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Locomotor Coordination Assay in Rats
大鼠中运动器官协调性的试验

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Abstract

Neonatal hypoxia-ischemia (HI) affects 60% of low birth weight infants and up to 40% of preterm births. Cell death and brain injury after HI have been shown to cause long-lasting neurological deficits. Two motor coordination tests on rats that had been exposed to HI on postnatal day 7 (P7) showed that HI in the P7 rat is associated with significant motor coordination impairment. These results call attention to the risks associated with perinatal ischemia and the need for proper treatment to reverse HI-induced deleterious effects.

Keywords: Motor coordination(运动协调), Balance(平衡), Inclined plane(斜面), Rat(大鼠), Hypoxia(缺氧)

Materials and Reagents

  1. Rats

Equipment

Note: Both rod and incline wire mesh are self-made from supplies bought at hardware store.

  1. Rod (30.0 cm L x 1.0 cm)
  2. Wire mesh (69.5 cm W x 45 cm L with 3 cm gaps)

Procedure

Motor coordination is assessed 21 days (P28) after HI insult [see Vannucci et al. (1999) for a description of HI protocol] as modified by us (Ferrari et al., 2010). Briefly, pregnant Wistar rat dams at gestational age E17 are housed in 12 h light-dark cycle with ad libitum access to food and water. The day of birth is postnatal day 0 (P0) and on P1 the litters are culled to 10 pups per dam and randomly mixed amongst dams to yield 20 pups per experiment. On day P7 all pups are removed from dams, weighed, sexed and randomly assigned to a group: sham- or HI-treated. They are then anesthetized and the left carotid artery isolated and permanently ligated. Sham pups are only anesthetized, an incision is made, immediately sutured and the wound cleaned without any further manipulation. Upon recovery, pups are returned to dams for 90 min and then they are removed and exposed to 8% oxygen for 90 min at which point they are returned to dams.

  1. Bar holding test and wire mesh ascending test
    At P21 the pups are weaned and randomly separated into 5 pups per cage. All behavioral training and testing is carried between 8 am and 4 pm, and pups are brought to the testing room at least 30 min prior to the motor evaluations in order to minimize stress-related confounds. Each pup is trained 5 times per day for 3 consecutive days prior to each one of the two tests. On the day of testing, each test is performed with five separate trials per animal with a resting time of 5 min between trials. For the bar holding test, the pups spend their resting time in their home cage and for the wire mesh ascending test, the pups spend their resting time in the enclosed platform at the top of the mesh. All pups are placed back in their original cages immediately after each training and testing event.

  2. Bar holding test
    As previously described by Tchekalarova et al. (2005) and slightly modified by Ferrari et al. (2010) and Infante et al. (2013), pups are allowed to grasp a wooden bar of 1cm in diameter and 30 cm long that is suspended 50 cm above a padded soft surface. The time spent on the bar grasping bar with forelimbs is measured for a maximum of 120 sec. Results shown are an average of the 5 recordings per pup (Sham, n=11; HI, n=23). This assay addresses motor coordination and function impairment.

  3. Wire mesh ascending test
    As previously described (Tchekalarova et al., 2005; Thonhoff et al., 2007; Ferrari et al., 2010; Infante et al., 2013), a pup is placed at the bottom of a mesh and the time that it takes a pup to ascend the mesh is measured for up to 120 sec. The 1 cm thick plastic mesh is 45 cm long and 15 cm wide and is placed at an angle of 70° in contact with an elevated enclosed platform at the top and a padded soft surface at the bottom. As a stimulus to ascend, the littermates are placed on the enclosed platform above and allowed to acclimatize to the surroundings for 5 min before testing. Results shown are an average of the 5 recordings per pup (Sham, n=19; HI, n=25). This assay addresses both motor coordination impairment and social drive.

Representative data


Figure 1. Bar holding test


Figure 2. Wire mesh ascending test


Figure 3. HI impairs motor coordination (Ferrari et al., 2010)

Notes

  1. As with all behavioral experiments animals must be handled daily by the single individual performing the evaluations. The testing room must be quiet and only the individual performing the evaluations should be in the room.
  2. When doing the wire mesh ascending test, it is vital that there be siblings at the top to provide motivation in order to get more homogenous results.

Acknowledgments

This work was supported by Department of Defense, NIEHS and NICHD.

References

  1. Ferrari, D. C., Nesic, O. B. and Perez-Polo, J. R. (2010). Oxygen resuscitation does not ameliorate neonatal hypoxia/ischemia-induced cerebral edema. J Neurosci Res 88(9): 2056-2065.
  2. Infante, S. K., Rea, H. C. and Perez-Polo, J. R. (2013). Transgenerational effects of neonatal hypoxia-ischemia in progeny. Int J Dev Neurosci 31(6): 398-405.
  3. Tchekalarova, J., Kubova, H. and Mares, P. (2005). Postnatal caffeine exposure: effects on motor skills and locomotor activity during ontogenesis. Behav Brain Res 160(1): 99-106.
  4. Thonhoff, J. R., Jordan, P. M., Karam, J. R., Bassett, B. L. and Wu, P. (2007). Identification of early disease progression in an ALS rat model. Neurosci Lett 415(3): 264-268.
  5. Vannucci, R. C., Connor, J. R., Mauger, D. T., Palmer, C., Smith, M. B., Towfighi, J. and Vannucci, S. J. (1999). Rat model of perinatal hypoxic-ischemic brain damage. J Neurosci Res 55(2): 158-163.

简介

新生儿缺氧缺血(HI)影响60%的低出生体重婴儿和高达40%的早产。 HI后的细胞死亡和脑损伤已显示引起持久的神经缺陷。 对于在出生后第7天(P7)暴露于HI的大鼠的两个运动协调测试显示P7大鼠中的HI与显着的运动协调障碍相关。 这些结果提醒注意与围产期缺血相关的风险和需要适当的治疗以逆转HI诱导的有害影响。

关键字:运动协调, 平衡, 斜面, 大鼠, 缺氧

材料和试剂

  1. 大鼠

设备

注意:ro d和倾斜线网是由在五金店购买的材料自制的。

  1. 杆(30.0cm L×1.0cm)
  2. 丝网(69.5cm W×45cm L,3cm间隙)

程序

运动协调在HI损伤后评估21天(P28)[参见Vannucci等人(1999)关于由我们修改的HI方案的描述](Ferrari等人 >,2010)。简而言之,将怀孕的Wistar大鼠水坝(处于胎龄E17)置于12小时光照 - 黑暗循环中,随意获取食物和水。出生日是出生后第0天(P0),并且在P1上,将幼仔从每个水坝中剔除至10只幼崽,并在水坝中随机混合,每个实验产生20只幼崽。在第P7天,将所有幼仔从大坝中移出,称重,性别并随机分配到一组:假手术或HI-处理的。然后将它们麻醉,分离左颈动脉并永久结扎。假手术只麻醉,切口,立即缝合和伤口清洁没有任何进一步的操作。恢复后,幼崽返回大坝90分钟,然后将它们移除并暴露于8%氧气90分钟,此时它们返回坝。

  1. 试棒保持试验和丝网上升试验
    在P21,将幼仔断奶并随机分成每笼5只幼仔。所有行为训练和测试在上午8点和下午4点之间进行,并且幼崽在运动评估之前至少30分钟被带到测试室以最小化应激相关的混杂。每个小狗在两次测试中的每一次之前连续3天每天训练5次。在测试当天,每个动物进行五个独立试验的每个试验,试验之间的休息时间为5分钟。对于酒吧保持测试,幼仔在他们的家笼中花费他们的休息时间,并且对于丝网上升测试,幼崽花费其在网格顶部的封闭平台中的休息时间。所有幼犬在每次训练和测试后立即放回原来的笼子里。

  2. 酒吧举行测试
    如先前由Tchekalarova等人(2005)所述,并且由Ferrari等人(2010)和Infante等人(2013年)稍微修改的(2013年) ),允许幼崽抓住直径为1cm并且长30cm的木条,其悬挂在填充的软表面上方50cm处。测量在具有前肢的棒夹持棒上花费的时间最大为120秒。显示的结果是每只幼仔的5次记录的平均值(Sham,n = 11; HI,n = 23)。该试验解决运动协调和功能障碍
  3. 丝网上升测试
    如前所述(Tchekalarova等人,2005; Thonhoff等人,2007; Ferrari等人,2010; Infante等人, et al。,2013),将小狗放置在网格的底部,并且测量小狗上升网格的时间长达120秒。 1cm厚的塑料网是45cm长和15cm宽,并且以70°的角度放置,与顶部的升高的封闭平台接触,并且在底部具有填充的软表面。作为上升的刺激,同窝幼仔放置在上面的封闭平台上,并允许适应环境5分钟,然后测试。显示的结果是每只幼仔的5次记录的平均值(Sham,n = 19; HI,n = 25)。该试验解决运动协调障碍和社会驱动力

代表数据


图1.栏持有测试


图2. 丝网上升测试


图3. HI损害运动协调(Ferrari 等人 > 2010)

笔记

  1. 与所有行为实验一样,动物必须由执行评估的单个人每天处理。测试室必须安静,只有进行评估的个人才能在房间内
  2. 当进行丝网上升测试时,至关重要的是在顶部有兄弟姐妹,以提供动机,以获得更均匀的结果。

致谢

这项工作得到国防部,国家卫生研究院和NICHD的支持。

参考文献

  1. Ferrari,D.C.,Nesic,O.B.and Perez-Polo,J.R。(2010)。 氧气复苏不能改善新生儿缺氧/缺血引起的脑水肿。 J Neurosci Res 88(9):2056-2065。
  2. Infante,S.K.,Rea,H.C.和Perez-Polo,J.R。(2013)。 新生儿缺氧缺血在子代中的跨代效应。 Int J Dev Neurosci 31(6):398-405。
  3. Tchekalarova,J.,Kubova,H。和Mares,P。(2005)。 产后咖啡因暴露:对发育过程中运动技能和运动活动的影响 Behav Brain Res 160(1):99-106。
  4. Thonhoff,J.R.,Jordan,P.M.,Karam,J.R.,Bassett,B.L.and Wu,P。(2007)。 ALS大鼠模型中早期疾病进展的鉴定 Neurosci Lett 415(3):264-268。
  5. Vannucci,R.C.,Connor,J.R.,Mauger,D.T.,Palmer,C.,Smith,M.B.,Towfighi,J。和Vannucci,S.J。(1999)。 围产期缺氧缺血性脑损伤的大鼠模型 J Neurosci Res 55(2):158-163。
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引用:Perez-Polo, J., Rea, H. C. and Infante, S. K. (2015). Locomotor Coordination Assay in Rats. Bio-protocol 5(22): e1655. DOI: 10.21769/BioProtoc.1655.
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