An Experimental Model of Neonatal Nociceptive Stimulation in Rats

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Apr 2014



In order to survive, preterm and/or sick neonates need diagnostic and therapeutic measures that may cause discomfort, stress and pain during a critical period of intense growing and modeling of the central nervous system (Anand et al., 2013). Scientific interest in the long lasting effects of the Neonatal Intensive Care (NIC) experience, which provides a sensory experience completely different from the natural uterine environment, is growing (Jobe, 2014). The follow-up of critically ill newborn infants until adulthood indicated an association of early noxious stimuli with long lasting alterations in somatosensory and cognitive processing (Doesburg, 2013; Vinall et al., 2014; Vinall et al., 2013). However, one major limitation of the clinical studies is the difficulty to distinguish between long-term effects of pain suffered during neonatal intensive care and other confounding factors such as the presence of non-painful stress during hospital stay, the occurrence of acute and chronic morbidities, the post-natal environmental influences and family care. In this context, the understanding of the roles played by each factor and the interplay between these diverse variables require the use of animal models. The protocol described here is used to model the noxious stimulation in which premature newborns are subjected during treatment in the NIC. The current protocol models inflammatory nociceptive stimulation in neonatal rats, as previously demonstrated (Leslie et al., 2011; Lima et al., 2014; Malheiros et al., 2014). Complete Freund's adjuvant (CFA) is a solution of antigen emulsified in mineral oil and used as an immunopotentiator, causing a painful reaction that lasts 7-8 days after subcutaneous injection. It is effective in stimulating cell-mediated immunity. The rodent model of neonatal inflammatory stimulation with CFA is advantageous because at birth the formation of the central nervous system is incomplete in rat pups and corresponds to that of 24 week intra-uterine human preterm neonates (Anand et al., 1999), following similar patterns in the development of the pain system (Fitzgerald and Anand,1993). The first postnatal week in newborn rat pups corresponds to human premature infants from 24-36 weeks of gestation (Kim et al., 1996; Wilson, 1995), offering a suitable condition to model and compare preterm (rat pups on P1) to full term (rat pups on P8) infants subjected to noxious stimulation. In this paper, we present our methods to induce nociceptive inflammatory stimulation in neonatal rat pups as an attractive approach to study short- or long-term effects and the mechanisms underlying the behavioral repertoire of ex-premature infants or adolescents.

Keywords: Pain (疼痛), Neonatal pain (新生儿疼痛), Development (发展), Neurogenesis (神经发生), CFA (CFA)

Materials and Reagents

  1. Pregnant Wistar rats are acquired around the 14th day of gestation
    Note: It is important to determine the P0 (postnatal day 0).
  2. Complete Freund’s adjuvant (CFA) (25 µl) (Sigma-Aldrich, catalog number: F5881 )
    Note: The CFA is diluted in 0,9% sterile saline just before use (2:1, CFA:saline).


  1. Plastic cages (for maintaining the animals)
  2. Automatic temperature control system
  3. Insulin Syringe Ultra-Fine 6 mm (15/64”) x 31 G needle
  4. Electronic scale (sensitivity 0.1 g)


Ethical statement: All procedures discussed here are in accordance with and were approved by the Independent Ethics Committee of the Universidade Federal de São Paulo.

  1. Obtain approval from local ethics committee.
  2. Pregnant Wistar rats from the 14th day of gestation have to be housed individually in plastic cages and placed in the animal facility, equipped with an automatic temperature control system (23 ± 2 ºC), ventilation, a 12-h light-dark cycle (lights on at 7:00 AM) and unrestricted access to food and water.
  3. The day of birth is designated as P0.
  4. Each litter is restricted to 8 pups within 24 h after birth, excluding those pups with body weights lower than 0.5 g.
  5. From P1 (postnatal day one) on, animals from all litters have their body weight recorded on a weekly basis, with an electronic scale (sensitivity 0.1 g), until the end of the experimental protocol.
  6. To compare the effects of CFA injection during neural development, on postnatal days 1, 8 or 21 rat pups receive a single intraplantar subcutaneous injection of an inflammatory agent, the complete Freund’s adjuvant (CFA; 25 µl) into the left hindpaw.
    Note: Take care to avoid spending more than 2 min with each pup. This would cause higher stress levels in the dam.
  7. All animals in a single litter are assigned to a particular group (CFA-stimulated at P1, P8, P21 or control), to avoid the influence that handling of pups at different ages could have on maternal care towards the remainder of the litter.
  8. After CFA injection, the pups return to their home-cage with their dams.
  9. As a result, paws of stimulated pups show swelling, edema and redness, starting a few hours after injection, lasting 5-7 days. The edema can be visually observed, as in Figure 1.
  10. Control animals are subjected to needle insertion like the experimental pups, but without fluid infusion.
  11. All litters are weaned on P21, and from then on, housed in same-sex groups of 5 animals of the same experimental group at most.
  12. To avoid litter effects, pups from different litter were randomly assigned to different groups.

    Figure 1. In A the intraplantar subcutaneous injection of CFA on postnatal day 1. Just after the injection B the bleaching and swelling under the skin of the left paw is visible. At day 3 C and day 6 D after CFA injection, the left paw is still swollen (red arrow) compared with the right paw.


All procedures are performed before 10 AM when the levels of corticosterone are low.


This work was supported by FAPESP, by a grant to LC (09/53646-0).


  1. Anand, K. J., Coskun, V., Thrivikraman, K. V., Nemeroff, C. B. and Plotsky, P. M. (1999). Long-term behavioral effects of repetitive pain in neonatal rat pups. Physiol Behav 66(4): 627-637.
  2. Anand, K. J., Palmer, F. B. and Papanicolaou, A. C. (2013). Repetitive neonatal pain and neurocognitive abilities in ex-preterm children. Pain 154(10): 1899-1901.
  3. Doesburg, S. M., Chau, C. M., Cheung, T. P., Moiseev, A., Ribary, U., Herdman, A. T., Miller, S. P., Cepeda, I. L., Synnes, A. and Grunau, R. E. (2013). Neonatal pain-related stress, functional cortical activity and visual-perceptual abilities in school-age children born at extremely low gestational age. Pain 154(10): 1946-1952.
  4. Fitzgerald, M. and Anand, K. (1993). The developmental neuroanantomy and neurophysiology of pain. In: Schechter, N., Berde, C. and Yaster, M. (eds). Pain management in infants, children and adolescents. Baltimore: Williams & Williams, pp. 11-32.
  5. Jobe, A. H. (2014). A risk of sensory deprivation in the neonatal intensive care unit. J Pediatr 164(6): 1265-1267.
  6. Kim, J. J., Foy, M. R. and Thompson, R. F. (1996). Behavioral stress modifies hippocampal plasticity through N-methyl-D-aspartate receptor activation. Proc Natl Acad Sci U S A 93(10): 4750-4753.
  7. Leslie, A. T., Akers, K. G., Martinez-Canabal, A., Mello, L. E., Covolan, L. and Guinsburg, R. (2011). Neonatal inflammatory pain increases hippocampal neurogenesis in rat pups. Neurosci Lett 501(2): 78-82.
  8. Lima, M., Malheiros, J., Negrigo, A., Tescarollo, F., Medeiros, M., Suchecki, D., Tannus, A., Guinsburg, R. and Covolan, L. (2014). Sex-related long-term behavioral and hippocampal cellular alterations after nociceptive stimulation throughout postnatal development in rats. Neuropharmacology 77: 268-276.
  9. Malheiros, J. M., Lima, M., Avanzi, R. D., Gomes da Silva, S., Suchecki, D., Guinsburg, R. and Covolan, L. (2014). Repetitive noxious neonatal stimuli increases dentate gyrus cell proliferation and hippocampal brain-derived neurotrophic factor levels. Hippocampus 24(4): 415-423.
  10. Vinall, J., Miller, S. P., Bjornson, B. H., Fitzpatrick, K. P., Poskitt, K. J., Brant, R., Synnes, A. R., Cepeda, I. L. and Grunau, R. E. (2014). Invasive procedures in preterm children: brain and cognitive development at school age. Pediatrics 133(3): 412-421.
  11. Vinall, J., Miller, S. P., Synnes, A. R. and Grunau, R. E. (2013). Parent behaviors moderate the relationship between neonatal pain and internalizing behaviors at 18 months corrected age in children born very prematurely. Pain 154(9): 1831-1839.
  12. Wilson, D. A. (1995). NMDA receptors mediate expression of one form of functional plasticity induced by olfactory deprivation. Brain Res 677(2): 238-242.


为了存活,早产和/或生病的新生儿需要诊断和治疗措施,其可能在中枢神经系统的强烈生长和建模的关键时期期间引起不适,压力和疼痛(Anand等人 ,2013)。科学兴趣的新生儿重症监护(NIC)经验的长期影响,提供了一个完全不同于自然子宫环境的感觉经验,越来越多(Jobe,2014)。直到成年的危重新生儿的随访表明早期有害刺激与体长感觉和认知加工中的持久改变的关联(Doesburg,2013; Vinall等人,2014; Vinall等人, et al。,2013)。然而,临床研究的一个主要限制是难以区分在新生儿重症监护期间遭受的疼痛的长期影响和其他混杂因素,例如在住院期间存在非痛性压力,发生急性和慢性病态,产后环境影响和家庭护理。在这种情况下,理解每个因素所起的作用以及这些不同变量之间的相互作用需要使用动物模型。这里描述的协议用于模拟早产新生儿在NIC治疗期间遭受的有害刺激。目前的方案模拟了新生大鼠的炎性痛觉刺激,如先前所证实的(Leslie等人,2011; Lima等人,2014; Malheiros等人, ,2014)。完全弗氏佐剂(CFA)是在矿物油中乳化的抗原的溶液,用作免疫增强剂,引起皮下注射后持续7-8天的疼痛反应。它在刺激细胞介导的免疫中是有效的。使用CFA的新生儿炎症刺激的啮齿动物模型是有利的,因为出生时中枢神经系统的形成在大鼠幼仔中是不完全的并且对应于24周子宫内人早产新生儿的形成(Anand等人, >,1999),在疼痛系统的发展中遵循类似的模式(Fitzgerald和Anand,1993)。新生大鼠幼仔中的第一个出生后的周对应于来自妊娠24-36周的人早产儿(Kim等人,1996; Wilson,1995),提供了合适的条件来建模和比较早产大鼠幼仔在P1上)至完全期(在P8上的大鼠幼鼠)进行有害刺激。在本文中,我们提出我们的方法诱导伤害性炎症刺激新生大鼠幼崽作为一个有吸引力的方法来研究短期或长期的影响和基础的早产儿或青少年的行为汇辑的机制。

关键字:疼痛, 新生儿疼痛, 发展, 神经发生, CFA


  1. 怀孕Wistar大鼠是在妊娠14天左右获得的 注意:确定P0(出生后第0天)很重要。
  2. 完全弗氏佐剂(CFA)(25μl)(Sigma-Aldrich,目录号:F5881)


  1. 塑料笼(用于保持动物)
  2. 自动温度控制系统
  3. 胰岛素注射器超细6 mm(15/64")x 31 G针
  4. 电子秤(灵敏度0.1 g)



  1. 获得当地伦理委员会的批准。
  2. 来自妊娠第14天的怀孕Wistar大鼠必须单独饲养在塑料笼中并放置在配备有自动温度控制系统(23±2℃),通风,12 -h明暗循环(上午7:00点亮),并且不受限制地进入食物和水
  3. 出生日期指定为P0。
  4. 每只窝在出生后24小时内限制为8只幼仔,不包括体重低于0.5g的幼犬。
  5. 从P1(出生后第一天)开始,来自所有窝的动物每周用电子秤(灵敏度0.1g)记录体重,直到实验方案结束。
  6. 为了比较在神经发育期间CFA注射的效果,在出生后第1,8或21天,幼鼠接受单次足底皮下注射炎性试剂,即完全弗氏佐剂(CFA;25μl)到左后爪。 > 注意:注意避免每只小狗花费超过2分钟。这将导致坝中的应力水平更高。
  7. 将单个垫料中的所有动物分配到特定的组(在P1,P8,P21或对照中CFA刺激),以避免处理不同年龄的幼仔对于对其余的垫料的母体护理的影响。
  8. 在注射CFA后,幼仔与他们的大坝回到他们的家笼。
  9. 结果,刺激的小狗的爪子在注射后几小时开始显示肿胀,水肿和发红,持续5-7天。 水肿可以在视觉上观察到,如图1所示。
  10. 对照动物像实验幼崽一样进行针刺,但没有流体输注
  11. 所有的仔猪在P21断奶,然后从同一个实验组的5只动物的相同性别组。
  12. 为了避免凋落物的影响,来自不同垃圾的幼仔被随机分配到不同的组

    图1.在A中,在出生后第1天皮内注射CFA。刚注射后,左爪子皮肤下的漂白和肿胀是可见的。 在注射CFA后的第3天和第6天,与右爪相比,左爪仍然肿胀(红色箭头)。






  1. Anand,K.J.,Coskun,V.,Thrivikraman,K.V.,Nemeroff,C.B.and Plotsky,P.M。(1999)。 新生大鼠幼仔中重复性疼痛的长期行为影响生理Behav 66(4):627-637。
  2. Anand,K.J.,Palmer,F.B.and Papanicolaou,A.C。(2013)。 重复的新生儿疼痛和早产儿儿的神经认知能力。 /em> 154(10):1899-1901。
  3. Doburg,S.M.,Chau,C.M.,Cheung,T.P.,Moiseev,A.,Ribary,U.,Herdman,A.T.,Miller,S.P.,Cepeda,I.L.,Synnes,A.and Grunau, 新生儿疼痛相关的压力,功能性皮质活动和视觉感知能力在学龄儿童出生在极低的胎龄。疼痛 154(10):1946-1952。
  4. Fitzgerald,M。和Anand,K。(1993)。疼痛的发展神经切开术和神经生理学。 In:Schechter,N.,Berde,C.and Yaster,M。(eds)。 婴儿,儿童和青少年的疼痛管理。巴尔的摩:威廉姆斯& Williams,pp。11-32。
  5. Jobe,A. H.(2014)。 新生儿重症监护病房感觉剥夺的风险 164(6):1265-1267。
  6. Kim,J.J.,Foy,M.R.and Thompson,R.F。(1996)。 行为压力通过N-甲基-D-天冬氨酸受体激活修饰海马的可塑性。 em> Proc Natl Acad Sci USA 93(10):4750-4753。
  7. Leslie,A.T.,Akers,K.G.,Martinez-Canabal,A.,Mello,L.E.,Covolan,L.and Guinsburg,R。(2011)。 新生儿炎症性疼痛增加大鼠幼崽的海马神经发生。 Neurosci Lett < em> 501(2):78-82。
  8. Lima,M.,Malheiros,J.,Negrigo,A.,Tescarollo,F.,Medeiros,M.,Suchecki,D.,Tannus,A.,Guinsburg,R.and Covolan, 性别相关的长期行为和海马细胞改变后伤害性刺激整个产后发育大鼠。/a> Neuropharmacology 77:268-276。
  9. Malheiros,J.M.,Lima,M.,Avanzi,R.D.,Gomes da Silva,S.,Suchecki,D.,Guinsburg,R.and Covolan,L.(2014)。 重复的有害的新生儿刺激增加了齿状回脑细胞增殖和海马脑源性神经营养因子水平。 Hippocampus 24(4):415-423。
  10. Vinall,J.,Miller,S.P.,Bjornson,B.H.,Fitzpatrick,K.P.,Poskitt,K.J.,Brant,R.,Synnes,A.R.,Cepeda,I.L.and Grunau,R.E。(2014)。 早产儿的侵入性程序:学龄期的大脑和认知发展。 em> Pediatrics 133(3):412-421。
  11. Vinall,J.,Miller,S.P.,Synnes,A.R.and Grunau,R.E。(2013)。 父母的行为在出生非常早的儿童中矫正18个月矫正年龄的新生儿疼痛和内在化行为之间的关系。 疼痛 154(9):1831-1839。
  12. Wilson,D.A。(1995)。 NMDA受体介导由嗅觉剥夺诱发的一种形式的功能可塑性的表达。 Brain Res 677(2):238-242。
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引用:Malheiros, J. M., Amaral, C., Leslie, A. T., Guinsburg, R. and Covolan, L. (2014). An Experimental Model of Neonatal Nociceptive Stimulation in Rats. Bio-protocol 4(21): e1283. DOI: 10.21769/BioProtoc.1283.