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Creating a Rat Model of Chronic Variate Stress

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Journal of Neuroinflammation
Mar 2014



Stress is a condition of human experience and an important factor in the onset of various diseases. There are numerous studies showing how stress can accelerate cell aging, immune senescence and some age-related diseases such as neurodegenerative disorders and osteoporosis. However, the effects of stress have different consequences depending on the type, duration or severity and predictability of the stressor applied. Although stress can be beneficial in its acute phase, repeated and severe stressful stimuli produce adverse effects. There are different models of stress depending on the exposure time; acute (when the stressor is applied for a short time, e.g. hours or days, and intensely) or chronic (when the stressor is applied for a long time, e.g. weeks or months, and less intensely. In these cases, the stressor can be repeated each time or different stressors can be used). The latter model is most frequently used to achieve similar conditions to those found in human diseases related to stress. Also, there are several different paradigms depending on the purpose of the study [development of drug therapies or modeling depressive behaviors; for the different paradigms see Dagnino-Subiabre, (2012)]. Here, we describe a 9-day variable-stressor paradigm with repeated and prolonged stimulation and a random daily stressor over days or weeks to minimize its predictability. This protocol has been adapted from other models of variable stress with significant modifications. The absence of predictability of the stressor applied is an important characteristic of this model compared to other models in which repeated stress is used. We avoid the use of a strong stressor, such as foot shock or tail pinch, and describe an easily reproducible new chronic mild stress model. Some models of chronic mild stress have been reported to lead to a wide range of behavioral disturbances and have been proposed as models of depression in animal studies (Cryan et al., 2005).

Keywords: Stress (强调), Rat (大鼠), Protocol (协议), Chronic (慢性)

Materials and Reagents

  1. Male albino Wistar rats (250-270 g)


  1. Glass tank (44 x 33 x 30 cm)
  2. Plastic tube (21 x 6 cm, 6 cm diameter)
  3. Individual cages (47 x 32 x 20 cm)
  4. Cold room or refrigerator (4 °C)


The stressors schedule used in this protocol is listed in Table 1. Application of stress starts at a different time every day to minimize its predictability (from 8:00 a.m. to 8:00 p.m.).

Table 1. Schedule of stressors used during chronic variant stress treatment
Day    Stressor              Time
-----  -----------------------    ------------
1     Forced swimming   10 min
2     Restraint                 3 h
3     Water deprivation   24 h
4     Restrain at 4 °C      90 min
5     Isolation                  24 h
6     Food deprivation     24 h
7     Water deprivation    24 h
8     Restrain at 4 °C      2 h
9     Food deprivation     24 h

Day 1: Forced swimming

  1. Fill a glass tank 22 cm deep with water at 23 ± 2 °C (Figure 1).
  2. Place the animal in the glass tank for 10 min. While the animal is swimming it may try to get out of the tank, so to prevent escape it may be necessary to carefully close it with a heavy lid.
  3. Return the animal to a clean and dry cage with fresh bedding in order to avoid chills and colds.

    Figure 1. Picture of a typical glass tank filled with cold water

    Day 2: Restraint
  1. Place the animal in a 21 x 6 cm plastic tube; adjust it with plastic tape on the outside so the animal is unable to move (Figure 2). The tube must have a 6 cm hole at the far end to allow regular breathing. To place the animal in the plastic tube, it is necessary to place the head of the animal close to the entrance, after which it should enter by itself (Figure 3).

    Figure 2. Picture of the tube with tape and animal inside

    Figure 3. Pictures showing the restraint procedure using a plastic tube

  2. Wait for 3 h. Although the plastic tube should be sufficient to prevent the animal coming out is desirable to leave the immobilizer inside the cage.
  3. Return the animal to its cage. The best way to extract the animal is to make a sudden movement downwards, dropping it into the cage. Try to avoid pulling from the tail.

    Day 3: Water deprivation
  1. Remove the bottle of water from the cage during 24 h. If the animal house is supervised by staff, indicate that the water/food must not be replaced in that cage.
  2. Place the bottle of water back after the time point is reached.

    Day 4: Restraint at 4 °C
  1. Place the animal in the above-mentioned plastic tube.
  2. Place the tube with the animal inside a cold room or a refrigerator at 4 °C for 90 min. Cold is a well-documented stressor (Krishnamurthy et al., 2011). Therefore, it is necessary to find a place with these features to put the animals in.
  3. Return the animal to its cage at room temperature.

    Day 5: Isolation
  1. Place the animal alone in a new cage.
  2. Return the animal to the cage with their mates.

    Day 6: Food deprivation
  1. Remove the food from the cage for 24 h.
  2. Place the food back.

    Day 7: Water deprivation
  1. Remove the water container from the cage for 24 h.
  2. Place the bottle of water back.

    Day 8: Restraint at 4 °C
  1. Put the animal in the plastic tube described above.
  2. Place the tube with the animal inside a cold room or a refrigerator at 4 °C for 2 h. It has been observed that after the first cold-environment exposure, 90 min at 4 °C is not enough time any more to reach adequate stress levels, that is why 2 h at 4 °C is more convenient.
  3. Return the animal to its cage at room temperature.

    Day 9: Food deprivation
  1. Remove the food from the cage for 24 h.
  2. Place the food back.

Representative data

Validation of the stress model: Changes in body and adrenal glands weight, in the blood levels of corticosterone, and in dopamine (DA) and 3,4-Dihydroxyphenylacetic acid (DOPAC) levels in the prefrontal cortex are typical effect of stress and are used as methods to assess stress models. With this chronic variant stress protocol, the body weight of the animals decreases, whereas adrenal weight and the blood levels of corticosterone increase (Figure 4). It has also been found that increased levels of dopamine and DOPAC in the prefrontal cortex are observed (Table 2).
Notes about reproducibility and variability: Stress perception is very subjective and each animal, just as each person, is stressed to a different degree. Therefore, it is normal that the results present some variability. To avoid this, glucose preference test is recommended (Hu et al., 2010); then, only animals that have been actually stressed must be included in the study.

Table 2. DA and DOPAC amount in brain cortex in control and stressed rats

Animals were killed at different time points after treatment (0, 2, 4, 8, and 10 days respectively), and the prefrontal cortex was harvested and processed for DA and DOPAC quantification by HPLC as described in de Pablos et al. (2006). Numbers are expressed as nanograms per gram of wet tissue and are Mean ± SD of five independent experiments. *p<0.05, **p<0.01, statistical significance (Student’s t test) compared with control animals.

Figure 4. A. Body weight gain (g); B. Adrenal glands weight (mg, bars) and ratio between adrenal glands weight and body weight gain (scatter plot and line). C. Serum corticosterone (percentage of control animals). Statistical signification: Student’s t test comparing before C and after 10 days of variate stress (S10d); *, p< 0.05; **, p< 0.01; #, p< 0.01 (for the ratio adrenal glands weight/body weight gain). One-way ANOVA followed by the LSD post hoc test for multiple range comparisons, p< 0.01; *, compared with the control; a, compared with the previous time point (S1d to S10d indicate the days subjected to variate stress).


Chronic-variable stress was adapted from other models of variable stress (Gamaro et al., 2003; Konarska et al., 1990; Murua and Molina, 1992; Muscat et al., 1992; Papp et al., 1991; Willner et al., 1987) with significant modifications. This work was supported by grant SAF-2012-39029 from the Spanish Ministry of Economy and Competitiveness and P10-CTS-6494 (Proyecto de Excelencia of Junta de Andalucia).


  1. Cryan, J. F. and Holmes, A. (2005). The ascent of mouse: advances in modelling human depression and anxiety. Nat Rev Drug Discov 4(9): 775-790.
  2. Dagnini-Subiabre, A. (2012). Modelos animales para el estudio del estrés y las conductas depresivas. Rev Farmacol Chile 5(1):19.
  3. de Pablos, R. M., Herrera, A. J., Espinosa-Oliva, A. M., Sarmiento, M., Munoz, M. F., Machado, A. and Venero, J. L. (2014). Chronic stress enhances microglia activation and exacerbates death of nigral dopaminergic neurons under conditions of inflammation. J Neuroinflammation 11: 34.
  4. de Pablos, R. M., Villaran, R. F., Arguelles, S., Herrera, A. J., Venero, J. L., Ayala, A., Cano, J. and Machado, A. (2006). Stress increases vulnerability to inflammation in the rat prefrontal cortex. J Neurosci 26(21): 5709-5719.
  5. Gamaro, G. D., Manoli, L. P., Torres, I. L., Silveira, R. and Dalmaz, C. (2003). Effects of chronic variate stress on feeding behavior and on monoamine levels in different rat brain structures. Neurochem Int 42(2): 107-114.
  6. Hu, H., Su, L., Xu, Y. Q., Zhang, H. and Wang, L. W. (2010). Behavioral and [F-18] fluorodeoxyglucose micro positron emission tomography imaging study in a rat chronic mild stress model of depression. Neuroscience 169(1): 171-181.
  7. Konarska, M., Stewart, R. E. and McCarty, R. (1990). Predictability of chronic intermittent stress: effects on sympathetic-adrenal medullary responses of laboratory rats. Behav Neural Biol 53(2): 231-243.
  8. Krishnamurthy, S., Garabadu, D., Reddy, N. R. and Joy, K. P. (2011). Risperidone in ultra low dose protects against stress in the rodent cold restraint model by modulating stress pathways. Neurochem Res 36(10): 1750-1758.
  9. Murua, V. S. and Molina, V. A. (1992). Effects of chronic variable stress and antidepressant drugs on behavioral inactivity during an uncontrollable stress: interaction between both treatments. Behav Neural Biol 57(1): 87-89.
  10. Muscat, R., Papp, M. and Willner, P. (1992). Reversal of stress-induced anhedonia by the atypical antidepressants, fluoxetine and maprotiline. Psychopharmacology (Berl) 109(4): 433-438.
  11. Papp, M., Willner, P. and Muscat, R. (1991). An animal model of anhedonia: attenuation of sucrose consumption and place preference conditioning by chronic unpredictable mild stress. Psychopharmacology (Berl) 104(2): 255-259.
  12. Willner, P., Towell, A., Sampson, D., Sophokleous, S. and Muscat, R. (1987). Reduction of sucrose preference by chronic unpredictable mild stress, and its restoration by a tricyclic antidepressant. Psychopharmacology (Berl) 93(3): 358-364.



关键字:强调, 大鼠, 协议, 慢性


  1. 将雄性白化Wistar大鼠(250-270g)


  1. 玻璃槽(44 x 33 x 30厘米)
  2. 塑料管(21×6cm,直径6cm)
  3. 个人笼(47 x 32 x 20厘米)
  4. 冷室或冰箱(4℃)



日    压力              时间
-----  -----------------------   ------------
1     强迫游泳 10分钟
2     约束                   3小时
3    水剥夺   24小时
4     在4°C下保持      90分钟
5    隔离                   24小时
6    食物剥夺     24小时
7     水剥夺    24小时
8    在4°C下保持      2 h
9    食物剥夺     24小时


  1. 在23±2℃下用22厘米深的水填充玻璃罐(图1)。
  2. 将动物放在玻璃罐中10分钟。 而动物是 游泳它可能会试图离开坦克,所以防止逃脱它可能 必须用厚盖子仔细关闭它
  3. 将动物放回干净,干燥的笼子里,新鲜的床上用品,以避免寒战和感冒


  1. 将动物放在21 x 6厘米的塑料管; 用塑料调整 胶带在外面,所以动物不能移动(图2)。 管子   必须在远端有一个6厘米的孔,以允许定期呼吸。 至 将动物放置在塑料管中,必须放置头部 的动物靠近入口,之后它应该进入 本身(图3)。



  2. 等待3小时。 虽然塑料管应该足够 防止动物出来是需要离开防盗器 内部。
  3. 将动物回到笼子里。 最好的方法 提取动物是使突然向下运动,放下它 进入笼子。 尽量避免从尾部拉扯。

  1. 在24小时内从笼子里取出一瓶水。 如果动物 房子是由工作人员监督,表明水/食物不能 在该笼中更换
  2. 在到达时间点后将瓶子放回去。

  1. 将动物放在上述塑料管中。
  2. 放置 管与动物在冷室或冰箱在4°C 90 min。 冷是一种良好记录的应激源(Krishnamurthy等人,2011)。 因此,有必要找到一个具有这些特征的地方 动物在。
  3. 将动物在室温下放回笼子。

  1. 将动物单独放在一个新的笼子里。
  2. 将动物送回笼子与他们的伴侣。

  1. 从笼子里取出食物24小时。
  2. 将食物放回。

  1. 从笼子中取出水箱24小时。
  2. 将瓶子的水倒回来。

  1. 将动物放在上述塑料管中。
  2. 放置 管与动物在冷室或冰箱在4°C 2 H。 已经观察到,在第一冷环境暴露之后, 在4℃下90分钟没有足够的时间来达到足够的应力 水平,这就是为什么在4℃下2小时更方便。
  3. 将动物在室温下放回笼子。

  1. 从笼子里取出食物24小时。
  2. 把食物放回去。


关于再现性和变异性的注意:压力感知是非常主观的,并且每个动物,正如每个人,被强调到不同程度。因此,结果呈现一些变化是正常的。为了避免这种情况,推荐葡萄糖偏好试验(Hu et al。,2010);那么,只有实际上受压迫的动物必须包括在研究中。


在处理后的不同时间点(分别为0,2,4,8和10天)处死动物,收获前额皮质,并通过HPLC进行DAOP和DOPAC定量,如de Pablos et al。 (2006)。数字表示为每克湿织物的纳克数,并且是五次独立实验的平均值±SD。与对照动物相比,* p <0.05,** p <0.01,统计学显着性(Student's t检验)。

图4. A。体重增加(g); B.肾上腺重量(mg,条)和肾上腺重量和体重增加之间的比率(散点图和线)。 C.血清皮质酮(对照动物的百分比)。统计意义:在C和10天变异应激(S10d)之间比较的Student's 试验; *,p < 0.05; **,p < 0.01; #,p < 0.01(对于肾上腺重量/体重增加的比率)。单因素方差分析,随后是多范围比较的LSD事后检验,p < 0.01; *,与对照相比; a,与先前时间点(S1d至S10d指示经受变化应力的天数)相比。


慢性可变应激来自其他可变应激模型(Gamaro等人,2003; Konarska等人,1990; Murua和Molina,1992; Muscat et al。,1992; Papp et al。,1991; Willner et al。,1987),其具有显着的修饰。这项工作得到了来自西班牙经济和竞争力部的SAF-2012-39029和P10-CTS-6494(安达卢西亚安达卢西亚的Proyecto de Excelencia)的支持。


  1. Cryan,J.F。和Holmes,A。(2005)。 鼠标的上升:建模人类抑郁症和焦虑的进展。 Rev Drug Discov 4(9):775-790。
  2. Dagnini-Subiabre,A。(2012)。 Modelos animales para el estudio delestrésy las conductas depresivas。 < em> Rev Farmacol Chile 5(1):19。
  3. de Pablos,R.M.,Herrera,A.J.,Espinosa-Oliva,A.M.,Sarmiento,M.,Munoz,M.F.,Machado,A.and Venero,J.L。(2014)。 慢性应激增强小胶质细胞活化,并加重黑曲霉多巴胺能神经元在炎症条件下的死亡。 J Neuroinflammation 1 1:34.
  4. de Pablos,R.M.,Villaran,R.F.,Arguelles,S.,Herrera,A.J.,Venero,J.L.,Ayala,A.,Cano,J.and Machado,A。(2006)。 压力增加了大鼠前额叶皮层炎症的易感性。 J Neurosci 26(21):5709-5719。
  5. Gamaro,G.D.,Manoli,L.P.,Torres,I.L.,Silveira,R。和Dalmaz,C。(2003)。 慢性变异压力对不同大鼠脑结构中的摄食行为和单胺水平的影响。 Neurochem Int 42(2):107-114。
  6. Hu,H.,Su,L.,Xu,Y.Q.,Zhang,H.and Wang,L.W。(2010)。 在大鼠慢性轻度应激模型中的行为和[F-18]氟脱氧葡萄糖微正电子发射断层扫描成像研究 Neuroscience 169(1):171-181。
  7. Konarska,M.,Stewart,R.E.and McCarty,R。(1990)。 慢性间歇性压力的可预测性:对实验室大鼠的交感神经肾上腺髓质反应的影响。 Behav Neural Biol 53(2):231-243。
  8. Krishnamurthy,S.,Garabadu,D.,Reddy,N.R。和Joy,K.P。(2011)。 超低剂量的利培酮通过调节应激途径来保护啮齿动物冷冻抑制模型中的应激。 a> Neurochem Res 36(10):1750-1758。
  9. Murua,V.S.and Molina,V.A。(1992)。 慢性可变应激和抗抑郁药物对不可控压力下的行为不活动的影响:两种治疗之间的相互作用。 Behav Neural Biol 57(1):87-89
  10. Muscat,R.,Papp,M。和Willner,P。(1992)。 通过非典型抗抑郁药,氟西汀和马普替林来逆转压力诱导的快感缺乏。 Psychopharmacology(Berl) 109(4):433-438。
  11. Papp,M.,Willner,P。和Muscat,R。(1991)。 快感的动物模型:通过慢性不可预测的轻度压力减轻蔗糖消耗和地方偏好调节。/a> Psychopharmacology(Berl) 104(2):255-259。
  12. Willner,P.,Towell,A.,Sampson,D.,Sophokleous,S.and Muscat,R。(1987)。 通过慢性不可预测的轻度压力降低蔗糖偏好,并通过三环抗抑郁药恢复。 Psychopharmacology(Berl) 93(3):358-364。
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引用:de Pablos, R. M., Sarmiento, M. and Espinosa-Oliva, A. M. (2014). Creating a Rat Model of Chronic Variate Stress. Bio-protocol 4(23): e1315. DOI: 10.21769/BioProtoc.1315.