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Mouse Model of Dextran Sodium Sulfate (DSS)-induced Colitis

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May 2016



Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the intestinal tract and is primarily comprised of Crohn’s disease (CD) and ulcerative colitis (UC). Several murine models that include both chemical induced and genetic models have been developed that mimic some aspects of either CD or UC. These models have been instrumental in our understanding of IBD. Of the chemical induced colitis models, dextran sodium sulfate (DSS) induced colitis model is a relatively simple and very widely used model of experimental colitis.

Keywords: Dextran sodium sulfate (DSS) (葡聚糖硫酸钠(DSS)), Colitis (结肠炎), Inflammatory bowel disease (IBD) (炎性肠病(IBD))


Inflammatory bowel disease (IBD) is a complex and multifactorial disease of unknown etiology (Sartor, 2006). However, multiple factors are considered to be critical in conferring IBD susceptibility, e.g., defects in host genetics, environmental triggers, aberrant immune response against microbial and dietary antigens (Sartor, 2006). Several murine models that address specific aspects of the disease process are available (Mizoguchi, 2012). DSS induced experimental colitis is a rapid and widely used model of intestinal inflammation (Perse and Cerar, 2012). Although the exact mechanism of DSS induced colitis is not well understood, it is widely accepted that disruption of the epithelial monolayer resulting in exposure of the underlying immune system to the intestinal contents containing microbiota and microbial products (Perse and Cerar, 2012). DSS induced colonic inflammation can be adapted as acute, chronic or relapsing model of intestinal inflammation by changing the concentration, duration and cycles of administration of DSS in drinking water. In this protocol, we provide a detailed description of procedures, important considerations when performing the protocol. We have used this protocol to address the role of Muc4 in DSS induced colitis in our previous work (Das et al., 2015).

Materials and Reagents

  1. Sterile Eppendorf tubes (National Scientific, catalog number: CN1700-BP )
  2. Tissue Path Macrosette processing cassettes (Fisher Scientific, catalog number: 15-182-706 )
  3. Glass slides
  4. 8-10 week old mice generated in house or obtained from the commercial vendors
    Note: Mice used in the study of Das et al., (2015) were of 129/Sv and C57BL/6J mixed background. Mice in this study were littermates and included both males and females housed under specific pathogen free (SPF) conditions.
  5. Dextran sodium sulfate (DSS) (TdB Consultancy, catalog number: DB001 , molecular weight: 35-55 kDa)
    Note: Stored in a dry place at room temperature.
  6. Liquid nitrogen
  7. Autoclaved drinking water
  8. Anesthesia (Isofluorane or CO2)
  9. 10% buffered formalin (Fisher Scientific, catalog number: SF100-4 )
  10. Ethanol
  11. Xylene
  12. Paraffin
  13. mirVana miRNA isolation kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM1560 )
  14. Bioanalyzer (Agilent Technologies, Waldbronn, Germany)
  15. DNase I (QIAGEN, catalog number: 79254 )
  16. Oligo-dT or random hexamer primers (Thermo Fisher Scientific, InvitrogenTM, catalog number: 100023441 )
  17. SuperScript reverse transcriptase II (Thermo Fisher Scientific, InvitrogenTM, catalog number: 18064014 )
  18. Light Cycler 480 SYBR Green mix (Roche Molecular Systems, catalog number: 04707516001 )
  19. Hematoxylin and eosin (H & E)


  1. Measuring scale/Ruler or Vernier calipers
  2. Animal weighing balance
  3. Feeding bottles
  4. Tissue-Tek VIP processing machine
  5. Dissection equipment
  6. Mouse microisolator chambers
  7. Pestle and mortar


  1. On day 0, tag all the mice in both the control and experimental groups according to the appropriate method (ear tagging, ear punching etc.) approved by the institute. It is important to tag mice individually so that body weight, stool consistency, blood in the stool etc. after DSS administration can be monitored. Weigh all the mice before treating them (ideally the weight of mice should be approximately around 20 g) with either autoclaved water or DSS dissolved in autoclaved drinking water. Collect stool samples if required.
  2. Prepare an appropriate concentration of DSS (2-3% based on the question in study, please see step 3 below) in autoclaved drinking water by dissolving DSS powder until a clear solution is reached. Fill the water bottles in the cages with approximately 100 ml for 2 days per cage with 4-5 mice. Discard the remaining water after 2 days and replenish with freshly prepared DSS containing water.
  3. For an injury repair model, 2% DSS (i.e., 2 g in 100 ml) in the drinking water is administered for the first 7 days followed by regular drinking water for another 7 days. However, the number of days on DSS followed by regular water can be changed based on the question in study. For example, if the question in study does not involve the recovery or the repair phase, then the DSS administration can be terminated either on day 7 or can be continued if prolonged inflammatory phase is desired. In order to determine the survival rate, 3% DSS in the drinking water can be administered until all the mice in either the control or the experimental group reach a humane endpoint. Ideally, the humane endpoint is when the mice have arrived the stipulated body weight loss approved by the ethical committee. In addition, severe and bloody diarrhea accompanied by hunched position, ruffled fur and reduced mobility are considered to be humane endpoint and the mice should be sacrificed by approved methods.
  4. Mice treated with DSS will lose a significant amount of body weight and therefore, should be weighed daily to monitor the health and to ensure that mice are sacrificed at a humane endpoint based on the approved ethical standards. Additionally, the mice should be monitored daily for the stool consistency (0 = well formed stool pellet, 2 = semi-formed stool, 4 = liquid stool that adhere to anal region) and blood in the stool (0 = no blood, 2 = blood trace in the stool clearly visible, 4 = gross rectal bleeding). The individual recording this information should be blinded to the study. Loss of body weight, diarrhea and fecal bleeding score can be plotted to determine the effect of DSS induced colitis between mice of different genotypes and/or treatment groups. As a general guideline, mice start to lose body weight around day 6 of DSS treatment and continue to lose weight approximately till day 10 (3 days after DSS removal) that is approximately 15-20% less than the initial body weight. During this time, the mice display behavioral changes such as reduced appetite, hunched back posture, ruffled fur etc. Following day 10, mice usually start to gain body weight and reaches the initial body weight by around day 14. However, this can vary depending on microbiota composition, animal facility and therefore should be carefully interpreted.
  5. Following the cycles of DSS (7 days) and regular water (7 days), on day 14, mice can be euthanized according to the appropriate method approved by the ethical committee (IACUC). Colon length should be measured using a ruler or Vernier calipers as DSS administration leads to swelling and shortening of colon. Pieces of colon (~0.5 cm) can be snap frozen in liquid nitrogen to be further processed for RNA extraction and gene expression analysis. For histology, the rest of the tissues can be Swiss rolled and stored in 10% buffered formalin using Tissue Path Macrosette processing cassettes. Following 48-72 h of fixation in formalin, the tissue samples should be removed from formalin and placed in 70% ethanol until further tissue processing and embedding. Normally, tissue processing can be carried out overnight (Table 1) using a Tissue-Tek VIP processing machine followed by paraffin embedding.

    Table 1. Tissue processing protocol

  6. Snap frozen tissues in liquid nitrogen can be used for RNA extraction following homogenization (using sterile pestle and mortar) using mirVana miRNA isolation kit according to manufacturer’s instructions, which can be used to isolate total RNA as well as small RNAs. Further treatment of the extracted RNA with DNase I (QIAGEN) according to manufacturer’s instructions is advised to obtain pure RNA without DNA contamination. RNA is further converted to cDNA using either oligo-dT or random hexamer primers and SuperScript reverse transcriptase II (Thermo Fisher Scientific) according to the manufacturer’s instructions. Expression of pro inflammatory cytokines such as TNF-α and IL1-β that are upregulated during DSS colitis can be used as useful markers of DSS induced colitis.
  7. Fomalin fixed tissues can be further paraffin embedded, sectioned and mounted on glass slides for histological analysis. For histological score analysis tissue sections can be stained with hematoxylin and eosin (H & E) and scored by a mouse pathologist in a blinded fashion. Briefly, a score from 0-3 is assigned for infiltration of inflammatory cells and 0-3 is assigned for tissue damage. Finally, a combined score of ‘0’ would indicate healthy intestine compared to a score of 6 which will indicate extensive infiltration and tissue damage. Immuno-histochemical staining for immune cells such as Macrophages (F4/80), apoptotic cells (Caspase 3), loss of epithelial architecture (E-cadherin) are reliable indicators of DSS induced colitis.
  8. Since the effect of DSS is more pronounced in the distal compared to the proximal colon, a histological scoring of proximal and distal colon can be performed separately.

Data analysis

  1. Appropriate statistical methods should be applied for data analysis.
  2. For representative data including survival curves, fecal blood score, gene expression analysis and histologic images, we refer readers to Das et al., (2015) Figures 2, 3, 4 and 5.


  1. All procedures performed should be approved by the Institutional Animal Care and User Committee (IACUC) of the respective institutions. The animals involved in this study must be treated humanely, and in all situations in which the mice are used, pain and discomfort should be minimized.
  2. It is important not to mix mice from different sources as they may have different microbiome compositions that may alter the outcome of DSS induced colitis. Additionally, when using mice that are genetically modified, littermate controls are advised unless the role of maternal contribution (e.g., in-utero or vertical transfer) from a specific genotype is being studied.
  3. Molecular weight of DSS is highly critical (molecular weight: 35-55 kDa).


We would like to thank members of Batra lab for useful discussions. The authors on this protocols were, in parts, supported by the NCI Fred and Pamela Cancer Center Support Grant P30 CA036727.


  1. Das, S., Rachagani, S., Sheinin, Y., Smith, L. M., Gurumurthy, C. B., Roy, H. K. and Batra, S. K. (2015). Mice lacking Muc4 are more resistant to experimental colitis and colitis-associated colorectal cancer. Oncogene 35(20): 2645-2654.
  2. Mizoguchi, A. (2012). Animal models of inflammatory bowel disease. Prog Mol Biol Transl Sci 105: 263-320.
  3. Perse, M. and Cerar, A. (2012). Dextran sodium sulphate colitis mouse model: traps and tricks. J Biomed Biotechnol 2012: 718617.
  4. Sartor, R. B. (2006). Mechanisms of disease: pathogenesis of Crohn’s disease and ulcerative colitis. Nat Clin Pract Gastroenterol Hepatol 3(7): 390-407.


炎症性肠病(IBD)是肠道的慢性炎性疾病,主要由克罗恩病(CD)和溃疡性结肠炎(UC)组成。 已经开发了几种包含化学诱导和遗传模型的鼠模型,其模拟CD或UC的某些方面。 这些模型在我们对IBD的理解方面发挥了重要作用。 在化学诱导的结肠炎模型中,葡聚糖硫酸钠(DSS)诱导的结肠炎模型是相对简单且非常广泛使用的实验性结肠炎模型。
【背景】炎症性肠病(IBD)是一种复杂而多因素的疾病,病因不明(Sartor,2006)。然而,多种因素被认为是赋予IBD易感性的关键,例如宿主遗传学,环境触发,对微生物和膳食抗原的异常免疫应答的缺陷(Sartor,2006)。可以提供几种针对疾病过程特定方面的小鼠模型(Mizoguchi,2012)。 DSS诱导的实验性结肠炎是一种快速和广泛使用的肠道炎症模型(Perse和Cerar,2012)。虽然DSS诱导的结肠炎的确切机制还不太清楚,但广泛接受的是上皮单层的破坏导致潜在的免疫系统暴露于含有微生物和微生物产物的肠内容物(Perse和Cerar,2012)。 DSS诱导的结肠炎症可以通过改变饮用水中DSS的浓度,持续时间和周期来适应肠道炎症的急性,慢性或复发模型。在本协议中,我们提供了程序的详细描述,执行协议时的重要注意事项。我们已经使用这个协议来解决我们以前的工作中Muc4在DSS诱导性结肠炎中的作用(Das et al。,2015)。

关键字:葡聚糖硫酸钠(DSS), 结肠炎, 炎性肠病(IBD)


  1. 无菌Eppendorf管(国家科学,目录号:CN1700-BP)
  2. 组织路径Macrosette处理盒(Fisher Scientific,目录号:15-182-706)
  3. 玻璃幻灯片
  4. 8-10周龄的老鼠在家里生产或从商业供应商获得
    注意:Das等人(2015)研究中使用的小鼠是129 / Sv和C57BL / 6J混合背景。这项研究中的小鼠是同窝同胞,包括在特定无病原体(SPF)条件下的男性和女性。
  5. 葡聚糖硫酸钠(DSS)(TdB咨询,目录号:DB001,分子量:35-55 kDa)
  6. 液氮
  7. 高压灭菌饮用水
  8. 麻醉(异氟烷或CO 2 )
  9. 10%缓冲福尔马林(Fisher Scientific,目录号:SF100-4)
  10. 乙醇
  11. 二甲苯
  12. 石蜡
  13. Vana miRNA分离试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:AM1560)
  14. 生物分析仪(Agilent Technologies,Waldbronn,德国)
  15. DNase I(QIAGEN,目录号:79254)
  16. 寡核苷酸或随机六聚体引物(Thermo Fisher Scientific,Invitrogen TM,目录号:100023441)
  17. SuperScript逆转录酶II(Thermo Fisher Scientific,Invitrogen TM,目录号:18064014)
  18. Light Cycler 480 SYBR Green mix(Roche Molecular Systems,目录号:04707516001)
  19. 苏木精和伊红(H& E)


  1. 测量秤/标尺或游标卡尺
  2. 动物称重平衡
  3. 喂奶瓶
  4. Tissue-Tek VIP加工机
  5. 解剖设备
  6. 鼠标微型隔膜室
  7. 杵和砂浆


  1. 在第0天,根据研究所批准的适当方法(耳标,耳冲等)标记对照组和实验组中的所有小鼠。重要的是,可以监视DSS管理后,单独标记小鼠,以便体重,粪便稠度,粪便中的血液等。在处理它们之前称重所有的小鼠(理想情况下,小鼠的体重约为20g),将高压灭菌的水或DSS溶解在高压灭菌的饮用水中。如果需要,收集粪便样品。
  2. 通过溶解DSS粉末,在高压灭菌的饮用水中准备适当浓度的DSS(基于研究中的问题,请参见下面的步骤3),直到达到澄清溶液。每个笼子用4-5只小鼠将大约100毫升的水瓶装入笼子中2天。 2天后丢弃剩余的水,并补充新鲜制备的DSS含水。
  3. 对于损伤修复模型,饮用水中的2%DSS(即,在100ml中的2g)在前7天施用,然后经常饮用水另外7天。然而,DSS之后的常规用水天数可以根据研究中的问题进行更改。例如,如果研究中的问题不涉及恢复或修复阶段,则DSS管理可以在第7天终止,或者如果需要延长的炎症期,可以继续进行。为了确定存活率,可以给予饮用水中3%的DSS,直到对照组或实验组中的所有小鼠达到人道终点。理想情况下,人性化的终点是当小鼠达到道德委员会批准的规定体重减轻时。此外,严重和血腥的腹泻伴随着臀部位置,皱纹毛皮和流动性下降被认为是人道端点,小鼠应通过认可的方法处死。
  4. 用DSS治疗的小鼠将失去大量的体重,因此,应每天称重以监测健康状况,并确保根据批准的道德标准,在人性化终点处死小鼠。此外,每天应监测小鼠的粪便稠度(0 =良好形成的粪便丸,2 =半形式粪便,4 =粘附于肛门区域的液体粪便)和粪便中的血液(0 =无血液,2 =血液痕迹在粪便中清晰可见,4 =总直肠出血)。记录此信息的个人应该对学习感到失明。可以绘制体重减轻,腹泻和粪便出血评分,以确定DSS诱导的结肠炎在不同基因型和/或治疗组之间的作用。作为一般指导,小鼠在DSS治疗的第6天左右开始体重减轻,直到第10天(DSS去除后3天),比初始体重减少约15-20%,继续减肥。在此期间,小鼠显示行为改变,如食欲降低,背部姿势,皱纹毛皮等。在第10天后,小鼠通常开始体重增加并达到初始体重大约14天然而,这可以根据微生物群组成,动物设施而变化,因此应该仔细解释。
  5. 在DSS(7天)和常规水(7天)的循环后,在第14天,小鼠可以根据伦理委员会(IACUC)批准的适当方法进行安乐死。应使用标尺或游标卡尺测量结肠长度,因为DSS给药导致结肠肿胀和缩短。结肠块(〜0.5cm)可以在液氮中快速冷冻,进一步加工用于RNA提取和基因表达分析。对于组织学,其余组织可以使用组织路径Macrosette处理盒在瑞士卷发并储存在10%缓冲福尔马林中。在福尔马林固定48-72小时后,将组织样品从福尔马林中取出并置于70%乙醇中直到进一步进行组织处理和包埋。通常,使用Tissue-Tek VIP处理机,然后进行石蜡包埋,可以进行组织处理过夜(表1)。


  6. 使用Vano miRNA分离试剂盒根据制造商的说明书,可以将液氮中的冷冻组织用于匀浆后的RNA提取(使用无菌杵和研钵),可用于分离总RNA以及小RNA。建议使用DNase I(QIAGEN)根据制造商的说明进一步处理提取的RNA,以获得无DNA污染的纯RNA。根据制造商的说明书,使用oligo-dT或随机六聚体引物和SuperScript逆转录酶II(Thermo Fisher Scientific)将RNA进一步转化为cDNA。在DSS结肠炎期间上调的促炎细胞因子如TNF-α和IL1-β的表达可用作DSS诱导的结肠炎的有用标记物。
  7. Fomalin固定组织可以进一步石蜡包埋,切片并安装在载玻片上用于组织学分析。对于组织学评分分析,组织切片可用苏木精和曙红(H& E)染色,并以盲法方式由小鼠病理学家评分。简而言之,0-3的分数被指定为炎症细胞的浸润,0-3被指定为组织损伤。最后,“0”的综合得分将表明健康的肠道相比,得分6,这将表明广泛的浸润和组织损伤。免疫组织化学染色免疫细胞如巨噬细胞(F4 / 80),凋亡细胞(Caspase 3),上皮结构丧失(E-钙粘蛋白)是DSS诱导的结肠炎的可靠指标。
  8. 由于与近端结肠相比,DSS的作用在远端更明显,可以分开进行近端和远端结肠的组织学评分。


  1. 适用的统计方法应适用于数据分析
  2. 对于包括生存曲线,粪便血液评分,基因表达分析和组织学图像的代表性数据,我们将读者引用到Das等人,(2015)图2,3,4和5.


  1. 执行的所有程序应由各机构的机构动物护理和使用委员会(IACUC)批准。参与本研究的动物必须受到人道待遇,并且在使用小鼠的所有情况下,疼痛和不适应最小化。
  2. 重要的是不要混合来自不同来源的小鼠,因为它们可能具有可能改变DSS诱导的结肠炎结果的不同的微生物组合物。另外,当使用经遗传修饰的小鼠时,除非正在研究特定基因型的母体贡献(例如,子宫内或垂直转移)的作用,否则建议使用同窝出生对照。
  3. DSS的分子量非常关键(分子量:35-55 kDa)


我们要感谢Batra实验室的成员进行有益的讨论。该协议的作者部分地得到了NCI Fred和Pamela Cancer Center Support Grant P30 CA036727的支持。


  1. Das,S.,Rachagani,S.,Sheinin,Y.,Smith,LM,Gurumurthy,CB,Roy,HK和Batra,SK(2015)。  缺乏Muc4的小鼠对实验性结肠炎和结肠炎相关结肠直肠癌更具抗性。癌基因 35(20):2645-2654。
  2. Mizoguchi,A.(2012)。炎症动物模型肠病。 Prog Mol Biol Transl Sci 105:263-320。
  3. Perse,M.和Cerar,A.(2012)。  葡聚糖硫酸钠结肠炎小鼠模型:陷阱和技巧生物医学生物技术 2012:718617.
  4. Sartor,RB(2006)。疾病机制:发病机制克罗恩病和溃疡性结肠炎。 Nat Clin Pract Gastroenterol Hepatol 3(7):390-407。
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引用:Das, S., Batra, S. K. and Rachagani, S. (2017). Mouse Model of Dextran Sodium Sulfate (DSS)-induced Colitis. Bio-protocol 7(16): e2515. DOI: 10.21769/BioProtoc.2515.