点应根据动物指南牺牲 关心。 如果大多数小鼠失去超过20%的体重,DSS 应减少浓度以减轻死亡率。
    5. 在第6天, 用高压灭菌水替换含有DSS的瓶子。 在我们的 经验,用DSS 5天治疗足以诱导急性结肠炎 在C57BL/6背景小鼠,但结果是高度依赖不仅   在鼠标线上,但也在住房设施和批次 DSS,因此必须在每种情况下进行测试
    6. 为了研究急性结肠炎, 小鼠可以在第7天,一天后通过颈部脱臼被杀死 终止DSS治疗。 在我们的实验条件下,我们看到   在第7天的组织学分析中的最大结肠炎评分。 小鼠开始从DSS诱导的损伤中恢复。 如果的目的 研究是确定从DSS诱发的损伤的恢复,进一步的时间 应分析点,例如在第10天和第13天。在这种情况下, 小鼠应加权直到终点。

  2. AOM/DSS诱导的CAC
    1. 在第1天,称重小鼠并将它们分成对照组(未处理)和实验组(AOM/DSS处理)。
    2. 在无菌PBS中制备浓度为1mg/ml的AOM。 AOM是一个   致癌试剂,从而适当的预防和保护 应采取措施处理。
    3. 注射小鼠 使用1ml注射器用AOM溶液腹膜内。 最后 浓度为10mg/kg体重的小鼠,即100μlAOM溶液 每10克小鼠体重1mg/ml浓度
    4. 每隔一天称重小鼠,如步骤A4
    5. 在第6天,制备2%DSS溶液(wt/vol)并处理小鼠5 天,如步骤A。继续称量小鼠,直到它们恢复 DSS治疗的第1天的体重
    6. 在第11天,将含有DSS的瓶子用高压灭菌的饮用水替换,并放置14天
    7. 在第26-31天和第46-51天,重复如上所述的DSS治疗。
    8. 在第100天,评价肿瘤发展。

  3. 样品收集
    1. 牺牲小鼠颈部脱位终点 实验,例如在第7天进行结肠炎分析,在第100天进行   肿瘤分析
    2. 使用无菌镊子和剪刀打开小鼠   以小心地移除整个结肠并将其放置在10cm培养皿中   用15ml冷无菌PBS,这应该足以覆盖整个   冒号。
    3. 在帮助下小心握住结肠的远端部分   的镊子,然后冲洗去除粪便与5-10毫升冷无菌PBS 使用连接到5ml注射器的21G针。 冲洗后 粪便,把清洁的结肠放在一个新的培养皿中
    4. 小心地沿纵向打开结肠,用冷PBS再次冲洗,以清除残留的粪便

  4. 结肠炎和肿瘤发展的评价
    1. 对于结肠炎实验,将纵向打开的结肠固定 "瑞士卷"在10%福尔马林溶液中在室温下过夜   然后使用标准方案包埋在石蜡中。 简而言之,结肠组织   在用于脱水的自动组织处理器中处理 升序系列乙醇(70%乙醇,在96%乙醇中孵育2次,   在无水乙醇中孵育,每次1小时),然后用二甲苯(3 孵育1小时,每次),然后在石蜡中在3℃孵育 61℃,最后嵌入石蜡块中。为了评估结肠炎, 石蜡切片应该用H& E染色并通过a 经验丰富的病理学家以盲法的方式评分上皮 损伤和炎症。严重性的附加参数 结肠炎是DSS治疗期间体重减轻的百分比, 其通过比较不同时间的体重来计算  第1天的值,并且可以在图中表示。在我们手中,使用2%  DSS和大多数C57BL/6背景,WT小鼠体重减轻5-8% 而在肠上皮细胞中缺乏p38α的小鼠可能丧失 15%的体重,这与较高的结肠炎评分相关 (炎症和上皮损伤)在H& E染色的结肠中评估 部分。
    2. 对于AOM/DSS实验,计数宏观 肿瘤在纵向开放的结肠。在我们自己养殖的老鼠  主要是C57BL/6背景,在100%的肿瘤发展中观察到  WT动物(Gupta等人,2014)。肿瘤大小(平均直径)为 使用数字卡尺测量并计算每只小鼠的肿瘤负荷 通过对给定小鼠中所有肿瘤的大小进行求和。后 计数肿瘤数量和载量,纵向打开结肠可以 拍照(图1),然后将它们固定为"瑞士卷"在10% 福尔马林溶液,然后包埋在石蜡中。 H& E染色 石蜡切片(图2)应分析肿瘤分级和 免疫细胞浸润的经验病理学家。

代表数据


图1.在AOM/DSS治疗的第100天显示结肠肿瘤(箭头)的纵向开放结肠的实施例。使用数字卡尺测量肿瘤的肿瘤大小和肿瘤大小。

图2.在AOM/DSS治疗的第100天H& E染色的结肠切片的实例,显示结肠肿瘤(箭头)和相邻的正常结肠组织

笔记

这个协议高度依赖于小鼠遗传背景,动物住房条件和AOM和DSS批次。 DSS的工作浓度应在中试实验中根据经验确定。我们已经使用AOM/DSS方案在大多数C57BL/6背景和大部分FVB背景中以及在混合的C57BL/6-129v小鼠中诱导结肠肿瘤。在所有情况下,在AOM/DSS治疗的第100天在100%分析的小鼠中观察到肿瘤。如果在该时间点没有检测到肿瘤,应该增加AOM浓度和/或DSS浓度。对于结肠炎实验,2%和5%之间的DSS持续5天应足以诱导上皮损伤。或者,如果在第7天观察到很少或没有损伤,则可以延长DSS治疗。

致谢

我们的工作得到BBVA基金和西班牙MICINN(BFU2010-17850)和欧盟委员会FP7(INFLA-CARE 223151和ERC 294665)的资助。该协议是由Neufert等人(2007)发布的协议的修改。

参考文献

  1. Gupta,J.,del Barco Barrantes,I.,Igea,A.,Sakellariou,S.,Pateras,I.S.,Gorgoulis,V.G.and Nebreda,A.R。(2014)。 p38alpha MAPK在结肠癌中的双重功能:抑制结肠炎相关的肿瘤起始,但对癌细胞的需求 生存。癌细胞 25(4):484-500。
  2. Neufert,C.,Becker,C。和Neurath,M.F。(2007)。 结肠癌发生的诱导型小鼠模型,用于分析散发性和炎症驱动的肿瘤进展。 a> Nat Protoc 2(8):1998-2004。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Gupta, J. and Nebreda, A. R. (2014). Induction of Colitis and Colitis-associated Colorectal Cancer (CAC). Bio-protocol 4(22): e1290. DOI: 10.21769/BioProtoc.1290.
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